Silva Andrea 2500D

UNIVERSIDAD DE LA FRONTERA
Facultad de Ingeniería, Ciencias y Administración

Programa de Doctorado y Magíster en Ciencias de Recursos Naturales
Development of edible coatings and films based on

hydrocolloid blends with polyphenol-rich extract: rheology
and functionality
Doctoral Thesis
In Fulfillment of the
Requirements for the Degree
Doctor of Sciences in Natural Resources
by
Andrea Cristina Silva Weiss
TEMUCO – CHILE
2012
Supervisor: Professor Mg. Valerio Bifani Cosentini
Department of Chemical Engineering
Universidad de La Frontera - UFRO
Temuco, Chile.
Reviewers: Dr. Carmen Gómez Guillén

Instituto de Ciencia y Tecnología de Alimentos y Nutrición- ICTAN
Consejo Superior de Investigaciones Científicas - CSIC.
Madrid, Spain.
Professor Dr. Carolina Shene de Vidts

Center of Food Biotechnology and Bioseparations
Temuco, Chile.
Professor Dr. Ma Cristina Diez Jerez

Environmental Biotechnology Center
Scientifical and Technological Bioresource Nucleus - BIOREN
Temuco, Chile.
Professor Dr. Erick Scheuermann Salinas

Temuco, Chile.
Dr. Paulo José do Amaral Sobral

Food Engineering Department
University of São Paulo- USP
Pirassununga, SP, Brazil.
Development of edible coatings and films based on hydrocolloid
blends with polyphenol-rich extract: rheology and functionality
Esta tesis fue realizada por Andrea Silva Weiss, bajo la supervisión del Profesor Valerio Bifani
Cosentini, del Departamento de Ingeniería Química de la Universidad de La Frontera.
Tesis presentada como parte de los requisitos para optar al grado de
Doctor en Ciencias de Recursos Naturales, aprobada por el COMITÉ DE TESIS:
Dr. Francisco Matus Baeza Prof. Mg. Valerio Bifani Cosentini

Director del Programa de Doctorado en Supervisor
Ciencias de Recursos Naturales
Dra. Carmen Gómez Guillén
Dirección de Postgrado Prof. Dra. Carolina Shene de Vidts

Universidad de La Frontera
Prof. Dra. Ma Cristina Diez Jerez
Prof. Dr. Erick Sheuermann Salinas
Dr. Paulo José do Amaral Sobral

Y así me di cuenta que el éxito no es personal, es regido por la filosofía Ubuntu.
Hay que ver las señales, descubrir a las personas que nos envía la vida, aprender de ellas,
cultivarlas y entregarles lo mejor que tenemos. Solo así las recompensas llegan al corazón.
Le dedico esta tesis de grado a todos los ángeles que están y han dejando huellas en mi camino.
Esas almas que he descubierto y que me han "visto", seres únicos, que valoro y quiero.
AGRADECIMIENTOS / ACKNOWLEDGEMENTS
El desarrollo de esta tesis fue posible principalmente gracias al financiamiento de la Comisión

Nacional Científica y Tecnológica (CONICYT) del Gobierno de Chile: Beca de doctorado D-
21070302, beca de apoyo a la realización de tesis doctoral AT-24090134; beca de asistencia a
cursos cortos en el extranjero 29090088, beca de estadía de investigación en el extranjero
75100006; beca de término de tesis doctoral 23110089 y beca de participación en reuniones de
sociedades científicas.
Agradezco además:
Al Programa de Doctorado en Ciencias de Recursos Naturales y a la Dirección de Postgrado de la

Universidad de La Frontera, por la contribución brindada para asistir a congresos.
Al proyecto INNOVA-CORFO 06CN12PAT-57 del profesor Valerio Bifani, que permitió

solventar parte de los experimentos de esta tesis, así como mostrar los resultados obtenidos en
congresos nacionales e internacionales.
A la comisión FULBRIGHT por haberme otorgado la beca de pasantía doctoral, Fulbright

Scholarship Program., reconocer mi trabajo y permitirme participar del 2011 Fulbright
Enrichment Seminar: "Greening of the planet: Global Challenges, Local solutions". New York,
NY April 14 - 17, 2011.
A los Fulbrighters que me acogieron como familia en la estadía en Estados Unidos. Gracias
Goma Mavika, Mr. President!.
A la Dra. María de la Luz Mora, por darme la oportunidad de participar de este programa de
doctorado; poder desarrollarme dentro de este periodo, aconsejarme, comprenderme y apoyarme
en momentos difíciles y confiar en mí.
i
A mi supervisor tutor de tesis, Profesor Valerio Bifani Cosentini, por su apoyo, comprensión y la
oportunidad de participar en su proyecto INNOVA-CORFO 06CN12PAT-57. Además, destacar
la importante participación en esta tesis de la Profesora Mónica Ihl Piel y expresarle muy
sentidamente lo importante que ha sido su apoyo; sus palabras precisas, sus llamados de atención,
sus correcciones de inglés, y sobretodo destacar la calidad humana que me ha brindado, junto a
mi tutor, desde que los conozco. Aprovecho de felicitarlos y destacar su larga y comprometida
trayectoria como profesores de la Universidad de La Frontera.
Agradezco a los miembros de la comisión evaluadora de tesis de la Universidad de La Frontera,

Dra. Cristina Diez y Dra. Carolina Shene, así como al Dr. Erich Scheuermann por sus críticas
constructivas y aportes en los avances de tesis. En particular, agradezco el apoyo y aporte
contante, así como las recomendaciones de mi trabajo entregadas por la Dra. Carmen Gómez, del
Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN), España y el Dr. Paulo
Sobral, de la University of São Paulo (USP), Brasil; quienes siempre me dieron palabras de
aliento, me aconsejaron y guiaron en este camino.
My gratefully to Dr. Gustavo Barbosa-Cánovas and his research groups for give me the
opportunity to do a research stay in the Center of Nonthermal Processing of Food, Department
of Biological Systems Engineering and work in Pilot Plant of Food Science and Human Nutrition
Building of Washington State University. Pullman, WA, USA.
Mi reconocimiento al Profesor Fernando Pardo, gracias por sus consejos y enseñanzas. También
agradezco lo aprendido y la calidad humana de la Dra. Paula Jara, quien me apoyó y guió durante
el desarrollo de la unidad de investigación el año 2007.
Gracias a Patricia Frías de México, Claudia Lucia de Colombia, Marina Caballero de Argentina,
Nicol Uslar y Paulina Valenzuela de Chile; quienes fueron mi familia durante 10 meses y de las
cuales aprendí lo importante.
Agradezco a mis queridas amigas y compañeras, Paula Aguilera y Elizabeth Garrido; los coffe
break, salidas y conversaciones con Marcela Verdugo, Laura Azocar, Karen Maquién, Pilar
ii
Ulloa, Rayen Millaleo, Cecilia Paredes, Ligia Pinilla y Mariela Bustamante; así como a todos los
miembros del programa con los que disfruté de una buena conversación y orienté o me orientaron
en distintos momentos.
Quiero agradecer profundamente a mi familia, especialmente a mi papá, Benjamín Silva

Guerrero, y mis hermanas, Carolina y Paulina Silva Weiss, por su continuo apoyo, por sus
energías y por compartir conmigo las penas, ansiedades, alegrías y logros que he sentido en este
camino. Un abrazo a mis hermanos Mauricio y Maximiliano, y para mis sobrinos queridos
Sebastián y Camilo.
GRACIAS
A aquellos que creyeron en mí, por alegrarme y por apoyarme y animarme en los momentos
difíciles.
Por la comprensión que han tenido siempre cuando fui por un sueño y por dejarme volar…
A todo aquel que pasó por mi camino, porque algo aprendí de cada uno de ustedes.
A los que han llegado a mi corazón y no se van más.
Por todas las cosas buenas, por permitirme culminar esta etapa y por las nuevas experiencias que
están por venir.
iii
LIST OF PUBLICATIONS
ISI publications: Published
Silva-Weiss, A., Bifani, V., Ihl, M., Sobral, P. J. A., & Gómez-Guillén, M. C. (2013). Structural
properties of films and rheology of film-forming solutions based on chitosan and chitosan-starch
blend enriched with murtilla leaf extract, Food Hydrocolloids, 31(2), 458–466.
Gómez, C., Ihl, M., Bifani, V., Silva, A., & Montero, P. (2007). Edible films made from Tuna-
fish gelatin with antioxidant extracts of two different murtilla ecotypes leaves (Ugni molinae
Turcz). Food Hydrocolloids, 21(7): 1130 – 1143.
Chapter in Book:
García, M., Bifani, V., Campos, C., Martino, M.N., Sobral, P., Flores, S., Ferrero, C., Bertola, N.,
Zaritzky, N.E., Gerschenson, L., Ramírez, C., Silva, A., Ihl, M., & Menegalli, F. (2008). Edible
coatings as an oil barrier or active system. In: Gutiérrez-López, G.F., Barbosa-Cánovas, G.,
Welti-Chanes, J., Parada Arias, E. (Eds.) Food Engineering: Integrated Approaches, p.: 225 -241.
Secaucus, NJ.: Springer
Other publications: Published
Silva-Weiss, A.; Bifani, V.; Ihl, M.; Gómez-Guillén, C. (2011). Polyphenol-rich extract from
murtilla leaves on physical properties and microstructure of films based on hydrocolloid blends.
Agro Sur, 39(S): 5 - 6.
iv
ISI publications: Submitted
Silva-Weiss, A., Bifani, V., Ihl, M. & Barbosa-Cánovas, G. (2013). Recovery of antioxidant
polyphenols by conventional and ultrasound-assisted extraction from rosemary and murtilla
leaves. LWT - Food Science and Technology, LWT-D-13-00165.
Silva-Weiss, A., Bifani, V., Ihl, M., Sobral, P. J. A., & Gómez-Guillén, M. C. (2013). Natural
additives in bioactive edible films and coatings: Functionality and applications in foods ‒ A
Review. Food Engineering Review, FERE-D-13-00006.
Silva-Weiss, A., Bifani, V., Ihl, M., Sobral, P. J. A., & Gómez-Guillén, M. C. (2013).
Polyphenol-rich extract from murtilla leaves on rheological properties of film forming solutions
based on different hydrocolloid blend. Journal Food Engineering, JFOODENG-D-13-00515.
ISI publications: In preparation
Silva-Weiss, A., Garrido, E., Ihl, M., Bifani, V., Mora, M. L. & Theng, B. K. Chemical
interactions, mechanical properties and structure of starch/CMC/montmorillonite nanocomposite
films. Food Research International.
Silva-Weiss, A., Graü, R., Marx, F., Bifani, V., & Ihl, M. Free radical scavenging capacity and
inhibition of oxidation of ascorbic acid by six aqueous infusions of leaves. Food Chemistry.
v
DEVELOPMENT OF EDIBLE COATINGS AND FILMS BASED ON
HYDROCOLLOID BLENDS WITH POLYPHENOL-RICH EXTRACT:
RHEOLOGY AND FUNCTIONALITY
Abstract
The development of edible films and coatings has been considered for their capability to improve
quality and safety in fresh food. Natural abundant biodegradable polymeric materials such as
cellulose derivatives (including methyl cellulose (MC) and carboxymethylcellulose (CMC))
chitosan, gums, starches or proteins are convenient for films and coatings production, due to
lower environmental consequences compared with common synthetic plastic materials. The
properties of edible films and coatings can be improved by combining the characteristics of
hydrocolloids. Furthermore, incorporation of natural polyphenolic extract with bio-active
properties in these blend solutions and composite films can enhance their active properties such
as antioxidant and UV-barrier properties. However, the physical and chemical properties of
edible films and coatings could also be modified when polyphenol-rich extracts are added.
In these work, the ultrasound assisted-extraction was optimized and compared with conventional
extraction of phenolic compounds from murtilla (Ugni molinae Turcz) leaves (Chapter 3). Then,
the influence of polyphenol-rich extract from murtilla leaves on rheological properties of six
blends of hydrocolloids based solutions were analyzed (Chapter 4). In addition, resulting films
were analyzed in terms of FT-IR spectra, mechanical properties of the films, and film
morphology (Chapter 5).
The natural source of polyphenols that should be used in edible coatings based on hydrocolloid
blends was selected in previous studies. We analyzed inhibition of the copper-catalyzed oxidation
of ascorbic acid, as well as scavenging capacity of DPPH. and ABTS.+ free radical of six natural
aqueous extracts. The best antioxidant capacity was for murtilla leaves, which showed the lowest
concentration of aqueous extract required for 50% reduction of DPPH. and ABTS.+ free radicals,
and the greatest total phenolic compound concentration (P < 0.05). Murta or murtilla is an
endemic shrub found in central- southern Chile. Phenolic acids as gallic acid as well as
vi
flavonoids aglycones and glycosides of quercetin, myricetin and kaempferol are among the main
compounds found in murtilla leaf extract.
Ultrasound-assisted extraction allows a higher recovery of phenolic compounds from murtilla

leaves than conventional maceration. In addition, the operating cost of ultrasound process in the
determined optimal conditions is more efficient and has higher performance than extraction by
maceration. The efficiency of the phenolic compounds recovery by ultrasound-assisted extraction
depends on the effect of parameter interaction, exposure time/amplitude, as well as of
independent variables, duty cycle and time.
In this investigation, a viscosity between 3 and 11 Pa s at 25 °C proved to be adequate for

spreading the film-forming solutions and form the film, which also is reflected in a thickness
homogeneous film. The polyphenol-rich extract from murtilla leaf reduced viscosity of film-
forming solutions based on CMC and CMC-starch, being their viscosity ranges, with and without
extract, suitable for coatings and films production. Solutions based on gelatines of high and low
molecular weight leave both an unstable rheological behavior when blended with polyphenolic
compounds, due to pH reduction and consequently polyphenol/protein interaction form
aggregates. When incorporating murtilla extract in chitosan and chitosan-starch solutions, the
formation and detachment of 0.5 ‒ 1.5 cm long brown aggregates were observed. These brown
aggregates could be produced by strong hydrophobic interaction of chitosan chains as well as
chitosan‒polyphenols interaction. In addition, sol‒gel transition occurred when extract from
murtilla leaves was added to chitosan and chitosan-starch solutions, increasing strongly their
viscosity and elasticity. However, viscosity of chitosan solution with murtilla leaf extract at 100
s-1 shear rate (7.11 Pa⋅s) and chitosan-starch solution with murtilla leaf extract at 50 s-1 shear rate
(5.86 Pa⋅s) is suitable for casting process.
Chitosan-starch solution with murtilla leaf extract form a more continuous and homogeneous film
structure than the solution based on chitosan with murtilla leaf extract, due to corn starch
stabilized the interaction between chitosan and murtilla leaf extract. Moreover, the extract
increased thickness and yellow color of these films and reduced considerably the polymer chain
mobility and, consequently, the flexibility and force of the films. The interaction mechanism of
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polyphenol-rich extract from murtilla leaves with chitosan chains inside the films is due to
electrostatic interactions (ionic complexations under acidic conditions, pH: 4.5), ester linkages
and hydrogen bonds, whereas interaction of polyphenols from extract with starch chains is due to
hydrogel bonds and ester linkages.
All these results indicate that the incorporation of murtilla leaf extract modifies the physical and
chemical properties of edible coatings and film-forming solutions. Depending of the product on
which this film-forming solution will be applied, these modifications increase the shelf life of the
product.
viii
TABLE OF CONTENTS
Agradecimientos / Acknowledgements i
List of publications iv
Abstract vi
Table of contents ix
Table index xv
Figure index xvii
Abbreviations xxi
Nomenclature xxi
CHAPTER 1 1
Introduction and objectives
1.1. General introduction 1
1.2. Hypotheses 12
1.3. General objective 12
1.4. Specific objectives 13
References 13
CHAPTER 2 17
Theoretical background
Natural additives in bioactive edible films and coatings: functionality and
applications in foods ‒ A review
Abstract 17
2.1 Introduction 18
2.2 Edible films and coatings: concept, formulation and requirements 20
2.3 Interactions among additives, film and food 22
2.4. Antioxidant 26
2.4.1 Synthetic versus natural antioxidants 27
2.4.2 Antioxidant mechanism to prevent lipid oxidation 29
ix
2.4.3 Antioxidant plant extract and bioactive compounds as antimicrobial agents 31
2.5 Antibrowning 32
2.5.1 Browning mechanism 32
2.5.2. Browning in fruits and vegetables 33
2.5.3 Browning inhibition 34
2.5.4 Ascorbic acid and acidulants as inhibitors of browning 34
2.5.5 Polyphenols: inhibitors of browning? 35
2.5.6 Packaging, films or coating applications to prevent browning. 36
2.6 Functionalities of edible films 37
2.6.1 Mechanical properties 37
2.6.2 Gases barrier 37
2.6.3 Light barrier 38
2.7 Edible films incorporating with natural additives: Food applications 38
2.7.1 Vitamins, acidulants and salts 38
2.7.1.2 Mechanical properties 39
2.7.1.3 Gases barrier 39
2.7.1.4 Light Barrier 41
2.7.2 Plant-based products 41
2.7.2.1 Antioxidant and antibrowning effects 42
2.7.2.3 UV Light Barrier and appearance 45
2.7.2.4 Effect of murtilla leaf extract on the structure and properties of active 47
films
2.7.3 Chitosan 50
2.8 Conclusions and perspectives 52
References 53
x
CHAPTER 3 61
Recovery of polyphenols and flavonoid by conventional and ultrasound-assisted
extraction from rosemary and murtilla leaves
Abstract 61
3.1 Introduction 62
3.2 Materials and methods 65
3.2.1 Materials 65
3.2.2 Preparation of plant material 65
3.2.3 Extraction systems 66
3.2.3.1 Ultrasound-assisted extraction (UAE) 66
3.2.3.2 Conventional maceration extraction (ME) 66
3.2.3.3 Combined extraction process (UAE + ME) 66
3.2.4 Optimization of UAE conditions 67
3.2.4.1 Experimental design 67
3.2.4.2 Statistical analysis 69
3.2.5 Comparison of extraction systems 69
3.2.6. Analysis 70
3.2.6.1 Total phenolic compounds (TPC) 70
3.2.6.2 Flavonoid content 70
3.2.6.3 DPPH radical scavenging activity 71
3.2.6.4 Field emission scanning electron microscopy (FESEM) 72
3.3. Result and Discussion 72
3.3.1. Effect of UAE conditions on recovery of phenolic compounds 72
3.3.2. Comparison of three extraction systems 75
3.3.2.1. Recovery of phenolic compounds 75
3.3.2.2. DPPH.+ radical scavenging activity 77
3.3.2.3. Flavonoid content 77
3.3.2.4. Morphology of rosemary leaf residue 80
3.4. Conclusions 84
Acknowledgements 84
References 84
xi
CHAPTER 4 87
Polyphenol-rich extract from murtilla leaves on rheological properties of film-
forming solutions based on different hydrocolloid blends
Abstract 87
4.1. Introduction 88
4.2.1. Materials 90
4.2.2. Obtaining and characterization of PEML 91
4.2.3 Preparation of the film-forming solutions (FFS) 92
4.2.4. Formulation of blend of hydrocolloids solutions 92
4.2.5. Rheological measurements: Fundamentals and methods 94
4.2.5.1. Steady-shear measurements 94
4.2.5.2. Dynamic measurements of viscoelastic properties 95
4.2.6. Film forming capacity 96
4.2.7. Data analysis 96
4.3. Results and discussion 96
4.3.1. Steady shear behavior 96
4.3.1.1 FFS based on CMC, CS-CMC and MS-CMC 99
4.3.1.2 FFS based on CMC, HG-CMC and LG-CMC 101
4.3.1.3 FFS based on CH and CH-CS blend 103
4.3.1.4 FFS based on CH-CMC 106
4.3.2. Dynamic behavior 106
4.3.2.1 FFS based on CMC, MS-CMC and CS-CMC 107
4.3.2.2 FFS based on CMC, LG-CMC and HG-CMC 110
4.3.2.3 FFS based on CH and CH-CS 112
4.3.2.4 FFS based on CH-CMC 114
4.4 Conclusions 115
4.5 Remarks 116
Acknowledgements 117
References 117
xii
CHAPTER 5 122
Structural properties of films and rheology of film-forming solutions based on
chitosan and chitosan-starch blend enriched with murtilla leaf extract
Abstract 122
5.1 Introduction 123
5.2.1 Materials 124
5.2.2 Obtaining and characterization of PEML 124
5.2.3 Preparation of film-forming solutions (FFS) 125
5.2.4 Rheology measurement: Fundamentals and methods 126
5.2.4.1 Steady shear measurements 126
5.2.4.2 Dynamic measurement of viscosity properties 127
5.2.5 Data analysis 128
5.2.6 Film production and characterization 128
5.2.6.1 Film production and film‒forming capacity 128
5.2.6.2 Film thickness 128
5.2.6.3 Optical properties 128
5.2.6.5 Morphology of films 129
5.2.6.6 Fourier transform infrared (FTIR) spectra 129
5.3 Result and discussion 129
5.3.1 Rheology of FFS 129
5.3.1.1 Steady shear behavior 129
5.3.1.2 Dynamic behavior 131
5.3.2 Film analysis 136
5.3.2.1 Film forming capacity of the solutions 136
5.3.2.2 Optical properties 137
5.3.2.3 Thickness 137
5.3.2.5 Morphology of films 139
5.3.2.6 Chemical structure 141
xiii
5.4 Conclusions 144
Acknowledgements 147
References 147
CHAPTER 6 149
General discussion and conclusions
General discussion 149
General conclusions 152
CHAPTER 7 154
Appendix
7.1 Original papers of this thesis
7.2 Papers in collaboration
xiv
TABLE INDEX
Chapter 1
1.1 Phenolic content and antioxidant activity of leaf aqueous extracts 7
1.2 Phenolic compounds identified in murtilla (Ugni molinae Turcz) leaves 10
1.3 Operation conditions for the ultrasound-assisted extraction of phenolic 11
compounds
Chapter 2
2.1 Concentration of active compounds and their effect on biopolymer-based 23
edible films
2.2 Functionality and bioactive properties of films containing vitamins and 40
their application on foods.
2.3 Effects of the incorporation of plant extracts into edible films on 43
functional and bioactive properties of films and potential applications on
food
2.4 Effects of plant aqueous extracts and phenolic compounds on the physical 46
properties of edible films
2.5 Natural plant extracts as additives in edible films and their application on 51
foods.
Chapter 3
3.1 Factors and levels used for the evaluation of ultrasound-assisted extraction 68
parameters
Chapter 4
4.1 Structure of selected hydrocoloid studied for formulating blend FFS 91
4.2 Composition of FFS based on hydrocolloid (H) solution blends with or 93
without polyphenol-rich extract from murtilla leaves (PEML).
4.3 Parameters for the Power law model at shear rate above 0.4 s-1 at 25 ºC. 97
xv
Significance of difference between parameters K and n of FFS with and
without PEML using orthogonal contrasts and appare nt viscosity based
on form of the Power law model: η(γ& ) = K (γ& ) n −1 at 25 ºC.
4.4 Significance between hydrocolloid blends type and FFS without and with 99
PEML. Orthogonal contrasts of Power law parameters.
4.5 Viscoelastic behavior of FFS based on hydrocolloid blend without and 109
with PEML at 25 ºC.
Chapter 5
5.1 Composition of film-forming solutions, with or without polyphenol-rich 126
aqueous extract from murtilla leaves (PEML)
5.2 Parameters for the Power law model, at shear rate above 0.4 s-1 at 25 ºC. 133
Significance of difference between parameters K and n of FFS with and
without PEML using orthogonal contrasts and apparent viscosity based
on the form of the Power law model.
5.3 Physical properties of chitosan and chitosan-starch films formulated with 138
polyphenol-rich aqueous extract from murtilla leaves (PEML), and other
literature cited films
xvi
FIGURE INDEX
Chapter 1
1.1 Type and source of natural biopolymer use in edible and 2
biodegradable packaging films and composites (Modified from
Tharanathan, 2003)
1.2 Systematic approach for developing novel film using blends 3
(Lacoste et al., 2005).
1.3 Chemical structures of (a) cellulose, (b) amylose from starch, (c) 5
chitosan and (d) sodium carboxymethylcellulose, DS=10. (From
Tharanathan, 2003)
1.4 Murtilla or murtilla (Ugni molinae Turcz) shrub, endemic in central- 8
southern Chile.
1.5 Chemical structures of some polyphenols found in aqueous extract 9
from murtilla (Ugni molinae Turcz) leave.
Chapter 2
2.1 Properties of coatings, films and food that could be affected by 24
interactions between biopolymers and active agents or natural
additives.
2.2 Natural additive molecules ( ): e.g., bioactive compounds or plant 25
extracts, plant-based products, and vitamins; food molecules: e.g.,
flavorings ( ) and vitamins ( ); environmental molecules: e.g.,
oxygen and/or odor ( ).
2.3 Formation of free radicals in autoxidation and possibilities for their 30
control (Araujo, 1995).
2.4 General scheme of browning reactions in foods 33
2.5 Edible films based on fish skin gelatin with added polyphenol-rich 48
extract from murtilla leaves (PEML). Extracts from the two murtilla
leaf ecotypes (SC and SG), which contain different concentrations of
xvii
total phenolic compounds, were studied (PEML SC: 283.2 and PEML
SG: 224.2 µg equivalent gallic acid/mL extract). F-SG: Film with
PEML SG, F-SC: Film with PEML SC, F-C: the control film without
extract.
2.6 Structure of the quercetin and myricetin flavonols 49
Chapter 3
3.1 Ultrasound cavitation (Soria & Villamiel, 2010) 63
3.2 Scheme of process to comparison of three extraction systems 67
3.3 Response surface plot, showing the effect of: a) Time and wave 74
amplitude, b) Duty cycle and time, and c) Duty cycle and wave
amplitude on phenolic compound recovery from rosemary leaves.
3.4 Correlation between experimental and prediction of phenolic 75
compound recovery from rosemary, and their comparison with
phenolic compound recovery obtained through of conventional
maceration extraction (horizontal line).
3.5 Total phenol compounds (a) and DPPH radical scavenging capacity (b) 76
from murtilla and rosemary leaves using ultrasound-assisted extraction
(UAE), maceration extraction (ME) and combination of UAE + ME
systems. Different capital letters indicate significant difference (P <
0.05) between extraction systems and different lower letters indicate
significant difference (P < 0.05) between murtilla and rosemary leaves.
3.6 Wavelength spectrum of quercetin and murtilla extract, with and 78
without incorporation of AlCl3, obtained by UAE and UAE+ME: a)
Quercetin with Al3+, b) Murtilla UAE+ME with Al3+, c) Murtilla UAE
3+
with Al , d) Murtilla UAE without Al3+ and e) Murtilla UAE+ME
without Al3+.
3.7 Flavonoid content of murtilla and rosemary leaf extract measured by 79
AlCl3 reaction. Different lower letters indicate significant difference (P
< 0.05) between extraction systems.
3.8 Wavelength sweep of quercetin (a) and rosemary extract (b) 80
xviii
spectrum after to reaction with AlCl3.
3.9 Scanning electron microscopy of rosemary leaf surface after: a) and b) 81
control untreated 5.000 X, c) ME 5.000 X and d) UAE 5.000 X.
3.10 Cross-section of the rosemary cell walls: a) control 10.000 X, b) ME 82
10.000 X, c) and d) UAE 10.000 X.
3.11 Cross-section of the rosemary cell walls after to ultrasound-assisted 83
extraction (UAE). Different amplifications: a) UAE 2.000 X, b) UAE
5.000 X, c) UAE 8.000 X, d) UAE 15.000 X
Chapter 4
4.1 Viscosity curve of FFS with (filled symbols) and without (Open 98
symbols) PEML at 25 °C. a) CH-CMC (■,□) and CMC (▲,∆) b) CH-
CS (■,□) and CH (▲,∆), c) CS-CMC (■,□) and MS-CMC (▲,∆), d)
HG-CMC (■,□) and LG-CMC (▲,∆).
4.2 a) Precipitate formed in LG-CMC-PEML blend system for effect to 112
polyphenol-rich extract from murtilla leaves (PEML), b) Aggregates
obtained from CH-PEML, CH-CS-PEML and CH-CMC-PEML.
4.3 Mechanical spectra showing the frequency dependence of G’ (filled 108
symbols) and G’’ (Open symbols) for FFS without PEML (left) and
with PEML (right) at 25 °C. a) CH-CS (▲,∆), CH-CMC (●,○), CH
(■,□); b) CMC (▲,∆), MS-CMC (●,○), CS-CMC (■,□); and c) HG-
CMC (▲,∆), LG-CMC (●,○).
4.4 Elasticity properties of FFS prepared from hydrocolloids blends 111
(according to Table 2) without (open symbol) and with (filled symbol)
added PEML, during temperature sweep of heating ramp.
4.5 Dynamic viscoelastic properties of FFS prepared from LG-CMC (■, □) 113
and HG-CMC (▲, ∆); without (open symbol) and with (filled symbol)
added PEML, during cooling (a) and subsequent heating (b) ramps.
xix
Chapter 5
5.1 Viscosity curves of film-forming solutions with PEML (filled symbols) 130
and without (open symbols) at 25 °C.
5.2 Mechanical spectra showing the angular frequency (ω) dependence of 134
storage modulus (G′, filled symbols) and loss modulus (G′′, open
symbols) for FFS based on CH and CH-CS, with PEML (right) and
without (left) (25 °C).
5.3 Elasticity properties of FFS based on CH and CH-CS (according to 135
Table 2) with PEML (filled symbols) and without (open symbols),
during temperature sweep.
5.4 Sol-gel states in a colloidal solution. a) The sol state corresponds to 136
fluid colloidal solution when the dispersant (water) phase predominates
over the dispersed phase (macromolecules). b) In the gel state, the
dispersed phase predominates over the dispersant phase, thus the
colloidal solution is more viscous.
5.5 Scanning electron micrographs of film cross-section: chitosan films 140
with PEML (CH-PEML) and without (CH), chitosan-starch film with
PEML (CH-CS-PEML) and without (CH-CS). Scale bar 5 µm and
5000x magnification.
5.6 Scanning electron micrographs of film cross-section: chitosan films 142
with PEML (CH-PEML) and without (CH), chitosan-starch film with
PEML (CH-CS-PEML) and without (CH-CS). Scale bar 5 µm and
5000x magnification.
5.7 Interaction mechanisms at acid pH (4.5 ± 2) of A) Chitosan film with 145
PEML (CH-PEML): Chitosan chains (above and below), and B)
Chitosan-starch film with PEML (CH-CS-PEML): chitosan chain
(above), corn starch chain (below). As models of phenolic acids and
flavonoids from PEML, gallic acid and myricetin are presented.
Example of interactions: (1) ester linkage, (2) electrostatic interaction,
and (3) hydrogen bond.
xx
ABBREVIATIONS
DS : Degree of substitution
FFS : Film-forming solutions
HT : Total hydrocolloids in solution
ME : Maceration extraction
PEML : Polyphenol-rich extract from murtilla leaves
TPC : Total phenolic compounds
UAE : Ultrasonic-assisted extraction
Biopolymers
CMC : Sodium carboxymethylcellulose
CS : Unmodified corn starch
CH : Chitosan
HG : High molecular weight fish-skin gelatin
LG : Low molecular weight fish-skin gelatin
MS : Modified corn starch
NOMENCLATURE
G' : Storage or elastic modulus [Pa] δ : Phase angle [ º ]
G'' : Loss or viscous modulus [Pa] γ& : Shear rate [ s-1]
G* : Complex modulus [Pa] η : Apparent viscosity [Pas]
K : Consistency index [Pa s] η* : Complex viscosity [Pas]
n : Flow index [-] τ : Shear stress [Pa ]
tg : Gel point time [min] ω : Angular frequency [rad/s]
xxi
Chapter 1. Introduction and objectives
CHAPTER 1
INTRODUCTION AND OBJECTIVES
1.1. General introduction
Innovative packaging technologies, especially films and coatings, with complete biodegradability
are gaining importance for preserving safety and structural-nutritional integrity of foods. The
development of edible films and coatings has been considered for their capability to improve
global quality and safety in fresh food. Films can contribute to the prevention or delay of physical,
chemical and microbiological spoilage reactions of foods, preserving their quality and increasing
their shelf life. They may be applied directly on the food surface by dipping, spraying or brushing
to prevent water loss and gas exchange and to provide a modified atmosphere around fresh food,
which retards their deterioration (McHugh & Senesi, 2000).
Edible films or coatings are generally defined as thin layers of material, which can be eaten by
consumer as part of food (Guilbert et al., 1996). Films or coatings can be used to cover food
surface or used as pouches or wraps. These can provide food with different functionalities such as
a barrier that improves its mechanic resistance, also they hinders the loss of solutes and volatiles
or flavors, and controls the transfer of moisture, oil and gases such as oxygen and carbon dioxide.
It also important that films, improves appearance of the food product (Kester & Fenema, 1986;
Guilbert et al., 1997).
Currently in the food and pharmaceutical industry, edible coatings and films are developed from
proteins, lipids, resins and polysaccharides and other components or blends of them, called
composite films. Film production by natural and abundant biodegradable polymeric materials
such as cellulose and its derivatives (including methyl cellulose (MC) and
carboxymethylcellulose (CMC)) as well as chitosan, gums, starches or proteins, are convenient
due to lower environmental consequences compared with common synthetic plastic materials
(Figure 1.1). Film components in solution, need to constitute a sufficiently cohesive structural
matrix (Guilbert et al., 1997), in which this is both compatible and stable in the presence of
additives, under processing conditions.
1
Figure 1.1 Type and source of natural biopolymer use in edible and biodegradable packaging
films and composites (Modified from Tharanathan, 2003)
A systematic approach for developing new edible films based on hydrocolloid blends and active
additives is using the relationship of systems: composition and processing conditions for film-
forming solutions (FFS), besides of structure and physical properties of films (Figure 1.2).
With respect to process conditions, it is necessary to know the flow behavior of FFS to determine
the temperature, pressure, stirring speed and viscosity to reduce the energy used for transport.
Rheological behavior and surface tension of FFS are important factors that will affect coating
adhesion, spreadability, thickness and uniformity of liquid coating layer and film performance
(Cisneros-Zevallos & Krochta, 2003; García et al., 2009; Tzoumaki, Biliaderis & Vasilakakis,
2009). High viscosity, low surface tension and high flocculation rate of the FFS favor the
increase in water vapour resistance of the films (Villalobos-Carvajal et al., 2009). Rheological
2
characterization of food materials and their constituents are important, particularly to relate
structure and stability with processing design (Yang et al., 2004). Rheological parameters are
required for calculation of any process involving fluid flow (e.g. pump sizing, extraction,
filtration, extrusion, purification). They play an important role in quality control and sensory
assessment of foods (Steffe, 1996; Marcotte et al., 2001; Rao, 2007). Understanding rheological
behavior of FFS is critical for selecting the proper processing conditions, scaling up the process
and establishing the machines and accessories to ensure their application. A reduction in the
solution viscosity provides a processing advantage during high shear processing operations, such
as pumping, filling and spray application, and ; whereas high apparent viscosity at low shear rates
provides a desirable mouthfeel during mastication (Reilly, 1997) and a better application of FFS
by immersion or dipping (García et al., 2009). In addition, in the dipping process, viscosity of
coating is optimized to be high enough to overcome gravity but low enough to facilitate
capillarity-driven leveling (Peressini et al., 2003).
Figure 1.2 Systematic approaches for developing novel film using blends (Lacoste
et al., 2005).
The viscous and elastic properties of the FFS can be evaluated through dynamic rheological
studies. Frequency sweeps assess the effect of additives on changes in microstructure and stability
3
of the FFS during storage and transportation, whereas a temperature sweep can show phase
transitions and elasticity, allowing the appropriate temperature ranges for the formulation and
application of the coating film on the product (Moraes et al., 2009).
With respect to FFS composition, edible coatings can be produced from a single hydrocolloid
(polysaccharide or protein) and/or lipids or mixtures of them (composites) (Kester & Fennema,
1986). Hydrocolloids, such as carboxymethylcellulose (CMC), starch, chitosan and gelatin are
biodegradable and non toxic products based on renewable resources. Sodium
carboxymethylcellulose is a cellulose derivative that has received considerable attention with
applications in many fresh foods, providing efficient oxygen and lipid barrier properties and high
solubility (Park et al., 1993; Tzoumaki, Biliaderis & Vasilakakis, 2009).
Fish gelatin has the capacity to form transparent and resistant films, whereas starches has been
used to produce biodegradable films by partially replacing plastic polymers due to its low cost
and renewability; but its applications are limited by its solubility in water and fragility. Blending
two different hydrocolloids can change strongly both the physical and rheological properties of
FFS and consequently of coatings and films. These changes occur due to the
compatibility/incompatibility between two macromolecules, which depend on their molecular
weight, chemical structure, conformation and hydration behavior, as well as the addition of
various chemicals or additives, in minor amounts (Greener-Donhowe & Fennema, 1994; Phan
The et al., 2009). Figure 1.3 depicts the chemical structures of some biopolymers used as matrix
of films and coatings.
For studying the functionality of edible films, approaches traditionally used in material sciences
have been adapted and modified. Functional properties of an edible film or coating will be
directly related to the composition and structure of its component. These have been modified
using blend of different agro-bio-polymers (Arvanitoyannis, Psomiadou, & Nakayama, 1996;
Arvanitoyannis et al., 1998a,b; Peressini et al., 2003; Bangyekan, Aht-Ong, Srikulkit, 2006).
Blends of polysaccharide-polysaccharide such as CMC-starch blend film show improvement in
tensile strength (Li et al., 2008); whereas chitosan-starch blend film provides increased tensile
strengths and elongation, and decreased water vapor barriers, which are better for packaging film
4
application (Xu et al., 2005; Bangyekan et al., 2006; Mathew et al., 2006). Other blends of
proteins-polysaccharides have also been studied such as whey protein-CMC blend (Koupantsis &
Kiosseoglou, 2009) and soy protein isolate-CMC blend (Su et al., 2010), which possibly form
cross-links between the polymers in the blend.
Figure 1.3 Chemical structures of (a) cellulose, (b) amylose from starch, (c) chitosan and (d)
sodium carboxymethylcellulose, DS=10. (From Tharanathan, 2003).
5
An advantage of coatings is that several active ingredients can be incorporated into the polymer
matrix, carrier and consumed with food, thus enhancing safety or even nutritional and sensory
attributes (Rojas-Grau et al., 2019) and health benefits to consumers (Martín-Belloso et al., 2009).
An aspect providing great versatility and interest in edible coatings nowadays is incorporating
active natural compounds from plants that could act as antioxidant, antimicrobial and/or anti-
browning agents as well as cross-linking agents. The interactions of active natural compounds
with the polymeric matrix may not only affect their release into food, but also alter some
functionalities or physical and chemical properties of film, such as solubility, light and gas barrier
properties (Bifani et al., 2007; Gómez-Guillén et al., 2007), as well as elasticity, resistance and
appearance. Besides, some studies have shown that antioxidant power (reducing ability and free
radical-scavenging capacity) as well as light barrier properties of biodegradable and edible films
can be increased by adding polyphenol-rich oregano or rosemary water extracts (Gómez-Estaca
et al., 2009a) or murtilla extracts (Gómez-Guillén et al., 2007). The composition and antioxidant
properties of aqueous extract from rosemary (Wada et al., 2004; Silva et al., 2011) and oregano
(Cervato et al., 2000; Kuliŝilić et al., 2006) have been studied. However, the effect of these
polyphenol-rich extracts on flow behavior and properties that contributed to functionality of films
have been less studied.
The natural source of polyphenols that would be used in edible coatings based on hydrocolloid
blends was selected in previous studies. With this purpose, we analyzed inhibition of the copper-
catalyzed oxidation of ascorbic acid (% I.O.AS.A), as well as DPPH. (free radical 2,2-diphenyl-1-
picrylhydrazyl) and ABTS.+ (radical cation 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate)
radical scavenging capacity of six aqueous extracts from melissa (Melissa officinalis), murtilla
(Ugni molinae Turcz), rosemary (Rosmarinus officinalis), bay laurel (Laurus nobilis), lemon
verbena (Aloysia citrodora) and matico (Buddleja globosa) leaves (Table 1.1). Murtilla and
melissa leaf aqueous extract showed the highest % I.O.AS.A (P <0.05). The best antioxidant
capacity, with the lowest concentration of aqueous extract required for 50% reduction of DPPH.
free radicals, for murtilla followed by melissa and then rosemary. Moreover, murtilla leaf extract
showed the lowest concentration required for 50% reduction of ABTS.+ and the greatest total
phenolic compound concentration (P < 0.05).
6
Table 1.1 Phenolic content and antioxidant activity of leaf aqueous extracts
Leaf source Scientific name TPC 1 I.O.AS.A 2 IC 50 % 3 IC 50 % 3

[%] ABTS.+ DPPH
Murtilla Ugni molinae Turcz 50.51 ± 1.26a 84.60 ± 12.99a 0.99 ± 0.06e 2.55 ± 0.10f
Melissa Melissa officinalis 32.07 ± 2.22b 90.74 ± 11.78a 3.96 ± 0.57d 10.95 ± 1.10e
Rosemary Rosmarinus officinalis 19.41 ± 0.56c 41.61 ± 8.27b 8.03 ± 0.21c 30.26 ± 1.38c
Laurel Laurus nobilis 7.08 ± 0.85d 0.01 ± 0.01c 9.30 ± 0.10b 48.28 ± 1.48b
Lemon verbena Aloysia citriodora 13.45 ±0.56e 23.57 ± 8.40b.c 11.27 ± 0.81a 56.10 ± 3.60a
Matico Buddleja globosa 13.23 ±0.35e 2.80 ± 1.12c 11.39 ± 0.15a 22.36 ± 0.78d
1
TPC: Total Phenol Content (mg Eq. Galic acid/ g leaves d.w.)
2
I.O.AS.A.: Inhibition of ascorbic acid oxidation
3
IC50: The half maximal inhibitory concentration [mg leaves d.m/mL]
7
Murta or murtilla (Ugni molinae Turcz) is an endemic shrub found in central- southern Chile
(Figure 1.4), which belongs to the Myrtaceae family. The extract from murtilla leaves is used in
cosmetics for neutralizing the oxidative stress. In medicine, it has been found to cause a
protective effect against oxidative damage in human erythrocytes (Suwalsky et al., 2007). This
extract has anti-inflammatory (Aguirre et al., 2006), analgesic (Delporte et al., 2007) and
antimicrobial (Shene et al., 2009) activities. In addition, aqueous murtilla leaf extracts have
shown to have high antioxidant activity in vitro (Rubilar et al., 2006). Studies about the chemical
composition of murtilla leaf extracts show the presence of phenolic acids, flavonoids, and tannins
(Montecinos et al., 1991; Rubilar et al., 2006).
Figure 1.4 Murta or murtilla (Ugni molinae Turcz) shrub,

endemic in central-southern Chile.
Phenolic acids as gallic acid as well as flavonoids aglycones and glycosides of quercetin,
myricetin and kaempferol (Bifani et al., 2007) are among the main found compounds (Figure
1.5). In Table 1.2, compounds reported from murtilla leaves are summarized.
8
Plant extracts have drawn attention and there is a tendency of replacing synthetic agents. In
general, plant extracts which contain high concentrations of phenolic compounds have strong
antioxidant properties (Mayachiew & Devahastin, 2010). Finding new natural, safe and
economical natural health products in form of plant extracts, mainly from abundant and low-
value raw materials, represents a real challenge nowadays, especially in the context of sustainable
development (Diouf, Stevanovic & Boutin, 2009).
Figure 1.5 Chemical structures of some polyphenols found in aqueous extract

from murtilla (Ugni molinae Turcz) leave.
9
Table 1.2 Phenolic compounds identified in murtilla (Ugni molinae Turcz) leaves
Phenolic compounds Formule m/z RT (min)

Hidroxibensoic acid and derivated
Gallic acid C7H6O5
Vainillinic acid1 C8H8O4
Other phenolic acid 1
Flavonol and their glycosides
Kaemphenol2 C16H12O6
Quercetin2 C15H10O7
Quercetin Glycoside1 302
Quercetin Glucoside2
Quercetin Dirhamnoside 2,3 302 57.753 ‒58.123
Quercetin Rhamnoside3
Quercetin Xiloside 2 302 57.753
Myricetin C15H10O8
Myricetin Glycoside1 318
Myricetin Glucoside3
Myricetin Xyloside3
Myricetin Rhamnoside3
Myricetin Dirhamnoside 2 335, 318 48.052 ‒ 48.505
Myricetin Galactoside 2 318 49.655 ‒ 50.115
Rutin C27H30O16
Rutin Glycoside1 302
Flavan 3-ols 3
(+)-Catequina1 C15H14O6
(-)-Epicatequina, (epi)catechin3 C15H14O6
Epigalocatequin galato 1
(Epi)gallocatechin3
Polymeric flavan-3-ol (E)C:
(Epi)catechin 3
Extraction solvent: 1 ethanol : water 1:1 (v/v) (García, 2007), 2

water (Bifani et al., 2007),
3
ethanol (Shene et al., 2009). According to Shene et al., (2009) flavonol glycosides and
flavan-3-ols were exclusively extracted by alcohols. RT: Retention time peak (min).
10
Table 1.3 Operation conditions for the ultrasound-assisted extraction of phenolic compounds
Rate, Time F (kHz), A (%),

Resource Solvent Result Reference
T(°C) (min) P (W) DC
Phenolic compounds
Flavanone glycosides Ethanol-water 80% 4, 40 30 TPC: 275.8 mg GAE/100 g Khan et al., 2010
from orange peel a fresh weight
Extraction yield: 10.9 %
Carnosic acid from Ethanol 10, 40 15 - 45 40 ∼ 18 mg GAE/g d.w Albu et al., (2004)
Rosemary Ethanol-water, 9:1 ∼ 14 mg GAE/g d.w
Oregano Methanol 2, Tamb 10 24, 200 16.92 mg GAE /kg sample Kotsiou et al., 2010
Bayberry pomace Methanol-water, 80:20 20, Tamb 20 40, 200 27.7 – 47.4 mg GAE/g d.w Zhou et al., 2009
Coconut shell powder Ethanol-water, 50% 33, 30 20 - 60 25, 150 Rodrigues & Pinto, 2007
Rutin b Methanol 30 Recovery: Paniwnyk et al., 2001
From Flowers buds Water 10, 23 20 20, 27 Rutin:1.2 g, 30 min
Rutin: 0.25 g, 20 min
Rutin and quercetin Ethanol-water 70% 40 30* 3 50, 100 Recovery (%): Yang & Zhang, 2008
stalks c Rutin: 99, Quercetin: 100
Rutin and quercetin Methanol d 10, 35 30 35 Recovery (%): Gu et al., 2005
from Tobacco Quercetin: 85, Rutin: 97
Bearberry leaves Ethanol-water, 50 % 25, 20 10-45 60 Gribova et al., 2008
Grape Seeds Ethanol, 53 % 29 TPC: 5.44 mg GAE/100 mL Ghafoor & Choi, 2009
∼23% more efficiency that
solvent extraction
Tartaric acid Water 70 30 24, 200 30 /0.2 Recovery: Palma & Barroso, 2002
Malic acid Tartaric acid: 200 mg/L
from grape seeds Malic acid : 51 mgL
Others
Betula alleghaniensis Ethanol-water, 95% 30 20, 750 70 / 0.2 Diouf et al., 2009
Britton
β-carotene from n-heptane , 30 8 / 0.6 ‒1 Recovery: 1.1 mg/g Dey & Rathod, 2012
spirulina
Bold letters are parameters obtained after optimization. Rate: Solvent: sample rate (mL /g dry weight), T: Temperature (°C), F: Frequency (kHz). I:
Intensity (W/cm2), P: Power (W), A: Amplitude (% or um), DC: duty cycle (-)(pulse length of ultrasound in second per 1 second).a particle size of 2 cm2,
b
probe 0.5 in Tip, c UAE + maceration extraction, d Anhydrous methanol containing 0.5% w/v ascorbic acid, leaves 60–80 mesh.
11
Non-conventional systems as ultrasonic-assisted extraction (UAE) have been used for the
recovery of bioactive molecules from plant materials with a common goal of extracting complete
compounds of interest without altering their properties (Sonria & Villamiel, 2010). Besides, in
order to produce less toxic extracts, a reduction in energy costs has been observed when using
UAE compared with maceration extraction system (Diouf et al., 2009). However, UAE could
allow both positive (intensification of the release of biologically active substances) and negative
(inactivation of phenols) actions on extract quality (Gribova et al., 2008). Therefore, the
combination of UAE conditions for obtaining high efficiency of phenolic compound recovery
and maintaining active antioxidant properties of these compounds should be investigated,
comparing it with conventional maceration extraction systems. In Table 1.3, UAE conditions
reported on the phenolic compound recovery have been summarized.
1.2 Hypotheses
• The efficiency of the phenolic compounds recovery by ultrasound-assisted extraction (UAE)

depend of the effect of parameter interaction, exposure time/amplitude/cycle time. Thus,
applying UAE, in the optimal combination of these parameters, the efficiency of antioxidants
phenolic recovery is significantly higher than that obtained by conventional maceration
extraction.
• Polyphenol-rich extract from murtilla leaves (PEML), incorporated in biopolymer matrices

based on carbohydrates or proteins and their mixtures, will modify the rheological behavior
of these solutions, as well as the structure and functionality of the resulting films.
1.3 General objective
• To study the system and conditions of extraction needed for obtain a maximum of phenolic
compound recovery and antioxidant activity from aqueous extract of murtilla leaves; and to
evaluate the effect of polyphenol-rich extract from murtilla leaves and the type of
biopolymer matrix, based on single or blend of hydrocolloids, on the rheological behavior of
film-forming solutions, as well as on the chemical structure and functionality of the resulting
12
selected films.
1.4 Specific objectives
• To determine the parameter combination of ultrasound-assisted extraction (UAE); ultrasonic

wave amplitude, exposure time and duty cycle, in order to obtain the maximum phenolic
compounds recovery in water, using rosemary leaves as plant material model.
• To compare the recovery of total phenolic compounds (TPC) and flavonoids, as well as
DPPH.+ free radical scavenging activity from murtilla and rosemary, applying three different
systems of extraction, ultrasound-assisted extraction (UAE), maceration extraction (ME) and
combination of UAE + ME.
• To determine the effect of polyphenol-rich extract from murtilla leaves (Ugni molinae Turcz)
and hydrocolloid blend type on rheological properties of film-forming solutions based on
fish-skin gelatin, corn starch, sodium carboxymethylcellulose and/or chitosan.
• To evaluate the changes on the physical stability of film-forming solutions as well as

structure, chemical interactions and physical and mechanical properties of resulting films,
when polyphenol-rich extract from murtilla leaves is added.
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16
Chapter 2. Theoretical background
CHAPTER 2
THEORETICAL BACKGROUND
NATURAL ADDITIVES IN BIOACTIVE EDIBLE FILMS AND COATINGS:
FUNCTIONALITY AND APPLICATIONS IN FOODS ‒ A REVIEW
A. Silva‒Weissa, ∗, V. Bifanib, M. Ihlb, P.J.A. Sobralc, M.C. Gómez‒Guillénd
a
Doctoral Program in Science of Natural Resources, Scientific and Technological Bioresource Nucleus
(BIOREN‒UFRO), Universidad de La Frontera, PO Box 54‒ D, Temuco, Chile
b
Chemical Engineering Department, Universidad de La Frontera, PO Box 54‒ D, Temuco, Chile
c
Food Engineering Department, University of São Paulo, PB 23, 13635‒900 Pirassununga, SP, Brazil
d
Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN, CSIC), 28040 Madrid, Spain
Abstract
Natural additives from plant extracts, their isolated active compounds, plant-based products and
vitamins are good alternatives to synthetic additives in foods. This paper reviews the effect of the
incorporation of natural additives on the functionality of edible films and the application of these
films to foods. Incorporation of natural additives to active packaging systems or biopolymer-
based edible films can modify the film structure and, as a result, modify their functionality and
application to foods. The final functionality of edible films is related to their bioactive properties
(such as antioxidant, antimicrobial and antibrowning activities) and functional properties, such as
barrier of oxygen, carbon dioxide and UV-Vis light; water vapor permeability (WVP); tensile
stress (TS); elongation at break (EB). Several categories of natural antioxidants found in plant,
spices and herbs have been incorporated into edible films and coatings, resulting in an
improvement of the bioactive properties of the films, as well as the safety and quality of foods.
However, a wide range of plant natural sources with bioactive properties have not yet been
characterized with respect to their ability to be applied directly on foods and used to develop
active packaging or biopolymer-based edible films for preserving and adding value to foods. In
addition, in vivo studies of the use of bioactive films to preserve the quality, shelf life and
nutritional value of foods remain limited.
Keywords: Biopolymers, Bioactive compounds, Plant-based products, Vitamins, Interactions
17
2.1 Introduction
Food and Agriculture Organization (FAO) and food industry reports on recent food consumer
trends indicate an increasing demand for fresh food products that are natural and easy to
consume, have specific advantages for health, are microbiologically safe and lack synthetic
additives (Del Greco, 2010; James & Ngarmsak, 2010). In combination with this demand, there is
a global need to reduce the deleterious residues produced by synthetic packaging materials. To
address these issues, edible biopolymer films and coatings have been investigated as an
environmentally friendly technology that would permit a reduction of the environmental impact
and disposal costs associated with synthetic polymer films. In addition, this active packaging
technology offers substantial advantages for increasing the shelf life of foods, including fruits and
vegetables.
Active packaging aims to increase the shelf life of foods. The most beneficial characteristics of
edible films and coatings are their inherent biodegradability and edibility, which must be
maintained by the addition of food-grade ingredients in processing facilities that are acceptable
for food processing, in which the solvents used are restricted to water and ethanol (Zaritzky,
2011; p. 633-634). Edible films and coatings have great potential for use in a wide variety of
applications. They can be used to extend the shelf life of foods by preventing dehydration,
oxidative rancidity, surface browning and oil diffusion. They can preserve or enhance the
sensorial properties of foods and may have the ability to modify the internal atmosphere of foods.
Manufacturers choose the most appropriate type of packaging or edible films for a product
depending on the nature and requirements of the product, the degree and nature of the protection
needed, the method of distribution, the shelf life and the environmental impact.
The incorporation of edible coatings containing active additives can add value to food products
by increasing their shelf life. Synthetic additives are sometimes avoided because of their
potentially toxic effects (Barlow, 1990; Namiki, 1990; Hou et al., 2007). Bioactive plant-based
products with antioxidant, antimicrobial and/or antibrowning capabilities are prominent
alternatives to chemical preservatives and additives. Their use in foods helps to address consumer
18
demands for minimally processed natural products while providing some extra benefits to both
food and the consumer (Buta et al., 1999; Burt, 2004; Moradi et al., 2012).
Natural additives from plant extracts, derived products and isolated bioactive compounds have
begun to be incorporated in packaging, coating and edible film. Research has shown that certain
natural plant additives can improve the bioactive and functional properties of films, such as UV
light, water vapor and oxygen barrier (Gómez-Guillén et al., 2007; López-de-Dicastillo et al.,
2012). However, natural additives can also result in structural changes in the bioactive properties
of biopolymer-based films and coatings as well as physicochemical and mechanical
modifications. Further research is needed to evaluate the functionality of incorporating natural
additives into films and coatings as well as their effects on food (Wang et al., 2012b).
The development of active packaging systems and edible biopolymer films using natural
additives, particularly those with antimicrobial properties, has been studied extensively (e.g.,
Ponce et al., 2008; Portes et al., 2009; Iturriaga, Olabarrieta & Martínez de Marañón, 2012).
Further research is needed on the effects of natural additives from plant extract, their derived
plant-based products and isolated active compounds on film functionality. The ultimate
functionality of edible films is related to their bioactive properties (such as antioxidant,
antimicrobial and antibrowning activities) and functional properties such as their ability to serve
as a barrier to water vapor, oxygen, carbon dioxide and UV-Vis light and their mechanical (such
as tensile stress and elongation at break) and physical properties (such as opacity and color).
This paper discusses the general properties of antioxidants and antibrowning agents and reviews
the development of active edible films and coatings that incorporate natural additives from plant
extracts, plant-based products and vitamins. The topics of interest are as follows: (i) natural
additives that have been and can be applied in edible films and coatings; (ii) factors to consider
when interactions occur between the natural additive and the coating, the natural additive and the
film, and the active film and the food, and how these interactions can affect film properties; (iii)
the effects of natural additives on the structure and, consequently, on the functionality and
bioactive properties (antioxidant, antimicrobial and antibrowning) of active edible films; and (iv)
19
the effects of active edible films with natural additives on the bioactive properties and quality of
fresh food products.
2.2 Edible films and coatings: concept, formulation and requirements
Edible films are defined as a thin layer of edible material formed on a food as a coating or placed
on or between food components (Guilbert, 1986). The film-forming solution (FFS) is applied to
food to generate an edible coating. If the film-forming solution is applied as a thin layer on a flat
surface and is allowed to dry, it is referred to as an edible film, the physical and mechanical
properties of which can be studied separately from the coated material.
Solvent casting is the most common technique used to form hydrocolloid edible films. To create
an edible coating or biodegradable packaging film, a base material in solution must constitute a
sufficiently cohesive structural matrix (Guilbert, Cuq, & Gontard, 1997). The structural matrix is
composed of biopolymers, such as proteins and/or polysaccharides, which are widely found in
nature in the form of cellulose, cellulose derivatives, chitosan and starch. Fatty acids and other
lipidic compounds, including natural fats, surfactants and resins, can also be added. These
improve the water barrier properties of the hydrocolloid matrix (Villalobos, Hernández-Muñoz,
& Chiralt, 2006). Surfactants can maximize the adhesion of the coating to the product (Lin &
Zhao, 2007). To reduce the rigidity and glass transition temperature of the polymers, plastifiers
rich in hydroxyl groups (such as glycerol, sorbitol, acetylated monoglyceride, polyethylene
glycol, sucrose and inverted sugars) can be added. An appropriate concentration of plasticizer of
between 15 and 30% w/w of the total polymer weight is needed depending on the biopolymer
used (Mathew, Brahmakumar, & Abraham, 2006; Mayachiew & Devahastin, 2010; Siripatrawan
& Harte, 2010). Plastifiers increase flexibility but reduce the barrier to water vapor (Gontard,
Guielbert, & Cuq, 1993). This is undesirable in foods with intermediate moisture, which are
characterized by a high aw (from 0.65 to 0.90) and a generally soft texture. An increase in the
amount of glycerol used as a plasticizer leads to a gradually decreasing contact angle value of
starch films due to glycerol hydrophilicity (Bangyekan, Aht-Ong, & Srikulkit, 2006), which
enhances the distribution of the coating and contributes to the formation of a homogeneous
thickness above the food.
20
To improve the appearance of coated foods, the adhesion, cohesion and durability of edible
coatings should be controlled (Krochta & De Mulder-Johnston, 1997). The main functions of
edible films and coatings include the control of mass transfer (preventing the loss of solutes and
volatiles), mechanical protection and sensory appeal. The mechanical strength of an edible film
must be adequate to protect the integrity and thermal insulation of both food and packaging
during distribution (Kester & Fenema, 1986; Gennadios & Weller, 1990; Guilbert et al., 1997).
To maintain the quality of fruits and vegetables, edible films and coatings should create a semi-
permeable barrier against moisture migration, carbon dioxide (CO2), aromas and lipids from the
foodstuff and the absorption of oxygen (O2) by the foodstuff. This semi-permeable barrier
regulates the atmosphere around foods, preventing their desiccation and controlling the migration
of ingredients and additives in the food systems. The reduction of O2 availability and increased
CO2 concentrations on fruit reduces ethylene levels, consequently reducing the respiration rate
and water loss and increasing the shelf life of fruits and vegetables.
Ingredients in food packaging that may migrate to food are considered food additives and must
meet food additive standards. Additives are incorporated to contribute to the overall quality,
safety, nutritive value, organoleptic characteristics (color, smell, and taste), appeal, convenience
and economy of foods (IFT, 2010). Edible films and coatings can transport additives to food and
improve the mechanical integrity and/or handling characteristics of the foods (Krochta & De
Mulder-Johnston, 1997; Zaritzky, 2011). Packaging materials such as edible films or coatings
that incorporate and carry natural additives (e.g., antimicrobials, antioxidants and flavor
components) can prevent or delay the occurrence of physical, chemical and microbiological
spoilage reactions on foodstuff, preserving its quality and prolonging its shelf life (Kester &
Fennema, 1986). Edible films or coatings also can act as a vehicle to fortify foods with vitamins
such as folic acid and ascorbic acid as well as other nutrients such as omega-3 fatty acids
(Shrestha, Arcot, & Paterson; 2003; León et al., 2008). Thus, the new generation of edible films
and coatings or active packaging has been specifically designed to increase functionality by
incorporating bioactive/functional natural additives. The incorporation of antimicrobial,
antioxidant and/or antibrowning compounds into films or coating materials can improve
protection against microorganism growth, oxidation, browning reactions and vitamin loss
21
(Bonilla, Atarés, Vargas, & Chiralt, 2012), preserving the sensorial and nutritional quality of
foods. According to AIMPLAS (2012), new active compounds from plant extracts could improve
the shelf life of perishable fresh foods by 20 percent. The quality of a food product whose useful
life is short (four to five days) could be extended by one or two additional days through the use of
this new type of active packaging, minimizing losses from spoilage through better conservation
and durability. However, the incorporation of additives increases the number of stages required
for processing, implying a higher production cost.
2.3 Interactions among additives, film and food
Studies should specifically address the compatibility of the coating agent and active compounds
because the properties of the resulting film depend on their binary combination and the ratio
between them, which, in turn, depends on the required dosage of active compounds in the
material. In addition, depending on the plant extract type that is incorporated in the films, the
concentration of the plant extract added to the biopolymer films should be studied and limited, as
a high concentration could generate undesirable odors, turbidity and/or compound precipitation
on the films and may promote oxidation in lipid systems (Wambura et al., 2008), as indicated in
Table 2.1. According to Cosgrove (2008), the dose of the active compounds needs to be
relatively low. Typically, the composition of a film strip can include 30% active ingredient. This
equates to approximately 30 mg in a 100 mg strip.
The chemical or physical interactions between biopolymers and natural additives or bioactive
compounds can affect their structure and, consequently, their functionality. Properties of films,
coatings and foods that can be affected by the incorporation of natural additives are summarized
in Figure 2.1. Interactions between natural/active compounds and the film material may be
identified by the presence of new absorption bands in the UV–visible regions of the electronic
absorption spectra of the natural/active compounds (Portes et al., 2009). In a food packaging
system, three phases should be considered: the food, the edible coating/film and the environment
(Figure 2.2).
22
1 Table 2.1 Concentration of active compounds and their effect on biopolymer-based edible films
2
Active compounds Concentration on film Biopolymer-based Film Reference
Effects of active compounds
Vitamins and/or acidulants > 0.5 g/ 3g biopolymer Methylcellulose Ayranci & Tunc, 2004
Increases the WVP of films at higher concentrations
α-tocopherol <100 mg/kg meat Has pro-oxidative effects on meat at higher Georgantelis et al., 2007
concentrations
Gallic acid, ferulic acids and/or 3 mg/cm2 film Zein films Arcan et al., 2011
flavonoids such as catechin, flavone or Catechin, gallic acid, p-hydroxybenzoic acid and ferulic
quercetin acid eliminate film brittleness and increase their
flexibility to 189%
Flavonoids (catechin, epicatechin, Chitosan Sousa et al., 2009
.+
epigallocatechin gallate and fisetin) Effectively scavenges the DPPH radical
Rosemary extracts < 1.0 g/L FFS Chitosan Wambura et al., 2008
Confers an undesirable odor at higher concentrations
Tea extract < 2.5 g/L FFS Chitosan Wambura et al., 2008
Induces turbidity at higher concentrations
Rosemary 200 mg/kg meat Chitosan Georgantelis et al., 2007
a
Kiam wood 300 – 1500 mg/L HPMC Chana-Thaworn et al.,
2011
3
a
4 Aqueous extract., bEthanol:water extract (1:8). Sodium carboxymethylcellulose (CMC), Hydroxypropyl methylcellulose (HPMC).
23
Figure 2.1 Properties of coatings, films and food that could be affected by interactions between
biopolymers and active agents or natural additives.
Most biopolymers are semi-crystalline. Mass transport through semi-crystalline materials can be
considered to occur via the amorphous region of the material. Interactions or mass transfer among
the film/foodstuff/environment can be classified as permeation, sorption and migration. (1) In
permeation, compounds from the environment (such as volatiles, gases, vapors or liquids) are
transported to the film/material in contact with the food and vice versa. Transport of small
molecules or compounds through the film can be explained by sorption, diffusion and desorption
mechanisms. (2) The sorption of solutes from the environment or from foodstuff compounds,
such as flavor, aroma, or dyes, that interact with biopolymers depends on the charge density of
the polymer network and, consequently, on the acetylation degree and pH of the polymer network
as well as on the dielectric constant of the media (Vachoud, Zydowicz & Domard, 2001). This
24
reveals the importance of electrostatic interactions, in which negatively charged chain

biopolymers can absorb positively charged compounds. The sorption of solutes on the film
matrix can also occur by hydrophobic interactions and H-bonding on the extreme surface of the
film or coating. (3) Migration represents the mass transfer of compounds originally present in the
film to the food or vice versa (negative migration). A main function of films and coatings is to
inhibit or reduce the migration of moisture, oxygen, carbon dioxide, lipids and/or aromas, among
other compounds, from foods. By contrast, the migration of particular food additives, such as
antioxidants and anti-microbial agents, from the film could improve the shelf life of the product
while minimizing the direct use of these additives in the manufacturing of the food.
Figure 2.2 Natural additive molecules ( ): e.g., bioactive compounds or plant extracts, plant-
based products, and vitamins; food molecules: e.g., flavorings ( ) and vitamins ( );
environmental molecules: e.g., oxygen and/or odor ( ).
25
The interaction between the film and the foodstuff (solid/solid interface) depends on the
molecular size of the biopolymer (weight and volume), the chemical nature of the compounds
(polarity, planarity, etc.), the temperature and working conditions, the film structure (crystallinity,
plasticizing transition temperature glass, etc.), the thickness of the film and the nature of the
material. When a coating is in contact with a food, the coating evaporation flux in the interface
between the coating/environment (liquid/air) should be evaluated, as well as the coating
absorption flux on the food for the interface between the food/coating (solid/liquid) (Karbowiak
et al., 2009).
The nature of the interaction between biopolymers and additives for preservation depends on the
nature, chemical characteristics, concentration and pH of both the biopolymer and additives
(Duckova & Mandak, 1981) as well as on the structural parameters of the active compounds
(stereochemistry, conformational flexibility and molecular weight).
The use of natural additives from plant extracts, derived products or isolated bioactive
compounds and vitamins in various foodstuff applications is increasing in the food industry. The
selection of these plant extracts and their application depends on their bioactive properties
(antioxidant, antimicrobial and/or antibrowning), availability, cost effectiveness and effect on the
sensory attributes of the final product, as well as on consumer awareness (Perumalla &
Hettiarachchy, 2011).
2.4 Antioxidants
Antioxidants are necessary in many cases to delay degradation processes in foods and preserve
their nutritional and organoleptic qualities. For example, antioxidants are necessary to prevent the
oxidative rancidity of lipids, essential oils, liposoluble vitamins (vitamin A, D, E, and K) and
pigments and to inhibit microorganism growth, enzymatic browning and vitamin loss. Foods that
contain endogenous and/or exogenous antioxidants can be considered functional foods because
they could deliver a health benefit to the consumer (Valenzuela, Sanhueza, & Nieto, 2003).
26
Oxygen has a deleterious effect on the quality of a wide variety of food products. The application
of edible films and coatings to food products represents a new approach to addressing oxygen
effects via the inclusion of antioxidants that also represent a barrier to oxygen, resulting in
improved preservation of food quality. The effects of antioxidants on the oxygen permeability of
edible films have been reviewed by Bonilla et al. (2012). In addition, the moisture content of the
foods and the relative humidity in the ambient atmosphere should be considered when developing
effective films and coatings with antioxidant activity.
2.4.1 Synthetic versus natural antioxidants

Synthetic (e.g., BHA and BHT) and natural (e.g., ascorbic acid and tocopherols) antioxidants are
added directly to foods as primary antioxidants. Synthetic antioxidants have been approved as
additives for food and are classified as Generally Recognized as Safe (GRAS). However, the
application of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate is limited to 200 mg
per 1 kg of food by the Codex Alimentarius (FAO/ WHO Food Standards, 2005). In addition,
BHA and BHT promote carcinogenic effects (Barlow, 1990; Namiki, 1990). Because consumers
prefer products containing natural antioxidants, which are safer and provide health benefits
(Marinova & Yanishlieva, 1997), natural antioxidants found in plants have been studied as
alternatives or complements to synthetic antioxidants (e.g., Zheng & Wang, 2001; Ladrón de
Guevara et al., 2002; Bera et al., 2006; Hinneburg et al., 2006)
Polyphenols are the major, although not only, plant compounds with antioxidant activity.
Phenolic compounds fall into several categories: simple phenolic compounds, phenolic acids
(derivatives of cinnamic and benzoic acids), coumarins, flavonoids, stilbenes, tannins, lignans
and lignins. Natural polyphenols are valuable compounds that possess scavenging properties
toward radical oxygen species and the ability to complex proteins. They can retard the oxidative
degradation of lipids and improve the quality and nutritional value of food. Consequently, interest
in vegetables, fruits and herbs as important sources of polyphenolic bioactive compounds has
increased. Chief among these compounds are flavonoids, which have potent antioxidant activities
and positive effects on human health (Pereira et al., 2012). Flavonoids occur mainly in leaves and
in the other parts of plants as aglycones and glycosides. In flavonoids glycosides one or more
27
sugar groups is bound to the phenolic groups via a glycosidic bond (Wach, Pyrzynska, &
Biesaga, 2007), which may be hydrolyzed in the oral cavity by saliva to deliver biologically
active aglycones (Walle et al., 2005). One example of a flavonoid is flavonol quercetin, which is
an antioxidant, anticarcinogenic, anti-inflammatory and antimicrobial compound (Boots et al.,
2008). Flavonoids from the aqueous extract of peanut shells have been shown to efficiently
remove free radicals, chelate metals such as iron (Fe+2) and inhibit human erythrocyte hemolysis,
which is induced in vitro by peroxyl radicals (Wang et al., 2007b). Lu & Foo (2000) studied the
antioxidant activity of polyphenols such as epicatechin as well as quercetin glycosides and
chlorogenic acid from apple pomace and observed that the antioxidant activities of these
compounds are higher than those of vitamins C and E. This increased antioxidant activity was, in
some cases, as much as 2 to 3 times higher in terms of the capacity to remove free radicals and 10
to 30 times higher in terms of the capacity to remove superoxide radicals.
Similarly, extracts of certain species of herbs are promising alternatives to synthetic compounds
that prevent the lipid peroxidation of foods. Plant extracts, such as rosemary extract and tea leaf
(Camellia sinensis L.) extract, slow the oxidation of several oils more effectively than synthetic
antioxidants such as BHA and BHT (Wambura et al., 2008). The antioxidant activity of rosemary
extract is associated with the presence of several phenolic diterpenes such as carnosol, rosmanol,
carnosic acid and methyl carnosate, which break free radical chain reactions by hydrogen
donation. Carnosic acid and rosmarinic acid are the main bioactive compounds present in
rosemary extracts, but whole rosemary extract is more active than rosmarinic acid (Frankel et al.,
1996). The antioxidant capacity of plant extracts are directly related to the total phenolic
compound and anthocyanin content, as has been observed for a bayberry fruit extract (Myrica
rubra Sieb. et Zucc.) (Zhou et al., 2009) and the aqueous extracts of rosemary and oregano
(Gómez-Estaca et al., 2009a).
In recent years, plant and waste material extracts and various polyphenolic compounds have been
used to successfully improve the oxidative stability of foods such as seafood (e.g., Farvin,
Grejsen & Jacobsen, 2012) and meat products (e.g., Mitsumoto et al., 2005). Extracts of natural
plants and waste materials can contain mixtures of several compounds with antioxidant
properties. Synergy occurs when the combined effect of a mixture of compounds is higher than
28
the sum of the individual effects, while antagonism occurs when a combination of compounds
exhibits a reduced effect compared to the individual compounds (Burt, 2004). For some
combinations of antioxidants, the overall effect has been found to be more pronounced
(antioxidant synergism) than the effect expected from the simple addition of the individual effects
(Yin et al., 2012). The additive effects of natural plant extracts vary according to the food product
type and the bioactive properties of each extract. According to Farvin et al. (2012), 2.4 and 4.8 g
of potato peel extract per kg of minced horse mackerel (Trachurus trachurus) provided the best
protection against lipid and protein oxidation and reduced tocopherol loss, whereas for beef and
chicken meat patties, a concentration of 200–400 mg of tea catechins per kg of food had
inhibitory effects on lipid oxidation (Mitsumoto et al., 2005)
Several natural antioxidants have been shown to inhibit lipid oxidation, such as the hydrodistilled
extracts of basil and laurel (Hinneburg et al., 2006). A mix of natural compounds such as
rosemary oleoresins (0.059%,), sage extracts (0.063%) and citric acid (0.028%) have also been
shown to inhibit lipidic oxidation (Jaswir, Che Man, & Kitts, 2000). The hydrodistilled extracts
of basil and laurel efficiently inhibit the peroxidation of linoleic acid, acting as scavengers of free
radicals and iron reduction agents.
2.4.2 Antioxidant mechanisms to prevent lipid oxidation

Lipid peroxidation refers to the oxidative degradation of lipids. Lipid peroxidation is caused by
the autoxidation of unsaturated lipids (RH), which can be controlled by various mechanisms
(Figure 3). This reaction is catalyzed in the presence of light, oxygen, heat and traces of metallic
ions, generating free radicals (R.). Hydroperoxides (ROOH) are the first products formed, which
degrade and liberate new free radicals. This process propagates the oxidative reactions of oils and
fats, resulting in the formation of different aldehydes and volatile ketones (rancidity).
Antioxidants significantly extend the shelf life of foods containing lipids susceptible to oxidation,
such as vegetable oils, animal fats, flavorings, spices, nuts, processed meats and snack products.
Antioxidants (AH) are free radical scavengers, which block radicals as soon as they are formed
by donating one hydrogen atom (Eq. 2.1 and 2.2) to quench peroxyl radicals (ROO.) before they
29
can further react with unsaturated lipids or by forming a complex (ROO.AH) with ROO. (Eq.
2.3).
Figure 2.3 Formation of free radicals in autoxidation and possibilities for their control (Araujo,
1995).
To be effective, the phenolic antioxidant must be a relatively stable free radical so that it reacts
slowly with substrate RH but rapidly with ROO•. Free radical inhibition occurs through Eq. 2.2.
The phenoxyl radical (A.) can (i) transform into a stable free radical that does not have enough
energy to react with the lipids or can (ii) interact with a second ROO., forming a peroxydienone.
When exposed to temperature and UV light, the peroxydienone generates new radicals in the
form of alkoxy radicals (RO.) that react with lipids, compromising the efficiency of the
antioxidant. Therefore, the molecular composition of the antioxidants must include the ability to
act as a hydrogen donor as well as to generate a low-reactivity A.. In this way, the efficiency of
30
the mono- and polyphenolic antioxidants depends on the stabilization (resonance) of the
phenoxyl radicals, which is determined by the substituents present on the aromatic ring and their
size. Antioxidants with larger substituent groups are more stable because they obstruct the
reaction of A with ROO.. Synthetic antioxidants are phenolic structures with various degrees of
alkyl substitution, while natural antioxidants can be phenolic compounds such as tocopherols,
flavonoids, phenolic acids and phenolics, as well as carotenoids and ascorbic acid.
R. + AH → A. + RH [Eq. 2.1]]
ROO. + AH → ROOH + A. [Eq. 2.2]]
ROO. + AH → ROO. AH [Eq. 2.3]]
The peroxidation of food can be prevented by the use of packaging with a UV-light and oxygen
barrier, chelating agents for inactivating trace metals (Cu2+ and Fe2+) and free radical scavengers.
The combined effects of these synergistic factors prevent the oxidation of packaged foods.
2.4.3 Antioxidant plant extract and bioactive compounds as antimicrobial agents

The antioxidant activity of bioactive compounds and plant extracts can be correlated with the
control of browning and microbial spoilage (Almajano et al., 2008). Herbs and spices containing
essential oils in the range of 0.05–0.1% have demonstrated activity against foodborne pathogens
such as Salmonella typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, Bacillus
cereus and Staphylococcus aureus (Tajkarimi, Ibrahim & Cliver, 2010). It remains to be
determined whether the whole plant extract or a particular active component is required to
produce these bioactive properties. Serra et al. (2008) demonstrated that two natural and waste-
derived extracts from olive oil and wine production have greater antimicrobial activity than
antioxidant compounds alone (quercetin, hydroxytyrosol and oleuropein) against foodborne
microorganisms (Escherichia coli, Salmonella poona, Bacillus cereus, Saccharomyces cerevisiae
and Candida albicans). Aqueous extracts from murtilla leaves, which exhibit high antioxidant
activity in vitro (Rubilar et al., 2006), also decrease the growth of Pseudomonas aeruginosa,
Klebsiella pneumoniae and Staphylococcus aureus (Shene et al., 2009). In the last 10 years,
31
sources of natural antimicrobials in spices, herbs and oils for food systems (fresh and fresh-cut
fruits, vegetables, meat, poultry, fish, rice and dairy products) that can be potentially incorporated
into edible films and coatings have been reviewed (Rojas-Grau, Soliva-Fortuny, & Martín-
Belloso, 2009; Tajkarimi, Ibrahim & Cliver, 2010). Edible films or packaging containing
antimicrobials are a form of active packaging. The physical properties and applications of edible
films containing plant EOs in food systems to control pathogens and spoilage bacteria have also
been reviewed (Du et al., 2011).
2.5 Antibrowning
In fresh and fresh-cut fruit processing operations, reactions leading to browning are undesirable,
not only due to the dark pigmentation they cause but also because they produce off-flavors,
softening, a loss of nutritional value and the decay of fresh foods. This leads to organoleptic
modifications and to diminished nutritional value of foods, causing great losses to the food
industry. The application of antioxidant treatments by dipping after peeling and/or cutting is the
most common way to prevent browning in fresh foods.
2.5.1 Browning mechanism

The process of browning results from both the enzymatic and non-enzymatic oxidation of
phenolic compounds. Enzymatic browning consists of the oxidation of phenolic substrates to
colored ortho-quinones, depending on the type of polyphenol that acts as a substrate (Amiot et
al., 1992). The phenomenon is usually caused by the action of polyphenol oxidase (PPO)
enzymes (1, 2-benzenediol-oxygen oxido-reductase; EC 1.10.3.1), that are present in mammals,
some bacteria, fungi and arthropods, and in most plants (fruit and vegetables). The PPO in
presence of oxygen, converts phenolic compounds into dark colored pigments (Zawistowski,
Biliaderis, & Eskin, 1991). Polyphenoloxidase catalyses two basic reactions: hydroxylation and
oxidation. Both reactions utilize molecular oxygen (air) as a co-substrate. The reaction is not only
dependent on the presence of air, but also on the pH (acidity). The reaction does not occur at acid
(pH <5) or alkaline (pH >8) conditions. The PPO enzyme has an optimum pH between 5-7 and
an optimum temperature of about 40 ºC. The PPO has two copper atoms in their active site
(Mayer & Harel, 1979), which in contact with atmospheric oxygen, form a complex enzyme-
32
cofactor-substrate in which the distance between the copper atoms coincides with that of the OH
groups in the ortho positions of the diphenols, resulting from the oxidation of the corresponding
ortho-quinone (Martínez & Whitaker, 1995). The ortho-quinones rapidly condense when
combined with amino or sulphydril groups on the proteins and also with sugar reducers, leading
to large insoluble brownish-gray, reddish or black polymers called melanines (Lee et al., 2003).
The formation of ortho-quinones is a reversible reaction, which, in the presence of reducing
agents such as ascorbic acid, leads to colorless ortho-diphenols, while the latter polymerization is
also reversible (McEvily, Iyengar, & Otwell, 1992) (Figure 2.4).
Figure 2.4 General scheme of browning reactions in foods
2.5.2 Browning in fruits and vegetables

In the case of fruits and vegetables, browning occurs as part of senescence. In healthy, intact
tissues, the enzyme and phenols are stored in different compartments of the cell (chloroplast and
vacuole, respectively). During senescence; this separation is removed through a process of
decompartmentalization. The practice of fresh-cut processing causes wounding, increases
metabolic activities and decompartamentalizes enzymes and substrates (González-Aguilar et al.,
2004), and thus, browning occurs quickly.
The PPO enzyme is one of the major causes of browning in fruits and vegetables. The inhibition
of browning is achieved when PPO is not in contact with the components necessary for producing
33
browning, such as oxygen, copper or ortho-phenolic substrates (Lambrecht, 1995). Another factor
thought to inhibit enzymatic browning is the activity of the enzyme involved in the biosynthesis
of phenolic compounds PAL, at the site of the mechanic damages or injuries produced on the
fruits and vegetables during harvest or postharvest treatments (Martínez & Whitaker, 1995).
Phenylalanine ammonia lyase (PAL; EC 4.3.1.5), is enzyme involved in the biosynthesis of
phenolic compounds, responsible for the conversion of L-Phenylalanine to trans-cinnamic acid.
In minimally processed vegetables, browning is related to final visual quality and PAL and PPO
enzymatic activity, which is increased by ethylene presence (Couture et al., 1993; Lattanzio et
al., 1994).
2.5.3 Browning inhibition

To prevent browning by the chemical reduction of quinones to colorless ortho-diphenol reduction
agents, antioxidants and enzymatic inhibitors can be used effectively (McEvily et al., 1992).
Sulfites have long been used as reduction agents to avoid browning in food products, and they
also possess antioxidant and antimicrobial activities. However, their use in foods was banned by
the USA Food and Drug Administration (FDA) in 1986 in view of their potential health risk to
sensitive individuals (Buta et al., 1999).
2.5.4 Ascorbic acid and acidulants as inhibitors of browning

In its active form, L-(+)-ascorbic acid very efficiently inhibits enzymatic browning because it
decreases the pH and reduces o-quinones, which are generated by the action of PPO enzymes, to
their corresponding phenolic substrates (Araujo et al., 1995; Perez-Gago, Serra, & del Río, 2006).
In addition, ascorbic acid is a good indicator of the nutritional and organoleptic quality of fruits
and vegetables (León et al., 2008). Nevertheless, in the presence of oxygen, ascorbic acid will
degrade to dehydroascorbic acid after a certain time. This oxidative reaction is catalyzed by metal
ions and light and produces the hydroxylation of aromatic rings in a non-enzymatic reaction
(Sarma, Sreelakshmi, & Sharma, 1997). Once the ascorbic acid has been completely oxidized to
diketogulonic acid, it loses its beneficial activity for the organism, and the quinones condense and
produce browning. Other factors such as temperature, relative humidity, processing methods and
the oxygen permeability of the packaging or edible films also have an effect on the stability of the
ascorbic acid present in foods (Martinez & Whitaker, 1995; Kitts, 1997).
34
Another conventional method for inhibiting browning is by reducing the pH and/or copper
chelation with acidulants such as citric, malic or phosphoric acid. Son et al. (2001) observed that
the synergic effects of apple compounds such as oxalic acid, oxaloacetic acid, ascorbic acid 2-
phosphate, cysteine, tripeptide glutathione, N-acetylcysteine, kojic acid and 4-hexylresorcinol
exhibit high inhibitory browning activity. Similarly, Lee et al. (2003) observed a positive
synergic effect of ascorbic acid, citric acid, oxalic acid and calcium chloride (CaCl2) on the
prevention of browning of apples slices.
Although some phenols act as substrates of the PPO enzyme, which promotes browning
reactions, another group of phenolic compounds inhibit PPO activity by binding the active site of
the enzyme (Janovitz-Klapp et al., 1990). Plant phenolic compounds such as tocopherol,
flavonoids, derivatives of cinnamic acid and coumarin are natural compounds that have an
antioxidant effect that inhibits PPO activity (Marshall et al., 2000). Phenolic acids such as p-
coumaric acid, ferulic acid, cinnamic acid and gallic acid are a group of natural polyphenols that
exhibit antibrowning activity similar to that displayed by ascorbic acid (Son et al., 2001). Kubo,
Chen & Nihei, (2003) observed that the antibrowning effects of polyphenols such as gallates
(gallic acid and its esters) are due to their capacity to reduce the ortho-quinones produced by
enzymatic oxidation.
2.5.5 Polyphenols: inhibitors of browning?

Carrot peels, which possess bioactive compounds such as quercetin, can be used as an additive to
prevent browning (Kim, Kim, & Park, 2005; Roldán et al., 2008) due to their thiol functional
groups (-SH), which are responsible for the inhibition of carrot PPO.
Phenolic acids such as gallic acid exhibit antioxidant and antibrowning activity that has been
attributed to their capacity to reduce ortho-quinones (colored) to the corresponding colorless
phenols (Kubo et al., 2003). Thus, it may be reasonable to assume that flavonoids with one more
OH group in their structure will display similar antibrowning activity.
Studies have demonstrated that flavonoids such as quercetin, rutin and morin (Caillet et al., 2007;
Boots et al., 2008) have significant antioxidant activity. Because antioxidants control the
35
diffusion of oxygen through membranes or films by diminishing their permeability (Pokomý,

1999; Ayranci & Tunc, 2004), these antioxidant flavonoids, when incorporated in edible films or
coatings, might act as antibrowning compounds by delaying the contact of oxygen with the
enzyme PPO. In addition, some flavonoids have also been found to protect ascorbic acid from
degradation. As indicated in Sarma et al. (1997), hydroxylated flavonoids, such as anthocyanins,
can inhibit the oxidation of ascorbic acid by means of two mechanisms: (i) metal chelation at pH
2 – 4, at which anthocyanins are susceptible to nucleophilic attack on positions 2 and 4, thereby
protecting ascorbic acid from metal-induced oxidation; and (ii) copigmentation, in which
ascorbic acid acts as a copigment and directly interacts with the metal-chelated anthocyanins to
form a stable anthocyanins-metal-copigment coordinated complex.
Makris & Rossiter (2002) have investigated the oxidation of quercetin and the corresponding
glycoside flavonol, rutin (quercetin-3-O-rutinoside), by the major enzymes involved in flavonol
degradation, PPO and POD. The oxidation of these polyphenols proceeds through different
pathways due to their structural differences. The incubation of quercetin with PPO produces
changes in its UV spectrum due to the formation of two major products that are less polar than
quercetin. This finding indicates that the flavonol skeleton might not be hydrolyzed during
enzymatic oxidation. Rutin is not oxidized by PPO because it is not a substrate of this enzyme;
the oxidation of quercetin by POD was not feasible in this experiment.
These findings, which are inconclusive regarding the ability of these polyphenols to inhibit
browning in foods when incorporated in edible films, provide a number of open questions for
future researchers in this field.
2.5.6 Packaging, films or coating applications to prevent browning

Another possibility to prevent browning in fresh products is to use packaging, films or coatings
that prevent the oxygen diffusion produced by mechanical damage during transport, storage and
distribution. This packaging can be effective in inhibiting the browning mediated by PPO in
various fruits and vegetables fresh-cut at an atmosphere containing less than 1 kPa of oxygen
(Gorny, 1997). The incorporation of antibrowning compounds in films or coatings is expected to
enhance the oxygen and light barrier, preventing oxygen from coming in contact with PPO and
36
maintaining the stability of ascorbic acid in the food. In addition, films providing a semi-
permeable barrier to oxygen/carbon dioxide could prevent the leaching of vitamins during food
washing (Shrestha et al., 2003). However, most antibrowning compounds are hydrophilic
compounds, which could increase the WVP of films and water loss from food products (Rojas-
Graü et al., 2007; López-de-Dicastillo,2012).
2.6 Functionality of edible films
2.6.1 Mechanical properties

Tensile strength and elongation at break are parameters that are related to the film’s mechanical
properties and chemical structure (McHugh & Krochta, 1994). The tensile strength (TS, MPa) is
the maximum strength measuring the resistance of the film, whereas the percentage of elongation
at break (EB, %) is a measure of the stretching capacity or flexibility of the film prior to breaking
(Krochta & DeMulder-Johnston, 1997).
2.6.2 Gases barrier properties

For food applications, the oxygen (O2), carbon dioxide (CO2) and water vapor permeability
(WVP) of biomaterials should be considered. Due to the hydrophilic nature of films, its barrier
properties are dependent on the surrounding relative humidity conditions (López-de-Dicastillo et
al., 2012). For this reason, tests should be carried out at storage conditions constant, % RH and
temperature. Increasing the polarity, hydrogen bond forces and crystallinity decreases the free
volume of the film, enhance the cohesion and reduce the flexibility thereby increasing the barrier
properties of the polymer (Mali et al., 2006). However, the differences in the oxygen barrier
properties depend mainly on the chemical composition, molecular structure of the polymers
(Mokwena & Tang, 2012), as well as water content, which reduce the interactions of hydrogen
bonds between the hydroxyl groups of the polymer chains.
In general, most biopolymers possess good properties as semi-permeable barriers to gases, but
they are weak barriers to water vapor. The transference of water vapor is generally due to the
hydrophilic portion of the film and depends on the ratio of hydrophilic to hydrophobic
compounds in the film (Hernandez, 1994). To prevent or diminish the dehydration of foods, films
37
used as packaging or coatings must control the transference of humidity from the product to the
environment; hence, the WVP of these films must be as low as possible (Ma et al., 2008).
In relation to maturity, fruit and vegetable products require a coating that presents a semi-
permeable barrier that permits the release of CO2 and diminishes O2 penetration. Total anoxia is
not recommended in the product because this encourages a fermentative metabolism that
generates ethanol and acetaldehyde, which produce off-flavors (Kester & Fennema, 1986).
2.6.3 Light barrier properties

Transparent packaging induces the oxidation and degradation of nutritional compounds because
light acts as a catalyst for these processes. Opaque packaging that absorbs light in the visible-UV
spectrum has been developed to prevent these reactions. The light barrier property is related to
the color and opacity of the edible films. In fruits and vegetables, gloss is an expected sensorial
characteristic of edible coatings. Therefore, the relation between the opacity and gloss factors
must be evaluated in vivo on the product to obtain an equilibrium between the sensory and
nutritional qualities of the food.
2.7 Edible films incorporating with natural additives: Food applications
Edible films based on polysaccharides (such as chitosan, starch, carrageenin, pectin and gellan),
proteins (such as whey protein concentrate and fish skin, pigskin, and bovine skin gelatin) and
carboxymethylcellulose (CMC) reduce the spoilage of some fresh foods, but a potential storage
improvement has been observed when antioxidant, antimicrobial and/or antibrowning compounds
are introduced into these biopolymers.
2.7.1 Vitamins, acidulants and salts

Vitamins such as α-tocopherol did not have a significant effect on the inhibition of the oxidation
of lipids included in fish skin gelatin films (Jongjareonrak et al., 2008). Ouattara et al. (2002)
studied the shelf life of ground meat after the addition of 5% (w/w) ascorbic acid and a protein-
based edible coating containing a mix of 3% (w/w) of natural spices (thyme, rosemary and sage).
They found that the combination of ascorbic acid and edible films with natural spices
38
significantly inhibited the lipid oxidation of ground meat during storage when compared to
samples containing ascorbic acid or control samples without additives. In addition, under
refrigeration in the dark, films containing ascorbic acid have been shown to retain their ascorbic
acid concentration (Dda, Araújo & Leão, 2009). In Table 2.2, the effects of vitamins added to
edible films on the functional and bioactive properties of films and their application on foods are
shown.
2.7.1.1 Mechanical properties

The addition of vitamin E to films based on fish skin gelatin from Priacanthus macracanthus and
Lutjanus vitta modifies film mechanical properties over time (Jongjareonrak et al., 2008).
Initially, vitamin E decreases the film tensile strength to 14%, reaching 16% after 6 weeks,
depending on the fish gelatin source. The interaction between gelatin and vitamin E reduces the
elongation of the gelatin-based film to 33% after 6 weeks of storage. The reduction in the film’s
mechanical properties occurs because the interaction between vitamin E and fish gelatin
decreases the movement of macromolecules in the biopolymer film (Jongjareonrak et al., 2008).
2.7.1.2. Gases barrier

α-Tocopherol (vitamin E), a hydrophobic molecule, can reduce the WVP of biopolymeric films,
as has been proven with calcium caseinate (Mei & Zhao, 2003) and fish skin gelatin
(Jongjareonrak et al., 2008). In the last case, incorporating vitamin E into a fish skin gelatin film
initially reduces the WVP by 38%, achieving a further reduction of approximately 11‒16% in 6
weeks. This reduction in the film’s WVP could be caused by an increase in the crosslinks
between protein chains and/or the protein and vitamin E during storage and the re-crystallization
of the gelatin in the matrix of the film (Arvanitoyannis et al., 1997), which produces a matrix that
is less permeable and less dense. Thus, some coatings enriched with vitamin E or calcium can
prolong the shelf life and increase the nutritional value of the fruits and vegetables to which they
are applied, such as raspberries and strawberries with a chitosan coating (Han et al., 2004) and
carrots with a xanthan gum coating (Mei et al., 2002).
39
1 Table 2.2 Functionality and bioactive properties of films containing vitamins and their application on foods.
Natural additive Biopolymer base Effect of vitamin on film Active film application References
properties
Ascorbic acid Methylcellulose Increases O2 barrier and Mushrooms and cauliflower: Controls browning and reduces Ayranci & Tunc, 2003;
and citric acid reduces PPO activity. vitamin C loss. Ayranci & Tunc, 2004
Ascorbic acid WPC Fresh-cut potatoes and apples: Controls browning. Coating Perez-Gago et al., 2006
(1% w/w) application did not reduce weight loss in fresh-cut apples, most
likely due to the high relative humidity of the product.
Ascorbic acid CMC Apple and potato: Controls browning. Baldwin et al., 1996
α-Tocopherol Fish skin gelatin Increases TS, Bacon: Lipidic oxidation control. Jongjareonrak et al., 2008
(0.02% w/w) transparency and WVP.
Reduces EB.
α-Tocopherol Chitosan Strawberries and raspberries: Reduces weight loss, increases Han et al., 2004
nutritional value, prolongs shelf life.
α-Tocopherol Xanthan gum Baby carrot: Improves desirable surface color without affecting Mei et al., 2002
(0.2% w/w) and taste, texture, fresh aroma and flavor, except for a slightly
5% calcium slippery surface. The β-carotene level is not affected by the
edible coating.
α-Tocopherol Chitosan Interacts with chitosan, Martins, Cerqueira, &
increasing the UV light Vicente, 2012
barrier, AOX activity
and WVP.
α-Tocopherol Whey protein O2 barrier. Han & Krochta, 2007
α-Tocopherol Calcium caseinate Reduces the WVP and Mei & Zhao, 2003
increases the EB.
2 Shown in parentheses: vitamin concentration. AOX: antioxidant, TS: Tensile strength (MPa), EB: Elongation at break (%), WVP: Water vapor permeability,
3 CMC: Sodium carboxymethylcellulose, WPC: whey protein concentrate.
40
Ascorbic acid has a hydrophilic character, an acidic pH and antioxidant properties. When added
to edible films and coatings, this vitamin can improve the oxygen barrier, reduce PPO activity
and thus enhance the efficiency of inhibition of enzymatic browning of fresh and fresh-cut fruit
and vegetables (Ayranci & Tunc, 2003; Ayranci &Tunc, 2004). However, greater inhibition of
enzymatic browning was observed when apple was dipped directly into ascorbic acid than when a
whey protein coating containing ascorbic acid was applied (Perez-Gago et al. 2006).
Vitamin and/or acidulant concentrations in an edible film should not be higher than 0.5 g/3 g
biopolymer because higher concentrations of these molecules increase the WVP (Ayranci &
Tunc, 2004). Therefore, the antibrowning effects of edible films and coatings containing ascorbic
acid depend on the biopolymer type and the vitamin concentration added.
2.7.1.3. Light barrier

Liposoluble vitamins such as vitamin E increase the transparency of films during storage,
allowing the normal passage of light, which catalyzes the rancidity of fats. However, film gloss is
not affected by the incorporation of vitamin E. In hydrocolloid matrices of fish gelatin, vitamin E
interacts with proteins through bridging hydrogen links, causing an increase in the absorption of
UV-visible light (Jongjareonrak et al., 2008).
2.7.2 Plant-based products

Plant extracts are complex systems that contain bioactive compounds at different concentrations.
When included in edible films, these extracts can exhibit antimicrobial, antibrowning and/or
antioxidant capacities. The primary purpose of adding natural extracts containing antioxidants to
a solution for coating lipidic food systems is to delay the onset of oxidation and the accumulation
of oxidative products (Wambura et al., 2008).
The addition of plant extracts has been proven to provide certain physicochemical modifications
or bioactivity to biopolymer materials such as chitosan (Wang et al., 2012a; Silva-Weiss et al.,
2013) and thus could expand their overall applications. However, the choice of the bioactive
agent could be restricted by the incompatibility of that agent with the packaging material or by its
heat instability during processing (Perez-Perez et al., 2006). The effects of aqueous plant extracts
41
and plant-based products on the functional and bioactive properties of films as well as their
potential applications on food are presented in Table 2.3.
Film antioxidant activity increases when flavonoids are added to the films. The increase in the
antioxidant activity of the film depends on the compound type used (e.g., flavonoid type) and
their concentration and quantity (Sousa, Guebitz & Kokol, 2009). However, the antioxidant
activity (free radical scavenging activity) of films may decrease, as observed for a chitosan-based
film containing a tea polyphenol extract whose antioxidant activity was reduced by 33‒43% after
3 to 4 weeks (Wang et al., 2012b). This behavior is most likely due to the loss of total phenolic
compounds in the film by oxidation. Therefore, kinetic studies of active compound delivery from
films and of the film’s antioxidant activity should be performed under the conditions of the
application and food storage.
When incorporating a plant extract into films, the main components of the extract that could
influence the antioxidant activity of the biopolymer matrix should be analyzed. Films with
natural additives have the potential to control lipid oxidation, which causes browning and/or
microbial spoilage, as has been demonstrated for some foods. These characteristics warrant the
consideration of some plant extracts and/or their active compounds as natural antioxidants and/or
anti-browning components for use as additives in edible coatings to prolong the quality shelf life
of fresh products.
2.7.2.1 Antioxidant and antibrowning effects

The strong scavenging activity observed on chitosan films containing an aqueous extract of tea
polyphenols (Wang et al., 2012b), grape seed (Moradi et al., 2012) and rosemary (Georgantelis et
al., 2007) might be due to an increase in intermolecular interactions, primarily weak interactions
such as hydrogen bonding between phenolic compounds and chitosan (Siripatrawan & Harte,
2010). Films containing tea and rosemary aqueous extracts display greater antioxidant effects on
the lipidic food storage stability of foods such as peanuts than that displayed by synthetic α-
tocopherol (Wambura et al., 2008), and films containing a borage extract display higher
antioxidant activities than films containing α-tocopherol and BHT (Gómez-Estaca et al., 2009b).
42
1 Table 2.3 Effects of the incorporation of plant extracts into edible films on functional and bioactive properties of films and potential applications
2 on food
Plant extract Biopolymer base Effects of natural additive on film properties/Potential applications References
a
Kiam wood HPMC Increases WVP and film solubility. Decreases TS and EB. Darkens film color Chana-Thaworn et al.,
and decreases transparency./Antimicrobial film to extend the shelf life of foods. 2011
Betacyanin HPMC Enhances barrier to oxygen and WVP. Akhtar et al., 2012
Oregano and rosemary a Fish skin and Increases AOX activity and UV light barrier./Inhibiting oxidative rancidity.
Bovine gelatin
Murtilla leaves SC and SG Fish skin gelatin SC: Increases AOX activity and UV light barrier. Increases cohesiveness and Gómez-Guillén et al., 2007
(283.2 and 224.2 µg eq. reduces TS, EB and WVP. SG: Increases AOX activity and UV light
gallic acid/mL, respectively) barrier./Inhibiting oxidative rancidity.
a
Green tea Agar and Increases film water solubility and decreases TS and EB in both films. Giménez et al., 2013
Agar–fish gelatin
b
Ginseng Alginate /Retarding lipid oxidation induced by UV light in food systems. Norajit, Kim & Ryu, 2010
b
Borage Sole skin gelatin/ Increases AOX activity, irrespective of the type of gelatin employed, to a level Gómez-Estaca et al., 2009b
(1542 g eq. caffeic acid/mL ) and commercial higher than films with tocopherol and BHT. Minor changes in physicochemical
fish gelatin properties.
a
Murtilla leaves SG CMC Decreases WVP, forms a selective barrier to gases (CO2/O2)./Increasing the Bifani et al., 2007
shelf life of fresh products with a high respiratory metabolism.
a
Murtilla leaves SC CMC Increases O2 barrier. Bifani et al., 2007
a
Green tea Chitosan Increases AOX activity and polyphenolic content. Increases mechanical Siripatrawan & Harte, 2010
properties and reduces the WVP. /Active packaging for food products.
a
Tea polyphenols Chitosan Increases AOX activity and water solubility. Decreases the WVP and increases Wang et al., 2012b
the opacity and color of chitosan films.
a
Grape seed Chitosan Strong scavenging activity. Moradi et al., 2012
a
Acacia seed Galactomannan Increases radical scavenging activity and the phenolic content of the film. Cerqueira et al., 2010
3 a b
Aqueous extract., Ethanol:water extract. Shown in parentheses: total phenolic content (TPC). AOX: antioxidant, TS: Tensile strength (MPa), EB: Elongation at
4 break (%), WVP: Water vapor permeability, CMC: Sodium carboxymethylcellulose, HPMC: Hydroxypropyl methylcellulose.
43
The inclusion of oregano and rosemary extracts in pigskin gelatin-based edible films (Gómez-
Estaca et al. 2007) yields antioxidant activities similar to those of the original film, in spite of the
difference in the polyphenol content. This result indicates that the antioxidant activity of the film
depends on the type and concentration of the polyphenols present in the extracts. These active
films increase the shelf life of sardines (Sardina pilchardus) by slowing lipid oxidation.
Furthermore, when combining these coatings with high pressures, microbial spoilage is delayed
due to the liberation of bioactive compounds from the film to the food.
Green tea extract has been added to films based on chitosan (Siripatrawan & Harte, 2010,
Siripatrawan & Noipha, 2012) and agar-gelatin (Giménez et al., 2013). Green tea-derived
polyphenols have been reported to exhibit strong antioxidant properties in numerous studies.
They can act as antioxidants by donating a hydrogen atom, accepting free radicals and
interrupting chain oxidation reactions or by chelating metals.
Spice oleoresins are natural plant extracts that constitute the essence of spices in their most
concentrated form and contain volatile as well as non-volatile components (such as carotenoids,
steroids, alkaloids, anthocyanins and glycosides). Food-grade oleoresins are obtained by alcohol
steam distillation from fresh vegetables. Ponce et al. (2008) studied chitosan coatings containing
different natural oleoresins. Films applied to butternut squash slices (Cucúrbita moschata)
displayed an antioxidant effect that inhibited POD activity when the films contained oleoresins of
rosemary, olive or blueberry, in decreasing order, while PPO activity was significantly reduced
when the chitosan coating contained olive, garlic and pepper oleoresins. The chitosan coatings
containing rosemary and olive oleoresins improved the antioxidant properties of fresh-cut
butternut squash, delaying the browning reactions that generally result in fruit and vegetable
quality loss. These chitosan films containing oleoresins also significantly inhibit PPO enzymatic
activity in lettuces, although the independent application of chitin and rosemary is more efficient.

Changes in the mechanical properties of biopolymer-based films when natural extracts are added
are presented in Table 2.4. Green tea water extract (GTE) slightly increases the mechanical
resistance (or TS) and extensibility (or EB) of chitosan-based films (Siripatrawan & Harte, 2010)
44
but decreases the TS and EB of both agar and agar‒gelatin films (Giménez et al., 2013),
negatively affecting the film’s mechanical properties. The mechanical properties of chitosan film
did not significantly change when the GTE concentration increased from 0 to 5% but significantly
increased when the GTE concentration increased from 5 to 20% (Siripatrawan & Harte, 2010).
The improvement in the mechanical properties of the films containing GTE may be attributed to
the interaction between the chitosan matrix and the polyphenolic compounds from the GTE. In
general, the incorporation of polyphenolic-rich aqueous extracts reduces the mechanical
properties (TS and EB) of the films (Moradi et al., 2012; Giménez et al., 2013; Silva-Weiss et al.,
2013). However, the addition of phenolic acid to the film has been shown to slowly increase
these properties (Mathew & Abraham, 2008; Rivero, García & Pinotti, 2010). However, there is a
negative relationship between the concentration of phenolic compounds in the plant extract and
the film’s mechanical properties. Extracts containing a higher concentration of phenolic
compounds (PEML SC) strongly reduced the tensile stress and elongation at break of gelatin-
based films (Gómez-Guillén et al., 2007), while films containing the PEML SG behaved
similarly to the control film .
2.7.2.3 UV Light barrier and appearance

Plant extracts are commonly used to provide color and opacity to polymers (Hong et al., 2000),
and as a result, films containing plant extracts are less transparent than films without plant
extracts (Gómez-Guillén et al., 2007, Wang et al., 2012b). Thus, the incorporation of plant
extracts in films provides an adequate barrier to light, which is also important for preventing the
degradation of ascorbic acid and, consequently, browning. These findings confirm the beneficial
effects of combining packaging with antioxidant applications. For instance, films of fish skin and
bovine gelatin enriched with polyphenol-rich aqueous extracts from rosemary and oregano
display improved antioxidant activity and light barrier properties, irrespective of the type of
gelatin employed (Gómez-Estaca et al., 2009a). Tuna gelatin films enriched with PEML display
similar effects (Gómez-Guillén et al., 2007). The light absorption in the limit of the visible
spectrum at a wavelength of 400 nm was 6 times higher for the film containing the murtilla
extract than that without the extract, while in the UV spectrum; this difference was even more
pronounced.
45
Table 2.4 Effects of plant aqueous extracts and phenolic compounds on the physical properties of
edible films
Biopolymer Thickness Mechanical properties

References
(µ
µm) TS (MPa) EB (%)
Additive
Chitosan
‒ 70.7 ± 5.0 29.69 ± 1.89 45.10 ± 1.40 Silva-Weiss et al. 2013
Murtilla leave a 77.5 ± 3.5 n.d. n.d. Silva-Weiss et al. 2013
‒ 62.1 ± 6.3 23.66 ± 2.63 54.62 ± 3.12 Siripatrawan & Harte, 2010
a
Green tea 62.1 ± 6.3 25.13 ± 1.91 58.14 ± 4.24 Siripatrawan & Harte, 2010
‒ ∼ 80 24.00 ± 3.00 29.00 ± 2.00 Moradi et al., 2012
a
Grape seed ∼ 80 16.00 ± 0.60 21.00 ± 3.00 Moradi et al., 2012
‒ ∼ 75 ∼ 18 Rivero et al., 2010
Tannic acid
∼ 95 ∼ 25 Rivero et al., 2010
(40 mg /g chitosan)
Chitosan‒Starch
‒ 72.0 ± 2.4 20.56 ± 1.78 13.52 ± 2.85 Silva-Weiss et al., 2013
Murtilla leave a 126 ± 12 17.13 ± 1.22 6.57 ± 2.43 Silva-Weiss et al., 2013
‒ 73.5 ± 3.9 ∼ 42 ∼ 57 Mathew & Abraham, 2008
Ferulic acid 78.9 ± 4.3 ∼ 49 ∼ 60 Mathew & Abraham, 2008
Agar
‒ 18.48 ± 1.28 62.35 ± 2.49 Giménez et al., 2013
a
Green tea 7.88 ± 1.02 51.69 ± 3.92 Giménez et al., 2013
Agar‒fish gelatin
‒ 15.03 ± 1.50 77.60 ± 5.76 Giménez et al., 2013
a
Green tea 6.88 ± 1.46 59.05 ± 3.20 Giménez et al., 2013
a
Aqueous extract, TS: Tensile strength (MPa), EB: Elongation at break (%), HPMC: Hydroxypropyl
methylcellulose. Different superscript letters in the same column represent significant differences (P < 0.05).
n.d.: non-determinate.
46
Therefore, the light barrier properties that these extracts of murtilla leaves, rosemary and oregano
confer to the edible films contribute to reducing the rancidity of lipids (Table 2.5) and preserving
the nutritional value of foods, particularly ascorbic acid, which helps to inhibit browning.
2.7.2.4 Effect of murtilla leaf extract on the structure and properties of active films
Murtilla or murtilla (Ugni molinae Turcz) is an endemic shrub in central‒southern Chile that
belongs to the Myrtaceae family. Polyphenol-rich aqueous extracts from murtilla leaves (PEML)
display high antioxidant activity in vitro (Rubilar et al., 2006) and antimicrobial activity against
foodborne microorganisms (Shene et al., 2009). Phenol acids such as gallic acid, as well as
flavonoid aglycones and glycosides from quercetin and myricetin, are among the main
compounds found in those extracts (Bifani et al., 2007; Rubilar et al., 2006); the proportion of
phenolic acid in the PEML is higher than that of flavonoids.
The increase in the total phenolic compounds and radical-scavenging activity is generally
proportional to the amount of extract added to the edible films (Cerqueira et al., 2010). However,
a high concentration of phenolic compounds can change the film structure and consequently
modify its functionality by affecting its barrier, mechanical and thermal properties. The effect of
the PEML of two leaf ecotypes, SC and SG, on the barrier and mechanical properties of films
based on fish skin gelatin (Gómez-Guillen et al., 2007) (Figure 2.4) and carboxymethylcellulose
(CMC) (Bifani et al., 2007) have been studied. A higher antioxidant activity and concentration of
total phenolic compounds is present in PEML SC (283.2 µg equivalent gallic acid/mL extract)
compared to PEML SG (224.2 µg equivalent gallic acid/mL extract) (Gómez-Guillen et al.,
2007). PEML SC also possesses a larger amount of myricetin than of quercetin, while PEML SG
contains a higher proportion of quercetin than of myricetin (Bifani et al., 2007). This is relevant
because myricetin (3 OH groups on the B ring) contains one more OH group than does quercetin
(2 OH groups on the B ring) (Figure 2.5), which allow that PEML SC, with a high myricetin
concentration, to react with other compounds
The extracts, PEML SC and PEML SG, affected the structure of gelatin films in different ways.
In comparison with the gelatin film-PEML SG and with the film without extract, the gelatin film-
PEML SC presented a compact structure, which denotes a greater cohesiveness between the
47
polymeric chains and compatibility between the biopolymer and the polyphenols of the PEML
SC. The compositions of PEML SC and PEML SG are quantitatively and qualitatively different.
Figure 2.5 Edible films based on fish skin gelatin with added polyphenol-rich extract from
murtilla leaves (PEML). Extracts from the two murtilla leaf ecotypes (SC and SG), which contain
different concentrations of total phenolic compounds, were studied (PEML SC: 283.2 and PEML
SG: 224.2 µg equivalent gallic acid/mL extract). F-SG: Film with PEML SG, F-SC: Film with
PEML SC, F-C: the control film without extract.
Both extracts affect the WVP of a CMC-based film, although in different ways (Bifani et al.,
2007). The WVP decreased significantly upon the incorporation of PEML SG but not PEML SC
in the film (P > 0.05). These results could be related to the structural modification of the CMC
network produced by the polyphenol extract because the WVP of the film increases with the
number of available polar (–OH) groups on the polymer (Cheng et al., 2002). This could explain
the lower WVP of the films containing PEML SG. The edible film based on the CMC-PEML SC
does not have any effect on the WVP, while the fish gelatin film-PEML SC slightly reduces the
WVP, though not significantly (Gómez-Guillen et al., 2007). The stability of the properties of the
WVP in both biopolymers can be explained by means of the higher cohesion of the polymeric
chains that can be obtained via the active compounds, quantitative and qualitative, of PEML SC.
48
In other studies, red natural pigment (betalain) added to HPMC-based films increased their WVP
when they were measured at 38°C and at a 90% RH gradient, but this permeability did not
change significantly after 20 days of light exposure (Akhtar et al., 2012). Active compounds in
the film could remain constant over time because the red color of betalain increased the film’s
light barrier, thereby maintaining the stability of the film properties over time.
The PEML SC and PEML SG increase the oxygen barrier properties of CMC-based films, while
the PEML SG reduces the film’s CO2 barrier properties (Bifani et al., 2007). This indicates that a
CMC-based film with PEML SG possesses a selective permeability to CO2 and O2, and thus
films containing PEML SG permit the release of a higher amount of CO2 than entering oxygen.
This suggests that films with PEML SG could be used as coatings for fruits with high respiration
to achieve a delay in ripening.
Figure 2.6 Structure of the quercetin and myricetin flavonols
PEML also has been added to chitosan and chitosan-starch composite films. Pure chitosan films
are generally cohesive and compact, with a smooth surface without pores or cracks. In the cross-
sections of chitosan films, chains are arranged in a horizontally ordered form at pH < 5.0.
However, upon the incorporation of the polyphenol-rich aqueous extract from murtilla leaves,
several elongated horizontal pores of approximately 1 ‒ 4 µm are observed (Silva-Weiss et al.,
2013). This indicates that an increase in the diffusion of gases such as oxygen and carbon dioxide
could occur, as the diameter of oxygen is 1.2 x 10-4 µm while that of carbon dioxide is 3.6 x 10-4
µm.
49
2.7.3 Chitosan
Chitosan offers real potential for applications in the food industry as a preservative coating due to
its specific physicochemical properties (such as good oxygen and carbon dioxide barrier and
mechanical properties), rapid biodegradability, biocompatibility with human tissues,
antimicrobial and antifungal activities and non-toxicity. The applications for active bio-based
chitosan film production and its potential in the food industry have been reviewed by Aider et al.
(2010).
Chitosan coatings have been widely used in postharvest studies of fruits and vegetables because
they maintain food quality and reduce the respiration rate (O2 barrier), ethylene production and
transpiration (El Ghaouth et al., 1991; 1992a,b; Li & Yu, 2000). As another benefit, the addition
of chitosan reduces the WVP and solubility of starch films (Bangyekan et al., 2006; Vasconez et
al., 2009). Optimum chitosan films can be prepared from 2% chitosan in 1% acetic acid
(Siripatrawan & Harte, 2010). In contrast to plant extracts, the antioxidant activity of chitosan is
not a result of its reduction power but rather its capacity to prevent the formation of reactive
oxygen species (ROS) through the chelation of metals (Kamil et al., 2002) and direct capture of
ROS (Xing et al., 2004). Chitosan-gelatin composite coatings, in contrast to gelatin coatings
containing rosemary or oregano extracts, do not inhibit oxidative rancidity but instead reduce the
growth of microorganisms in sardines (Gómez-Estaca et al., 2007). Zhang & Quantick (1997)
reported that the application of a coating of 1 to 2% w/v of chitosan in litchi (Litchi chinensis
Sonn cv. Huaizhi) partially delays the decomposition of fruits and delays the changes produced
by browning. This is reflected in the preservation of the anthocyanin contents, flavonoids and
total phenolic compounds, as well as the decrease in PPO activity and weight loss of the food.
However, an increase in the chitosan concentration in the coating did not significantly increase
the beneficial effects of the chitosan on spoilage and browning of the fruit. In addition, chitosan
coatings (2% w/v) help to preserve the concentration of ascorbic acid in tissues, which inhibits
enzymatic browning (Pen & Jiang, 2003; Jiang et al., 2005).
50
1 Table 2.5 Natural plant extracts as additives in edible films and their application on foods.
2
Natural additive Biopolymer Effects of plant extracts on film characteristics/ References
Food: Protective effects of active natural films on food
b
Borage seed Fish gelatin Horse mackerel (Trachurus trachurus) patties: Protective effects on lipid oxidation Giménez et al., 2011
throughout frozen storage.
Oregano and Pigskin Antioxidant activity. / Gómez-Estaca et al., 2007
a
Rosemary Sardines: Controls lipid oxidation.
a
Murtilla leaves CMC Increases oxygen barrier./ Ihl et al., 2006
Apricot: Reduces weight loss.
a
Rosemary CMC Antioxidant activity. / Wambura et al., 2008
Roasted peanuts: Increases oxidative stability compared to coating containing tea
leaf extract and vitamin E.
a
Green tea Chitosan Enhances the antimicrobial and antioxidant properties of the film. Siripatrawan & Noipha, 2012
95% DS Pork sausages: Inhibits lipid oxidation and microbial growth, extends shelf life.
Rosemary Chitosan Antioxidant activity. / Georgantelis et al., 2007
Beef Burgers: Retains red color, best antioxidant effects.
Tea polyphenols Chitosan Mango (Mangifera indica L): Enhances their sensory quality, reduces weight loss Wang et al., 2007a
90–95% DS and respiration rate and inhibits chlorophyll degradation in fruit peels.
Olive, garlic and Chitosan Butternut squash: Controls PPO activity. Ponce et al. (2008)
c
pepper
Rosemary and Chitosan Butternut squash (Cucurbita moschata Duch): Improves antioxidant protection, Ponce et al., (2008)
c
olive prevents browning reactions, reduces quality loss and does not introduce
deleterious effects on the sensorial acceptability of squash.
c
Rosemary Chitosan Romaine lettuce: Controls PPO activity. Ponce et al. (2008)
a b c
3 Aqueous extract, Ethanol:water 50:50 (v:v), Oleoresins, AOX: antioxidant, TS: Tensile strength (MPa) and EB: Elongation at break (%).
51
2.8 Conclusions and perspectives
Food safety and quality can be influenced by the chemical interactions between biopolymer
packaging materials and foods. There is evidence that natural additives such as polyphenol-rich
extracts from plants can change the structure and morphology of edible films and coatings and
that there is a close relationship between the incorporation of plant extracts and physicochemical
modifications of edible films and coatings.
A combined analysis of functional and bioactive properties is crucial for predicting the
functionality of edible films incorporating natural additives. Polyphenolic-rich plant extracts can
modify the film structure and film functional properties depending on the concentration of
phenolic compounds, the nature of the biopolymer in which they are incorporated and the
conditions (e.g., pH, moisture, temperature) in which the film is developed, processed and/or
stored. The antioxidant activity of the film incorporating bioactive natural additives depends on
the polyphenol type and concentration present in the extracts. However, compatibility with the
biopolymer, stability during processing and storage and the conditions of the bioactivity of the
bioactive natural additives should also be evaluated. Because the antioxidant activity of films
with active natural additives can decrease over time, controlled delivery studies of natural
additives or their main compounds in films/materials should be performed.
Because plant extracts are complex systems, to evaluate their contribution to the bioactive and
functional properties of edible films, the main bioactive compounds from the plant extracts must
be identified, and the properties of the films containing the plant extract must be evaluated in two
ways: (i) incorporating the main bioactive compounds separately and in different concentrations
and (ii) incorporating a plant extract concentration equivalent to the concentration of the
bioactive compounds present in the extract. Such an evaluation establishes whether the changes
in the functional properties and bioactivity of the edible films are due to the chemical structure,
the active compound concentration in the extracts and/or a synergic effect of the plant extract
compounds.
52
The incorporation of natural extracts from plants, spices and herbs and the incorporation of
vitamins C and E, alone or combined, represent a promising approach for the development of
bioactive edible films/coatings with new and improved bioactive, mechanical and
physicochemical properties and applications.
When incorporated in biopolymer matrices, several natural plant additives provide antioxidant
activity, barriers to UV-visible light and beneficial properties for the reduction of lipid oxidation
and the browning of foods. However, in vivo studies of their capacity to preserve the quality,
shelf life and nutritional value of foods remain limited. Furthermore, there are a wide range of
plant natural sources with bioactive properties that remain to be investigated, regulated for
application directly on foods and used for the development of active packaging or biopolymer-
based edible films for preserving and adding value to foods.
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Chapter 3. Recovery of antioxidant polyphenolics by conventional and ultrasound-assisted
CHAPTER 3
RECOVERY OF ANTIOXIDANT POLYPHENOLICS BY CONVENTIONAL AND
ULTRASOUND-ASSISTED EXTRACTION METHODS FROM ROSEMARY AND
MURTILLA LEAVES
A. Silva-Weiss a*, V. Bifani b, M. Ihl b, G.V. Barbosa-Cánovas c
a
Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Doctoral Program in Science of
Natural Resources, Universidad de La Frontera, PO Box 54‒ D, Temuco, Chile.
b
c
Center for Nonthermal Processing of Food, Washington State University,
Pullman, WA 99164-6120, USA
Abstract
Optimal operation conditions of ultrasound-assisted extraction (UAE), duty cycle, wave
amplitude, and exposure time were determined for maximizing recovery of total phenolic
compounds (TPC) from rosemary and murtilla leaves. The UAE; conventional maceration
extraction (ME); and combination of UAE+ME methods in TPC recovery, free radical
scavenging activity, and flavonoid concentration were investigated and compared. The structure
of the leaf residues from UAE, ME and the control (water at 30°C, 1 min, without stirring) were
examined. The optimal conditions determined, in order to obtain the highest TPC recovery from
rosemary, were 20% amplitude, duty cycle 0.8 – 1 and 45 min exposure time. UAE of antioxidant
phenolic compounds from rosemary and murtilla leaves was more effective than ME and
UAE+ME. After UAE, the rosemary residue or cake showed noticeable changes on their
microstructure. On the surface, trichomes were disintegrated and holes between 18.2 - 804 nm
were observed, while on the cross-section, cell walls were broken, and microscopic channels of
1.01 - 1.24 µm were observed.
Keywords: Ultrasound-assisted extraction, Phenolic compounds, Rosemay leaves, Murtilla

leaves, Optimization, Antioxidant activity.
61
3.1 Introduction
Phenolic compounds, ubiquitous in plants, are an essential part of the human diet, and are of
considerable interest due to their antioxidant properties. However, many phenolic compounds are
easily hydrolyzed and oxidized; therefore, their recovery can be negatively influenced by long
extraction times and high temperature (Dai & Mumper, 2010) used on conventional extraction by
maceration. Other parameters that affect extract quality and process efficiency in the
conventional extraction by maceration are solid-liquid ratio and sample particle size.
Ultrasound-assisted extraction (UAE) has been recognized as an efficient and environmental-

friendly process for the extraction of phenolic compounds from a wide range of plants, herb and
spices. According to Vilkhu et al. (2008), the higher yield obtained in these UAE processes are of
major interest for the industry, since the technology is an “add on” step to the existing process
with a minimum alteration. In addition, UAE allows a reduction in solvent usage, and shortening
the extraction time; also the application in aqueous extraction where organic solvents can be
replaced with generally recognized as safe (GRAS) solvents. However, depending on the process
conditions and the matrix, UAE could allow both positive and negative effects on extract quality.
These effects include intensification of the release of biologically active substances and
inactivation of phenolics, respectively (Gribova et al., 2008; Soria & Villamiel, 2010). Sound
waves are defined as mechanical vibrations in a solid, liquid or gas.
The ultrasonic processor generates longitudinal mechanical vibrations by means of electric

excitation. Ultrasonic waves of high power levels (low frequency, 20 – 100 kHz and high
intensity 10 – 1000 W/cm2) consist of alternating compression (high-pressure) and rarefaction
(low-pressure) cycles traveling through a medium (Figure 3.1). The ultrasound causes cavitation
in the liquid. It occurs when, in rarefaction cycles, the pressure of the liquid falls. Water
molecules in liquid state reach the vapor pressure of the liquid, forming vapor micro-bubbles or
cavities (cavitation). Then, in compression cycles, these vapor bubbles are crushed and abruptly
imploded, returning to liquid state. The collapse of these bubbles generates pressure waves,
which can be of very high frequencies. The collapses of smaller bubbles create higher frequency
waves, than larger bubbles. In addition, shear at high rates (up to 280 m/s) and temperature
62
gradients occur within a material during the implosion, which can physically disrupt its structure
or promote certain chemical reactions (Ashokkumar et al., 2008; Marcuzzo et al., 2010).
Advantages of the UAE are the disintegration of cell walls, better solvent penetration into cellular
materials and intensification of mass transfer (Vinatoru et al., 1997; Wang & Weller, 2006). In
applications of UAE, optimization of ultrasonic variables, the type of available ultrasonic
processor and plant matrix should be considered (Ma et al., 2008). The majority ultrasound
equipment has a constant output ultrasound frequency, and this power is represented by the probe
being used.
Figure 3.1 Ultrasound Cavitation (Soria & Villamiel, 2010)
63
At the same time, it is possible to modify the amplitude and duty cycle (percentage of ultrasound
application in the overall treatment) of ultrasound equipment, time and temperature of exposure
to these waves and the ratio solvent / sample. More effective recovery of cell contents can be
obtained by optimizing ultrasound application factors including frequency, sonication power and
time, as well as ultrasonic wave distribution (Wang & Weller, 2006; Khan et al., 2010).
According to Rawson et al. (2011), to maintain the stability of bioactive compounds such as
ascorbic acid, total phenolics and lycopene, it is necessary to control the amplitude of the
ultrasound wave, processing treatment temperature and time as well as ultrasonic power density
or intensity (W/m2) transferred to a medium.
Compounds present in vegetables, spices and herbs, with functional and technological properties,
have gained considerable importance in the market of food, pharmaceuticals and cosmetics.
Rosemary (Rosmarinus officinalis L.) extracts have exhibited potent antioxidant activity in both
aqueous and lipid systems and are widely used in the food industry (Del Baño et al., 2003;
Georgantelis et al., 2007). It has recently reported been that aqueous rosemary extract acts as
antioxidant in vivo, increasing antioxidant enzyme activities and decreasing diabetes level (Silva
et al. 2011) as well as inhibiting skin carcinogenesis (Parmar et al., 2011). Rosemary aqueous
extracts contain rosmarinic acid, and several phenolic diterpenes, such as carnosol, rosmanol,
carnosic acid, methyl carnosate and some flavonoids, such as cirsimaritin, genkwanin and 1-
methoxyluteolin-2-glucoside, among the most active compounds (Ibañez et al., 2002; Wada et al.,
2004). Carnosic and rosmarinic acid are the main bioactive compounds present in rosemary
extracts (Moreno et al., 2006), but whole rosemary extract has been shown to be more active than
rosmarinic acid (Frankel et al., 1996).
Murtilla (Ugni molinae Turcz) is an endemic shrub found in central - southern Chile, which
belongs to the Myrtaceae family. Aqueous extracts from murtilla leaves showed high antioxidant
activity in vitro (Rubilar et al., 2006) and decreased the growth of Pseudomonas aeruginosa,
Klebsiella pneumoniae and Staphylococcus aureus (Shene et al., 2009). Phenolic acids such as
gallic acid, as well as flavonoid aglycones and glycosides from quercetin, myricetin and
kaempferol are among the main compounds found in those extracts (Rubilar et al., 2006; Bifani
et al., 2007).
64
The focus of this research was to determine parameter combinations of UAE; ultrasonic wave
amplitude, exposure time and duty cycle; where it can be obtained a maximum recovery of
phenolic compounds from rosemary, a plant that was used as a model to validate the processing
approach. In addition, three extraction systems: UAE, maceration extraction (ME) and
combination of UAE + ME; were compared with the recovery of total phenolic compounds
(TPC) and flavonoids, as well as DPPH. free radical scavenging activity from rosemary and
murtilla leaves.
3.2 Materials and methods
3.2.1 Materials
Plant materials: Rosemary leaves (Rosmarinus officinalis) were purchased from a local
supermarket (Winco Market, Moscow, WA, USA). Murtilla or murtilla leaves (Ugni molinae
Turcz) were obtained from the Instituto de Investigaciones Agropecuarias (INIA Carillanca),
Temuco, Chile.
Chemicals: Anhydrous sodium carbonate (Na2CO3), Folin-ciocalteu phenol reagent (2 N, Sigma-

Aldrich), gallic acid 98% (3, 4, 5-Trihydroxybenzoic acid, Acros Organics), quercetin dihydrate
97%, DPPH free radical (2, 2-diphenyl-1-picrylhydrazyl Sigma-Aldrich), and aluminium chloride
hexahydrate 99% (AlCl3, Acros Organics).
3.2.2 Preparation of plant material
In order to obtain an efficient and effective ultrasound-assisted extraction, it is necessary to take

into account sample characteristics such as moisture content and particle size, as well as the
solvent used for the extraction (Wang & Weller, 2006). Leaf samples were air-dried for 25 h to
about 7% moisture content in a convection oven/shaking incubator (GFL-3032, Germany), at 35
ºC. Aqueous extracts were prepared by adding 10 g of milled leaves in 100 mL of distilled water
(ratio of plant material: solvent = 1:10) at 30 °C. In the rosemary case, the size used for the
extraction was between 1 – 5 mm, whereas murtilla was used as in the powder form. Leaf
65
moisture content was determined by gravimetric mass loss technique by drying the sample (3 – 4
g) to constant weight in an oven at 105 ºC.
3.2.3 Extraction systems
3.2.3.1 Ultrasound-assisted extraction (UAE)

The UAE was performed using an ultrasonic processor (Hielscher USA Inc., Ringwood, New
Jersey, USA) model UP-400S with a titanium probe H22D (22 mm diameter probe, height 100
mm, acoustic intensity: 85 W/cm2). The ultrasonic processor works at 300 W output power in
aqueous medium and 24 ± 1 kHz frequency. A double walled vessel of 500 ml (8 cm internal
diameter, 13.5 cm depth) was used as a treatment chamber. On this, a beaker glass of standard
geometry (75 mm height, 65 mm diameter) allowed processing at the same liquid height and
depth in the mixture probe (40 mm). Probe was centrally located in the mixture of water and
leaves. Temperature was set up and kept constant via a refrigerated bath (VWR Scientific Model
1166, Niles Illinois, USA) to reduce water temperature increase caused by exposure to ultrasound
waves. A thermo-couple was used in the beaker glass to monitor the temperature (30 ± 5 °C)
throughout the experiments.
3.2.3.2 Conventional maceration extraction (ME)

The ME was performed was performed at 170 rpm, 90 min, 30 ºC, according to Silva-Weiss et al.
(2013).
3.2.3.3 Combined extraction process UAE+ME

Combined extraction was obtained applying the UAE treatment and then the extraction was
prolonged in maceration extraction conditions (170 rpm, 90 min, 30 °C).
After extraction in the three systems Figure 3.2, the extracts were filtered with Whatman No. 1
paper in a Büchner funnel under vacuum, and sterilized through a PES membrane of 0.22 µm and
then stored at -20 °C until use. Treatments were performed according to the experimental design
presented in section 3.2.4.1.
66
Figure 3.2 Scheme of process to comparison of three extraction systems
3.2.4 Optimization of UAE conditions
3.2.4.1 Experimental design

Optimization of phenolic extraction conditions from rosemary was carried out using response
surface methodology (RSM). As presented in Table 3.1, a design for 3 factors with 3 levels, each
was applied. Sixteen experimental runs were used, including two replicates at the center point.
All experimental runs were carried out in triplicate with randomized order. The design variables
were ultrasound equipment duty cycle; ultrasound wave amplitude and time, where total phenolic
content (TPC) was the dependent variable. For this experiment, the amplitude of the oscillatory
system was set up between 20% and 100% (equivalent to 100 µm using probe H22).
67
Table 3.1 Factors and levels used for the evaluation of

ultrasound-assisted extraction parameters
Level
Factor Unit
-1 0 1
Cycle X1 0.2 0.6 1.0 [-]
Amplitude X2 20 60 100 [%]
Time X3 15 30 45 [min]
*Amplitude of ultrasound is the percentage of maximum ultrasonic power,

100% amplitude with H22 = 100 µm. cycle or duty cycle: pulse of
ultrasound in fractions of second.
Regarding duty cycle variable, Dey and Rathod (2012) explain that, the pulse duration and pulse
interval refer to ‘‘ON’’ time in seconds and ‘‘OFF’’ time in seconds, respectively. The total time
of a pulse duration period (ON) plus a pulse interval period (OFF) is the cycle time. A duty cycle
is the proportion of the pulse duration period to the cycle time, which can be expressed as
fraction [Eq. 3.1] or percentage.
[Eq. 3.1]
Experimental data were fitted to a second-order polynomial model [Eq. 3.2] and regression
coefficients were obtained.
k k k −1 k
Y = β 0 + ∑ β i X i + ∑ β ii X i2 + ∑ ∑ β ij X i X j [Eq. 3.2]]
i =1 i =1 i =1 j= 2
68
where Y is the estimated response associated with each variable level combination and β0, βj, βjj
y βij are the regression coefficients for intercept, linear, quadratic and interaction parameters,
respectively, whereas Xi and Xj are the independent variables.
The experimental and predicted values were compared to determine the validity of the model.
ME was used as a control for comparing the effect of the sixteen treatments.
3.2.4.2 Statistical analysis

Analysis of variance (ANOVA) was performed to identify the significance of the responses
among the treatments and model factors, and to support the polynomial equation. The results
were evaluated with the statistical program JMP ® 8 (Trial Version, SAS Institute Inc., Cary, NC,
USA).
3.2.5 Comparison of extraction systems
The evaluation and comparison of UAE, ME, and UAE+ME extraction systems was performed
by applying the sample UAE conditions previously obtained (cycle, amplitude and time). TPC
and flavonoids recovery and DPPH. antiradical capacity, in both rosemary and murtilla leaves,
were studied. The results were expressed as mean ± standard deviation from three replicates.
Significance of the variables at P < 0.05 was determined using ANOVA. The Duncan test was
applied for comparative differences among extraction systems (n = 3). The difference of means
between pairs leaf type was resolved by means of confidence intervals using a Tukey-b test at a
level of significance of p < 0.05.
3.2.6 Analysis
3.2.6.1 Total phenolic compounds (TPC)
Total phenolic assay includes monophenols and easily oxidized polyphenols, where aqueous
monophenol solutions may also act as antioxidants (Singleton et al., 1999). The phenolic content
69
was determined using the Folin-Ciocalteu reagent method (Singleton & Rossi, 1965).
Appropriate dilution of the extract (500 uL) was oxidized for 6 min with 250 µL of Folin-
Ciocalteu (1 N) and the reaction was neutralized with 1250 uL sodium carbonate (20 % w/v). For
the blank, sample volume was replaced by distilled water. The tubes were vortexed and covered
with parafilm. After two hours of incubation at dark, room temperature (∼20 ºC) the absorbance
of the resulting blue color was measured at 760 nm using a spectrophotometer (Shimatzu UV-
2550, with temperature controller, CPS-control). If the absorbance exceeded 1, the sample was
diluted until the reading was less than this value. Total phenolic content was expressed as
milligrams of gallic acid equivalents (GAE) per gram of dry weight sample. The standard curve
based on gallic acid provided the equation y = 0.0157x - 0.0357, R2 = 0.9957, where y is the
absorbance at 760 nm and x is gallic acid concentration (mg/L). For each treatment, the TPC
value was determined as the average of the three independent replicates (n = 3). The dry matter of
the extract was determined after drying 3.000 mL of extract in an oven at 105 ºC for 24 h. Values
are mean ± SD of samples analyzed in triplicate.
3.2.6.2 Flavonoid content

Flavonoid content was determined by the aluminum chloride colorimetric method according to
Chang et al. (2002). The content of flavonoids in the extracts was measured
spectrophotometrically at 415 nm through the formation of a bright yellow complex between
flavonoids and aluminium. Quercetin was used as standard for the calibration curve. Quercetin
(10 mg) was dissolved in 80% ethanol and then diluted to 25, 50, 75 and 100 µg/mL to build up
the calibration curve. Various dilutions were also tested in the rosemary and murtilla leaf extracts
under study. Dilutions of standard or sample (0.5 mL) were mixed with 1.5 mL 95% methanol,
0.1 ml of aluminium chloride 10%, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled
water, being incubated for 30 minutes at room temperature. Absorbance of the reaction mixture
was measured at 415 nm. For the blank, the amount of aluminum chloride was replaced by
distilled water.
3.2.6.3 DPPH radical scavenging activity

The DPPH method is suitable for testing antioxidant capacity of biological material, since it is
not interfered with glucose and it is used for measuring the antioxidant activity of the most
70
common natural antioxidants (Blois, 1958). DPPH radical scavenging was measured according to
the methodology described by Tepe (2008), with some modifications. Briefly, a stock solution of
aqueous extract (0.1 g wet basis/ mL) was diluted from 1:05 to 1:200. Aliquot of 1250 µL of each
extract solution was transferred to cuvettes, and 500 µL of a recently prepared methanolic
solution of DPPH (0.10 mM) was added to each. Then, the mixture was vortexed vigorously and
left for 20 min at room temperature in the dark. The absorbance was then measured at 517 nm
using a spectrophotometer (UV-2550, Shimatzu).
The tests were carried out in triplicate and DPPH scavenging activity was calculated from
[Eq. 3.3]].
 Abs0 − Abs1 
DPPH scavenging activity (%) =   × 100 [Eq. 3.3]]
 Abs0 
where Abs0 was the absorbance of control and Abs1 was the absorbance in the presence of the test
compound. The spectrophotometer was zeroed with distilled water. Gallic acid was used as the
positive control (standard). The results were expressed as IC50 value, and the concentration of
extract required for 50% scavenging of DPPH free radicals present in the reaction mixture.
3.2.6.4 Field emission scanning electron microscopy (FESEM)
For evaluating the effect of the extraction systems on the microstructure of rosemary leaves
(particle size between 5 – 12 mm), leaf residue of UAE and ME and of control (C) without
extraction (water at 30 °C, 1 min and no stirring) was used. Leaf residue was filtered (Whatman
Nº1) and lyophilized for 15 h at -50ºC and 250 Hg (Freeze Dry Systems, Labconco). The samples
were carefully cut into pieces of 2 – 3 mm using a scalpel to avoid damaging the structure. The
samples were coated with gold for 6 minutes at 150-100 Ǻ. According to Yang & Zhang (2008)
samples were examined under high vacuum and an acceleration voltage of 20 kV. Surface and
cross-section morphology of residue was evaluated.
71
3.3 Results and discussion
3.3.1 Effect of UAE conditions on recovery of phenolic compounds
According to ANOVA analysis, the recovered phenolic content from rosemary leaves varied
significantly (P < 0.01) among the UAE treatments. Depending on the combination of UAE
conditions applied, up to 35% difference in the TPC recovery from rosemary leaves can be
obtained. Duty cycle and exposure time, as well as amplitude-time interaction significantly
influenced TPC recovery (P < 0.01).
The UAE conditions that allowed a maximum recovery of phenolic content from leaves (71.54
mg GAE/g leaves d.m) were duty cycle of 0.8 – 1, 20% amplitude of ultrasonic wave and 45 min
of ultrasonic time. Under these conditions, the desirability function for maximize the TPC
recovery response, given by desirability profiler of JMP program, was 0.9976. This means that
the maximization was effective (on a scale of zero to one). Consistent with our results, Dey &
Rathod (2012) recenttly showed that the extraction yield of β-carotene from spirulina has a
maximum between 0.6 – 1 duty cycle. On the other hand, small p-values for the interaction
amplitude-time and the squared terms of amplitude and time indicated that there is curvature in
the response surface (Figure 3.3), which can be represented by a second order equation. The
predicted model is presented in the Eq. 3.4.
TPC = 20.1823 + 20.9135 X1 - 0.1604 X 2 + 1.9678 X 3 + 0.0626 X1X 2 + 0.1749 X1X 3

2 2 2
0.0054 X 2 X 3 - 17.9225 X1 + 0.0023 X 2 - 0.021 X 3 [Eq. 3.4]]
The lack of fit test did not present problems with the model (P > 0.05), showing that the
independent variables studied represented a 77% of the variation in TPC recovery from rosemary
in aqueous media. Consequently, this model can be applied for the estimation of the TPC
72
recovery response, by setting the duty cycle, amplitude and exposure time of ultrasound, within
the boundary conditions studied.
Experimental values were well correlated with predicted values by the model for total phenolic
recovery from rosemary (Figure 3.4). It was noted that if an adequate combination of factors for
the UAE (1 cycle, 20% amplitude and 45 min) is applied, it is possible to obtain an increase up to
14% in the TPC recovered from rosemary leaves (70.11 mg GAE/g leaves d.m.) compared with
conventional maceration extraction (60.45 mg GAE/g leaves d.m).
Similarly, for phenolic acids from Salvia miltiorrhiza root, it was reported by Dong et al. (2010)
that 15% more efficiency in the UAE (45 Hz; 60% aqueous ethanol at 30 ºC, 25 min and ratio of
material to solvent of 1:20) was obtained than when using reflux extraction. TPC recovery from
rosemary leaves in aqueous media with both ultrasound or maceration extraction were higher
than the value reported by Weerakkody et al. (2010) for rosemary leaves using ethanol as solvent
(56.76 mg GAE/g leaves d.m.) in a similar maceration extraction system (leaves/solvent: 1:10, 24
h and 28 ºC).
73
a) Duty cycle : 1
(-)
b) Wave amplitude: 20 %
c) Time: 45 min
Figure 3.3 Response surface plot, showing the effect of: a) Time and wave amplitude, b) Duty
cycle and time, and c) Duty cycle and wave amplitude on TPC recovery from rosemary leaves.
74
Figure 3.4 Correlation between experimental and prediction of TPC recovery from
rosemary, and their comparation with TPC recovery obtained through of conventional
maceration extraction (horizontal line).
3.3.2 Comparison of three extraction systems
3.3.2.1 Recovery of phenolic compounds from rosemary and murtilla leaves

Figure 3.5 depicts the TPC recovery and free radicals scavenging activity of DPPH from
rosemary and murtilla leaves in three different extraction systems (UAE, ME or UAE+ME). UAE
showed significantly higher TPC recovery from rosemary and murtilla leaves than ME or
UAE+ME systems. In these three extraction systems, the TPC recovery proved to be higher in
murtilla than in rosemary leaves.
75
120
Murta a,B
100 Rosemary
a,A
(mg GAE/g leave d.m.)

Total phenolic content
a,A
b,B
80
b,A
b,A
60
40
20
0
8.0 ME UAE UAE+ME
Murta
7.0 Extraction systems b,A
Rosemary
(mg GAE/g leaves d.m.)
6.0 b,A
IC50 DPPH
5.0 b,B
4.0
a,A a,A
3.0 a,B
2.0
1.0
0.0
ME UAE UAE+ME
Extraction systems
Figure 3.5 Total phenol compounds (a) and DPPH radical scavenging capacity (b) from murtilla
and rosemary leaves using ultrasound-assisted extraction (UAE), maceration extraction (ME) and
combination of UAE + ME systems. Different capital letters indicate significant difference (P <
0.05) between extraction systems and different lower letters indicate significant difference (P <
0.05) between murtilla and rosemary leaves.
76
3.3.2.2 DPPH. radical scavenging activity

The extract concentration that inhibits 50% of DPPH free radicals (IC50) was 32% lower in
murtilla extract and 28% lower in rosemary extract obtained by UAE than by use of ME. Similar
to the results of TPC recovery, the scavenging activity of rosemary and murtilla leaves extracted
under UAE were higher than with ME or UAE+ME extraction systems. These results can be
attributed to both long time and orbital stirring of ME, which could release a higher component
concentration of the leaves, thus reducing the antioxidant compound concentration of the extract.
Also, as it is known, antioxidant activity of phenolic compounds in biological systems and food
depends on the structure of the food, in particular the number and positions of the hydroxyl
groups and the nature of substitutions on the aromatic rings (Balasundram et al., 2006). Since
sonochemical reactions achieve targeted hydroxylation of polyphenolics and carotenoids to
increase bioactivity (Vilkhu et al., 2008), the ultrasound process acts as an efficient way of
enhancing the antioxidant activity of certain compounds such as flavonoids (Ashokkumar et al.,
2008). However, during acoustic cavitations, it is also possible that hydroxyl radicals can be
produced and these sonochemically generated radicals react with easily oxidizable compounds
such as phenols (Wan et al., 2005; Soria & Villamiel, 2010).
According to Chang et al. (2002), aluminium chloride forms acid stable complexes of yellow
color at 415 nm with the C-4 keto group and either the C-3 and C-5 hydroxyl groups of flavonols
(such as galangin, morin and kaempferol) and flavones that, in addition to C-3 and C-5, have C-4'
hydroxyl groups such as luteolin. Aluminium chloride also forms acid labile complexes with the
ortho-dihydroxy groups in the A-ring (C7 and C8) and B-ring (C3' and C4') of flavonoid
glycosides (such as rutin and quercitrin) and flavonoid aglycones (such as quercetin, and
myricetin) at 415-440 nm.
3.3.2.3 Flavonoid content

In murtilla leaf extract, a yellow complex of flavonoids with aluminium was observed at
wavelength maximum of 415 nm, while wavelength maximum of quercetin was at 428 nm (Fig.
3.6). Flavonoid content in murtilla extract obtained by ME was lower than that obtained by UAE
or UAE+ME (see Figure 3.7). This result is because ultrasound waves quench the radical
77
sonochemistry, especially in aqueous systems, avoiding degradation of bioactives (Vilkhu et al.,

2008).
Figure 3.6 Wavelength spectrum of quercetin and murtilla extract, with and without
incorporation of AlCl3, obtained by UAE and UAE+ME: a) Quercetin with Al3+, b) Murtilla
UAE+ME with Al3+, c) Murtilla UAE with Al 3+
, d) Murtilla UAE without Al3+ and e) Murtilla
UAE+ME without Al3+.
78
0.4
a
a
(mg QE/ g leave d.m.)

Flavonoid content 0.3
b
0.2
0.1
0.0
ME UAE UAE+ME
Extraction Systems
Figure 3.7 Flavonoid content of murtilla and rosemary leaf extract

measured by AlCl3 reaction. Different lower letters indicate
significant difference (P < 0.05) between extraction systems.
Rosemary extract formed no visible complex of flavonoid-Al at 415 nm (Figure 3.8). This result
could be due to the presence of flavanones and isoflavones that show no significant absorption at
415 nm. These results can be estimated by infusing the 2, 4-dinitrophenylhydrazine method
(Chang et al. 2002). Absorbance peak was observed at 340 nm in rosemary extract, which
interfered with flavonoid measurement. This band has been reported in flavones that, in addition
to C-3 and C-5, have C-4’ hydroxyl groups as in luteolin (Lock Sing de Ugaz, 1997). These
results are consistent with the presence of luteolin (1-methoxyluteolin-2-glucoside) in rosemary
leaves, which is one of the main components showing quenching effects (Wada et al., 2004).
79
Figure 3.8 Wavelength sweep of quercetin (a) and rosemary extract (b)
spectrum after to reaction with AlCl3.
3.3.3 Morphology of rosemary leaf residue
Field emission scanning electron microscopy (FESEM) has provided evidence of the mechanical
effects of ultrasound waves. As can be seen in Figure 3.9, physical changes were observed in
rosemary leaf surface, when UAE and ME were applied. In rosemary leaves, glandular trichomes
or oil-containing glands are found. In the control sample, whole trichomes were observed. In
contrast, trichome has been broken with ME and disintegrated with UAE. Thereby, the
mechanical effect produced on the leaf surface by the ultrasonic wave is greater than with orbital
agitation used in the maceration. Disintegration of rosemary leaf trichomes has also been
observed by applying microwave extraction (Bousbia et al., 2009). Unlike the control residual
80
material, holes can be observed in the surface of rosemary leaves from UAE (18.2 – 804 nm) and
ME (55.1 – 276 nm).
a) b
c) d)
Figure 3.9 Scanning electron microscopy of rosemary leaf surface after: a)

and b) control untreated 5.000 X, c) ME 5.000 X and d) UAE 5.000 X.
Microscopy of the cross-section indicated that cell wall was broken with ME and UAE (Figure
3.10). In addition, in the cell wall of rosemary leaves from UAE, holes between 1.01 – 1.24 µm
can be observed at higher microscopy magnification (Figure 3.11). These holes correspond to
microscopic channels (Soria & Villamiel, 2010), whereby the release of active compounds from
the cell could occur at a higher rate than in other extraction systems. As ultrasound breaks the cell
wall mechanically by the cavitation shear forces, it facilitates the transfer from the cell into the
solvent.
81
a) b)
c) d)
Figure 3.10 Cross-section of the rosemary cell walls: a) control 10.000 X,

b) ME 10.000 X, c) and d) UAE 10.000 X.
Phenolic compounds in the cell are concentrated mainly in the vacuole (97 %) and the cell wall
shows only (3%) (Toivonen et al., 2008). Phenolic compounds, when are released from vacuole,
can bind to cell walls (Padayachee et al., 2012a,b), forming complexes with polysaccharides
present in the cell walls. These complexes could be attacked by radicals formed in water by
ultrasonic waves, increasing the release of the phenolic compounds from plant material.
82
a) b)
c) d)
Figure 3.11 Cross-section of the rosemary cell walls after to ultrasound-assisted

extraction (UAE). Different amplifications: a) UAE 2.000 X, b) UAE 5.000 X, c) UAE
8.000 X, d) UAE 15.000 X
3.4 Conclusions
Ultrasound-assisted extraction is an efficient and low cost technology on the recovery of

antioxidant phenolic compounds. The optimal conditions to obtain the highest phenolic
compound recovery from ultrasound-assisted extraction were 20% wave amplitude, duty cycle of
0.8 – 1 s and 45 min. Using rosemary leaves in aqueous media, the ultrasound variable cycle,
time and amplitude, represents a 77% of the variation in the phenolic compound recovery. The
ultrasound-assisted extraction from rosemary leaves allowed 23% more phenolic compound
83
recovery than maceration extraction and 14% more than ultrasound-assisted extraction with
maceration extraction. On the other hand, the ultrasound-assisted extraction from murtilla leaves
recovered 24% more phenolic compound than maceration extraction and 19% more than
ultrasound-assisted extraction with maceration extraction. Structural changes in the cell wall of
leaves residues from ultrasound-assisted extraction support the results of higher antioxidant
activity and phenolic compund recovery in comparison to maceration extraction and ultrasound-
assisted extraction with maceration extraction.
Acknowledgements
We thank M.Sc. I. Seguel from Unidad de Recursos Genéticos, INIA Carillanca, Chile, for the
murtilla leaf ecotype; and Dr. Valerie Lynch -Holm from Franceschi Microscopy & Imaging
Center, Washington State University, Pullman, USA. This work was supported by CONICYT
CHILE grants Nº 21070302, 24090134, 29090088, FULBRIGHT/CONICYT grant to research
visit at Washington State University, USA and INNOVA-CORFO Project Nº 06N12PAT-57.
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Chapter 4. Polyphenol-rich extract from murtilla leaves on rheological properties of FFS
CHAPTER 4
POLYPHENOL-RICH EXTRACT FROM MURTILLA LEAVES ON RHEOLOGICAL
PROPERTIES OF FILM FORMING SOLUTIONS BASED ON DIFFERENT
HYDROCOLLOID BLENDS
a
Doctoral Program in Science of Natural Resources, Scientific and Technological
Bioresource Nucleus (BIOREN‒UFRO), Universidad de La Frontera, PO Box 54‒ D, Temuco, Chile
b
c
d
Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN, CSIC). 28040 Madrid, Spain
Abstract
The rheological properties of film forming solutions (FFS) were studied as a function of the
hydrocolloid blend type and as a function of polyphenol-rich extract from murtilla leaves (Ugni
molinae Turcz) (PEML) in FFS. The hydrocolloids used in the FFS were sodium
carboxymethylcellulose (CMC), low and high molecular weight fish-skin gelatin (LG and HG),
unmodified and modified corn starch (CS and MS) and chitosan (CH). Six binary blends were
evaluated (CH-CMC, CH-CS, CS-CMC, MS-CMC, HG-CMC and LG-CMC). Steady-state flow
tests and oscillatory measurements within the linear viscoelasticity region (LVR) were carried out.
The studied FFS are non-Newtonian, behaving as pseudoplastic fluids above 0.4 s-1 shear rate.
FFS flow behavior was significantly affected by the hydrocolloid blend type as well as presence
or absence of PEML in the matrix. With CH and CH-CS, the PEML formed aggregates leading to
a gel-like structure with thixotropic behavior. PEML induced gel thermostability as in CH-CMC.
LG-CMC and HG-CMC were unstable with the frequency when PEML was added. FFS based on
CS-CMC, MS-CMC, and CMC behaved as homogeneous solution, either with or without PEML.
The viscosity, ranging between 2.75 - 8.49 Pa⋅s to 2 s-1 at 25 °C, was suitable for the casting
process and provided stable solutions in the wide frequency range and temperature studied.
Keywords: Rheology properties, Edible coatings, Hydrocolloid blends, Polyphenols
87
4.1. Introduction
Innovative packaging technologies with complete biodegradability are growing in importance for
preserving food safety and structural-nutritional integrity (Tharanathan, 2003). The development
of edible films and coatings has been considered for their capability to improve a global food
quality and safety.
Edible coating formulations must wet and spread uniformly on the food product’s surface and,
upon drying, a coating that has adequate adhesion, cohesion, and durability must be formed to
function properly (Krochta & Mulder-Johnston, 1997). Film forming solutions (FFS) based on
hydrocolloids can be used to obtain either edible and biodegradable coatings or films.
Rheological behavior understanding of FFS is critical for the scale-up process, since they have to
ensure that processing requirements and machinability issues can be properly addressed (López,
Zaritzky & García, 2010). In addition, flow properties of FFS are of primary importance for
coating quality in the solid state (Peressini et al., 2003). Rheological data obtained from
characterization of food materials are required for the calculation of any process involving fluid
flow (e.g. pump sizing, extraction, filtration, extrusion, purification), playing an important role in
process design, quality control and sensory assessment of food materials (Steffe, 1996; Marcotte
et al., 2001; Rao, 2007), since these can relate to their structure and physical stability (Yang et al.,
2004; Tzoumaki et al., 2011). The viscosity at high shear rates is an indicator of product viscosity
during processing operations (Bourbon et al., 2010), while sample viscosity at low shear rates is
an indicator of the consistency in mouth (Morris & Taylor, 1982). Thus, a reduction in the
solution viscosity provides a processing advantage during high-shear processing operations, such
as pumping and filling (Tada, Matsumoto & Masuda, 1998), whereas high apparent viscosity
during mastication provides a desirable mouthfeel when consuming it (Reilly, 1997).
Edible coatings can be produced from a single hydrocolloid (polysaccharide or protein) and/or
lipids or mixtures of them (Kester & Fennema, 1986). Hydrocolloids, in particular carboxymethyl
cellulose (CMC), starch, chitosan and gelatins are biodegradable and non toxic products based on
renewable resources. Blending two different hydrocolloids can change strongly both the physical
and rheological properties of FFS and consequently of coatings and films. These changes occur
88
due to the compatibility/incompatibility between two macromolecules, which depend on their

molecular weights, chemical structures, conformations and hydration behaviors, as well as the
addition of various chemicals or additives (Greener-Donhowe & Fennema, 1994; Phan The,
Debeaufort, Voilley & Luu, 2009).
Incorporation of antioxidants additives into packaging or coating materials has become popular
since oxidation is a major problem affecting the food quality (Siripatrawan & Harte, 2010).
Natural compounds from plants incorporated into edible films and coatings could act not only as
antioxidant, but also as antimicrobial, cross-linking and/or anti-browning agents (Mathew &
Abraham, 2008; Wambura, Yang & Mwakatage, 2008; Rahman, Kim & Kang, 2009; Mayachiew
& Devahastin, 2010, Siripatrawan & Harte, 2010). Crosslinking is an important step in the
preparation of hydrocolloid films to ensure their stability and mechanical resistance (Mathew &
Abraham, 2008).
Plant extracts, in terms of active agents, received much attention since there is a tendency of
replacing synthetic agents. There has been shown that the antioxidant power (reductor ability and
free radical-scavenging capacity) as well as physical properties of biodegradable and edible films
and film forming solutions can be increased or vary with adding polyphenol-rich aqueous extract
from oregano or rosemary (Gómez-Estaca et al., 2009a), murtilla leaves (Gómez-Guillén et al,
2007) and ginseng extract (Norajit, Kim & Ryu, 2010). However, the compatibility and stability
of edible coatings enriched with these phenolic compounds have hardly been studied. Royo,
Fernández-Pan & Maté (2010) reported that stability of whey protein-based edible coating
decreased as the concentration of natural compounds from oregano increased. In contrast, gelatin-
based edible coating reacted only slightly with the polyphenols from aqueous oregano and
rosemary extracts as shown by dynamic viscoelastic properties (Gómez-Estaca et al., 2009b).
Murtilla (Ugni molinae Turcz) is an endemic shrub in central-south Chile which belongs to the
Myrtaceae family. Murtilla leaf extract is used in cosmetics for neutralizing the oxidative stress.
In medicine, it has been found to cause a protective effect against oxidative damage in human
erythrocytes (Suwalsky et al., 2007), which has anti-inflammatory (Aguirre et al., 2006),
analgesic (Delporte et al., 2006) and antimicrobial (Shene et al., 2009) activities. In addition,
89
PEML have been shown to have high antioxidant activity in vitro (Rubilar et al.,
2005). Furthermore, studies on the chemical composition of murtilla leaf extracts show the
presence of phenolic acids, flavonoids, and tannins (Montecinos et al., 1991; Rubilar et al., 2005).
Among the main compounds found are phenolic acids like gallic acid, as well as flavonoids
aglycones and glycosides of quercetin, myricetin and kaempferol (Rubilar et al., 2005; Bifani et
al., 2007).
The purpose of this work was to investigate the impact of the polyphenol-rich aqueous extract
from murtilla leaves (PEML) and hydrocolloid blend type on the oscillatory and state-steady flow
behavior of film forming solutions based on fish-skin gelatins, corn starches, sodium
carboxymethylcellulose and/or chitosan.
4.2.1. Materials
Fresh murtilla leaves (Ugni molinae Turcz) of ecotype 27-1 were sampled near Temuco, Chile
(38º35’39’’ South latitude) at the Instituto de Investigaciones Agropecuaria, INIA Carillanca.
Sodium carboxymethylcellulose (CMC), molecular weight 280–400 kDa, degree of substitution
(DS) 0.7–0.9, was purchased from Prinal S.A. (Santiago, Chile). Fish gelatine samples for food,
pharmaceutical grade, were provided by Norland Products Inc. (Cranbury, NJ, USA). Low
molecular weight fish-skin gelatin (LG, ∼55 kDa) and high molecular weight fish-skin gelatin
(HG, 120 kDa) were obtained from the skins of deep water fish such as cod, haddock and pollock,
and contain ∼60 hydroxyproline and ∼96 proline residues per 1000 total residues. Waxy corn
starch (MS, Clearam CH-20, acetylated distarch adipate), containing ∼1% amylose, was
purchased from Roquette Fréres (Lestrem, France). This type of starch was chosen for its stability
at low temperatures (Alvarez et al., 1997). Unmodified corn starch (CS) containing ∼73%
amylopectin and 27% amylose) and medium molecular weight chitosan (CH) (with a
deacetylation degree of 75%) were purchased from Sigma-Aldrich. Glacial acetic acid (98%
purity) and glycerol (1, 2, 3-propanetriol, 87% purity, Merck) were also used to obtain FFS. The
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macromolecules used have different functional groups that can act as potential binding moieties
to PEML (Table 4.1).
Table 4.1 Structure of selected hydrocolloid studied for formulating blend FFS
Type Used Source Structure Moiety c
Starch ⇒ Amylose + Plant α-(1→4)-D-glucose + α-(1→4) and -H, -OH

-CH3 (in MS)
Amylopectin α-(1→6)-D-glucose
Cellulose ⇒ CMC Plant β-(1→4)-D-glucose ⇒ β(1→4)-D- -COOH
-OH
glucopyranose
Chitin ⇒ Chitosan Animal β-(1→4)-D-(N-acetyl)glucosamine + -NH2
-H, -OH
β-(1→4)-D-glusamine
-COCH3
-NHCOCH3
Protein ⇒ Gelatin a Animal α-chain composed of peptide triplets -COOH
-NH2
(Gly-X-Y-Gly-X-Y)n, n∼170b
-H, -OH
a
Gelatin obtained by hydrolyzing collagen. Three α-chains intertwined in the collagen triple helix.
b
X and Y attached to glycine (Gly) can be any of the amino acids but proline (Pro), alanine (Ala) and
hydroxyproline (Hyp) are more abundant. In fish gelatin, the abundance of X and Y is 330-360 Gly, 50-
79 Hyp, 95-120 Pro and 95-125 Ala (residues/1000 total amino acid residues) (Karim & Bhat, 2009;
Gómez-Guillén et al., 2002).
c
Depending of medium pH.
4.2.2. Obtaining and characterization of PEML
Leave samples were air-dried for 48 h to about 7% moisture content in a convection oven/shaking
incubator (GFL-3032, Germany) at 35 ºC. Dried and powdered Ugni molinae T. leaves were used
in the preparation of PEML. Leaves (10 grams) in 100 mL distilled water were extracted at 25 ºC
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for 90 min at 170 oscillation min-1 and filtered through Whatman No 1 filter paper to obtain the
water extract. The PEML showed 1.4% solids and 0.98 g/mL density. Antioxidant capacity,
expressed as the PEML concentration required to scavenge 50% of ABTS.+ (radical cation 2,2'-
azinobis-(3-ethylbenzothiazoline-6-sulfonate)) free radical was 0.93 ± 0.14 mg dm leaves/mL.
Total phenol content, including monophenols as well as more easily oxidized polyphenols
(Singleton, Orthofer & Lamuela-Ravent, 1999) was 40.67 mg gallic acid equivalent (GAE)/g dm
murtilla leaves.
4.2.3 Preparation of the film-forming solutions (FFS)
The concentration of components in each FFS for hydrocolloid blends formulation is shown in
Table 4.2. Gelatin-based FFS: For aqueous solutions of HG and LG (1.0 % w/w), dry gelatins
were dissolved in distilled water and maintained in a water bath at 45 ºC for 30 min, then solutions
were magnetically stirred for 30 min. CMC-based FFS: CMC (1.0, 1.5 and 2% w/w ) was
completely dispersed in distilled water with the shaking incubator at 170 oscillation min-1 and 25
ºC for 24 h, followed by stirring using an Ultra-Turrax T25 (Janke & Kunkel, Germany) with
four-blade propellers, at 500 rpm and 35 ºC for 30 min. Starch-based FFS: Aqueous solutions
from CS and MS (0.5% w/v) were prepared by heating beyond their gelatinization temperature (70
± 5 ºC) for 20 min under gentle magnetic stirring, then cooling at 25 ºC at an approximate rate of
2–3 ºC/min. Chitosan-based FFS: Chitosan was dispersed in acetic acid (1% v/v) to prepare
solutions of 1.0, 1.5 and 2% w/w. This dispersion was stirred using an Ultra-Turrax T25 with four-
blade propellers at 500 rpm and 65 ºC for 2 hrs. After the chitosan was completely dissolved, the
solutions were filtered through cheese-cloth.
4.2.4. Formulation of blend of hydrocolloids solutions
The concentration of total hydrocolloids in solution (HT) was maintained at a constant 2% w/v
value. Blended solutions were prepared by mixing the FFS shown above, without or with added
PEML (20 mL/g HT, 81.33 mg GAE/g HT), and glycerol (25 g/g HT) previously dissolved in
distilled water and gently controlled stirring for 30 min at 45 ºC.
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Table 4.2 Composition of FFS based on hydrocolloid (H) solution blends

with or without polyphenol-rich extract from murtilla leaves (PEML).
Glycer
Hydrocolloid PEML b
ol
FFS [% w/w] [g/g HT] [mL/g HT]
H1-H2 H1 H2 HT a Without With c
CH 2.00 - 2.00 0.25 - 20

CH-CS 1.50 0.50 2.00 0.25 - 20
CH-CMC 1.00 1.00 2.00 0.25 - 20
CMC 2.00 - 2.00 0.25 - 20

CS-CMC 0.50 1.50 2.00 0.25 - 20
MS-CMC 0.50 1.50 2.00 0.25 - 20
HG-CMC 1.00 1.00 2.00 0.25 - 20

LG-CMC 1.00 1.00 2.00 0.25 - 20
a
HT: Total concentration of hydrocolloids in solution, b Polyphenol-rich extract
c
from Murtilla leaves, Total phenol content from PEML expressed as gallic
acid equivalent (GAE): 81.33 mg GAE/ g HT
The incorporation of PEML was performed as part of the dissolution solvent in the individual
preparation of CMC or CH, depending on the blend type. In the blend of CH and CMC, the
amount of PEML was divided in two portions and incorporated into CH and CMC in equal
proportions. The FFS based on HG, LG, CS and MS were formulated initially without PEML and
then blended with CMC or CH FFS, with or without PEML, at the indicated concentrations in
Table 4.2. Finally, vacuum was applied to remove air from the systems and prevent air bubble
formation in blend FFS. The pH value of blend FFS, with and without added PEML, was
determined at 23 ºC. Samples for rheological analysis were stored at 5 ºC for 3 days, until use.
93
4.2.5. Rheological measurements: Fundamentals and methods
Dynamic viscoelasticity and steady-state flow measurements were carried out in a controlled-
stress rheometer Bohlin CVO (Bohlin Instruments, Inc. Grandbury, NJ) with a cone-plate
geometry (cone angle 4º, diameter = 40 mm, gap = 150 µm). Before analysis, the sample was
placed into the rheometer, which was equilibrated at 25 ºC.
4.2.5.1. Steady-shear measurements

Flow curves and thixotropic properties were obtained by registering the shear rate when shear
stress was increased from 0 to 250 Pa and decreased from 250 to 0 Pa at 25 ºC. Experimental data
were fitted to Ostwald–de Waele model or rheological Power law model (Eq. 4.1).
τ = K γ& n [Eq. 4.1]
Where τ is shear stress (Pa), K is consistency index (Pa⋅s), n is flow index (-) and γ& is shear rate
(s-1). For n = 1, the Power law model shows a Newtonian fluid model i.e. a fluid which exhibits a
viscosity that is shear rate-independent. For non-Newtonian material (n ≠ 1), when n > 1 material
has shear-thinning or dilatant behavior i.e. viscosity increases with shear rate, whilst that when
n < 1 material has shear-thickening or pseudoplastic behavior i.e. viscosity decreases with shear
rate.
In addition, to elucidate the behavior of hydrocolloids blend solutions, with or without PEML,
the changes in the solution’s apparent viscosity or steady-shear viscosity (η, Pa⋅s) were
investigated. Apparent viscosity for Power law model was calculated as seen in Eq. (2). Three
repetitions were performed for each sample.
η = Κ γ& n −1 [Eq. 4.2]

( γ& )
94
4.2.5.2. Dynamic measurements of viscoelastic properties

Three dynamic studies were performed: (1) An oscillatory stress sweep test from 0.03 to 400 Pa,
at a constant frequency of 0.1 Hz and 25 ºC was made to set the upper limit of the linear
viscoelastic region (LVR). (2) Frequency sweep over a range of 0.01–50 Hz at 25 ºC was
performed at an oscillatory stress within LVR for each solution. Viscoelastic parameters, storage
or elastic modulus (G’, Pa), loss or viscous modulus (G’’, Pa), Complex modulus (G*, Pa) [Eq.
4.3], complex viscosity (η*, Pa⋅s) [Eq. 4.4] and tangent of the phase angle (Tan δ, -) [Eq. 4.5] as
a function of angular frequency (ω, rad/s) were measured, obtaining the typical mechanical
spectra. FFS behavior with respect to frequency was classified as predominantly viscous (G' <
G'') or predominantly elastic (G' > G''), as well as by the presence of crossover point (G' = G''),
which means the frequency at which the typical behavior of gel shifts from elastic to viscous (Lo,
Robbins, Argin-Soysal & Sadar, 2003). (3) Temperature ramps were performed at a scan rate of 1
ºC/min and 0.1 Hz from 40 - 2 ºC and back to 40 ºC. Here the phase transitions with temperature
and elasticity of the FFS was evaluated through the phase angle. The elasticity is the behavior of
stress/strain reversible, which is measured as the reciprocal of the phase angle.
G* = G'2 + G' '2 [Eq. 4.3]
G*
η* = [Eq. 4.4]
ω
G' '
Tanδ = [Eq. 4.5]
G'
where G’ is a measure of the energy stored and recovered in a cyclic deformation whereas G’’ is a
measure of the dissipated energy, Tan δ is the tangent of the phase angle (δ) and represents the
ratio of viscous modulus to elastic modulus and is a useful quantifier of the presence and extent
of elasticity in a fluid, η* is the frequency-dependent viscosity function determined during forced
harmonic oscillation of shear stress; contains both real and imaginary parts.
95
4.2.6. Film forming capacity
To evaluate the film forming capacity of hydrocolloids blend solutions, such as in López et al
(2010), 40 g of FFS 2% w/w were casted onto plexiglass plates (12.5 × 12.5 cm) and dried at
40 ºC for 24 h in a ventilated oven (Binder FD 240, Tuttlingen, Germany). Homogeneity and
appearance of the films were examined as well as the film remotion easiness and adherence at
50% RH.
4.2.7. Data analysis
JMP® 8.0. Software (SAS Institute, Version 6.09, Cary, NC) was used for all statistical analyses.
Rheological parameters were analyzed using factor analysis ANOVA related to factors: X1: the
presence or absence of PEML and X2: the hydrocolloid blend type. Significance was accepted at
the 5% confidence level. Significant differences between FFS were performed using orthogonal
contrasts, since the blends are different in nature.
4.3. Results and discussion
4.3.1. Steady shear behavior
The values from flow curves of FFS at 25 °C were modeled using the Power law. Flow
parameters are show in Table 4.3.
96
Table 4.3 Parameters for the Power law model at shear rate above 0.4 s-1 at 25 ºC. Significance of difference between parameters K
and n of FFS with and without PEML using orthogonal contrasts and apparent viscosity based on form of the Power law model:
η(γ& ) = K (γ& ) n −1 at 25 ºC.
Sample K ( Pa⋅⋅s n) n(-) r2 ( - ) η (γ& = 2) ( Pa⋅⋅s ) pH
PEML Without With Without With Without With Without With Without With
CH 4.23 164 ** 0.70 0.32 ** 0.993 0.984 3.45 102 ** 4.74 4.47 NS
CH-CS 3.15 150 ** 0.63 0.17 ** 0.990 0.915 2.44 84.5 ** 4.47 4.43 NS
CH-CMC 64.0 36.3 ** 0.16 0.37 ** 0.917 0.982 35.8 23.4 * 4.64 4.59 NS
CMC 12.6 6.26 ** 0.40 0.50 ** 0.976 0.984 8.49 4.44 ** 6.73 5.42 **
CS-CMC 10.6 3.89 * 0.43 0.50 ** 0.983 0.990 7.02 2.75 ** 6.22 5.44 **
MS-CMC 7.00 4.53 ** 0.45 0.49 ** 0.985 0.990 4.77 3.19 ** 5.87 5.43 *
HG-CMC 2.45 1.53 NS 0.52 0.57 ** 0.996 0.996 1.75 1.14 * 6.70 5.40 **
LG-CMC 4.75 0.60 ** 0.47 0.65 ** 0.992 0.998 3.30 0.48 ** 6.18 5.16 **
* P < 0.05, ** P < 0.01, NS: Not significant
97
The FFS studied had a non-Newtonian behavior (n ≠ 1). Viscosity curves (Figure 4.1) showed
that apparent viscosity was highly dependent on the shear rate at which shear stress was
measured. Values and statistical significance of power law parameters for FFS with and without
PEML and different hydrocolloid blends are shown in Table 4.3 and Table 4.4, respectively.
1000 1000
a) b)
100 100
Viscosity (Pa s)
Viscosity (Pa s)
10 10
1 1
0.1 0.1
0.01 0.1 1 10 100 1000 0.01 0.1 1 10 100 1000
10 10
c) d)
Viscosity (Pa s)
Viscosity (Pa s)
1 1
0.1 0.1
0.1 1 10 100 1000 0.1 1 10 100 1000
Shear rate (1/s) Shear rate (1/s)
Figure 4.1 Viscosity curve of FFS with (filled symbols) and without (Open symbols)
PEML at 25 °C. a) CH-CMC (■,□) and CMC (▲,∆) b) CH-CS (■,□) and CH (▲,∆), c)
CS-CMC (■,□) and MS-CMC (▲,∆), d) HG-CMC (■,□) and LG-CMC (▲,∆).
Multifactorial analysis of variance (ANOVA) indicates that the interaction between the
hydrocolloid blend type and the presence of PEML in coatings affect significantly the response
98
of K and n (P < 0.05). This means that both the incorporation of PEML as well as hydrocolloid
blend type affects the rheological behavior of the FFS studied.
4.3.1.1 FFS based on CMC, CS-CMC and MS-CMC

In relation to coating and film apareance, FFS based on CMC was homogeneus, without bubbles,
such as their blend with normal corn starch (CS-CMC) and modified waxy corn starch (MS-
CMC). The dried films were smooth, homogeneous, easy to peel and to handle them, neither
adhering to the plate nor sticking itself. Besides, these films were transparent, although CS-CMC
film was visually more opaque than CMC and MS-CMC. Excellent stability without phase
separation was observed in CMC and MS-CMC solutions; but in CS-CMC, a slight
retrogradation was observer after a rest period.
Table 4.4 Significance between hydrocolloid blends type and FFS without and with PEML.
Orthogonal contrasts of Power law parameters.
K ( Pa⋅⋅s n) n (-)
Contrast
Without With Without With
CH-CS vs CH NS ** ** **
CH-CMC vs CH ** ** ** **
CH-CMC vs CH-CS ** ** ** **
CS-CMC vs CMC NS * ** NS
MS-CMC vs CMC ** NS ** NS
MS-CMC vs CS-CMC ** NS ** NS
HG-CMC vs CMC ** ** ** **
LG-CMC vs CMC ** ** ** **
HG-CMC vs LG-
* NS ** **
CMC
*P<0.05, ** P<0.01, NS: Not significant
99
Pseudoplastic behavior over the entire range of shear rates was observed. Similarly, pseudoplastic
behavior has been reported in cellulose derived solutions, like cellulose-based biohydrogel
(Fatimi, Tassin, Turczyn, Axelos & Weiss, 2009; Edali, Esmail & Vatistas, 2001) and
methylcellulose-starch (Peressini et al., 2003), although Cross model can be more appropriate
than Ostwald-de Waele model to describe flow behavior (Benchabane & Bekkour, 2008;
Peressini et al., 2003). When comparing the pseudoplastic behavior among FFS based on CMC,
CS-CMC and MS-CMC, it can be observed that n varies significantly (P < 0.01) among these
hydrocolloid blends (0.40, 0.43 and 0.45, respectively). In the same way, Techawipharat,
Suphantharika & BeMiller (2008) found that n was higher in FFS based on normal rice starch
(n=0.48) and waxy rice starch (n=0.45) than on starch-CMC blends (n=0.43). Whereas, recently
Arancibia, Jublot, Costell & Bayarri (2011) reported a value of n= 0.44 for CMC solution with
5% sunflower oil. In relation to consistency index, K varies significantly among MS-CMC vs
CMC and MS-CMC vs CS-CMC, whereas CS-CMC vs CMC did not show differences (P < 0.01).
The viscosity in CMC, CS-CMC and MS-CMC decreased with the shear rate applied. A shear
rate of 10-1 to 101 s-1 is typical for draining under gravity, when dispersion is applied as coating.
In general, at 2 s-1shear rate, starch addition decreases viscosity of single CMC solution, due to
partly reduced interactions of CMC chains by competition for hydrogen bonds between CMC-
CMC chains and starch-CMC chains, showing their compatibility. Aparent viscosity of FFS
based on CMC, CS-CMC and MS-CMC (η2 = 8.49, 7.02, 4.77 Pa⋅s, respectively) facilited casting
process since a uniform layer of solution was spread on the plate, covering the entire surface at
25 ºC. Because starch with more amylopectin is more easily to react with CMC (Li, Shoemaker,
Ma, Shen & Zhong, 2008), modified waxy corn starch (MS) strongly decreased the viscosity of
the mixture with CMC compared with the use of normal unmodified corn starch (CS). Thus, the
MS-CMC blend could contain more intermolecular hydrogen bonds between MS and CMC,
while in CS-CMC blend prevailed hydrogen bonds between CMC-CMC chains. Remarkable
decrease in viscosity has also been reported in blends of waxy rice starch and CMC (Li et al.,
2008). The viscosity of FFS based on CMC was 17% higher than CS-CMC and 44% higher than
MS-CMC. The results were in accordance with Techawipharat et al. (2008) who reported that
CMC increases in 33% and 45% the viscosity of FFS based on normal rice starch and waxy rice
starch, respectively. Moreover, CMC FFS (2% w/v, DS=1.5) showed slightly thixotropic
100
behavior, also reported by Arancibia et al. (2011); Benchabane & Bekkour (2008) and by Edali et
al., (2001).
When PEML was added, CMC, MS-CMC and CS-CMC were visually homogeneous and easy
distributed above plexiglass plates during casting process. In the films, PEML generates a shiny
yellow-brown color. These films were easily manipulated and have low adhesion with the
plexiglass plate and with itself. Addition of PEML reduced viscosity of these samples (P < 0.01).
Du et al. (2011) also reported a decrease in viscosity when apple skin polyphenols were added to
FFS based on apple pure. The decrease in viscosity of starch-CMC-PEML could be produced
because the polyphenols rich in –OH groups acted as a space producer between CMC and starch
polymeric chains, increasing the free volume and mobility of the matrix. However, an increase in
n was observed when including PEML into all CMC-based FFS, which was in agreement to
previous work (García et al., 2008).
4.3.1.2 FFS based on CMC, HG-CMC and LG-CMC

The FFS based on gelatin-CMC blends (LG-CMC and HG-CMC) contained a high amount of
foam and bubbles, attributed to the high surface properties of gelatin polypeptides, which should
be removed to overcome a negative effect on homogeneity, thickness and barrier properties of the
resulting film. Both films obtained with LG-CMC and HG-CMC showed a highly transparent
appearance, stable and also adherent, specifically in the case of HG-CMC, where adhesion
produced by the films was difficult to handle at 58% humidity.
The viscosity of gelatin-CMC blend was less dependent on the applied shear rate than the
viscosity of CMC solution alone. Although FFS had the same 2% w/w of total hydrocolloid
concentration (see Table 4.2), when 1% hydrocolloid solution of CMC has been replaced by fish
gelatin, the viscosity of CMC (η2=8.49 Pa⋅s) was strongly reduced when mixed with LG and HG,
being the apparent viscosity of LG-CMC blend (η2=3.30 Pa⋅s) greater than the viscosity of HG-
CMC blend (η2=1.75 Pa⋅s).
When adding PEML, HG-CMC blend showed a slight decrease in viscosity (P < 0.05), while LG-
CMC reduced its viscosity to a greater extent (P < 0.01), due to the formation of visible
101
precipitate when mixed with PEML (Figure 4.2a). As LG was relatively more surface active than
HG (Surh et al., 2006), the observed precipitation in the presence of PEML could be possible due
to a higher amount of moiety free in LG to react with PEML. Contradictory work shows that
there is a low affinity of polyphenolic compounds to proteins with low molecular weight
(Kosińska, Karamać, Penkacik, et al., 2011). Hovewer, Gómez-Guillén et al. (2007) also reported
an interaction and visual precipitation when fish gelatin film forming solutions were mixed with
murtilla leaves extract. In contrast, polyphenol–gelatin interactions did not produced any visible
protein precipitation in FFS based on tuna-fish gelatin with added water extracts from oregano or
rosemary, since these extracts do not contain high-molecular-weight polyphenol complexes
(Gómez-Estaca et al., 2009 a,b).
Figure 4.2 a) Precipitate formed in LG-CMC-PEML blend system for effect to polyphenol-rich
extract from murtilla leaves (PEML), b) Aggregates obtained from CH-PEML, CH-CS-PEML
and CH-CMC-PEML.
Interactions between gelatin and phenols/polyphenols from PEML can be explained by different
mechanisms: (1) initially hydrophobic interactions subsequently augmented by formation of
hydrogen bonds between the polyphenol –OH groups and the gelatin –COOH groups (Gómez-
102
Estaca et al., 2009c); (2) the presence of glycosidic polyphenols in PEML may affect
compatibility with gelatin by promoting interactions through –OH groups (Spyropoulos, Portsch
& Norton, 2010); (3) covalent cross-links (C-N) formed by polyphenols reacting under oxidizing
conditions with gelatin side chains (Strauss & Gibson, 2004) so (4) the precipitating capacity of
gelatin by the condensed tannin fraction of polyphenol-rich extracts, as previously reported with
a blueberry leaves extract (Naczk, Grant, Zadernowski & Barre, 2006). Kosińska et al. (2011)
found a linear relationship between the amount of protein–polyphenol complex precipitated and
the amount of tannin fraction added, being gelatin precipitated by tannin fraction over a wide
range of pH (3–7). The reduction in pH of gelatin-CMC-PEML blend suggests that both amino
groups of the protein and the hydroxyl groups of polyphenols could be ionized producing
precipitation of the mixture through charge interactions between -NH3+ and -O-, respectively. In
the latter case, the polyphenols present in PEML generate a process of complex coacervation with
fish gelatin producing aggregates that contain polyphenols. Positive effects of this phenomenon
can be used in the encapsulation and microencapsulation. Complex gelatin coacervates
containing antioxidant polyphenols in the form of microcapsules are of special interest as carrier
and release systems of functional components to provide protection against oxidation or
degradation during storage (Gómez-Guillén, Giménez, López-Caballero & Montero, 2011).
The precipitation of components in gelatin-CMC blend with PEML incorporated was consistent
with rheological parameters, since PEML substantially altered flow parameters n and K of LG-
CMC (P< 0.01), whereas it increased n but not K in HG-CMC; and significant differences in flow
behavior were observed between HG-CMC and LG-CMC when PEML was added. Despite
changes in the viscosity of gelatin-CMC-PEML solutions, the resulting films were visually
homogeneous and continuous with better adherence compared to other films studied, but with a
darker color due to the extract. LG-CMC-PEML turned out to be a weak film because it breaks
easily.
4.3.1.3 FFS based on CH and CH-CS blend

The CH-CS blend solution was homogeneous, with a pseudoplastic behavior similar to the one
obtained with CH solution alone. The same behavior has been reported earlier (Garcia, Pinotti &
Zaritzky, 2006). Chitosan solution presents pseudoplastic behavior without thixotropy, which was
103
in agreement to previous work (Madrigal-Carballo, Seyler, Manconi, Mura et al., 2008). In corn
starch-based solutions has been previously observed a thixotropic behavior (Bertuzzi, Armada &
Gottifredi, 2007). This behavior decreases with starch concentration, since the application of
shear that breaks or deforms the hydrated granules forming aggregates. In our study, given the
low concentration of corn starch (0.5% w/w) in CH-CS, the hysteresis loops are mild, which can
be well observed in non log-log flow curve (not show) among 210-253 Pa of shear stress and
900-1300 s-1. However, non-significant statistical differences between K of CH-CS versus CH
were observed. This could indicate that CS did not affect CH-CS behavior, despite n decreased
when CS was added into CH solutions, indicating that CS increased the shear-thinning behavior
of CH dispersion (see Figure 4.1).
When incorporating PEML in CH, the formation and detachment of brown aggregates 0.5-1.5 cm
long were observed (Figure 4.2b). Brown aggregated could be produced by strong interaction of
chitosan chains as well as chitosan-polyphenols interaction. Popa et al., (2000) also observed
chitosan-polyphenols complex formation. In the same way, interaction between chitosan and
polyphenolic compounds from green tea (Siripatrawan & Harte, 2010) and indian gooseberry
extract Mayachiew & Devahastin (2010), as well catechin (Zhang & Kosaraju, 2007), tannic acid
(Rivero, García & Pinotti, 2010) and gallic acid have also been observed (Curcio et al., 2009).
According to Kosaraju, D'Ath & Lawrence (2006), reversible complexation of polyphenols may
be considered as a two-stage process, of which in the first stage, chitosan and polyphenols are at
equilibrium in a soluble complex due to the development of non-covalent binding forces. As
equilibrium changes to a second stage, these soluble complexes may aggregate and precipitate
from the solution. Production of aggregates from chitosan reacting with several other compounds
has been extensively reviewed by Kumar et al., (2005).
Solutions based on CH and CH-CS containing PEML presented a chewy consistency that
hindered their spreading on the plate for forming the corresponding film at 2 s-1 shear rate, being
the viscosity of CH-PEML (102 Pa⋅s) and CH-CS-PEML (85 Pa⋅s) too high for their processing
and casting process. In these conditions, films with varying thickness were obtained from CH-
PEML and CH-CS-PEML affecting film integrity. However, according to viscosity curve, CH-
PEML solution at 100 s-1 shear rate (7.11 Pa⋅s) and CH-CS-PEML solution at 50 s-1 shear rate
104
(5.86 Pa⋅s) shown suitable viscosity for casting process. Differences between CH-CS blend with
or without PEML were in accordance with chitosan-starch blend when phenolic compounds such
as ferulic acid are incorporated (Mathew & Abraham, 2008).
Flow parameters of CH-PEML and CH-CS-PEML dispersions were not adjusted properly to
Power law model in the whole shear rate range studied (r2 = 0.778 and 0.550 respectively). When
the Power law was applied by shear rate sections, it was observed that at a shear rate below 0.4
s-1 CH-PEML and CH-CS-PEML blend FFS behave as dilatant fluids (see Figure 4.1), whereas
at higher shear rates, FFS behave as pseudoplastic fluids (see Table 4.3). The dilatant behavior of
CH-PEML (n = 2.00 and K = 823 Pa⋅s) and CH-CS-PEML (n = 2.68 and K = 1243 Pa⋅s) at low
shear rates indicated that the formation of new linkages between CH and PEML predominated
over destroying the structure, given to a network restructuring below 0.4 s-1. According to
Triantafillopoulos (1988), poorly stabilized systems develop dilatant flow at relatively lower
shear rates, where dilatant flow depends on particle size and shape. In suspensions containing
dispersed solids above 40% the interpretation of rheological measurements is difficult. In
addition, unstable flow is characterized by zig-zag patterns at high shear rate, as it could be
observed in flow and viscosity curves of CH-CS-PEML and CH-PEML (see Figure 4.1). In
relation to pseudoplastic behavior at shear rate above 0.4 s-1, molecules align in the direction of
flow and apparent viscosity decreases under increasing shear rate. The PEML increased K and
decreased n in both CH and CH-CS with PEML and dramatic change in apparent viscosity was
observed (P < 0.01). In addition, a strong time-dependent flow (thixotropic flow) was observed in
flow curve of CH and CH-CS when PEML were added. Flavonoids, mainly myricetin and
quercetin glucosides with many hydroxyl group (–OH), are present in aqueous murtilla leaf
extract (Bifani et al., 2007). Thus, –OH group in chitosan chains can be forming hydrogen bonds
with the -OH groups present in flavonoids. In another work, it is observed a thixotropic behavior
when chitosan dilutions were mixed with lecithin, since methyl group (–CH3) present in lecithin
forms aggregates with chitosan (Madrigal-Carballo et al., 2008). Besides, methyl group also is
present in rhamnoside flavonols of murtilla leaf extract (Shene et al., 2009), which could also
generate more hydrogen bonds between chitosan and PEML.
105
4.3.1.4 FFS based on CH-CMC

As the CH-CMC blend was rubbery, affecting the casting process, it had to be spread on the plate
using a spatula for making the film. Despite this, the resulting film was stable, but thick and
rough. The CH-CMC blend was not adjusted properly to the Power law model in the whole shear
rate range studied (r2 = 0.728). CH-CMC blend solutions show pseudoplastic behavior above 0.4
s-1 (r2 = 0.917), whereas between 0 to 0.4 s-1, CH and CMC chains links are still forming,
remaining the viscosity constant.The viscosity of CH-CMC blend was a 76%, 90% and 93%
higher than CMC, CH and CH-CS viscosity, respectively. The high viscosity observed in the CH-
CMC blend solution can be due to electrostatic interactions between oppositely charged groups,
i.e -NH3+ of chitosan and -COO− of CMC, considering the chemical structure, pKa and pH of CH
and CMC solutions.
Film obtained from CH-CMC-PEML was rough to the touch and showed low adhesion to the
contact. The viscosity of CH-CMC-PEML at 2 s-1 (η2 =23.4 Pa⋅s) was lower than CH-CMC
(η2= 35.4 Pa⋅s). Moreover, PEML incorporated into CH-CMC decreased K and increased n (P <
0.01), becoming its flow behavior less dependent on shear rate. As a result, when shear rate
increases, polysaccharide chains of CH and CMC moved in flow direction. Therefore, random
coil structure of chains were more exposed to moiety allowing more shift free in CH and CMC
for PEML to generate junction. This behavior can explain that CH-CMC-PEML overcome
viscosity of CH-CMC at shear rate over 50 s-1 as can be seen in flow curve.
4.3.2. Dynamic behavior
Dynamic rheological tests study the viscoelastic properties of materials, providing

complementary information to the steady shear studies. Frequency sweeps show the effects of
additives on the properties of a polymeric sample. Mechanical spectra within LVR provided
information about structure (viscous or elastic material) and stability material at rest and upon
during transport (Figure 4.3). Beside, classification of the sample structure as gel-like (strong or
weak gel), concentrated solution (entanglement network) or dilute solution according to Ross-
Murphy (1984), Clark and Ross-Murphy (1987), and Steffe (1996) were obtained (Table 4.5.).
106
Finally, temperature sweep show the transitions occurring in the material as well as changes in its
elasticity when PEML is added (Figure. 4.4).
The LVR used in this study was maximum stress value in the flat region of G' and stress curve.
As expected, since different sources of hydrocolloids were used in each FFS, high variability in
stress value among 0.1- 2.0 Pa (0.01-1 % strain) were found for LVR, increasing stress
proportionally to strain. Structure like-gels were more sensitive to strain than solutions.
According to Clark & Ross-Murphy (1987), LVR region is a characteristic of a material; whereas
the strain value at the LVR rarely exceeds 0.1 for colloidal gels, a larger LVR region with a strain
≥ 1 is usually observed for biopolymer gels. Our study showed that stress was ≥ 1 for FFS with
viscous or gel-like behavior, while stress was < 1 in dilute solutions. The strain had opposite
trend that stress, in agreement with Peressini et al. (2003).
4.3.2.1 FFS based on CMC, MS-CMC and CS-CMC

At low frequencies, a dominant viscous behavior was observed in CS-CMC, MS-CMC and CMC
solutions. At higher frequencies, above 19, 13 and 25 rad/s respectively, the inter-chain
entanglements did not have sufficient time to slide showing a physical gel-like behavior.
Therefore these FFS could be classified as concentrated solutions, in agreement with García et al.
(2008). During heating the phase angle (δº) of MS-CMC, containing a larger amount of
amylopectin, revealed a less elasticity than in CS-CMC.
The PEML incorporated into CMC, CMS-CS and CMC-MS reduced viscoelastic parameters,
reaching gel point at higher frequencies than without PEML (see Table 4.5). Despite their
behavior these FFS with and without PEML were classified as concentrated solutions.
107
1000 1000
100 100
10 10
10010 100 10010 100
G' (Pa) , G'' (Pa)
10
10
100 10 100
100 10 100
10 10
1 1
10 100 10 100
ω (rad/s) ω (rad/s)
Figure 4.3 Mechanical spectra showing the frequency dependence of G’ (filled symbols) and G’’
(Open symbols) for FFS without PEML (left) and with PEML (right) at 25 °C. a) CH-CS (▲,∆),
CH-CMC (●,○), CH (■,□); b) CMC (▲,∆), MS-CMC (●,○), CS-CMC (■,□); and c) HG-CMC (),
LG-CMC (●,○).
108
The crossover point of CMC-PEML occurred at 54 Pa, whereas Garcia et al. (2008) reported it
was observed at ∼ 40 Pa for CMC solution with murtilla extract of SG ecotype. In addition, CS-
CMC, MS-CMC, CMC systems with and without PEML showed good stability at the studied
frequency range. However, the viscoelastic behavior in MS-CMC-PEML proved to be more
stable than CS-CMC-PEML (see Figure 4.3).
Table 4.5 Viscoelastic behavior of FFS based on hydrocolloid blend without and
with PEML at 25 ºC.
Crossover point
Sample PEML G' = G'' ω η* tg Classification
(Pa) (rad/s) ( Pa⋅⋅s ) (min)
Without
CH 123 104 1.27 4.4 DS
CH-CS 131 105 0.77 4.6 DS
CH-CMC - - - - GL
CMC 38 19 2.80 2.2 CS
CS-CMC 29 13 3.20 2.3 CS
MS-CMC 22 25 2.30 2.3 CS
HG-CMC - - - - DS
LG-CMC 27 54 0.50 2.9 CS
With
CH - - - - GL
CH-CS - - - - GL
CH-CMC - - - - GL
CMC 53 86 0.87 3.7 CS
CS-CMC 33 101 0.46 4.0 CS
MS-CMC 32 64 0.70 3.2 CS
HG-CMC - - - - DS
LG-CMC - - - - DS
DS: Diluted solution, CS: Concentrate solution and GL: gel-like
109
As can be observed in Figure 4 4, slight change in elasticity of MS-CMC at 25 ºC was observed

with and without PEML added, whereas PEML reduced the elasticity of CMC and CS-CMC.
During heating from 2 to 40 ºC the elasticity of these solutions decreased, however, no
remarkable transition in this range of temperature was observed.
4.3.2.2 FFS based on CMC, LG-CMC and HG-CMC

The HG-CMC blend solution showed a predominantly viscous behavior at 25 ºC up to 9 rad/s
(see Figure 4.3c). After this point, unstable behavior was observed. At the same time, LG-CMC
blend showed a prevailing viscous mechanical spectrum at frequencies under 50 rad/s, whereas at
higher frequencies it reached crossover point (Table 4.5).
A similar consideration was made by Surh et al. (2006), when comparing stability of emulsions
based on LG and HG, where LG was more stable over time and thus less susceptible of
separation than the HG solution. When PEML was added, LG-CMC and HG-CMC showed a
predominantly liquid-like viscous behavior at 25 ºC; however, it was unstable along frequency.
The gel and melting points were determined as reported by Gómez-Guillén, Turnay, Fernández-
Diaz et al. (2002), who indicated that gelling temperature of different marine gelatin species
varied from 11 to 19 ºC (δ ∼50 - 45º) and melting temperature from 13 to 21 ºC (δ ∼1-10 º).
During cooling down (Figure 4.5), 1 g gelatin in 100 g FFS LG-CMC was sufficient to induce
renaturation of the polypeptide chains into triple helix, producing onset of gelling starting around
15 ºC (δ ∼71º). The same gel point at 15 ºC have been reported in gelatin solution (6.7 w/v) from
dried channel catfish skin gelatin alone (Liu, Li & Guo, 2008), whereas in gelatin from bigeye
snapper skin was considered as 10 ºC (Binsi, Shamasundar, Dileep et al., 2009). The results
shown below indicate that CMC reduced gel ability of LG. During subsequent heating, melting
point of LG-CMC was observed at a temperature around 13 ºC (δ ∼32º) as indicated by a sudden
drop in G’ values. This is lower than the reported value for gelatin alone from dried channel
catfish skin (23 ºC, δ ∼10º) (Liu et al., 2008), but higher than gelatin from bigeye snapper skin
(Binsi et al., 2009). Temperature transition was not clearly observed in HG-CMC blend during
heating. Because α-chains cross-links makes firmer and more stable gels (Liu et al., 2008 ), it can
110
be deduced that the formation of the triple helical structure by the larger polypeptide gelatin
chains was greatly interfered by CMC.
90 CH
CH-E
CH-CS
80
CH-CS-E
CH-CMC1
70 CH-CMC2
CH-CMC-E
CMC
60 CM-E
CS-CMC
Phase angle (º)
CS-CMC-E
50
MS-CMC
MS-CMC-E
40
30
20
10
0
0 5 10 15 20 25 30 35 40 45
Temperature (ºC)
Figure 4.4 Elasticity properties of FFS prepared from hydrocolloids blends

(according to Table 2) without (open symbol) and with (filled symbol) added PEML,
during temperature sweep of heating ramp.
111
The LG-CMC-PEML showed a higher elasticity than HG-CMC-PEML during heating at

temperature between 2 to 23 ºC. The HG-CMC-PEML blend overcame the elasticity of LG-
CMC-PEML blend at temperature over 23 ºC. In turn, LG-CMC-PEML reduced quickly their
elasticity from 17 ºC (δ ∼58º) to 27 ºC (δ ∼88º), indicating that its melting process was occurring.
Onset melting temperature was around 17 ºC with G’∼0.12 and G’’∼ 0.19 Pa. During cooling,
onset gel point around 21 ºC (δ ∼87º, G’∼0.006 and G’’∼0.14 Pa) was observed, with a strong
reduction of phase angle to 58º at 2 ºC. This thermal transition occurred at significant higher
temperature in LG-CMC without PEML and lower values of G’ and G’’. In another fish gelatin
solution with rosemary and oregano extract (Gómez-Estaca et al., 2009b), the same strong
reduction in phase angle was observed with onset gel point around 15 ºC (δ ∼60-70º), but
insignificant differences in respect to control sample without extract was observed. However, in
both tuna-fish gelatin FFS with added murtilla extracts (Gómez-Guillén, et al., 2007) and oregano
or rosemary extract (Gómez-Estaca et al., 2009b) were reported that gelatin polypeptide chains
produce interference with polyphenols upon cold renaturation and subsequent melting, resulting
in G’’ value rises proportionate to the amount of polyphenols added to the FFS. In contrast to
these studies, our results show that δ, G’ and G’’ values strongly decreased during cooling and
heating when PEML was added into LG-CMC. In this case, polyphenols in PEML could be
bound to both gelatin and CMC chains, consequently increasing distance between these chains.
4.3.2.3 FFS based on CH and CH-CS

The CH-CS and CH reached crossover point at frequencies above 100 rad/s, being classified as
diluted solutions (see Table 4.5). The FFS based on CH alone reached a crossover point at 104
rad/s (100 Pa, pH 4.5), which was consistent with ∼104 rad/s (123 Pa, pH 5.4.) reported by
Chenite, Buschmann, Wang, Chaput & Kandani (2001). On the other hand CH and CH-CS
without PEML, the viscoelastic behavior moved from a more liquid behavior to a more solid or
elastic behavior by incorporating PEML in the formulations. Thus, CH-PEML and CH-CS-
PEML behaved like gels with features of physical gel.
112
90 90
80 80
Phase angle (º)
70 70
60 60
50 50
40 40
30 30
40 35 30 25 20 15 10 5 0 100 100 0 5 10 15 20 25 30 35 40
10 10
1
G' (Pa)
0.1 0.1
0.01 0.01
0.001 0.001
40 35 30 25 20 15 10 5 0 100 100 0 5 10 15 20 25 30 35 40
10 10
G'' (Pa)
1 1
0.1 0.1
0.01 0.01
40 35 30 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40
Temperature (ºC) Temperature (ºC)
Figure 4.5 Dynamic viscoelastic properties of FFS prepared from LG-CMC (■, □) and HG-CMC
(▲, ∆); without (open symbol) and with (filled symbol) added PEML, during cooling (a) and
subsequent heating (b) ramps.
113
In CH solution, sol-gel transition occurred over 27 ºC, where δº was reduced from 73º at 27 ºC to
56º at 40 ºC. This transition was not observed during heating of CH-CS, being the solution CH-
CS less elastic than CH alone, in the whole studied temperature range. Incorporating PEML
drastically increased the elasticity of CH and CH-CS (see Figure 4.4). Phase angle decreased
with presence of PEML from 78º to 10º for CH-CS and 71º to 21º for CH at 2 ºC.
Elastic solid form by CH-PEML and CH-CS-PEML indicates that these have a definite shape,
which was visualized in this study. Consequently, when an external force is applied in an elastic
solid, this changes its shape instantaneously, but returning to its original shape after force
removal. Such results imply a rearrangement of polymers in presence of PEML, modifying the
network structure of CH and CH-CS solutions. Considering the effect of PEML on the elasticity
data and the frequency sweep data, the results seemed to suggest the presence of hydrogen-bonds
and hydrophobic interactions which get stronger with temperature particularly in CH-PEML.
4.3.2.4 FFS based on CH-CMC

Macromolecular dispersions of CH-CMC showed a predominantly elastic behavior in the
frequency range explored at 25 ºC (Figure 4.3a). When incorporating PEML in CH-CMC, a
slight decrease in elastic behavior was observed. Anyway, these FFS were classified as gels in
whole range frequency. In general, gelation arises either from chemical cross-linking by way of
covalent reactions or from physical cross-linking through polymer-polymer interactions (Tabilo-
Munizaga & Barbosa-Cánovas, 2005).
A transition to strong gel occurred in CH-CMC above 23 ºC (Figure 4.5), where phase angle was
reduced from ∼11º at 23 ºC to ∼1.8º at 40 ºC. This behavior was consistent with the phenomenon
observed by Chen & Fan (2008), in dynamic rheological analysis of CH-CMC hydrogels, but at
∼35 ºC. Another transition was also observed in CH-CMC at 23 º C. It was possible to
discriminate two different regions over 23 ºC. One part of the blend became less elastic and
another part became a strong elastic gel. This suggests that some CH chains may remain attached
to CMC and other CH chains may form strong hydrophobic interactions, because according to
Chen & Fan, (2008), hydrophobic interactions seem to be the main driving force to form a
chitosan gel at higher temperatures in the presence of CMC. In addition, higher elasticity of both
114
FFS based on CH-CMC and CH-CMC-PEML suggests a strong interaction between CMC and
CH. However, CH-CMC-PEML behavior was more stable than CMC-CH at temperature over 23
ºC. This effect may be attributed to the fact that junction zones between CH and CMC chains
were formed through interaction with polyphenols present in PEML. In CH-CMC-PEML, some
CH chains could allow free amine moiety for interacting with food and body tissues, with
potentially good application in pharmaceutical and food products.
4.4 Conclusions
The studied hydrocolloidal dispersions shown a typical non-Newtonian behavior in the studied
range of shear stress (0 – 250 Pa⋅s) and shear rates (0 - 100 s-1). At shear rate over 0.4 s-1, all the
tested film-forming solutions behaved as pseudoplastic fluids. Pseudoplastic behavior was
affected by hydrocolloid blend types as well as by the presence or absence of polyphenol-rich
extract from murtilla leaves in film-forming solutions.
Polyphenol-rich extract from murtilla leaves can act in a different way on rheological properties
depending on hydrocolloid blends that have been added.
Polyphenol-rich extract from murtilla leaves turns chitosan and chitosan-corn starch solutions
from a diluted solution into gel-like structure, strongly increasing the elastic character of these
film-forming solutions. The chitosan solution with polyphenol-rich extract from murtilla leaves at
100 s-1 shear rate (7.11 Pa⋅s) and chitosan-corn starch solution with polyphenol-rich extract from
murtilla leaves at 50 s-1 shear rate (5.86 Pa⋅s) are suitable for perform coatings and edible films
by casting process.
Polyphenol-rich extract from murtilla leaves changes the flow behavior of the film-forming
solution based on carboxymethylcellulose, cornstarch-carboxymethylcellulose and modified
starch-carboxymethylcellulose, becoming these from an entangled network to a less viscous
solution. However, all these solutions, with and without polyphenol-rich extract from murtilla
115
leaves, behaved as concentrate solution. Their viscosity ranges between 2.75 and 8.49 Pa⋅s at 2 s-1
and 25 ºC was suitable for the casting process.
The gel obtained from CH-CMC improves its thermostability when polyphenol-rich extract from
murtilla leaves is incorporated. However, film forming solution based on 2% w/w of CH-CMC
with and without polyphenol-rich extract from murtilla leaves has characteristics that make
casting process difficult, with viscosity over 25 Pa⋅s at 2 s-1 and 25 ºC.
Both gelatin of high and low molecular weight, incorporated on solution based on
carboxymethylcellulose and polyphenol-rich extract from murtilla leaves have an unstable
behavior in the studied frequency range. Polyphenol-rich extract from murtilla leaves generates
the formation of aggregates with gelatin of low molecular weight, which could be dragging the
antioxidant activity conferred by polyphenol-rich extract from murtilla leaves active components
outwards the resulting coating or film.
4.5 Remarks
The behavior of various films-forming solution blends, with added polyphenol-rich extract from
murtilla leaves, open the possibility of various applications in food, cosmetic and pharmaceutical
industries. The polyphenol-rich extract from murtilla leaves into chitosan and chitosan-corn
starch solutions could produce a desirable mouth sensation, whereas this extract forming
aggregates in gelatin of low molecular weight with carboxymetylcellulose blend could have
interesting applications for the production of microcapsules acting as carrier and release systems
of functional components. The carboxymethylcellulose, corn starch-carboxymethylcellulose and
modified corn starch-carboxymethylcellulose solutions, with and without polyphenol-rich extract
from murtilla leaves, showed suitable viscosity for the application as edible coating as well as
film- forming solution for the casting process.
In the future, in these edible films and coatings should be studied the antioxidant capacity,
antioxidant delivery, permeability to gases and thermal properties. These would help
understanding if the properties of the film are active and functional.
116
Acknowledgements
We thank M.Sc. I. Seguel from Unidad de Recursos Genéticos, INIA Carillanca, for the murtilla
leaf ecotype. This work was supported by CONICYT CHILE grants Nº 21070302, 24090134
and 29090088; Doctoral Program in Science of Natural Resources, Universidad de La
Frontera, Temuco, Chile; DIUFRO grant EP 120617 ( DI06-0001) and INNOVA-CORFO
Project Nº 06N12PAT-57, and the Spanish Ministerio de Ciencia e Innovación under project
AGL2008-00231/ALI.
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Chapter 5. Structural properties of films and rheology of film‒forming solutions based on
chitosan and chitosan‒starch blend enriched with murtilla leaf extract
CHAPTER 5
STRUCTURAL PROPERTIES OF FILMS AND RHEOLOGY OF FILM‒FORMING
SOLUTIONS BASED ON CHITOSAN AND CHITOSAN‒STARCH BLEND ENRICHED
WITH MURTILLA LEAF EXTRACT
a
Doctoral Program in Science of Natural Resources, Scientific and Technological Bioresource Nucleus
(BIOREN‒UFRO), Universidad de La Frontera, PO Box 54‒D, Temuco, Chile
b
c
d
Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN, CSIC). 28040 Madrid, Spain
Abstract
Chitosan (CH) and chitosan‒corn starch (CH‒CS) film‒forming solutions (FFS) and films, with
or without polyphenol‒rich aqueous extract from murtilla (Ugni molinae Turcz) leaves (PEML)
were prepared. The impact of the FFS type and PEML, considering pH values of the FFS, on
dynamic and steady‒shear behavior of FFS, interaction mechanisms of PEML with polymer
chains and changes on infrared spectra of films were investigated. Mechanical properties,
thickness and color from films were also evaluated. Blending CH with PEML produced huge
aggregates that were visible to the naked eye. Rheological parameters, K and n, were affected
significantly by the FFS type and the PEML presence. Viscosity of both FFS increased with the
addition of PEML, leading a gel‒like structure and thixotropic behavior. Sol‒gel transition
occurred when PEML was added to FFS, increasing strongly their elasticity. The addition of
PEML to CH‒CS blend film leads to a reduction (p < 0.05) of elongation at break and tensile
strength, increasing its thickness and yellow color. The PEML formed electrostatic interactions
with chitosan. Ester linkages and hydrogen bonds were also formed between PEML and both
chitosan and starch blends.
Keywords: Rheology, Physical properties, Structure, Edible films, Polyphenols, Ugni molinae T.
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5.1 Introduction
Hydrocolloids, such as chitosan and starch, are biodegradable and non‒toxic biopolymers based
on renewable resources, used as coatings and films in food and pharmaceutical applications. The
cationic character of chitosan offers an opportunity to establish electrostatic interactions with
other compounds, depending on the pH and on the acid type used to dissolve it (Alvarado et al.,
2007). Due to these characteristics, chitosan has been widely used for the production of edible
films with adequate barrier to water vapor (Aider, 2010; Alvarado et al., 2007; Rivero, García, &
Pinnoti, 2010). However, considering the cost of chitosan preparation, it seems economically
feasible to combine it with other film forming biopolymers (Aider, 2010). One of them, starch, is
the most common carbohydrate in the human diet. Blending two different hydrocolloids can
strongly change both the physical and rheological properties of the composite film‒forming
solutions (FFS), affecting the functionality of the resulting coatings and films. These changes
occur due to the compatibility/incompatibility between both macromolecules, which depend on
their molecular weight, chemical structure, pH, conformation and hydration behavior among
others.
Incorporation of antioxidant and/or antimicrobial compounds into films or coating materials can
provide food protection against oxidation, microorganism growth, enzymatic browning and
vitamin losses (Bonilla et al., 2012). In previous works, the polyphenol‒rich aqueous extracts
from murtilla leaves have been shown to induce antioxidant capacity to fish gelatin films
(Gómez-Guillén et al., 2007) and also to carboxymethylcellulose‒montmorillonite
nanocomposite films (Quilaqueo Gutiérrez et al., 2012). The composition of polyphenol‒rich
aqueous extract could modify the physical properties and chemical structure of films and coatings.
The effect of those additives on the film properties will depend on their chemical structure,
concentration, dispersion degree in the film and interaction with the polymers (Kester &
Fennema, 1986).
Murta or murtilla (Ugni molinae Turcz) is an endemic shrub in central‒southern Chile, which
belongs to the Myrtaceae family. Aqueous extracts from murtilla leaves showed high antioxidant
activity in vitro (Rubilar et al., 2005) and decreased the growth of Pseudomonas aeruginosa,
123
Klebsiella pneumoniae and Staphylococcus aureus (Shene et al., 2009). Phenol acids like gallic
acid, as well as flavonoid aglycones and glycosides from quercetin, myricetin and kaempferol are
among the main compounds found in those extracts (Bifani et al., 2007; Rubilar et al., 2005).
The rheological properties of biopolymer solutions can affect spread ability, thickness and
uniformity of liquid coating layer and film performance. A reduction in the solution's viscosity
provides a processing advantage during high shear processing operations, such as pumping,
filling and spraying application; whereas high apparent viscosity at low shear rates provides a
desirable mouthfeel during mastication and a better application of FFS by dipping (García et al.,
2009).
In this context, the purpose of the present work was to investigate the effect of the
polyphenol‒rich aqueous extract from murtilla leaves (PEML) on the dynamic and steady‒shear
rheological behavior of FFS based on chitosan and chitosan‒starch blend, and on chemical
structure, morphology and physical properties of the resulting films.
5.2.1 Materials
Fresh murtilla leaves (Ugni molinae Turcz) of ecotype 27‒1 were sampled near Temuco, Chile
(38º35′39′′ South latitude) at the Instituto de Investigaciones Agropecuarias, INIA Carillanca.
Corn starch (CS), containing 27% amylose, and medium molecular weight chitosan (CH), 190–
310 kDa, with a deacetylation degree (DD) of 75%, were purchased from Sigma‒Aldrich Co.
Glacial acetic acid (CH3 COOH, 98% purity, Merck) and glycerol (CH2 OH‒CHOH‒CH2 OH,
87% purity, Merck) were also used for obtaining FFS.
5.2.2 Obtaining and characterization of PEML
Murtilla leaf samples were air‒dried for 48 h to about 7% moisture content at 35 ºC. Dried leaves
were milled and the leaf powder (10 g) was macerated with distilled water (ratio = 1:10) at 170
124
rpm for 90 min at 25 ºC. The mixture was filtered (Whatman No 1 paper) and sterilized through a
membrane PES of 0.22 µm to obtain the polyphenol‒rich aqueous extract from murtilla leaves
(PEML). The PEML showed 1.4% solids and 0.98 g/ml density. Antioxidant capacity, expressed
as the PEML concentration required to scavenge 50% of ABTS.+ (2,2′‒azinobis‒3‒
ethylbenzothiazoline‒6‒sulfonate) free radical, was 0.038 mg gallic acid equivalent (GAE)/mL,
and the total phenol content was 40.67 mg GAE/g d.m. murtilla leaves.
5.2.3 Preparation of film‒forming solutions (FFS)
The concentration of components in each FFS for hydrocolloid blend formulation is shown in
Table 5.1. Starch‒based FFS: Aqueous solution from CS (0.5% w/w) was prepared by heating
beyond their gelatinization temperature (70 ± 5 ºC) for 20 min under gentle magnetic stirring, and
cooled until 25 ºC at a rate of 2–3 ºC/min. Chitosan‒based FFS: Best chitosan films were
prepared from 2% chitosan in 1% acetic acid, as described before (Siripatrawan & Harte, 2010).
Chitosan was dispersed in acetic acid (1% v/v) to prepare solutions of 1.5 and 2% w/w. This
dispersion was mechanically stirred at 500 rpm for 2 h (Ultra‒Turrax T25, Janke & Kunkel,
Germany), and filtered through cheese‒cloth. The incorporation of PEML was 40 % of the
dissolution solvent of CH solution. Considering the results obtained before (Bifani et al., 2007) in
relation to the reduction in the oxygen permeability of films obtained, after incorporation of 40
mL of aqueous extract of murtilla leaves in 100 mL of film‒forming solution of
carboxymethylcellulose. Chitosan‒starch blend FFS: The concentration of total hydrocolloids in
solution (HT) was maintained at a constant 2% w/w value. CH‒CS blend FFS was prepared by
mixing the CH and CS solutions shown above, with or without added PEML (20 mL/g HT, 81.33
mg GAE/g HT). The addition of 25 % w/w glycerol was reproduced from reported formulations
of films based on chitosan and chitosan‒starch films (Mathew, Brahmakumar, & Abraham, 2006;
Mayachiew & Devahastin, 2010) and a gentle stirring of the solution at controlled conditions for
30 min at 45 ºC was performed. Vacuum was applied to remove air bubble formation in blend
FFS. The pH value of blend FFS was determined at 23 ºC. Samples for rheological analysis were
stored at 5 ºC for 3 days, until use.
125
5.2.4 Rheological measurements: Fundamentals and methods
Dynamic viscoelasticity and steady state flow measurements were carried out in a
controlled‒stress rheometer (Bohlin CVO Instruments, Inc. Grandbury, NJ) with a cone‒plate
geometry (cone angle 4º, diameter = 40 mm, gap = 150 µm). Before analysis, the sample was
placed into the rheometer, which was equilibrated at 25 ºC (Gómez‒Guillén et al., 2007).
Table 5.1 Composition of film-forming solutions, with or without

polyphenol-rich aqueous extract from murtilla leaves (PEML).
Hydrocolloid Glycerol PEML

FFS [% w/w] [g/g HT] [mL/g HT]
H1‒H2 H1 H2 HT a Without With b
CH 2.00 - 2.00 0.25 0 20

CH-CS 1.50 0.50 2.00 0.25 0 20
a b
HT: Total concentration of hydrocolloids in solution, Total phenol content
from PEML expressed as gallic acid equivalent (GAE): 81.33 mg GAE/ g HT
5.2.4.1 Steady shear measurements

Flow curves and thixotropic properties were obtained by registering the shear rate when shear
stress was increased from 0 to 250 Pa and decreased from 250 to 0 Pa, at 25 ºC. Experimental
data were fitted to Ostwald–de Waele model or rheological Power law model according to
Equation 5.1.
τ = K γ& n [Eq. 5.1]
126
where τ is shear stress (Pa), K is consistency index (Pa⋅s), n is flow index (‒) and γ& is shear rate
(s‒1).
The changes in the solution’s apparent viscosity or steady‒shear viscosity (η, Pa⋅s), were
investigated according to Equation 5.2. Three repetitions were performed for each sample.
η( γ& ) = Κ γ& n −1
[Eq. 5.2]
5.2.4.2 Dynamic measurements of viscoelastic properties

Three dynamic studies were performed: (a) An oscillatory stress sweep test from 0.03 to 400 Pa,
at a constant frequency of 0.1 Hz and 25 ºC was made to set the upper limit of the linear
viscoelastic region (LVR). (b) Frequency sweep over a range of 0.01–50 Hz at 25 ºC was
performed at an oscillatory stress within LVR for each solution. Viscoelastic parameters, storage
or elastic modulus (G′, Pa), loss or viscous modulus (G′′, Pa), complex modulus (G*, Pa),
complex viscosity (η*, Pa⋅s) and tangent of the phase angle (Tanδ = G′′/G′, Equation (3)) as a
function of angular frequency (ω, rad/s) were measured, obtaining the typical mechanical spectra.
Classification of the sample structure as gel‒like (strong or weak gel), concentrated solution
(entanglement network) or diluted solution according to Clark and Ross‒Murphy (1987) was
applied. (c) Temperature ramps were performed at a scan rate of 1 ºC/min and 0.1 Hz from 40 to
2 ºC and back to 40 ºC. The phase transitions with temperature and elasticity of the FFS were
evaluated through the phase angle (δ, º) according to Equation (3). Elasticity is the reversible
behavior of stress/strain, which is measured as the reciprocal of δ, where purely elastic solids
have a phase angle of 0° and purely viscous fluids have a phase angle of 90°.
127
5.2.5. Data analysis
Rheological parameters were analyzed using factor analysis ANOVA related to factors: X1 (the
presence or absence of PEML) and X2 (the FFS type). Significance was accepted at 5%
confidence level. Mean difference among FFS was performed using orthogonal contrasts.
Duncan’s multiple range test was applied to compare the means for thickness, color and
mechanical properties of the films. JMP® 8.0. Software (SAS Institute, Version 6.09, Cary, NC)
was used for all statistical analyses.
5.2.6. Films production and characterization
5.2.6.1. Film production and film‒forming capacity

The FFS (40 g, 2% w/w) were cast onto plexiglass plates (12.5 × 12.5 cm) and dried at 40 ºC for
24 h in a ventilated oven (Binder FD 240, Tuttlingen, Germany). All the films were equilibrated
for three days to 48.0 ± 2.8% RH using saturated sodium bromide (NaBr) solution at 22.0 ±
0.5 °C. The film‒forming capacity of the solutions, remotion easiness, superficial appearance,
homogeneity and continuity of the films were visually examined.
5.2.6.2. Film thickness

Film thickness was measured using a digital micrometer (Mitutoyo, Tokyo, Japan). Five
measurements were taken on different positions around each sample.
5.2.6.3. Optical properties

Film surface color was measured using a Minolta Chromameter (CR‒400, Konica Minolta
Business Technologies, Inc., Tokyo, Japan). Films specimens were placed on a white standard
plate and five measurements were performed at different locations for each one. CIE‒Lab
tristimulus values, lightness (L*: black [‒] to light [+]), redness (a*: green [−] to red [+]) and
yellowness (b*: blue [−]) to yellow [+]) were measured.
128
5.2.6.4. Mechanical properties

Tensile strength (TS, MPa) and elongation to break (EB, %) of the films were measured using
Instron 4501 Universal Testing Machine (Instron Co., Canton, MA, USA) with tensile grip probe.
The conditioned films were cut into 100 × 20 mm strips for testing and re‒conditioned for two
days to 48% RH at 22 °C. Initial grip separation was set at 60 mm (initial length). Force and
distance were recorded during the stretch of strip at a rate of 1 mm/s until breaking using Texture
exponet 32 program.
5.2.6.5. Morphology of films

Scanning electron microscopy (SEM) was performed on a JEOL scanning electron microscope
(JSM‒6380 LV, Japan Electron Optics Limited, Tokyo, Japan) at 20 kV. Film samples between
20 ‒ 28% moisture were examined for cross‒section characteristics and their thickness. Samples
were coated with gold using a sputter‒coating system (Edwards 5150 sputter‒coating unit) for
180 s/ 30 mA.
5.2.6.6. Fourier transform infrared (FTIR) spectra

FTIR spectra of films were recorded in a Perkin Elmer Spectrum 400 FT‒IR with an attenuated
total reflectance (ATR) accessory from 650–4000 cm‒1. Measurements were performed at room
temperature and 48% RH. An interactive baseline correction at 1812 cm‒1 was applied. Signal
averages were obtained for 50 scans at a resolution of 4 cm‒1. No specific preparation was
required for film infrared analysis.
5.3 Results and discussion
5.3.1 Rheology of FFS
5.3.1.1 Steady shear behavior

As can be seen in Table 5.2, the studied FFS had a non‒Newtonian behavior (n ≠ 1). Orthogonal
contrast comparison of K and n parameters from samples showed significant differences (P <
0.01) between FFS with and without PEML. The FFS type and the presence of PEML in coatings
affected significantly the response of K and n (P < 0.05).
129
Viscosity curves (Figure 5.1) showed that apparent viscosity was highly dependent on the shear
rate at which shear stress was measured. Similar to CH solution alone, CH‒CS blend solution
was homogeneous, with a pseudoplastic behavior (n < 1). Thixotropic behavior was observed in
CH‒CS solution due to that the application of shear breaks or deforms the starch‒hydrated
granules, forming aggregates. Because of the low concentration of corn starch (0.5% w/w) in
CH‒CS solution, mild hysteresis loops were observed in non log‒log flow curve (not shown).
When incorporating PEML in CH, the formation and detachment of 0.5‒1.5 cm long brown
aggregates were observed. Brown aggregates could be produced by strong hydrophobic
interaction of chitosan chains as well as chitosan‒polyphenols interaction. Popa et al., (2000) also
observed chitosan‒polyphenol complex formation which, as reported by Kosaraju, D'Ath and
Lawrence (2006), could be reversible.
1000
100
Viscosity (Pa s)
10
CH
1 CH-CS
CH-PEML
CH-CS-PEML
0.1
0.01 0.1 1 10 100 1000
Shear rate (1/s)
Figure 5.1 Viscosity curves of film-forming solutions with PEML

(filled symbols) and without (open symbols) at 25 °C.
130
When the Power law was applied by shear rate sections, it was observed that at a shear rate below
0.4 s‒1 CH‒PEML and CH‒CS‒PEML blend FFS behaved as dilatant fluids (see Figure 5.1),
whereas at higher shear rates, FFS behaved as pseudoplastic fluids. The dilatant behavior (n > 1)
indicated that the formation of new linkages between hydrocolloids and PEML predominated
over destroying the structure, with the result of a restructured network. Flavonoids, mainly
myricetin and quercetin rhamnosides, with many hydroxyl groups (–OH) and methyl groups (–
CH3) are present in murtilla leaf extract (Bifani et al., 2007; Shene et al., 2009), which can be
forming more interactions by hydrogen bonds with the ‒OH groups present in chitosan. In
relation to pseudoplastic behavior, it was due to that molecules align in the direction of flow and
apparent viscosity decreases under increasing shear rate. The PEML decreased n, increased K,
and increased apparent viscosity in both FFS (Table 5.2). As can be observed in viscosity curves,
time‒dependent flow and an unstable flow, characterized by zigzag patterns at high shear rate,
were also observed with PEML.
Solutions based on CH and CH-CS containing PEML presented a chewy consistency that
hindered their spreading on the plate for forming the corresponding film at 2 s-1 shear rate, being
the viscosity of CH-PEML (102 Pa⋅s) and CH-CS-PEML (85 Pa⋅s) too high for their processing
and casting process. In these conditions, films with varying thickness were obtained from CH-
PEML and CH-CS-PEML affecting film integrity. However, according to viscosity curve, CH-
PEML solution at 100 s-1 shear rate (7.11 Pa⋅s) and CH-CS-PEML solution at 50 s-1 shear rate
(5.86 Pa⋅s) shown suitable viscosity for casting process. Differences between CH-CS blend with
or without PEML were in accordance with chitosan-starch blend when phenolic compounds such
as ferulic acid are incorporated (Mathew & Abraham, 2008).
5.3.1.2 Dynamic behavior

The viscous and elastic properties of the FFS can be evaluated through dynamic rheological
studies. Frequency sweeps assess the effect of additives on changes in microstructure and
stability of the FFS during storage and transportation, whereas a temperature sweep can show
phase transitions and elasticity, allowing the appropriate temperature ranges for the formulation
and application of the coating film on the product.
131
The limits of linear viscoelastic region (LVR) used in this study was the maximum stress value in
the flat region of G′ and the stress curve. Stress values of 1.0 and 2.0 Pa (0.2 and 0.05% strain)
were found for LVR in FFS with and without PEML, respectively. The strain had an opposite
trend to stress.
According to the mechanical spectra (Figure 5.2), FFS based on CH and CH‒CS without PEML
shows predominantly viscous behavior (G′′ > G′), but crossover point was reached at frequencies
above 100 rad/s, being classified as diluted solutions.
132
Chapter 5. Structural properties of films and rheology of film‒forming solutions based on chitosan and chitosan‒starch blend enriched
with murtilla leaf extract
Table 5.2 Parameters for the Power law model, at shear rate above 0.4 s-1 at 25 ºC. Significance of difference between parameters K
and n of FFS with and without PEML using orthogonal contrasts and apparent viscosity based on the form of the Power law model:
η(γ& ) = K (γ& ) n −1
.
Sample K ( Pa⋅⋅s n) n(-) r2 ( - ) η (γ& = 2) pH

( Pa⋅⋅s )
Withou With Without With Without With Without With Without With
PEML
CH 4.23 164 ** 0.70 0.32 ** 0.993 0.984 3.45 102 * 4.74 4.47 NS
*
* 4.47 4.43 NS
CH-CS 3.15 150 ** 0.63 0.17 ** 0.990 0.915 2.44 84.5
*
** P < 0.01, NS: Not significant
133
Without PEML With PEML

1000 1000
G' : CH-CS
G'' : CH-CS
G' : CH
G'' : CH
G' (Pa); G'' (Pa)
G' (Pa); G'' (Pa)

Diluted solution
100 100
gel-like
10 10
10 100 10 100
Crossover point
Sample G′ = G′′ ω η* tg
(Pa) (rad/s) ( Pa⋅⋅s )(min)
CH 123 104 1.27 4.4
CH-CS 131 105 0.77 4.6
Figure 5.2 Mechanical spectra showing the angular frequency (ω) dependence of storage
modulus (G′, filled symbols) and loss modulus (G′′, open symbols) for FFS based on CH and
CH-CS, with PEML (right) and without (left) (25 °C).
The FFS based on CH alone reached a crossover point at 104 rad/s (123 Pa, pH 4.7), which was
consistent with ∼104 rad/s (100 Pa, pH 5.4.) reported by Chenite et al., (2001). In contrast to CH
and CH‒CS without PEML, by incorporating PEML in the FFS, G′ was always higher than G′′
over the whole frequency range. The PEML reacted with biopolymers in solution generating a
gel‒like behavior. The frequency dependence of the viscoelastic parameters has a typical
134
structure of physical gels, which are reversible gels, depending on the environmental and process
factors such as pH, moisture, shear rate and temperature (Rinaudo, 2006). This restructuring
ability of CH‒PEML and CH‒CS‒PEML physical gels could make them an important class of
materials with many applications, such as in drug delivery. Furthermore, gelling hydrocolloids
could enhance the hardness, adhesiveness, and viscoelastic properties of the FFS.
According to results of temperature sweep essays (Figure 5.3), CH and CH‒CS FFS behave as a
sol (Phase angle > 45º), which means that the sol‒gel transition (Figure 5.4) could not be
determined in the temperature domain studied. During heating of the CH solution, phase angle (δ,
º) was reduced from 74º at 25 ºC to 56º at 40 ºC, which indicated that its dynamic elasticity
increased a 24% between 25 to 40 ºC. This change was not observed when heating CH‒CS
solution. Over the full temperature range studied of 2 ‒ 40 °C, CH‒CS solution was less elastic
than the CH solution alone.
90
CH
80 CH-CS
CH-PEML
70
CH-CS-PEML
Phase angle (º)
60
50
40
30
20
10
0
0 5 10 15 20 25 30 35 40 45
Temperature (ºC)
Figure 5.3 Elasticity properties of FFS based on CH and CH-CS (according to Table 2)
with PEML (filled symbols) and without (open symbols), during temperature sweep.
135
The high temperature can reduce the intermolecular hydrogen bonding interactions and
accelerate the mobility of chitosan molecules in solution, removing energized water molecules
surrounding the chitosan chains (Tang, Du, Hu, Shi, & Kennedy, 2007). The dewatered
hydrophobic chitosan chains were associated with each other, which increased the chitosan
solution elasticity over 25 ºC. In CH and CH‒CS FFS, gel (Phase angle < 45º) occurred when
incorporating PEML, increasing their elasticity in 71 and 88%, respectively. Such results imply a
rearrangement of polymers in the presence of PEML, modifying the network structure of both
CH and CH‒CS solutions. The CH‒CS‒PEML remains as a gel along temperature, reducing its
elasticity in 60%. In the same temperature range, CH‒PEML gel increased its elasticity in 39%.
a) b)
Figure 5.4 Sol-gel states in a colloidal solution. a) The sol state corresponds to
fluid colloidal solution when the dispersant (water) phase predominates over the
dispersed phase (macromolecules). b) In the gel state, the dispersed phase
predominates over the dispersant phase, thus the colloidal solution is more viscous.
5.3.2. Film analysis
5.3.2.1. Film‒forming capacity of the solutions
The CH and CH‒CS films were easy to remove from the plate, with a homogenous surface
appearance and a continuous matrix were formed, with good structural integrity. The CH‒CS film
136
was smoother than CH film, exhibiting CH film surface undulations. When the CH and CH‒CS
solutions contain PEML, a chewy consistency hindered their spreading on the plate for forming
the corresponding film. The CH‒CS‒PEML film was more homogeneous and continuous than
the CH‒PEML film, but air bubbles on the surface of both films were observed.
5.3.2.2 Optical properties

Color parameters a* (+: red; ‒: green) and b* (+: yellow; ‒: blue) were good indicator of film
color (Table 5.3). CH and CH‒CS films had slightly yellow color, being CH‒CS films less
yellow than the CH film. In contrast to CH and CH‒CS, a slightly orange color (brownish) was
observed in CH and CH‒CS films containing PEML, being yellow color of CH‒CS‒PEML film
significantly higher that of the CH‒PEML (P < 0.05). Similar changes in chitosan film color have
been found by adding green tea extract (Siripatrawan & Harte, 2010), but in grape seed extract
yellow color decreases (Moradi et al. 2012).
5.3.2.3 Thickness
The incorporation of starch in the chitosan film did not change significantly the thickness of CH
and CH‒CS films (P < 0.05). Compared with the films without PEML, CH‒CS‒PEML film
strongly increases their thickness, whereas CH‒PEML film does it only slightly (Table 5.3).

Comparison of mechanical properties results is difficult since in general there are a great
dispersion in deacetylation degree (DD), molecular weight (MW), concentration of chitosan and
plasticizer, film preparation methods, conditioning and tensile test conditions.
As can be seen in Table 5.3, the TS and EB values of CH films (2% w/w, 75% DD) were close to
results obtained in chitosan films (2% w/w, 90% DD) by Siripatrawan and Harte (2010) and
Pereda et al., (2011), for similar chitosan and glycerol concentration, and the same film
conditioning and tensile tests. Whereas, TS was lower and EB was between the values reported
by Mathew et al. (2006), and Mathew and Abraham (2008) in chitosan films.
137
Table 5.3 Physical properties of chitosan and chitosan-starch films formulated with polyphenol-rich aqueous extract from murtilla
leaves (PEML), and other literature cited films
Thickness Color Mechanical properties

Additive References
(µ
µm) a* b* TS (MPa) EB (%)
Chitosan
‒ 70.7 ± 5.0 c -1.01 ± 0.11 b 4.52 ± 0.39 c 29.69 ± 1.89 a 45.10 ± 1.40 a This work
PEML 77.5 ± 3.5 b 14.99 ± 2.98 a 35.11 ± 2.14 b ̶ ̶ This work
‒ 97.0 ± 8.0 -0.01 ± 1.19 44.67 ± 2.39 17.34 ± 2.43 44.20 ± 7.90 Pereda et al. (2011)
‒ 62.1 ± 6.3 1.11 ± 0.16 1.37 ± 0.10 23.66 ± 2.63 54.62 ± 3.12 Siripatrawan & Harte (2010)
Green tea extract 62.1 ± 6.3 3.32 ± 0.66 28.87 ± 0.83 25.13 ± 1.91 58.14 ± 4.24 Siripatrawan & Harte (2010)
‒ 80 0.70 ± 1.00 29.00 ± 2.00 24.00 ± 3.00 29.00 ± 2.00 Moradi et al. (2012)
Grape seed extract 80 21.0 ± 0.90 16.00 ± 0.20 16.00 ± 0.60 21.00 ± 3.00 Moradi et al. (2012)
Chitosan-starch
‒ 72.0 ± 2.4 c -0.41 ± 0.07 c 2.08 ± 0.19 d 20.56 ± 1.78 b 13.52 ± 2.85 b This work
a
PEML 126 ± 12 12.15 ± 1.23 a 63.63 ± 1.12 a 17.13 ± 1.22 c 6.57 ± 2.43 c This work
‒ 98.9 ± 5.1 37.50 27.95 Mathew et al. (2006)
‒ 73.5 ± 3.9 ~ 42 ~ 57 Mathew and Abraham (2008)
Ferulic acid 78.9 ± 4.3 ~ 49 ~ 60 Mathew and Abraham (2008)
Different superscript letters in the same column represent significant differences (P < 0.05). TS: Tensile strength (MPa) and EB: Elongation at break (%).
138
The mechanical properties of chitosan‒starch films can depend on chitosan‒starch ratio and
starch source, in addition to characteristics and conditions listed above. The CH film shows
higher TS and EB than CH‒CS film (ratio = 1.5:0.5). According to Mathew et al. (2006),
chitosan‒potato starch films (ratio = 2:3) show higher EB than chitosan films alone, whereas TS
values of chitosan‒potato starch films (ratio = 1:0.5) have a maximum of 37.5 MPa, due to the
formation of intermolecular hydrogen bonds between the chitosan and starch.
The addition of PEML in chitosan film induces the development of structural discontinuities,
producing a highly brittle film structure that can be attributed to less chain mobility, due to
excessive linkages between PEML and chitosan chains causing polymer aggregation. Hence,
lower resistance and ductility did not allow measuring of mechanical properties of CH films. In
CH‒CS‒PEML films, PEML significantly decreased their TS and EB, also seen on chitosan film
with grape seed extract added (Moradi et al., 2012). The impaired mechanical behavior in
CH‒CS‒PEML films could also be attributed to linkages between PEML and chitosan‒starch
associations, leading to an increased film volume and decreased chains mobility.
5.3.2.5 Morphology of films

The cross‒sections of scanning electron micrographs for films formed from CH and CH‒CS with
and without PEML are shown in Figure 5.5. It is clear from the images that distinctive film
structures formed were dependent of presence or absence of PEML and FFS type used. Chains in
CH film were arranged in a horizontally ordered form, because there are repulsive forces between
CH chains when the pH is below 5.0. The CH‒CS film forms a structure with more fibrous
appearance than the CH film. Besides, it can be observed some aggregates of starch on the
surface of the CH‒CS film.
The PEML incorporated produced a remarkable change in the structure of the films. In the CH
film cross‒section, PEML creates several elongated horizontal pores of approximately 1 ‒ 4 µm.
In the CH‒CS film with PEML, the formation of horizontal layers and some zones with
discontinuity or disorder, and pores were observed. Contrary, ferulic acid into chitosan‒starch
blend films forms compact structure (Mathew & Abraham, 2008) while chitosan‒starch blend
films without ferulic acid show a structure with certain discontinuous zones and small pores. The
139
physical properties of films are also depending on its microstructure and unfortunately, the
PEML provoked some discontinuity in films. It’s known that cross linking increases the overall
molecular weight and glass transition of polymers. And, in these cases, more important
plasticizer concentration could be used to avoid the discontinuity.
CH CH-PEML
CH-CS CH-CS-PEML
Figure 5.5 Scanning electron micrographs of film cross-section: chitosan films with PEML (CH-
PEML) and without (CH), chitosan-starch film with PEML (CH-CS-PEML) and without (CH-
CS). Scale bar 5 µm and 5000x magnification.
140
5.3.2.6. Chemical structure

In order to investigate potential structural differences between CH and CH‒CS films with and
without PEML, IR spectra were recorded (Figure 5.6). Chitosan with a pKa ~ 6.3 is polycationic
when dissolved in acid and presents –NH3+ sites. In the chitosan film, absorption bands at 902,
1024, 1058 cm‒1 (C–O stretching), and 1151 cm‒1 (asymmetric stretching of C–O–C bridge,
glycosidic linkage) were characteristics of its saccharide structure (Ávila et al., 2012; Monier et
al., 2010; Rivero, García, & Pinotti, 2010). The peak at 1380 cm‒1 was assigned to C–O
stretching of primary alcoholic group (–CH2OH) (Ávila et al., 2012; Sionkowska et al., 2010)
and C‒O‒H stretching at 1455 cm‒1. The bands at 2924, 1407, 1327 and 1257 cm‒1 were assigned
to C‒H bending of –CH2 due to pyranose ring and band at 2878 cm‒1 was assigned to C‒H
bending of –CH3 from –NHCOCH3 (Mathew & Abraham, 2008; Sionkowska et al., 2010; Yan
et al., 2010). Broad band from 3680‒2990 cm‒1 represents polymeric hydrogen bonds, being
identified the peaks at 3344 cm‒1 (stretching O‒H symmetric) and 3263 cm‒1 (stretching N‒H
asymmetric). The band at 1651 cm‒1 (C=O stretching, amide I band from –NHCOCH3) overlap
with band at 1635‒1638 cm‒1 (NH‒bending of the glucosamine unit, amide II). The bands from
1557‒1538 cm‒1 were N–H bending in primary amine groups (‒NH2/‒NH3+) and 1334 cm‒1 were
C‒N stretching vibration (amide III).
As can be seen in CH‒CS film, new peak at 760 cm‒1 was attributed to C–H deformation
vibration. This was coupled with an increase of the C‒O stretching of –CH2OH group. Moreover,
the bands characteristics of the crystallinity of starch (1042, 1015 and 960 cm‒1) (Vicentini et al.,
2005) were not observed in these spectra. These results indicated that chitosan and starch form
hydrogen bonds. A characteristic absorption peak assigned to C‒O‒C in glycosidic linkage at
1151 cm‒1 was similar in both CH and CH‒CS films, which indicated that the glycosidic chain
length was not modified.
141
115,0
110
1203 902 707
851 816
818
105 2163 2113
1651
1635
1257
CH
1635
100
1462 816
1205 707
95 2163 2113 760
852
1651 1258 926 818 CH-CS
90 1455 1380
1638 1555 1327
85 1550 1406 1334 817 706
2161 2111 1207
2924 1715 1462 852 764 CH-PEML
80 2878 1455 861 778
1651 1639 1331 1251
927
75 3344 1254
3263 2161 2111 1207 817 764 706
2925
70 2878 1715 852 778
1614
1455
1251
926
861 CH-CS-PEML
3345 1651 1380
65
1254
3268 1555 1407 1339
60 2926
1058
%T 2880 926
1614 1379 1151
55 3259
1455
1555 1406 1339
50 2926
2880
45 1379
3259
40 1555 1406 1024
35
1072
30 996
25 1020
20
15 1022
10
5,0 1022
3550,0 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650,0
cm-1
Figure 5.6 Scanning electron micrographs of film cross-section: chitosan films with PEML (CH-PEML) and without (CH), chitosan-
starch film with PEML (CH-CS-PEML) and without (CH-CS). Scale bar 5 µm and 5000x magnification.
142
When PEML was added in CH and CH‒CS film, various small modifications were revealed in
the spectra. Band of N‒H bond shifted to 3259 cm‒1 and O‒H linkage stretching of phenolic and
hydroxyl groups, that appears near 3330 cm‒1 (Božič, Gorgieva & Kokol, 2012b), overlapping
band of N‒H bond stretching. The C‒H stretch vibrations of ‒CH3 decreased and moved from
2878 cm‒1 to 2880 cm‒1, suggesting that hydrogen bonds could occur between hydrogen from –
CH3 or –OH of chitosan groups and oxygen from polyphenol –OH groups.
The FTIR‒ATR spectra of PEML‒loaded CH and CH‒CS films show some differences in the
range 1800‒1500 cm‒1, when characteristic bands of substituted amino groups and carbonyl
groups are located (Mayachiew & Devahastin, 2010). Band of C=O from N‒acetyl group of
chitosan at 1651 cm‒1 was show only as a shoulder. Band of –NH bending of amide II decreased
and overlap with a new peak that appeared at 1627, 1622 and 1614 cm‒1 in CH‒PEML and at
1614 cm‒1 in CH‒CS‒PEML. Three new peaks appeared, representing C=C stretching (1614
cm‒1) and H‒atoms (861 and 778 cm‒1) of aromatic rings (Yan et al., 2010); and reflect the
presence of both phenolic acids (as gallic acid) and flavonols (myricetin quercetin, kaempferol),
present in PEML and therefore, in the films (Bifani et al., 2007; Rubilar et al., 2005). In
CH‒PEML, a new peak around 1620 cm‒1 and 1627 cm‒1 have been related to intermolecular
interactions between chitosan and phenolic compounds, resulting in a lower percentage of
antioxidant release (Mayachiew & Devahastin, 2010). Band at 1627 cm‒1 becomes evident
(overlapping with that of amide I), suggesting that the reaction between the chitosan and carbonyl
groups from polyphenols occurred via ester linkage. Changes around 1200‒1400 cm‒1 were due
to –OH bending of the phenolic compounds and stretching vibrations of C–O and C=O from
polyphenols (Božič et al., 2012b). Besides, band of C–N stretching vibration shifted to 1339 cm‒1
in CH‒PEML and CH‒CS‒PEML. This result suggests that amino groups from chitosan interact
with polyphenols from PEML.
Characteristic carbonyl groups (–COOH) of phenolic acids have been found about 1700 cm‒1
(Monier et al., 2010). A new small peak was found in PEML‒loaded CH and CH‒CS films
around 1715 cm‒1. This has been also observed in CH film loaded with quercetin (1715 cm‒1)
(Božič et al., 2012b), polyphenolic compounds from olive‒leaf extract (1700 cm‒1) (Kosaraju et
al., 2006), green tea (Siripatrawan & Harte, 2010), and Indian gooseberry extract (1720 cm‒1)
143
(Mayachiew & Devahastin, 2010). These last authors have attributed this peak to ester linkage
between chitosan and polyphenolic extract, which was sharper with an increase in the extract
concentration. Ester linkages have been also reported between chitosan (pH ~ 4.5, pKa ~ 6.3)
loaded with tannic acid (pKa COOH = 4.4) at 1730 cm‒1 (Rivero et al., 2010), 4‒hydroxycinnamic
acid at 1700 cm‒1 (Monier et al., 2010), caffeic acid (pKa COOH = 4.0), and gallic acid (pKa COOH =
4.4, pKa OH = 10) around 1715 cm‒1 (Božič, Gorgieva & Kokol, 2012a). These last authors
showed that chitosan with phenolic acids at pH 4.5 had a higher activity as antioxidant and
antimicrobial, against Escherichia coli and Listeria monocytogenes, that chitosan at other pHs or
without polyphenols
As a way to integrate our results and previous studies mentioned above, the model shown in
Figure 5.7 is proposed, which represents interaction mechanisms of gallic acid and myricetin, as
models of phenolic acids and flavonoids from PEML, with CH and CH‒CS films. The model
indicates that in CH‒PEML (pH = 4.4) and CH‒CS‒PEML films (pH = 4.5), ‒COOH groups
from gallic acid are partially dissociated. It suggest that the part of non‒dissociated gallic acid
from PEML forms ester linkages when it reacts with ‒CH2OH groups of starch and chitosan
hydrocolloids; while dissociated gallic acid with COO‒ moiety may be bonded ionically with
protonated amino groups (NH3+) of chitosan, establishing electrostatic interactions capable of
forming complexes. In the case of myricetin from PEML, loaded in chitosan solution at pH 4.5,
only interactions by hydrogen bonds can be shown. These facts can explain some changes
occurring in the CH and CH‒CS film properties loaded with PEML and the relationship between
physical properties and changes of peak pattern of chitosan and chitosan‒starch films.
5.4 Conclusions
The presence of both corn starch and polyphenol‒rich extract from murtilla leaves (PEML) affect
rheological behavior of chitosan solutions. The studied film-forming solutions show a typical
non‒Newtonian behavior at 25 ºC, and these behaved as pseudoplastic fluids at shear rate over
0.4 s‒1.
144
A B
Figure 5.7 Interaction mechanisms at acid pH (4.5 ± 2) of A) Chitosan film with PEML (CH-PEML): Chitosan chains (above and
below), and B) Chitosan-starch film with PEML (CH-CS-PEML): chitosan chain (above), corn starch chain (below). As models of
phenolic acids and flavonoids from PEML, gallic acid and myricetin are presented. Example of interactions: (1) ester linkage, (2)
electrostatic interaction, and (3) hydrogen bond.
145
The polyphenol-rich extract from murtilla leaves produces a strong reduction of the fluidity of
chitosan and chitosan‒starch solutions. Besides, viscoelastic properties show that polyphenols
from murtilla leaf extract turns chitosan and chitosan-cornstarch solutions from a diluted solution
into a gel‒like structure, increasing their elasticity in 71 and 88% respectively, which is a clear
indication of the improvement on the three‒dimensional cross‒linked networks stability. This
restructuring ability of gels of chitosan and chitosan-cornstarch with polyphenol-rich extract from
murtilla leaves make them an important class of materials with applications such as in drug
delivery or as natural‒based wound dressings impregnated with bioactive compounds.
The chitosan film was more ductile and elastic than chitosan-cornstarch film. Starch stabilized
the interaction between chitosan and polyphenol-rich extract from murtilla leaves, and then
chitosan-cornstacrh with polyphenol-rich extract from murtilla leaves was a more continuous and
homogeneous film structure than chitosan with polyphenol-rich extract from murtilla leaves. The
polyphenol-rich extract from murtilla leaves incorporated to chitosan-cornstacrh film increases
the thickness and turns from yellow to slight orange color. The polyphenol-rich extract from
murtilla leaves interacts with chitosan and starch chains in the films.
The polyphenolic nature of the polyphenol-rich extract from murtilla leaves extract makes it an
extremely highly reactive agent with chitosan. Thus, polyphenol-rich extract from murtilla leaves
provoked great discontinuity in chitosan with polyphenol-rich extract from murtilla leaves film
matrix due to excessive chitosan cross‒linking, which was favored during dehydration of film
formation. The polyphenols from murtilla leaves also produced noticeable linkages in the
presence of chitosan and starch, which reduced considerably polymer chains mobility and
consequently, the film’s mechanical properties changes. However, chitosan‒starch associations
prevented to some extent the excessive polyphenol‒polymer interactions.
Supported by the film characterization, mechanical properties, SEM and, FTIR analysis, the
interaction mechanism of polyphenols from murtilla leaves with chitosan chains, inside the films,
was due to electrostatic interactions (ionic complexations in acidic conditions, pH: 4.5), ester
linkages and hydrogen bonds, while polyphenols from murtilla leaves with starch chains was due
to hydrogel bonds and ester linkages.
146
Acknowledgments
We thank Mrs I. Seguel (M.Sc.) from Unidad de Recursos Genéticos, INIA Carillanca, Temuco,
Chile for the murtilla leaf ecotype. This work was supported by CONICYT CHILE grants Nº
21070302, 24090134 and 29090088; Doctoral Program in Science of Natural Resources,
Universidad de La Frontera, Temuco, Chile; DIUFRO project EP 120617 (DI06‒0001); CYTED
project 309 AC0382; and the Spanish Ministerio de Ciencia e Innovación project
AGL2008‒00231/ALI.
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148
Chapter 6. General discussion and conclusions
CHAPTER 6
GENERAL DISCUSSION AND CONCLUSIONS
6.1 General discussion
Film production with natural and abundant biodegradable polymeric materials, such as cellulose
derivatives (including methyl cellulose (MC) and carboxymethylcellulose (CMC)), chitosan,
gums, starches or proteins, are convenient due to lower environmental consequences compared
with common synthetic plastic materials. The properties of edible films and coatings can be
improved by combining the characteristics of hydrocolloids. Besides, incorporation of natural
polyphenolic extract with bio-active properties in these blend solutions and composite films can
enhance their active properties such as antioxidant and UV-barrier properties. However, the
physical and chemical properties of edible films and coatings also could be modified when
polyphenol-rich extracts are added.
In these work the ultrasound assisted-extraction was optimized and compared with conventional
extraction of phenolic compounds from murtilla leaves (Chapter 3). Then, the influence of
polyphenol-rich extract from murtilla leaves on rheological properties of six blends of
hydrocolloids based solutions were analyzed (Chapter 4). In addition, resulting films were
analyzed in terms of FT-IR spectra, mechanical properties of the films, and film morphology
(Chapter 5).
Ultrasound-assisted extraction allows higher recovery of phenolic compounds from murtilla

leaves than the conventional maceration. In addition, the operating cost of ultrasound process in
the optimal conditions determined is more efficient and has higher performance than extraction
by maceration. The particle size reduction by the ultrasonic cavitation increases the surface area
in contact between the solid and the liquid phase, whereby the release of active compounds from
the cell occur at a higher rate than in other extraction systems. In addition, phenolic compounds,
when are released from the vacuole, can bind to cell walls (Padayachee et al., 2012a,b), forming
complexes with polysaccharides present in the cell walls. These complexes could be attacked by
149
radicals formed in water by ultrasonic waves, increasing the release of the phenolic compounds
from plant material
From rheological point of view, the viscosity of film-forming solution should be sufficiently high
to prevent sagging by gravity effects. However, high viscosities are not good during preparation
of liquid films because it is more difficult to disperse the ingredients on the plates and eliminate
visible air bubbles, which are responsible for discontinuities in solid films (Peressini, et al.,
2003). In addition, lower viscosities allows consuming less energy at stirring of the solutions and
makes easier the spreading of the film-forming solution during the casting process. On the other
hand, uniformity of the final coated product is often a critical aspect of film quality (Peressini, et
al., 2003). The desired mechanical properties of an edible film depend on its applications and the
subsequent transportation and handling of the coated foods. Yet, appropriate deformability is also
desired for easy handling in some applications (Gontard, Guilbert, & Cuq, 1992).
In this investigation, a viscosity between 3 and 11 Pa s at 25 °C proved to be adequate for the

film-forming solutions. It was spread easily and cover the molding plate to form the film, which
also is reflected in a thickness homogeneous film.
The waxy corn starch added on the film-forming solution was chosen for its stability when frozen
(Alvarez, 1997), since food as beef, fish and fruit can be distributed and stored at freezing
temperatures. The film-forming solutions based on CMC, CMC-cornstarch, and CMC-waxy
cornstarch, with or without murtilla leaf extract, require a low shear rate, between 2 and 10 s-1 at
25 °C, to achieve a viscosity suitable for their spreading and homogeneity on the molding plate.
This means that these solutions need a low requirement of agitation energy, resulting in lower
processing costs. However, CMC-waxy cornstarch (waxy starch with 1% amylose) shows lower
viscosity that CMC-cornstarch (starch with 27% amylose). The differences in the film-forming
properties with more linear or branched polysaccharides are clearly indicated by linear amylose
molecules, which form strong and flexible films whereas branched amylopectin-based films are
weak and brittle (Zobel, 1988; Peressini, et al., 2003).
150
The film-forming solutions based on chitosan and chitosan-cornstarch without extract also
achieve an adequate viscosity at low shear rate of 2 s-1 at 25 °C. However, chitosan-cornstarch
solution with extract needed a 50 s-1 shear rate and the chitosan solution with extract needed a
100 s-1 shear rate to achieve a viscosity of required magnitude. Thus, developing a workable
solution of chitosan and chitosan-starch with polyphenolic-rich extract has a higher energy cost,
compared with the solutions based on CMC and CMC-starches. However, application of
chitosan-based coatings has, besides antimicrobial properties, other advantages as natural
antioxidant, which can delay the post-harvest enzymatic browning of fruits and vegetables (Pen
& Jiang, 2003; Ponce et al., 2008). Because film-forming solutions containing about 1.5% w/w of
chitosan have been shown to effectively reduce the growth of molds and yeasts (Durango, Soares
& Andrade, 2006), solutions based on chitosan and chitosan-starch are also chosen in this work.
On the other hand, Chile has a lot of shellfish, which generate as byproduct crustacean shells, raw
material for chitosan. Finally, chitosan coatings, containing natural phenolic compounds, can
inhibit lipid oxidation of meats, as have been observed by Siripatrawan & Noipha (2012) and
Krkić et al. (2013).
Although the chitosan and gelatin macromolecules have the same functional groups (-COOH
and-NH2), these two film-forming solutions behaved differently when murtilla leaf extract
polyphenols are incorporated. This occurs due to the murtilla extract reduces the pH of gelatin
solutions, while the pH of chitosan solutions remained constant. The dissociation of protein and a
stronger binding occur at lower pH since protein leads to more binding sites at lower pH values
(Ozdal, Capanoglu, & Altay, 2013).
In this case, the lower pH produced a strong binding between protein and polyphenols, which
precipitated part of the protein. In addition, conditions of temperature, types of proteins, protein
concentration, types and structures of phenolic compounds and several other factors affect
protein–phenolic interactions. Protein–phenolic interactions influence the structure, functional
and nutritional properties, and digestibility of proteins (Ozdal, Capanoglu & Altay, 2013).
151
6.2. General conclusions
The results obtained demonstrated that the efficiency of the phenolic compounds recovery by
ultrasound-assisted extraction (UAE) depends on the effects of parameter interaction,
time/amplitude/duty cycle, as well as of independent variables, duty cycle and exposure time.
This implies that applying UAE, in the optimal combination of these parameters (1 duty cycle ,
20% amplitude or 20 µm) and 45 min ultrasonic time at constant 30 °C), the efficiency of
recovery of phenolic antioxidants is significantly higher (23%) than that obtained by
conventional extraction by maceration (250 rpm, 30 °C and 90 min).
Rheological behavior of film-forming solutions depends of the molecular weight of hydrocolloid,

type of hydrocolloid and hydrocolloid blend. Moreover, rheological behavior is affected by the
presence of polyphenol-rich extract from murtilla leaves on the film-forming solution.
The polyphenol-rich extract from murtilla leaf reduces viscosity of film-forming solutions based
on CMC and CMC-starch blends, being their viscosity ranges, with and without extract, suitable
for coating and film production.
Both solutions based on gelatines of high and low molecular weight, leave an unstable
rheological behavior when blended with polyphenolic compounds, due to reduced pH and the
consequently polyphenol/protein interaction form aggregates. However, these aggregates could
have interesting applications for the production of microcapsules, acting as carrier and release
systems of functional components.
The polyphenols from murtilla extract form bindings with chitosan and chitosan-starch chains,
generating gel-like behavior and reaching high viscosity. However, chitosan-starch solution with
murtilla leaf extract was a more continuous and homogeneous film structure than solution based
on chitosan with murtilla leaf extract. This is due to corn starch that stabilized the interaction
between chitosan and murtilla leaf extract. This improved chitosan-starch with murtilla extract
added, allowed their processing and casting process. Moreover, the restructuring ability of gel
based on chitosan-starch with murtilla leaf extract makes them an important class of materials
152
with potential applications in drug delivery, edible film/coating or as natural‒based wound

dressings impregnated with bioactive compounds.
The interaction mechanism of polyphenol-rich extract from murtilla leaves with chitosan chains
in the films is due to electrostatic interactions (ionic complexations under acidic conditions, pH
4.5), ester linkages and hydrogen bonds, whereas interaction of polyphenols from extract with
starch chains is due to hydrogel bonds and ester linkages. Starch stabilizes the interaction
between chitosan and polyphenolic compound from murtilla leaf extract, therefore chitosan-
starch blend with extract form a continuous and homogeneous film structure. Moreover, the
extract reduced considerably the polymer chain mobility and consequently, the flexibility and
force of the film.
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153
Chapter 7. Appendix
CHAPTER 7
APPENDIX
154
Food Hydrocolloids 31 (2013) 458e466
Contents lists available at SciVerse ScienceDirect
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
Structural properties of films and rheology of film-forming solutions based

on chitosan and chitosan-starch blend enriched with murta leaf extract
A. Silva-Weiss a, *, V. Bifani b, M. Ihl b, P.J.A. Sobral c, M.C. Gómez-Guillén d
a
Doctoral Program in Science of Natural Resources, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera,
Av. Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile
b
Chemical Engineering Department, Universidad de La Frontera, Av. Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile
c
Food Engineering Department, University of São Paulo, PB 23, 13635-900 Pirassununga, SP, Brazil
d
Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN, CSIC) (formerly Instituto del Frío), C/ José Antonio Novais 10, 28040 Madrid, Spain
a r t i c l e i n f o a b s t r a c t
Article history: Chitosan (CH) and chitosan-corn starch (CH-CS) film-forming solutions (FFS) and films, with or without
Received 19 September 2012 polyphenol-rich aqueous extract from murta (Ugni molinae Turcz) leaves (PEML) were prepared. The
Accepted 26 November 2012 impact of the FFS type and PEML, considering pH values of the FFS, on dynamic and steady-shear
behavior of FFS, interaction mechanisms of PEML with polymer chains and changes on infrared
Keywords: spectra of films were investigated. Mechanical properties, thickness and color from films were also
Rheology
evaluated.
Physical properties
Blending CH with PEML produced huge aggregates that were visible to the naked eye. Rheological
Structure
Edible films
parameters, K and n, were affected significantly by the FFS type and the PEML presence. Viscosity of both
Polyphenols FFS increased with the addition of PEML, leading a gel-like structure and thixotropic behavior. Solegel
Ugni molinae T. transition occurred when PEML was added to FFS, increasing strongly their elasticity.
The addition of PEML to CH-CS blend film leads to a reduction (p < 0.05) of elongation at break and
tensile strength, increasing its thickness and yellow color. The PEML formed electrostatic interactions
with chitosan. Ester linkages and hydrogen bonds were also formed between PEML and both chitosan
and starch blends.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction rheological properties of the composite film-forming solutions

(FFS), affecting the functionality of the resulting coatings and films.
Hydrocolloids, such as chitosan and starch, are biodegradable These changes occur due to the compatibility/incompatibility
and non-toxic biopolymers based on renewable resources, used as between both macromolecules, which depend on their molecular
coatings and films in food and pharmaceutical applications. The weight, chemical structure, pH, conformation and hydration
cationic character of chitosan offers an opportunity to establish behavior among others.
electrostatic interactions with other compounds, depending -on Incorporation of antioxidant and/or antimicrobial compounds
the pH and -on the acid type used to dissolve it (Alvarado et al., into films or coating materials can provide food protection against
2007). Due to these characteristics, chitosan has been widely oxidation, microorganism growth, enzymatic browning and
used for the production of edible films with adequate barrier to vitamin losses (Bonilla, Atarés, Vargas, & Chiralt, 2012). In previous
water vapor (Aider, 2010; Alvarado et al., 2007; Rivero, García, & works, the polyphenol-rich aqueous extracts from murta leaves
Pinnoti, 2010). However, considering the cost of chitosan prepara- have been shown to induce antioxidant capacity to fish gelatin
tion, it seems economically feasible to combine it with other film films (Gómez-Guillén, Ihl, Bifani, Silva, & Montero, 2007) and also to
forming biopolymers (Aider, 2010). One of them, starch, is the most carboxymethylcellulose-montmorillonite nanocomposite films
common carbohydrate in the human diet. Blending two different (Quilaqueo Gutiérrez, Echeverría, Ihl, Bifani, & Mauri, 2012). The
hydrocolloids can strongly change both the physical and composition of polyphenol-rich aqueous extract could modify the
physical properties and chemical structure of films and coatings.
The effect of those additives on the film properties will depend on
* Corresponding author. Tel.: þ56 45 241411; fax: þ56 45 241439. their chemical structure, concentration, dispersion degree in the
E-mail addresses: acsilva@ufro.cl, asilvaweiss@gmail.com (A. Silva-Weiss). film and interaction with the polymers (Kester & Fennema, 1986).
0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2012.11.028
A. Silva-Weiss et al. / Food Hydrocolloids 31 (2013) 458e466 459
Murta or murtilla (Ugni molinae Turcz) is an endemic shrub in Table 1

central-southern Chile, which belongs to the Myrtaceae family. Composition of film-forming solutions, with or without polyphenol-rich aqueous
extract from murta leaves (PEML).
Aqueous extracts from murta leaves showed high antioxidant
activity in vitro (Rubilar et al., 2005) and decreased the growth of Hydrocolloid Glycerol PEML
Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus FFS [% w/w] [g/g HT] [mL/g HT]
aureus (Shene et al., 2009). Phenol acids like gallic acid, as well as
H1eH2 H1 H2 HTa Without Withb
flavonoid aglycones and glycosides from quercetin, myricetin and
CH 2.00 e 2.00 0.25 0 20
kaempferol are among the main compounds found in those CH-CS 1.50 0.50 2.00 0.25 0 20
extracts (Bifani et al., 2007; Rubilar et al., 2005). a
HT: Total concentration of hydrocolloids in solution.
The rheological properties of biopolymer solutions can affect b
Total phenol content from PEML expressed as gallic acid equivalent (GAE):
spreadability, thickness and uniformity of liquid coating layer and 81.33 mg GAE/g HT.
film performance. A reduction in the solution’s viscosity provides
a processing advantage during high shear processing operations, Considering the results obtained before (Bifani et al., 2007) in
such as pumping, filling and spraying application; whereas high relation to the reduction in the oxygen permeability of films ob-
apparent viscosity at low shear rates provides a desirable mouth- tained, after incorporation of 40 mL of aqueous extract of murta
feel during mastication and a better application of FFS by dipping leaves in 100 mL of film-forming solution of carboxymethylcellu-
(García et al., 2009, pp.169). lose. Chitosan-starch blend FFS: The concentration of total hydro-
In this context, the purpose of the present work was to inves- colloids in solution (HT) was maintained at a constant 2% w/w
tigate the effect of the polyphenol-rich aqueous extract from murta value. CH-CS blend FFS was prepared by mixing the CH and CS
leaves (PEML) on the dynamic and steady-shear rheological solutions shown above, with or without added PEML (20 mL/g HT,
behavior of FFS based on chitosan and chitosan-starch blend, and 81.33 mg GAE/g HT). The addition of 25% w/w glycerol was repro-
on chemical structure, morphology and physical properties of the duced from reported formulations of films based on chitosan and
resulting films. chitosan-starch films (Mathew, Brahmakumar, & Abraham, 2006;
Mayachiew & Devahastin, 2010) and a gentle stirring of the solution
2. Materials and methods at controlled conditions for 30 min at 45 C was performed.
Vacuum was applied to remove air bubble formation in blend FFS.
2.1. Materials The pH value of blend FFS was determined at 23 C. Samples for
rheological analysis were stored at 5 C for 3 days, until use.
Fresh murta leaves (U. molinae Turcz) of ecotype 27-1 were
sampled near Temuco, Chile (38 350 3900 South latitude) at the
2.4. Rheological measurements: Fundamentals and methods
Instituto de Investigaciones Agropecuarias, INIA Carillanca. Corn
starch (CS), containing 27% amylose, and medium molecular weight
Dynamic viscoelasticity and steady state flow measurements
chitosan (CH), 190e310 kDa, with a deacetylation degree (DD) of
were carried out in a controlled-stress rheometer (Bohlin CVO
75%, were purchased from SigmaeAldrich Co. Glacial acetic acid
Instruments, Inc. Grandbury, NJ) with a cone-plate geometry (cone
(CH3COOH, 98% purity, Merck) and glycerol (CH2OHeCHOHe
angle 4 , diameter ¼ 40 mm, gap ¼ 150 mm). Before analysis, the
CH2OH, 87% purity, Merck) were also used for obtaining FFS.
sample was placed into the rheometer, which was equilibrated at
25 C (Gómez-Guillén et al., 2007).
2.2. Obtaining and characterization of PEML
2.4.1. Steady shear measurements

Murta leaf samples were air-dried for 48 h to about 7% moisture
Flow curves and thixotropic properties were obtained by
content at 35 C. Dried leaves were milled and the leaf powder
registering the shear rate when shear stress was increased from 0 to
(10 g) was macerated with distilled water (ratio ¼ 1:10) at 170 rpm
250 Pa and decreased from 250 to 0 Pa, at 25 C. Experimental data
for 90 min at 25 C. The mixture was filtered (Whatman N 1 paper)
were fitted to Ostwaldede Waele model or rheological Power law
and sterilized through a membrane PES of 0.22 mm to obtain the
model according to Equation (1).
polyphenol-rich aqueous extract from murta leaves (PEML). The
PEML showed 1.4% solids and 0.98 g/ml density. Antioxidant
s ¼ K g_ n (1)
capacity, expressed as the PEML concentration required to scavenge
50% of ABTS$þ (2,20 -azinobis-3-ethylbenzothiazoline-6-sulfonate)
where s is shear stress (Pa), K is consistency index (Pa s), n is flow
free radical, was 0.038 mg gallic acid equivalent (GAE)/mL, and the
index () and g_ is shear rate (s1).
total phenol content was 40.67 mg GAE/g d.m. murta leaves.
The changes in the solution’s apparent viscosity or steady-shear
viscosity (h, Pa s), were investigated according to Equation (2).
2.3. Preparation of film-forming solutions (FFS)
Three repetitions were performed for each sample.
The concentration of components in each FFS for hydrocolloid hðg_ Þ ¼ K g_ n1 (2)
blend formulation is shown in Table 1. Starch-based FFS: Aqueous
solution from CS (0.5% w/w) was prepared by heating beyond their
gelatinization temperature (70 5 C) for 20 min under gentle 2.4.2. Dynamic measurements of viscoelastic properties
magnetic stirring, and cooled until 25 C at a rate of 2e3 C/min. Three dynamic studies were performed: (a) An oscillatory stress
Chitosan-based FFS: Best chitosan films were prepared from 2% sweep test from 0.03 to 400 Pa, at a constant frequency of 0.1 Hz
chitosan in 1% acetic acid, as described before (Siripatrawan & and 25 C was made to set the upper limit of the linear viscoelastic
Harte, 2010). Chitosan was dispersed in acetic acid (1% v/v) to region (LVR). (b) Frequency sweep over a range of 0.01e50 Hz
prepare solutions of 1.5 and 2% w/w. This dispersion was at 25 C was performed at an oscillatory stress within LVR for
mechanically stirred at 500 rpm for 2 h (Ultra-Turrax T25, Janke & each solution. Viscoelastic parameters, storage or elastic modulus
Kunkel, Germany), and filtered through cheese-cloth. The incor- (G0 , Pa), loss or viscous modulus (G00 , Pa), complex modulus (G*, Pa),
poration of PEML was 40% of the dissolution solvent of CH solution. complex viscosity (h*, Pa s) and tangent of the phase angle (tan
460 A. Silva-Weiss et al. / Food Hydrocolloids 31 (2013) 458e466
d ¼ G0 /G00 , Equation (3)) as a function of angular frequency (u, rad/s) separation was set at 60 mm (initial length). Force and distance
were measured, obtaining the typical mechanical spectra. Classifi- were recorded during the stretch of strip at a rate of 1 mm/suntil
cation of the sample structure as gel-like (strong or weak gel), breaking using Texture exponet 32 program.
concentrated solution (entanglement network) or diluted solution
according to Clark and Ross-Murphy (1987) was applied. (c) 2.6.5. Morphology of films
Temperature ramps were performed at a scan rate of 1 C/min and Scanning electron microscopy (SEM) was performed on a JEOL
0.1 Hz from 40 to 2 C and back to 40 C. The phase transitions with scanning electron microscope (JSM-6380 LV, Japan Electron Optics
temperature and elasticity of the FFS were evaluated through the Limited, Tokyo, Japan) at 20 kV. Film samples between 20 and
phase angle (d, ) according to Equation (3). Elasticity is the 28% moisture were examined for cross-section characteristics
reversible behavior of stress/strain, which is measured as the and their thickness. Samples were coated with gold using
reciprocal of d, where purely elastic solids have a phase angle of a sputter-coating system (Edwards 5150 sputter-coating unit) for
0 and purely viscous fluids have a phase angle of 90 . 180 s/30 mA.
2.5. Data analysis 2.6.6. Fourier transform infrared (FTIR) spectra

FTIR spectra of films were recorded in a Perkin Elmer Spectrum
Rheological parameters were analyzed using factor analysis 400 FT-IR with an attenuated total reflectance (ATR) accessory from
ANOVA related to factors: X1 (the presence or absence of PEML) and 650 to 4000 cm1. Measurements were performed at room
X2 (the FFS type). Significance was accepted at 5% confidence level. temperature and 48% RH. An interactive baseline correction at
Mean difference among FFS was performed using orthogonal 1812 cm1 was applied. Signal averages were obtained for 50 scans
contrasts. Duncan’s multiple range test was applied to compare the at a resolution of 4 cm1. No specific preparation was required for
means for thickness, color and mechanical properties of the films. film infrared analysis.
JMPÒ 8.0. Software (SAS Institute, Version 6.09, Cary, NC) was used
for all statistical analyses. 3. Results and discussion
2.6. Films production and characterization 3.1. Rheology of FFS
2.6.1. Film production and film-forming capacity 3.1.1. Steady shear behavior
The FFS (40 g, 2% w/w) were cast onto plexiglass plates As can be seen in Table 2, the studied FFS had a non-Newtonian
(12.5 12.5 cm) and dried at 40 C for 24 h in a ventilated oven behavior (n s 1). Orthogonal contrast comparison of K and n
(Binder FD 240, Tuttlingen, Germany). All the films were equili- parameters from samples showed significant differences (P < 0.01)
brated for three days to 48.0 2.8% RH using saturated sodium between FFS with and without PEML. The FFS type and the pres-
bromide (NaBr) solution at 22.0 0.5 C. The film-forming capacity ence of PEML in coatings affected significantly the response of K
of the solutions, remotion easiness, superficial appearance, homo- and n (P < 0.05).
geneity and continuity of the films were visually examined. Viscosity curves (Fig. 1) showed that apparent viscosity was
highly dependent on the shear rate at which shear stress was
2.6.2. Film thickness measured. Similar to CH solution alone, CH-CS blend solution was
Film thickness was measured using a digital micrometer homogeneous, with a pseudoplastic behavior (n < 1). Thixotropic
(Mitutoyo, Tokyo, Japan). Five measurements were taken on behavior was observed in CH-CS solution due to that the applica-
different positions around each sample. tion of shear breaks or deforms the starch-hydrated granules,
forming aggregates. Because of the low concentration of corn starch
2.6.3. Optical properties (0.5% w/w) in CH-CS solution, mild hysteresis loops were observed
Film surface color was measured using a Minolta Chromameter in non logelog flow curve (not shown).
(CR-400, Konica Minolta Business Technologies, Inc., Tokyo, Japan). When incorporating PEML in CH, the formation and detachment
Films specimens were placed on a white standard plate and five of 0.5e1.5 cm long brown aggregates were observed. Brown
measurements were performed at different locations for each one. aggregates could be produced by strong hydrophobic interaction of
CIE-Lab tristimulus values, lightness (L*: black [] to light [þ]), chitosan chains as well as chitosanepolyphenols interaction. Popa,
redness (a*: green [] to red [þ]) and yellowness (b*: blue []) to Aelenei, Popa, and Andrei (2000) also observed chitosane
yellow [þ]) were measured. polyphenol complex formation which, as reported by Kosaraju,
D’Ath, and Lawrence (2006), could be reversible.
2.6.4. Mechanical properties When the Power law was applied by shear rate sections, it was
Tensile strength (TS, MPa) and elongation to break (EB, %) of observed that at a shear rate below 0.4 s1 CH-PEML and CH-CS-
the films were measured using Instron 4501 Universal Testing PEML blend FFS behaved as dilatant fluids (Fig. 1), whereas at
Machine (Instron Co., Canton, MA, USA) with tensile grip probe. The higher shear rates, FFS behaved as pseudoplastic fluids. The dilatant
conditioned films were cut into 100 20 mm strips for testing and behavior (n > 1) indicated that the formation of new linkages
re-conditioned for two days to 48% RH at 22 C. Initial grip between hydrocolloids and PEML predominated over destroying
Table 2
Parameters for the Power law model, at shear rate above 0.4 s1 at 25 C. Significance of difference between parameters K and n of FFS with and without PEML using orthogonal
contrasts and apparent viscosity based on the form of the Power law model: hðg_ Þ ¼ Kðg_ Þn1 .
K(Pa sn) n () r2 () hðg_ ¼2Þ (Pa s) pH
Without With Without With Without With Without With Without With
CH 4.23 164 ** 0.70 0.32 ** 0.993 0.984 3.45 102 ** 4.74 4.47 NS
CH-CS 3.15 150 ** 0.63 0.17 ** 0.990 0.915 2.44 84.5 ** 4.47 4.43 NS
**P < 0.01, NS: Not significant.

1000 3.1.2. Dynamic behavior

The viscous and elastic properties of the FFS can be evaluated
through dynamic rheological studies. Frequency sweeps assess the
100 effect of additives on changes in microstructure and stability of the
Viscosity (Pa s)
FFS during storage and transportation, whereas a temperature

sweep can show phase transitions and elasticity, allowing the
appropriate temperature ranges for the formulation and applica-
10
tion of the coating film on the product.
The LVR used in this study was the maximum stress value in
CH
the flat region of G0 and the stress curve. Stress values of 1.0 and
1 CH-CS
2.0 Pa (0.2 and 0.05% strain) were found for LVR in FFS with and
CH-PEML
without PEML, respectively. The strain had an opposite trend to
CH-CS-PEML
stress.
0.1 According to the mechanical spectra (Fig. 2), FFS based on CH
0.01 0.1 1 10 100 1000 and CH-CS without PEML shows predominantly viscous behavior
(G00 > G0 ), but crossover point was reached at frequencies above
Shear rate (1/s) 100 rad/s, being classified as diluted solutions. The FFS based on
Fig. 1. Viscosity curves of film-forming solutions with PEML (filled symbols) and
CH alone reached a crossover point at 104 rad/s (123 Pa, pH
without (open symbols) at 25 C. 4.7), which was consistent with w104 rad/s (100 Pa, pH 5.4.)
reported by Chenite, Buschmann, Wang, Chaput, and Kandani
(2001). In contrast to CH and CH-CS without PEML, by incor-
the structure, with the result of a restructured network. Flavonoids, porating PEML in the FFS, G0 was always higher than G00 over the
mainly myricetin and quercetin rhamnosides, with many hydroxyl whole frequency range. The PEML reacted with biopolymers in
groups (eOH) and methyl groups (eCH3) are present in murta leaf solution generating a gel-like behavior. The frequency depen-
extract (Bifani et al., 2007; Shene et al., 2009), which can be forming dence of the viscoelastic parameters has a typical structure of
more interactions by hydrogen bonds with the eOH groups present physical gels, which are reversible gels, depending on the
in chitosan. In relation to pseudoplastic behavior, it was due to that environmental and process factors such as pH, moisture, shear
molecules align in the direction of flow and apparent viscosity rate and temperature (Rinaudo, 2006). This restructuring ability
decreases under increasing shear rate. The PEML decreased n, of CH-PEML and CH-CS-PEML physical gels could make them an
increased K, and increased apparent viscosity in both FFS (Table 2). important class of materials with many applications, such as in
As can be observed in viscosity curves (Fig. 1), time-dependent flow drug delivery. Furthermore, gelling hydrocolloids could enhance
and an unstable flow, characterized by zigzag patterns at high shear the hardness, adhesiveness, and viscoelastic properties of the
rate, were also observed with PEML. FFS.
Without PEML With PEML

1000 1000
G' : CH-CS
G'' : CH-CS
G' : CH
G'' : CH
G' (Pa); G'' (Pa)
100 Diluted solution 100
gel-like
10 10
10 100 10 100
Crossover point
Sample G' = G'' ω η* tg
(Pa) (rad/s) ( Pa⋅⋅s )(min)
CH 123 104 1.27 4.4
CH-CS 131 105 0.77 4.6
Fig. 2. Mechanical spectra showing the angular frequency (u) dependence of storage modulus (G0 , filled symbols) and loss modulus (G00 , open symbols) for FFS based on CH and CH-
CS, with PEML (right) and without (left) (25 C).
According to results of temperature sweep essays (Fig. 3), CH 3.2.3. Thickness

and CH-CS FFS behave as a sol (Phase angle > 45 ), which means The incorporation of starch in the chitosan film did not change
that the solegel transition could not be determined in the significantly the thickness of CH and CH-CS films (P < 0.05).
temperature domain studied. During heating of the CH solution, Compared with the films without PEML, CH-CS-PEML film strongly
phase angle (d, ) was reduced from 74 at 25 C to 56 at 40 C, increases their thickness, whereas CH-PEML film does it only
which indicated that its dynamic elasticity increased a 24% slightly (Table 3).
between 25 and 40 C. This change was not observed when heating
CH-CS solution. Over the full temperature range studied of 2e40 C, 3.2.4. Mechanical properties
CH-CS solution was less elastic than the CH solution alone. The high Comparison of mechanical properties results is difficult since in
temperature can reduce the intermolecular hydrogen bonding general there are a great dispersion in deacetylation degree (DD),
interactions and accelerate the mobility of chitosan molecules in molecular weight (MW), concentration of chitosan and plasticizer,
solution, removing energized water molecules surrounding the film preparation methods, conditioning and tensile test conditions.
chitosan chains (Tang, Du, Hu, Shi, & Kennedy, 2007). The dewa- As can be seen in Table 3, the TS and EB values of CH films (2% w/
tered hydrophobic chitosan chains were associated with each other, w, 75% DD) were close to results obtained in chitosan films (2% w/w,
which increased the chitosan solution elasticity over 25 C. In CH 90% DD) by Siripatrawan and Harte (2010) and Pereda, Ponce,
and CH-CS FFS, gel (Phase angle < 45 ) occurred when incorpo- Marcovich, Ruseckaite, and Martucci (2011), for similar chitosan
rating PEML, increasing their elasticity in 71 and 88%, respectively. and glycerol concentration, and the same film conditioning and
Such results imply a rearrangement of polymers in the presence of tensile tests. Whereas, TS was lower and EB was between the values
PEML, modifying the network structure of both CH and CH-CS reported by Mathew et al. (2006), and Mathew and Abraham
solutions. The CH-CS-PEML remains as a gel along temperature, (2008) in chitosan films.
reducing its elasticity in 60%. In the same temperature range, CH- The mechanical properties of chitosan-starch films can depend
PEML gel increased its elasticity in 39%. on chitosan-starch ratio and starch source, in addition to charac-
teristics and conditions listed above. The CH film shows higher TS
3.2. Film analysis and EB than CH-CS film (ratio ¼ 1.5:0.5). According to Mathew et al.
(2006), chitosan-potato starch films (ratio ¼ 2:3) show higher EB
3.2.1. Film-forming capacity of the solutions than chitosan films alone, whereas TS values of chitosan-potato
The CH and CH-CS films were easy to remove from the plate, starch films (ratio ¼ 1:0.5) have a maximum of 37.5 MPa, due to
with a homogeneous surface appearance and a continuous matrix the formation of intermolecular hydrogen bonds between the
were formed, with good structural integrity. The CH-CS film was chitosan and starch.
smoother than CH film, exhibiting CH film surface undulations. The addition of PEML in chitosan film induces the development
When the CH and CH-CS solutions contain PEML, a chewy consis- of structural discontinuities, producing a highly brittle film struc-
tency hindered their spreading on the plate for forming the cor- ture that can be attributed to less chain mobility, due to excessive
responding film. The CH-CS-PEML film was more homogeneous linkages between PEML and chitosan chains causing polymer
and continuous than the CH-PEML film, but air bubbles on the aggregation. Hence, lower resistance and ductility did not allow
surface of both films were observed. measuring of mechanical properties of CH films. In CH-CS-PEML
films, PEML significantly decreased their TS and EB, also seen on
3.2.2. Optical properties chitosan film with grape seed extract added (Moradi et al., 2012).
Color parameters a* (þ: red; : green) and b* (þ: yellow; : The impaired mechanical behavior in CH-CS-PEML films could be
blue) were good indicator of film color (Table 3). CH and CH-CS also attributed to linkages between PEML and chitosan-starch
films had slightly yellow color, being CH-CS films less yellow than associations, leading to an increased film volume and decreased
the CH film. In contrast to CH and CH-CS, a slightly orange color chains mobility.
(brownish) was observed in CH and CH-CS films containing PEML,
being yellow color of CH-CS-PEML film significantly higher than 3.2.5. Morphology of films
that of the CH-PEML (P < 0.05). Similar changes in chitosan film The cross-sections of scanning electron micrographs for films
color have been found by adding green tea extract (Siripatrawan & formed from CH and CH-CS with and without PEML are shown
Harte, 2010), but in grape seed extract yellow color decreases (Fig. 4). It is clear from the images that distinctive film structures
(Moradi et al., 2012). formed were dependent of presence or absence of PEML and FFS
type used. Chains in CH film were arranged in a horizontally
90 ordered form, because there are repulsive forces between CH
CH
chains when the pH is below 5.0. The CH-CS film forms a structure
80 CH-CS
CH-PEML
with more fibrous appearance than the CH film. Besides, it can be
70 observed some aggregates of starch on the surface of the CH-CS
CH-CS-PEML
Phase angle (º)
60 film.
50 The PEML incorporated produced a remarkable change in the
structure of the films. In the CH film cross-section, PEML creates
40
several elongated horizontal pores of approximately 1e4 mm. In the
30 CH-CS film with PEML, the formation of horizontal layers and some
20 zones with discontinuity or disorder, and pores, were observed.
10 Contrary, ferulic acid into chitosan-starch blend films forms
compact structure (Mathew & Abraham, 2008) while chitosan-
0
starch blend films without ferulic acid show a structure with
0 5 10 15 20 25 30 35 40 45
certain discontinuous zones and small pores. The physical proper-
Temperature (ºC)
ties of films are also depending on its microstructure and unfor-
Fig. 3. Elasticity properties of FFS based on CH and CH-CS (according to Table 2) with tunately, the PEML provoked some discontinuity in films. It’s
PEML (filled symbols) and without (open symbols), during temperature sweep. known that cross linking increases the overall molecular weight
Table 3
Physical properties of chitosan and chitosan-starch films formulated with polyphenol-rich aqueous extract from murta leaves (PEML), and other literature cited films.
Thickness (mm) Color Mechanical properties References
a* b* TS (MPa) EB (%)
Chitosan
c b c a a
e 70.7 5.0 e1.01 0.11 4.52 0.39 29.69 1.89 45.10 1.40 This work
b a b
PEML 77.5 3.5 14.99 2.98 35.11 2.14 n.d. n.d. This work
e 62.1 6.3 1.11 0.16 1.37 0.10 23.66 2.63 54.62 3.12 Siripatrawan & Harte (2010)
Green tea extract 62.1 6.3 3.32 0.66 28.87 0.83 25.13 1.91 58.14 4.24 Siripatrawan & Harte (2010)
e 80 0.70 1.00 29.00 2.00 24.00 3.00 29.00 2.00 Moradi et al. (2012)
Grape seed extract 80 21.0 0.90 16.00 0.20 16.00 0.60 21.00 3.00 Moradi et al. (2012)
e 97.0 8.0 -0.01 1.19 44.67 2.39 17.34 2.43 44.20 7.90 Pereda et al. (2011)
Chitosan-starch
e 72.0 2.4 c -0.41 0.07 c
2.08 0.19 d
20.56 1.78 b
13.52 2.85 b
This work
PEML 126 12 a 12.15 1.23 a
63.63 1.12 a
17.13 1.22 c
6.57 2.43 c
This work
e 73.5 3.9 n.d n.d w 42 w 57 Mathew and Abraham (2008)
Ferulic acid 78.9 4.3 n.d n.d w 49 w 60 Mathew and Abraham (2008)
e 98.9 5.1 n.d n.d 37.50 27.95 Mathew et al. (2006)
Values of this work in the same column with different superscripts mean that the values are significantly different (P < 0.05). TS: Tensile strength (MPa) and EB: Elongation at
break (%), e : without additive, n.d.: non determinated. Mean standard deviation values.
and glass transition of polymers. And, in these cases, more impor- Sarhan, & Ayad, 2010; Rivero, García, & Pinotti, 2010). The peak at
tant plasticizer concentration could be used to avoid the 1380 cm1 was assigned to CeO stretching of primary alcoholic
discontinuity. group (eCH2OH) (Ávila et al., 2012; Sionkowska, Planecka,
Kozlowska, Skopinska-Wisniewska, & Los, 2010) and CeOeH
3.2.6. Chemical structure stretching at 1455 cm1. The bands at 2924, 1407, 1327 and
In order to investigate potential structural differences between 1257 cm1 were assigned to CeH bending of eCH2 due to pyranose
CH and CH-CS films with and without PEML, IR spectra were ring and band at 2878 cm1 was assigned to CeH bending of eCH3
recorded (Fig. 5). Chitosan with a pKa w6.3 is polycationic when from eNHCOCH3 (Mathew & Abraham, 2008; Sionkowska et al.,
dissolved in acid and presents eNHþ 3 sites. In the chitosan film, 2010; Yan et al., 2010). Broad band from 3680 to 2990 cm1
absorption bands at 902, 1024, 1058 cm1 (CeO stretching), and represents polymeric hydrogen bonds, being identified the peaks at
1151 cm1 (asymmetric stretching of CeOeC bridge, glycosidic 3344 cm1 (stretching OeH symmetric) and 3263 cm1 (stretching
linkage) were characteristics of its saccharide structure (Ávila, NeH asymmetric). The band at 1651 cm1 (C]O stretching, amide I
Bierbrauer, Pucci, López-González, & Strumia, 2012; Monier, Wei, band from eNHCOCH3) overlap with band at 1635e1638 cm1
Fig. 4. Scanning electron micrographs of film cross-section: Chitosan films with PEML (CH-PEML) and without (CH), chitosan-starch film with PEML (CH-CS-PEML) and without
(CH-CS). Scale bar 5 mm and 5000 magnification.
115,0
110
1203 902 707
851 816
105 818
2163 2113
1651
1635
1257
CH
1635
100
1462 816
1205 707
95 2163 2113 760
852
1651 1258 926 818 CH-CS
90 1455 1380
1638 1555 1327
85 1550 1406 1334 817 706
2161 2111 1207
2924 1715 1462 852 764 CH-PEML
80 2878 1455 861 778
1651 1639 1331 1251
927
75 3344 1254
3263 2161 2111 1207 817 764 706
2925
70 2878 1715
926 852 778
1614
1455
1380 1251 861 CH-CS-PEML
65 3345 1651
1254
3268 1555 1407 1339
60 2926
1058
%T 2880 926
1614 1379 1151
55 3259
1455
1555 1406 1339
50 2926
2880
45 1379
3259
40 1555 1406 1024
35
1072
30 996
25 1020
20
15 1022
10
5,0 1022
3550,0 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650,0
cm-1
Fig. 5. FTIR spectra of: Chitosan films with PEML (CH-PEML) and without (CH), chitosan-starch film with PEML (CH-CS-PEML) and without (CH-CS) after conditioning to 43% RH and
25 C.
(NH-bending of the glucosamine unit, amide II). The bands acids (as gallic acid) and flavonols (myricetin quercetin, kaemp-
from 1557 to 1538 cm1 were NeH bending in primary amine ferol), present in PEML and therefore, in the films (Bifani et al., 2007;
groups (eNH2/eNH3þ) and 1334 cm1 were CeN stretching Rubilar et al., 2005). In CH-PEML, a new peak around 1620 cm1 and
vibration (amide III). 1627 cm1 have been related to intermolecular interactions
As can be seen in CH-CS film, new peak at 760 cm1 was between chitosan and phenolic compounds, resulting in a lower
attributed to CeH deformation vibration. This was coupled with an percentage of antioxidant release (Mayachiew & Devahastin, 2010).
increase of the CeO stretching of eCH2OH group. Moreover, the Band at 1627 cm1 becomes evident (overlapping with that of amide
bands characteristics of the crystallinity of starch (1042, 1015 and I), suggesting that the reaction between the chitosan and carbonyl
960 cm1) (Vicentini, Dupuy, Leitzelman, Cereda, & Sobral, 2005) groups from polyphenols occurred via ester linkage. Changes
were not observed in these spectra (Fig. 5). These results indicated around 1200e1400 cm1 were due to eOH bending of the phenolic
that chitosan and starch form hydrogen bonds. A characteristic compounds and stretching vibrations of CeO and C]O from poly-
absorption peak assigned to CeOeC in glycosidic linkage at phenols (Bo zi
c et al., 2012b). Besides, band of CeN stretching
1151 cm1 was similar in both CH and CH-CS films, which indicated vibration shifted to 1339 cm1 in CH-PEML and CH-CS-PEML. This
that the glycosidic chain length was not modified. result suggests that amino groups from chitosan interact with
When PEML was added in CH and CH-CS film, various small polyphenols from PEML.
modifications were revealed in the spectra. Band of NeH bond Characteristic carbonyl groups (eCOOH) of phenolic acids have
shifted to 3259 cm1 and OeH linkage stretching of phenolic and been found about 1700 cm1 (Monier et al., 2010). A new small
hydroxyl groups, that appears near 3330 cm1 (Bo zic, Gorgieva, & peak was found in PEML-loaded CH and CH-CS films around
Kokol, 2012b), overlapping band of NeH bond stretching. The Ce 1715 cm1, which has been also observed in CH film loaded with
H stretch vibrations of eCH3 decreased and moved from quercetin (1715 cm1) (Bo zic et al., 2012b), polyphenolic
2878 cm1 to 2880 cm1, suggesting that hydrogen bonds could compounds from olive-leaf extract (1700 cm1) (Kosaraju et al.,
occur between hydrogen from eCH3 or eOH of chitosan groups and 2006), green tea (Siripatrawan & Harte, 2010), and Indian goose-
oxygen from polyphenol eOH groups. berry extract (1720 cm1) (Mayachiew & Devahastin, 2010). These
The FTIR-ATR spectra of PEML-loaded CH and CH-CS films show last authors have attributed this peak to ester linkage between
some differences in the range 1800e1500 cm1, when characteristic chitosan and polyphenolic extract, which was sharper with an
bands of substituted amino groups and carbonyl groups are located increase in the extract concentration. Ester linkages have been also
(Mayachiew & Devahastin, 2010). Band of C]O from N-acetyl group reported between chitosan (pH w 4.5, pKa w 6.3) loaded with
of chitosan at 1651 cm1 was show only as a shoulder. Band of eNH tannic acid (pKaCOOH ¼ 4.4) at 1730 cm1 (Rivero et al., 2010), 4-
bending of amide II decreased and overlap with a new peak that hydroxycinnamic acid at 1700 cm1 (Monier et al., 2010), caffeic
appeared at 1627, 1622 and 1614 cm1 in CH-PEML and at 1614 cm1 acid (pKaCOOH ¼ 4.0), and gallic acid (pKaCOOH ¼ 4.4, pKaOH ¼ 10)
in CH-CS-PEML. Three new peaks appeared, representing C]C around 1715 cm1 (Bo zic, Gorgieva, & Kokol, 2012a). These last
stretching (1614 cm1) and H-atoms (861 and 778 cm1) of aromatic authors showed that chitosan with phenolic acids at pH 4.5 had
rings (Yan et al., 2010); and reflect the presence of both phenolic a higher activity as antioxidant and antimicrobial, against
Fig. 6. Interaction mechanisms at acid pH (4.5 2) of A) Chitosan film with PEML (CH-PEML): Chitosan chains (above and below), and B) Chitosan-starch film with PEML (CH-CS-
PEML): Chitosan chain (above), corn starch chain (below). As models of phenolic acids and flavonoids from PEML, gallic acid and myricetin are presented. Example of interactions:
(1) ester linkage, (2) electrostatic interaction, and (3) hydrogen bond.
Escherichia coli and Listeria monocytogenes, that chitosan at other important class of materials with applications such as in drug
pHs or without polyphenols. delivery or as natural-based wound dressings impregnated with
As a way to integrate our results and previous studies mentioned bioactive compounds.
above, the model shown in Fig. 6 is proposed, which represents The CH film was more ductile and elastic than CH-CS film. Starch
interaction mechanisms of gallic acid and myricetin, as models of stabilized the interaction between CH and PEML, and then CH-CS-
phenolic acids and flavonoids from PEML, with CH and CH-CS films. PEML was a more continuous and homogeneous film structure than
The model indicates that in CH-PEML (pH ¼ 4.4) and CH-CS-PEML CH-PEML. The PEML incorporated to CH-CS film increases the
films (pH ¼ 4.5), eCOOH groups from gallic acid are partially thickness and turns from yellow to slight orange color. The PEML
dissociated. It suggest that the part of non-dissociated gallic acid interacts with chitosan and starch chains in the films. The poly-
from PEML forms ester linkages when it reacts with eCH2OH groups phenolic nature of the PEML extract makes it an extremely highly
of starch and chitosan hydrocolloids; while dissociated gallic acid reactive agent with chitosan. Thus, PEML provoked great disconti-
with COO moiety may be bonded ionically with protonated amino nuity in CH-PEML film matrix due to excessive chitosan cross-
groups (NHþ 3 ) of chitosan, establishing electrostatic interactions linking, which was favored during dehydration of film formation.
capable of forming complexes. In the case of myricetin from PEML, PEML also produced noticeable linkages in the presence of chitosan
loaded in chitosan solution at pH 4.5, only interactions by hydrogen and starch, which reduced considerably polymer chains mobility
bonds can be shown. These facts can explain some changes occur- and consequently, the film’s mechanical properties changes.
ring in the CH and CH-CS film properties loaded with PEML and the However, chitosan-starch associations prevented to some extent
relationship between physical properties and changes of peak the excessive polyphenolepolymer interactions.
pattern of chitosan and chitosan-starch films. Supported by the film characterization, mechanical properties,
SEM and, FTIR analysis, the interaction mechanism of PEML with
chitosan chains inside the films, was due to electrostatic interac-
4. Conclusions
tions (ionic complexations in acidic conditions, pH: 4.5), ester
linkages and hydrogen bonds, while PEML with starch chains was
The presence of both corn starch and PEML affect rheological
due to hydrogel bonds and ester linkages.
behavior of chitosan FFS. The studied FFS show a typical non-
Newtonian behavior at 25 C. The FFS behaved as pseudoplastic
fluids at shear rate over 0.4 s1. The PEML produces a strong Acknowledgments
reduction of the fluidity of chitosan and chitosan-starch solutions.
Besides, viscoelastic properties show that polyphenols from murta We thank Mrs I. Seguel (M.Sc.) from Unidad de Recursos
leaf extract turns CH and CH-CS solutions from a diluted solution Genéticos, INIA Carillanca, Temuco, Chile for the murta leaf ecotype.
into a gel-like structure, increasing their elasticity in 71 and 88% This work was supported by CONICYT CHILE grants N 21070302,
respectively, which is a clear indication of the improvement on the 24090134 and 29090088; Doctoral Program in Science of Natural
three-dimensional cross-linked networks stability. This restructur- Resources, Universidad de La Frontera, Temuco, Chile; DIUFRO
ing ability of CH-PEML and CH-CS-PEML gels make them an project EP 120617 (DI06-0001); CYTED project 309 AC0382; and
the Spanish Ministerio de Ciencia e Innovación project AGL2008- Mathew, S., Brahmakumar, M., & Abraham, T. E. (2006). Microstructural imaging
and characterization of the mechanical, chemical, thermal, and swelling
00231/ALI.
properties of starchechitosan blend films. Biopolymers, 82(2), 176e187.
Mayachiew, P., & Devahastin, S. (2010). Effects of drying methods and conditions on
release characteristics of edible chitosan films enriched with Indian gooseberry
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ARTICLE IN PRESS
FOOD
HYDROCOLLOIDS
Food Hydrocolloids 21 (2007) 1133–1143
www.elsevier.com/locate/foodhyd
Edible films made from tuna-fish gelatin with antioxidant extracts of

two different murta ecotypes leaves (Ugni molinae Turcz)
M.C. Gómez-Guilléna,, M. Ihlb, V. Bifanib, A. Silvab, P. Monteroa
a
Instituto del Frıó (CSIC), C/José Antonio Nováis, 10, 28040 Madrid, Spain
b
Departamento de Ingenierıá Quı´mica, Universidad de La Frontera, Avda, Francisco Salazar 01145, P.O. Box 54 D, Temuco, Chile
Received 9 June 2006; accepted 4 August 2006
Abstract
The aqueous extract of two ecotypes of murta (Ugni molinae Turcz) leaves (Soloyo Grande ‘‘SG’’ and Soloyo Chico ‘‘SC’’), were
analysed for their antioxidant capacity, SC extract exhibiting a higher antioxidant capacity than SG extract. This difference affects
physical properties of tuna-fish (Thunnus tynnus) gelatin-based edible films with incorporated murta leaves extract. Dynamic viscoelastic
studies, scanning electron microscopy (Cryo-SEM) and SDS-PAGE denoted a certain degree of interference of polyphenolic compounds
in the arrangement of gelatine molecules. The addition of the murta extracts leads to transparent films with increased protection against
UV light as well as antioxidant capacity. The puncture test showed no significant differences ðp40:05Þ between the Control film and the
SG film, whereas puncture force and puncture deformation were significantly lower for SC ecotype. Water vapour permeability of tuna-
fish skin gelatin films with SG was 2.87 108 g mm h1 cm2 Pa1, significantly different ðpp0:05Þ to 1.83 108 g mm h1 cm2 Pa1
of the film with SC.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Fish skin gelatin; Leaves extracts; Murta ecotypes; Edible films; Water vapour permeability; Antioxidant properties
1. Introduction abundance, relatively low price and excellent functional

properties. There has been recently an increasing interest
Soluble gelatin, obtained industrially from collagen related to the filmogenic capacity and applicability as food
present in bones or skins, is extensively used as an additive packaging (Arvanitoyannis, 2002). The type of packaging
to enhance the elasticity, consistency, and stability of food is an important factor to enhance the conservation and
products. It may also be used as an outer film, to protect protection of perishable foods, specifically in those cases
against drying, light, and oxygen. Gelatin has the property where oxidative and microbiological deterioration occurs.
of forming a reversible gel in cold, because of the partial The majority of the packaging materials used to be of
renaturalization of the single collagen chains into triple synthetic origin, but nowadays, because of environmental
helical structures, which upon heating, melts from helix to motivations, there is an increasing effort in finding
coil transition (Ledward, 1986). The quality of a food biodegradable edible materials, when possible coming from
grade gelatin depends to a large extent on its rheological recycling industrial wastes or from renewable resources
properties (mainly gel strength and viscosity), but it is also (Tharanathan, 2003). This same sensitivity can be seen in
determined by other characteristics, particularly transpar- the recent scientific literature focused to edible and/or
ency, absence of colour and flavour, and easy dissolution. biodegradable films, with numerous references related to
Because of all these properties, gelatin has been one of the gelatine, either pure or mixed with other biopolymers
first materials used as matrix for bioactive components, (Arvanitoyannis & Biliaderis, 1998; Bertan, Tanada-Palmu,
and this biopolymer remains still of interest because of its Siani, & Grosso, 2005; Menegalli, Sobral, Roques, &
Laurent, 1999; Simon-Lukasik & Ludescher, 2004; Sobral,
Corresponding author. Tel.: +34 91 5492300; fax: +34 91 5493627. Menegalli, Hubinger, & Roques, 2001). Nevertheless all
E-mail address: cgomez@if.csic.es (M.C. Gómez-Guillén). these studies have been done on commercial mammalian
0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2006.08.006
ARTICLE IN PRESS
1134 M.C. Gómez-Guillén et al. / Food Hydrocolloids 21 (2007) 1133–1143
gelatin. There are various reasons for considering the 2.2. Antioxidant activity measurement
marine gelatin as an alternative to the terrestrian mammals
gelatine (bovine and pigs); among them, the possibility of Leaf extracts, previously air dried until 10% wet base
bovine gelatine to transmit infectious illness like spongy- (w.b.), were obtained as described before (Bifani et al.,
form encephalopathy, or from a social-cultural approach, 2006).
some cultures reject it because of their religious beliefs.
Othersides, the main problem of marine gelatins are their (a) DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scaven-
inferior rheological properties, which may limit their ging capacity, based on the bleaching of this stable
application field (Leuenberger, 1991; Norland, 1990). radical: the hydrogen-donating ability of the crude
There has been an increased interest these last years on extract was determined by the method described by
the knowledge focused on fish gelatin (Gómez-Guillén & Hatano, Kagawa, Yasuhara, and Okuda (1988). An
Montero, 2001; Gudmundsson & Hafsteinsson, 1997; aliquot of the extract (100 mL of 1:100 dilution) was
Haug, Draget, & Smidsrod, 2004; Muyonga, Cole, & added to ethanol 80% (final volume 900 ul), shaked
Duodu, 2004). However, there is not enough information and 2.7 mL of DPPH (final concentration 2.0
available about the application on films (Jongjareonrak, 104 M) added. After vigorous shaking, the mixture
Benjakul, Visessanguan, & Tanaka, 2006) and no informa- was left to stand for 20 min and the absorbance
tion at all about antioxidant-fish gelatin active films. In measured at 520 nm. A control with 100 mL water in
general in foods, development of rancid flavor and 900 mL ethanol 80%, and a standard curve of the water
undesirable chemical compounds are the results of lipid soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-
oxidation, leading to quality deterioration and reduction of tetramethylchroman-2-carboxylic acid from Aldrich),
shelf-life. Marine proteins, due to their high content on with 20–80 mL Trolox solution of 2.5 mg/10 mL and
polyunsaturated fatty acids, are especially vulnerable in 880–820 mL ethanol 80%, respectively) were performed
this aspect. simultaneously. Antioxidant capacity was expressed as
There is a growing interest to identify antioxidative mg Trolox/g leaves (w.b.).
properties in many natural sources of polyphenolic (b) SDS–linoleic acid. While most of the synthetic antiox-
compounds for food preservation (Peschel et al., 2006). idants designed for stabilisation of polymers and other
Recently, the germoplasm of murta or murtilla (Ugni organic materials are lipid soluble, the majority of
molinae Turcz), a wild shrub growing in the South of Chile, natural antioxidants are water soluble; to test natural
has been collected and characterised (Seguel, Peñalosa, water-soluble antioxidants, microheterogeneous sys-
Gaete, Montenegro, & Torres, 2000) and the leaves tems like micelles, are used. The SDS–linoleic acid
extracts obtained using methanol, ethanol or water as method was chosen here to determine the reduction of
solvents, has been reported as a source of antioxidant lipid oxidation. The conjugated diene hydroperoxides
polyphenols (Rubilar et al., 2006) and physiological are products of linoleic acid peroxidation induced into
benefits (Montenegro, 2002). Therefore, in the last the micellar phase by a radical initiator AAPH (2,20 -
years there has been an increasing interest towards the azo-bis(2-amidinopropanedihydrochloride), which on
commercial use of this plant as application for foods and thermolysis, provides radicals at constant rate. These
cosmetics. Bifani et al. (2006), using the aqueous extracts hydroperoxides in the micelles of SDS in buffer solution
of two different ecotypes of murta leaves, and applying at pH 7.4 have strong UV absorption with a maximum
them to edible films of carboxymethylcellulose, found at 234 nm, as described by Counet, Callemien, and
distinguished permeabilities between the films, related to Collin (2006), Foti, Piattelli, Baratta, and Ruberto
water vapour, CO2 and O2, because of different quantities (1996), Roginsky and Lissi (2005), Sánchez-Moreno
of the flavonols myricetin and quercetin found in both and Larrauri (1998): just before measurement, the
ecotypes. reaction mixture of 2.6 mM linoleic acid in 0.1 M SDS
The objective of the present work was to obtain and solution, made in 10 mM phosphate buffer pH 7.4, 2%
characterize edible films with antioxidant capacity based on of AAPH, and the solution of 0.25 mg Trolox in 8 mL
tuna-fish gelatin with extracts of two ecotypes grown in of ethanol+2 mL distiled water, were prepared. To
Pumalal, near Temuco, whose different flavonols content 4.0 mL of reaction mixture, 20 mL of APPH were added,
was studied before (Bifani et al., 2006). dissolved and 100, 200 and 300 mL of 1:100 dilution of
sample solution (or water for the control) added, kept at
2. Materials and methods 50 1C for 90 min with lid, cooled rapidly to room
temperature and absorbance read at 233 nm, before and
2.1. Quantification of polyphenols after the 90 min incubation. The Trolox (0.25 mg/
10 mL) was used as standard substance and the results
Quantification of polyphenols was carried out in were expressed as Trolox equivalent (mg Trolox/g
aqueous extracts of murta leaves. Extractable polyphenols leaves (w.b.).
were analysed with the Folin-Ciocalteu method and (c) Ferric-reducing/antioxidant power (FRAP): The FRAP
expressed as mg gallic acid/mL extract. assay was used as a measure of the reducing ability of
ARTICLE IN PRESS
M.C. Gómez-Guillén et al. / Food Hydrocolloids 21 (2007) 1133–1143 1135
murta extracts following the method of Benzie and solutions of NaBr for two days before analysis. Film
Strain (1996). It is based on the increase in absorbance thickness was measured with a digital micrometre (Mitu-
at 595 nm due to the formation of the complex toyo MDC-25 M, Kanagawa, Japan) averaging nine
tripiridiltriazine (TPTZ)–Fe(II) in the presence of different positions.
reducing agents at 37 1C. The aqueous murta extracts
were diluted with distiled water (1:10 v/v), and then 2.4. Mechanical properties
30 mL were incubated with 90 mL distiled water and
900 mL FRAP reagent (containing TPTZ and FeCl3). A puncture test was performed to determine puncture
Absorbance values were read after 4 and 30 min. A force and puncture deformation of films. Films were fixed
calibration curve was performed with FeSO47H2O. in a 5.6 cm diameter cell and perforated to breaking point
Results were expressed as equivalent concentration of using a texturometer (Instron 4501, Instron Engineering
FeSO47H2O mM at 4 and 30 min. Corp., Canton, MA, USA) with a round-ended stainless
steel plunger (+ ¼ 3 mm). Cross-head speed was 60 mm/
The FRAP assay was used also as a measure of the min and a 100 N load-cell was used. Breaking force was
reducing ability of the edible films and filmogenic solutions. expressed in N, and breaking deformation was expressed in
For this purpose, conditioned films (0.125 g) were dissolved %, according to Sobral et al. (2001). All determinations
in 5 mL distiled water at 45 1C. When the dissolution of the were carried out at least in quadruplicate.
film was performed in acid medium (after 10 days of
storage at room temperature and 58% relative humidity), 2.5. Water vapour permeability
distiled water was replaced by 0.5 M acetic acid. Dissolved
films and filmogenic solutions were measured as described Water vapour permeability was determined following the
above. method described by Sobral et al. (2001). Films were fixed
onto the opening of cells (permeation area ¼ 5.31 cm2)
2.3. Film formation containing silica gel and then the cells were placed in
dessicators with distiled water. The cells were weighed daily
Gelatin was extracted from approximately 1-month- during 7 days at 22 1C. Water vapour permeability was
frozen cleaned tuna-fish (Thunnus tynnus) skins following calculated from the equation WVP ¼ wxt1 A1 DP1,
basically the procedure reported in Gómez-Guillén and where w is the weight gain (g), x is the film thickness (mm),
Montero (2001). Gelatin filmogenic solutions were pre- t is the time of gain (h) and DP is the difference of partial
pared at a protein concentration (Nx5.4) of 2 g/100 mL vapour pressure of the atmosphere with silica gel and pure
distiled water, using glycerol (0.25 g/g protein) as plastici- water (2642 Pa at 22 1C). Results were expressed as
zer. Dry gelatin was hydrated at 18–20 1C overnight and g mm h1 cm2 Pa1. All tests were made in duplicate.
dissolved later in water at 45 1C. After complete solubilisa-
tion, the remaining water and glycerol were added. 2.6. Light absorption
In active films, aqueous extracts from murta (Ugni
molinae TURCZ) leaves were added in a proportion 1:1 v/v The light barrier properties of gelatine films were
of gelatine solution+glycerol/extract). Two Pumalal eco- measured by exposing the films to light absorption at
types of murta leaves (Soloyo Grande, SG; Soloyo Chico, wavelengths ranging from 690 to 200 nm, using a UV-1601
SC) were sampled near Temuco (381350 3900 South latitude) spectrophotometer (Model CPS-240, Shimadzu, Kyoto,
at the Instituto de Investigación Agropecuaria INIA Japan). The transparency of the films was calculated by the
Carillanca, Chile). Aqueous extracts were obtained by equation T ¼ Abs600/x, where Abs600 is the value of
mixing 1.5 g cut up leaves with 20 mL distiled water, absorbance at 600 nm and x is the film thickness (mm).
heating at 35 1C for 20 min and filtering later with According to this equation, higher values of T would
Whatman N1 4 filter paper. Before casting, filmogenic indicate lower degree of transparency.
solutions were left at 35 1C for one hour. During this time,
solutions containing murta extracts showed some floccula- 2.7. Electrophoretic analysis (SDS-PAGE)
tion, attributed to the interaction of the polyphenols of the
extracts with the gelatine polypeptides chains (Haslam, Films were dissolved in distiled water at 60 1C and then
1998). Solutions were then filtered through a 1 mm pore size mixed with a 2-fold concentrated loading buffer (2% SDS,
glass fibre filter. Cleaned filmogenic solutions were applied 5% mercaptoethanol and 0.002% bromophenol blue) until
on plexiglass plates (11.5 11.5 cm2) in a calculated reaching a final concentration of 2 mg/mL of protein.
amount to obtain films of around 100 mm in thickness, Filmogenic solutions were mixed directly with loading
and were dehydrated at 42 1C in an oven with air renewal buffer at the same protein concentration. Samples were
and circulation (Binder FD 240, Tuttlingen, Germany) for heat-denatured 5 min at 90 1C and analysed by PAGE-SDS
18–20 h. according to Laemmli (1970) using 3% stacking gels and
The resulting films were conditioned at room tempera- 5% resolving gels in a Mini Protean II unit (Bio-Rad
ture at 58% relative humidity in dessicators with saturated Laboratories, Hercules, CA) at 25 mA/gel. The loading
ARTICLE IN PRESS
volume was 15 mL in all lines. Protein bands were stained should be higher than of the SG extract. As can be seen in
with Coomassie brilliant Blue R250. Type I collagen from Table 1, if the radical scavenger activity of the extracts
foetal calf skin was used as markers of a-, b- and g- chain were measured using DPPH, the SC extract showed the
component mobilities. highest activity: for 1 mL extract (corresponding to 100 mL
of 1:100 dilution), 22.14 mg/g Trolox Equivalent for SC
2.8. Dynamic viscoelastic properties extract versus 10.35 mg/g Trolox Equivalent for SG
extract, being the ratio of SC/SG of 2.1.
Dynamic viscoelastic studies of clear filmogenic solu- The antioxidant activity of SG and SC extracts,
tions were performed on a Bohlin CSR-10 rheometer examined as their protective action toward linoleic acid
rotary viscometer (Bohlin Instruments Ltd., Gloucester- peroxidation in micelles of sodium dodecyl sulphate in
shire, UK) using a cone-plate geometry (cone angle 41, buffer solution, pH 7.4, can be appreciated also in Table 1.
gap ¼ 0.15 mm). Cooling and heating from 40 to 6 1C and The best result was observed with 1 mL extract (corre-
back to 40 1C were performed at a scan-rate of 1 1C/min, sponding to 100 mL of the 1:100 dilution) of the SC
frequency 1 Hz, and oscillating applied stress of 3.0 Pa. The extract (Table 1): 4.76 mg/g Trolox Equivalent for SC and
elastic modulus (G0 ; Pa) and viscous modulus (G00 ; Pa) 2.68 mg/g Trolox Equivalent for SG, showing the SC
were represented as a function of temperature. Several extract 1.8 fold more antioxidant capacity than the SG
determinations were performed for each sample, being the extract. There can also be appreciated that 300 mL of
experimental error always below 6%. the extracts were too much for the micelles in the
conditions of the experiment and therefore showed lower
2.9. Film microstructure antioxidant values for both extracts (1.73 mg/g Trolox
Equivalent for SG and 2.82 mg/g Trolox Equivalent for
Cryoscanning electron microscopy (Cryo-SEM) was SC). Foti et al. (1996) did also not found significant
used to examine representative film surfaces and also the interferences from low concentrations of tested com-
corresponding filmogenic solutions. Samples were mounted pounds; besides, they found that the method is extremely
with OCT compound (Gurr) and mechanically fixed onto sensitive and allows the study of the oxidation process at
the specimen holder using the Oxford CT1500 Cryosample low conversions (less than 10%), at which steady-state
Preparation Unit (Oxford Instruments, Oxford, England). kinetic analysis better applies.
Samples were frozen in subcooled liquid nitrogen for 2 min There can be seen in Table 2, that the aqueous extracts of
and then transferred to the preparation unit. After ice both ecotypes show also a high antioxidant capacity when
sublimation, the surfaces were gold sputter coated, and measured through the FRAP method (4 min: 902.87
subsequently transferred into the cold stage of the SEM 15.2 mmol FeSO4 in SC and 670.279.0 mmol FeSO4 in
chamber. Specimens were observed with a DSM960 Zeiss SG; 30 min: 1475.1711.1 mmol FeSO4 in SC and
SEM microscope (Zeiss, Oberkochen, Germany) at 1148.2735.7 mmol FeSO4 in SG). Therefore, also in this
135 1C under a 15 kV acceleration potential. method the SC ecotype showed more antioxidant capacity
than the SG ecotype (1.3 fold) and the results present the
2.10. Statistical analysis same relationship than the total polyphenol concentration,
expressed as gallic acid content: 283.3 mg mL1 in SC and
Statistical tests were performed using the SPSS computer 224.2 mg mL1 in SG. When comparing with the phenol
programme (SPSS Statistical Software, Inc., Chicago, Ill.) antioxidant coefficient (PAC) shown by Katalinic, Milos,
One-way analysis of variance was carried out. The Kulisic, and Jukic (2006) for 70 infusates of medicinal
difference of means between pairs was resolved by means plants, calculated as the ratio of FRAP (mM/L)/total
of confidence intervals using a Tukey test. Level of phenolics (mM of catechin or gallic acid equivalent/L), SC
significance was set for pp0:05.
3. Results and discussion Table 1

Antioxidant capacity of Soloyo Grande (SG) and Soloyo Chico (SC)
Pumalal ecotypes of aqueous murta leaves extracts, diluted 1:100,
3.1. Antioxidant properties of murta leaves extracts
measured as Trolox Equivalent (mg/g), with DPPH (mean of three
determinations) and SDS–linoleic acid (SDS–L) (mean of two determina-
The water extracts of murta leaves are rich in derivatives tions) methods
of gallic acid, myricetin and quercetin (Rubilar et al.,
2006), which are very polar molecules, with several –OH Antioxidant Sample volume Trolox equivalent (mg/g)
capacity as (1:100) (mL)
groups. The identification of some of the phenolic Soloyo Grande Soloyo Chico
substances showed that SC extract has a higher concentra-
tion of some flavonols than SG extract, like the myricetin DPPH 100 10.3570.40 22.1471.14
derivatives: myricetin dirhamnoside, myricetin glucoside or SDS-L 100 2.68 4.76
SDS-L 200 2.15 3.47
galactoside (Bifani et al., 2006). In correlation to these, one SDS-L 300 1.73 2.82
can expect that the antioxidant capacity of the SC extract
ARTICLE IN PRESS
extract presents a PAC of 3.2 and SG extract of 2.3. ably remain in solution. After the elimination of the
Although Katalinic et al. (2006) reported total phenolics as aggregates, the protein concentration remaining in the film
catechin equivalent, and in the present work as gallic acid forming solutions with murta extract was of 19.427
equivalent, these authors, of the 70 infusates studied, found 0.60 mg/mL for the SG ecotype and 17.8070.23 mg/mL
that Melissae folium infusions showed the highest anti- for the SC ecotype. The difference between these protein
oxidant capacity and the highest phenol content, with a concentrations obtained with the addition of both water
PAC of 3.3 and discussed the possibility that the molar extracts in reference to 20 mg/mL protein of the Control
response of the total phenol content method could be film forming solution was attributed to the removal of
roughly proportional to the number of phenolic hydroxyl polyphenol–protein complexes.
groups in a given substrate. The reducing capacity is After the drying process, very transparent films were
enhanced when two phenolic hydroxyl groups are oriented obtained, with a mean thickness of 100 mm. The incorpora-
ortho or para, which is consistent with the myricetin tion of murta extracts decreased the transparency of the
structure, and the high concentration of this compound resulting films, especially in the case of the SC ecotype
found in SC extract (Bifani et al., 2006). (Table 3). Also Jongjareonrak et al. (2006) found
differences in the transparency of gelatin films, slightly
3.2. Addition of murta extracts into filmogenic solutions higher for tuna-fish skin than those obtained from bigeye
snapper and brownstripe red snapper skin. No significant
It is well known that polyphenols present the ability to differences ðp40:05Þ were found on the thickness of the
form complexes with proteins causing protein precipitation films made with both ecotypes, neither with the film
(Haslam, 1998). But there has been shown that tannin– without extract (Table 3).
gelatin complexes retain a certain capacity as radical
scavengers (Riedl & Hagerman, 2001). The partial floccu- 3.3. Light absorption
lation of the protein in the film forming solution is
attributed to the polyphenols content of the murta extracts, Because of a slightly darker hue of the murta films than
mainly when SC ecotype was used, because of the higher the Control film, there could be a modification of their
concentration of the flavonols myricetin and quercetin properties, like light barrier. To confirm this, a spectro-
found, in relation to the SG extract (Bifani et al., 2006). scopic scanning of the films at wavelenghts between 690 y
The visible aggregates were eliminated through filtration, 200 nm was performed (Fig. 1). At the beginning of the
but the smaller polyphenol–gelatin complexes most prob- visible spectrum, at wavelengths of about 400 nm, both
types of films with antioxidant extracts presented an
absorbance level of approximately 6 fold higher than the
Table 2 Control gelatin film. The differences in the ultraviolet
Antioxidant capacity of aqueous extracts of Soloyo Grande (SG) and
Soloyo Chico (SC), filmogenic solutions (S) and films (F) with Soloyo
spectrum are even higher. This could also mean, besides the
Grande (SG) and Soloyo Chico (SC) Pumalal ecotypes, through the results seen in Table 3, that the tuna-fish skin gelatin films,
FRAP method. (S-C Filmogenic solution control without extract, F-C especially those enriched with murta extracts, could be an
Film control without extract) excellent barrier to prevent UV light-induced lipid oxida-
tion, when applied in food systems. At the wavelength
Samples mmol FeSO4 7 H2O
range between 298 and 380 nm the water extract of SC
4 min 30 min presented absorbance values higher than the SG extract,
which was attributed to differences in polyphenolic
SG 670.1678.97 1148.21735.74
SC 902.79715.24 1475.09711.15
composition. However, no differences in the spectrums
S-C 81.31714.67 200.14738.03 and level of light absorption were found in the films made
S-SG 496.20730.95 908.22738.81 with both ecotypes. Films from bigeye snapper and
S-SC 568.27711.64 908.27729.52 brownstripe red snapper skin gelatin have been also
F-C 299.8175.96 424.6178.35 reported to exhibit a high absorption to light in the UV
F-SG 394.65743.13 667.38770.42
F-SC 542.43745.70 943.50744.71
range (200–280 nm) (Jongjareonrak et al., 2006). The films
obtained with other protein systems offered also a high UV
Table 3
Physical properties of edible films with added murta extracts (F-SG : with Soloyo Grande; F-SC: with Soloyo Chico; F-C: Control without extract)
Samples Transparency Thickness (mm) Puncture force (N) Puncture WVP

deformation (%) (108 g mm h1 cm2 Pa1)
F-C 0.479 0.09870.02 5.9171.49 13.7770.30 2.1670.19

F-SG 0.500 0.10170.01 4.7871.06 11.3972.65 2.8770.40
F-SC 0.804 0.09770.01 2.7571.08 3.5670.03 1.8370.11
ARTICLE IN PRESS
F-SC glycerol, has been reported to increase tensile strength in

4 F-SG films and decrease elongation at break, due to the capacity
to act as a complexing agent of proteins (Orliac, Rouilly,
3 Silvestre, & Rigal, 2002). However, these authors observed
4
SC that, beyond a certain percentage of tannin addition, tensile
ABS
3 SG
2 strength decreased considerably due to weakening interac-
Abs
2 tions stabilizing the protein network. Therefore, the lower
1 mechanical properties of the film containing the SC
1 F-C
0 extracts, compared to the SG films, could be attributed to
200 300 400 500 600
both quantitative and qualitative differences in polyphenols
0 content, which is in accordance with all the other results.
200 300 400 500 600 700
wavelength (nm)
3.5. Water vapour permeability
Fig. 1. Changes in absorbance at wavelengths ranging from 200 to 700 nm
of films with added murta extracts (F-SG : with Soloyo Grande; F-SC: Water vapour permeability of tuna-fish skin gelatin films
with Soloyo Chico; F-C: Control without extract). Inner graphic shows ranged from 2.87 108 g mm h1 cm2 Pa1 in the film
absorbance changes of murta leaves water extracts (SG: Soloyo Grande
containing SG to 1.83 108 g mm h1 cm2 Pa1 in the film
ecotype and SC: Soloyo Chico ecotype).
with SC (Table 3). Therefore the SC film, even showing lower
mechanical properties, presents a reduced permeability to
protection, as were the case of Alaska pollack surimi water vapour in relation to the Control film (without extract),
proteins (Shiku, Hamaguchi, Benjakul, Visessanguan, & and the SG film. Sobral et al. (2001) reported in pigskin
Tanaka, 2004) or whey protein (Fang, Tung, Britt, Yada, & gelatin the linearly increase in WVP from 1.8 to
Dalgleish, 2002), being considerably higher than in many 3.2 108 g mm h1 cm2 Pa1 , between 15 and 65 g sorbi-
synthetic polymer films (Shiku et al., 2004). tol/100 g gelatin. When using 25 g glycerol/100 g pigskin
gelatine, WVP was found to be considerably higher (around
3.4. Mechanical properties 7 108 g mm h1 cm2 Pa1) (Thomazine et al., 2005).
Glycerol is well recognised to present a higher plasticizing
Puncture force and puncture deformation values are effect than sorbitol, causing an increase in film flexibility but
shown in Table 3. In this work, the puncture force in the reduced resistance and water vapour permeability (Cuq et al.,
tuna-fish skin gelatin films is lower than the values reported 1995; Gennadios, Weller, Hanna, & Froning 1996). Thoma-
by Sobral et al. (2001) and Thomazine, Carvalho, and zine et al. (2005) explained this behaviour in terms of
Sobral (2005) in mammalian (pigskin and bovine hide) molecular weight and number of molecules of plasticizers in
gelatin films (E14–16 N), using a comparable amount of the films. Similar findings have been recently reported
plasticizer (25 g of sorbitol or glycerol/100 g gelatine), and working with different concentrations and type of plasticizers
also taken into account that the thickness of the in films from skin gelatin of bigeye snapper and brownstripe
mammalian gelatin films was considerably lower (Earound red snapper (Jongjareonrak et al., 2006).
42 mm). These last authors report a puncture deformation The results of WVP obtained in the present study with
in pigskin gelatin films noticeably lower than in tuna-fish tuna-fish skin gelatin films are noticeably lower than the
gelatin films (E3.5%). Films obtained with other proteins reported above in pigskin gelatin. A possible explanation
and similar concentrations of glycerol as plasticizer have lies in differences in aminoacid composition between both
been shown to achieve similar or lower force values than gelatins, since fish gelatins are known to be less rich in
with tuna-fish gelatin, as with films based on gluten proline and hydroxyproline (Norland, 1990), thus increas-
(Gontard, Guilbert, & Cuq, 1993), fish myofibrilar protein ing their hydrophobicity as compared to mammals gelatins.
(Cuq, Aymard, Cuq, & Guilbert, 1995), or muscle proteins WVP in tuna-fish gelatin films was also lower than in films
from Nile Tilapia (Paschoalick, Garcia, Sobral, & Habi- based on myofibrillar proteins of Atlantic Sardine (Cuq
tante, 2003). Besides in these films the puncture deforma- et al., 1995) or muscle proteins from Nile Tilapia
tion has also been shown around three times lower than in (Paschoalick et al., 2003). When compared to other
tuna-fish gelatin films. The addition of the murta extracts biopolymers, fish gelatin films have been found also to be
at the films produces a lower puncture force and puncture less permeable to water vapour than films based on
deformation, being significantly different ðpp0:05Þ only for cellulose (Psomiadou, Arvanitoyannis, & Yamamoto,
SC ecotype, and no significantly different ðp40:05Þ 1996) or starch (Arvanitoyannis, Psomiadou, Nakayama,
between the Control film and the SG film. A possible Aiba, & Yamamoto, 1997).
explanation lies in the alteration of the plasticizer/gelatin
ratio in the murta added films, especially in the SC film, as a 3.6. Viscoelastic properties
consequence of the slightly lower protein content. On the
other hand, the presence of vegetable tannins in films On the cleaned film forming solutions with added extracts,
produced from sunflower protein isolate plasticized with the changes observed on the dynamic viscoelasticity upon
ARTICLE IN PRESS
cooling and subsequent heating may serve to show possible triple helix of the SC extract in the cold renaturation
interactions between the components of those extracts and process of gelatin.
the gelatin molecules. Fig. 2 shows the viscoelastic proper-
ties of the different filmogenic solutions. Slight differences 3.7. Electrophoretic analysis
can be appreciated both on the elastic modulus (G0 ) and
viscous modulus (G00 ), when the temperature decreases The electrophoretic analysis of the filmogenic solutions
from 40 to 6 1C and during the subsequent heating from 6 showed a molecular weight distribution very similar to the
to 40 1C (Fig. 2). The minimal critical gelling concentration pure tuna-fish gelatin, with predominance of a-chains
of the filmogenic solution was exceeded, since 2 g gelatin/ (E100 kDa) and their dimmers (b-components) (Fig. 3).
100 mL was sufficient to induce renaturation of the The g-components (E300 kDa) appeared only at trace
polypeptide chains into triple helix during cooling down, levels. In the filmogenic solutions with murta extract,
being the gelation temperature around 13 1C in the three independently of the ecotype used, a protein fraction of
cases assayed. Either the elastic modulus (G0 ) or the gelling/ lower molecular weight than the a-chains appeared, as well
melting temperature of the gelatin is highly dependant on as a trace fraction at the end of the polyacrilamide gel. The
protein concentration. The fact that gel could be obtained presence of these lower molecular weight proteins could be,
with such a low protein concentration, indicates a high in part, responsible for the interference of the murta
gelling capacity of the tuna-fish skin gelatin, as reported extracts in the formation of the gelatin triple helical
previously (Gilsenan & Ross-Murphy, 2000). The addition structure upon cooling, as observed previously by means
of both extracts did not modify perceptibly the thermal of dinamic viscoelastic studies.
transition temperatures of the gelatin, because the melting When looking at the molecular composition of the dried
temperature Tm (around 22 1C) was the same for the three films, the incorporation of murta extract modifies percept-
samples analysed. At temperatures below Tm a major ibly the electrophoretic profile compared to the control
contribution of the viscous component (G00 ) in the samples film, because g-trimmers traces disappeared and the
with murta extracts than in the Control were found, presence of b components and a2 chains are notably
showing the interference of both extracts with the reduced. These probably could be due to the contact of the
polypeptide chains, to form the protein net. Nevertheless, murta polyphenols with certain protein fractions that
the solution containing the SC extract reached a smaller G0 further interact during the drying of the film, suffering a
value at 6 1C than with the SG extract, the latter being noticeable aggregation, and thus not present in the
similar to the gelatin solution without murta extract. After electrophoretic gel. In the profile of the film made with
few minutes of maturation at 6 1C, these differences in G0 the SC ecotype, it can be appreciated a certain amount of
were more pronounced, showing a bigger interference into degraded low molecular weight protein at the end of the
200
S-C
S-SG
150 S-SC
G' (Pa)
100
50
S-C
7 S-SG
6 S-SC
5
G'' (Pa)
4
3
2
1
0
40 35 30 25 20 15 10 5 5 10 15 20 25 30 35 40
(a) Temperature (°C) (b) Temperature (°C)
Fig. 2. Viscoelastic properties of film forming solutions (S-SG: with Soloyo Grande; S-SC: with Soloyo Chico; S-C: Control without extract). Changes in
the modulus of elasticity G0 and modulus of viscosity G00 were monitored during cooling from 40 to 6 1C (a) and subsequent heating from 7 to 40 1C (b).
ARTICLE IN PRESS
F-SG F-SC F-C S-SG S-SC S-C Gelatin STD
α1
α2
Fig. 3. SDS-PAGE of film forming solutions with added murta extracts (S-SG: with Soloyo Grande; S-SC: with Soloyo Chico; S-C: Control without
extract) and resulting edible films (F-SG : with Soloyo Grande; F-SC: with Soloyo Chico; F-C: Control without extract).
electrophoretic gel. This can support the reduced rheolo- 3.9. Antioxidant capacity
gical properties of the film formed with this ecotype.
The antioxidant capacity through the FRAP method
3.8. Scanning electron microscopy were measured both for the filmogenic solutions and the dry
films (Table 2). The solution without murta extract shows
About the electron scanning microscopy at low tem- some antioxidant capacity, attributed to the gelatine,
peratures (cryo-SEM), in Fig. 4 are shown the micro- because there has been reported that that fish gelatin
structures of both the filmogenic solutions and the surfaces hydrolysates exhibit high antioxidant and radical-scanven-
of the dried films. On the Control (without extract) ging capacity, due to their aminoacids content like glycine,
filmogenic solution a fibril structure was observed, proline (Mendis, Rajapakse, & Kim, 2005). After the drying
compartmentalised like beehives into cells, with a fine process, the film shows higher values of FRAP than the
network inside. On the SG ecotype solution, the arrange- filmogenic solution. In the solutions containing murta
ment of the cells is similar, although thinner, showing an extracts, the antioxidant capacity increased in more than
apparent increase in free volume in relation to the solution 5 fold in relation to the Control sample, and the SC values
without murta extract. With the SC ecotype, the filmogenic even slightly more. In the murta extract films, the
solution shows a more aggregated, irregular and diffuse antioxidant capacity is also higher than in the Control
structure. films and again, the values with SC ecotype were higher.
The microscopic image of the surface of the film These findings are in direct relation with the higher
containing the SG ecotype is similar to the Control film, concentration of polyphenols and the higher antioxidant
with a uniform granular structure, typical of its protein capacity of the SC extract, as compared to SG extract. After
nature. A similar granular and uniform microstructure has 10 days of storage, the films loose their easy-solubility
been reported in other protein-based films (Fang et al., capacity in water at 45 1C (for the FRAP assay). Therefore,
2002; Tang, Jiang, & Wen, 2005). The SC film presents a the antioxidant capacity measurements decreased drasti-
very even and compacted structure, which denotes notice- cally for all the film samples (Table 4). But, with a better
able greater cohesiveness than both others. The micro- solubilisation in acidic media, the FRAP values were similar
structure images found, either in the filmogenic solutions to that obtained the first day, most probable do to the
containing SC or in the corresponding dried films, are polyphenol content and antioxidant capacity of the murta
indications of a greater protein aggregation suffered as a extracts added. When looking to the FRAP values of the
consequence of the qualitative and quantitative differences Control film (gelatin without extract), FRAP values
in the polyphenol content of both extracts, as seen before remained low, due to the formation of non-acid-reducible
(Bifani et al., 2006). The more compacted and diffuse bonds among gelatin polipeptide chains. These is important
surface of the SC film might be responsible for the lower to take into account when thinking on food applications of
permeability to water vapour of the film, although with the studied films, because it seems to be of critical
inferior mechanical properties. importance that the application should be done rapidly if
ARTICLE IN PRESS
Fig. 4. Scanning electron microscopy at low temperature (Cryo-scanning) of film forming solutions with added murta extracts (S-SG: with Soloyo
Grande; S-SC: with Soloyo Chico; S-C: Control without extract) and resulting edible films (F-SG : with Soloyo Grande; F-SC: with Soloyo Chico; F-C:
Control without extract).
an adequate diffusion of the polyphenol content into the ties to water vapour and UV light. It is possible to increase
food surface should be allowed. Further studies would be significantly the antioxidant properties of the film, when
needed to avoid protein aggregation during film storage. natural extracts with high polyphenols content are added,
producing only minor modifications of the film properties.
4. Conclusions This would be the case of films with Soloyo Grande
ecotypes. When using an extract with a bigger content of
The edible films of tuna-fish gelatin are transparent and polyphenols, like the Soloyo Chico ecoptype, the antiox-
show acceptable mechanical properties and barrier proper- idant capacity of the film increases, but the mechanical
ARTICLE IN PRESS
Table 4 Fang, Y., Tung, M. A., Britt, I. J., Yada, S., & Dalgleish, D. G. (2002).
Antioxidant capacity, measured through the FRAP method, of edible Tensile and barrier properties of edible films made from whey proteins.
films with added murta extracts (F-SG : with Soloyo Grande; F-SC: with Journal of Food Science, 67(1), 188–193.
Soloyo Chico; F-C: Control without extract) after 10 days of storage at Foti, M., Piattelli, M., Baratta, M. T., & Ruberto, G. (1996). Flavonoids,
room temperature and 58% relative humidity coumarins and cinnamic acids as antioxidants ina micellar system.
Structure-activity relationship. Journal of Agricultural and Food
Samples mmol FeSO4 7 H2O Chemistry, 44, 497–501.
Gennadios, A., Weller, C. L., Hanna, M. A., & Froning, G. W. (1996).
Solubilized in water Solubilized in acetic acid Mechanical and barrier properties of egg albumen films. Journal of
Food Science, 61(3), 585–589.
4 min 30 min 4 min 30 min
Gilsenan, P. M., & Ross-Murphy, S. B. (2000). Rheological characterisa-
F-C10d 13.7378.56 47.2272.61 14.3475.52 81.47718.60 tion of gelatins from mammalian and marine sources. Food Hydro-
F-SG10d 174.3272.61 305.92717.75 433.56738.78 693.08756.21 colloids, 14(3), 191–195.
F-SC10d 174.8875.19 273.63723.47 595.2278.29 958.87741.12 Gómez-Guillén, M. C., & Montero, P. (2001). Method for the production
of gelatin of marine origin and product thus obtained. International
Patent PCT/S01/00275.
Gontard, N., Guilbert, S., & Cuq, J. L. (1993). Water and glycerol as
plasticizers effect mechanical and water vapour barrier properties of an
properties decreases, due to a greater interaction between edible wheat gluten film. Journal of Food Science, 58(1), 206–211.
polyphenols and proteins. Gudmundsson, M., & Hafsteinsson, H. (1997). Gelatin from cod skins as
affected by chemical treatments. Journal of Food Science, 62 37–39, 47.
Haslam, E. (1998). Practical polyphenolics. From structure to molecular
Acknowledgements recognition and physiological action. Cambridge: Cambridge University
Press.
Hatano, T., Kagawa, H., Yasuhara, T., & Okuda, T. (1988). Two new
We are very grateful to Mrs. I. Seguel (M.Sc.) from flavonoids and other constituents I licorice root: their relative
Unidad de Recursos Genéticos INIA Carillanca, Temuco, astringency and radical scavenging effects. Chemical and Pharmaceu-
Chile, that provided us with the murta ecotypes for the tical Bulletin, 36(6), 2090–2097.
study, and to Mrs Yoshie Motomura (Ph.D.), Senior Haug, I. J., Draget, K. I., & Smidsrod, A. (2004). Physical and rheological
properties of fish gelatine compared to mammalian gelatin. Food
Volunteer of the Japan International Cooperation Agency
Hydrocolloids, 18(2), 203–213.
(JICA) at Universidad de La Frontera, for her help in Jongjareonrak, A., Benjakul, S., Visessanguan, W., & Tanaka, M. (2006).
antioxidant measurements on the murta extracts. This Effects of plasticizers on the properties of edible films from skin gelatin
work has been performed within the framework of the of bigeye snapper and brownstripe red snapper. European Food
project CYTED XI.20 and sponsored by Universidad de Research and Technology, 222(3-4), 229–235.
La Frontera, projects DIUFRO 140502 and 120617, and Katalinic, V., Milos, M., Kulisic, T., & Jukic, M. (2006). Screening of 70
medicinal plant extracts for antioxidant capacity and total phenols.
by the Spanish Ministry of Education and Science under Food Chemistry, 94, 550–557.
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assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Ledward, D. A. (1986). Gelation of gelatin. In J. R. Mitchell, & D. A.
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Food Engineering:
Integrated Approaches
Edited by:
Gustavo F. Gutiérrez-López
Instituto Politécnico Nacional
México, DF, México
Gustavo V. Barbosa-Cánovas
Washington State University
Pullman, WA, USA
Jorge Welti-Chanes
Universidad de Las Américas
Cholula, Puebla, México
and
Efrén Parada-Arias
Instituto Politécnico Nacional
México, DF, México
ISBN: 978-0-387-75429-1 e-ISBN: 978-0-387-75430-7
Librery of Congreso Control Number: 2007938046
© 2008 Springer Science + Business Media, LLC

233 Spring Street, New York, NY 10013, USA.
Springer
(Capítulo 12: Pág: 225 – 241)

12
Edible Coating as an Oil Barrier or
Active System
M. GARCIA, V. BIFANI, C. CAMPOS, M.N. MARTINO, P. SOBRAL,
S. FLORES, C. FERRERO, N. BERTOLA, N.E. ZARITZKY,
L. GERSCHENSON, C. RAMÍREZ, A. SILVA, M. IHL AND F. MENEGALLI
12.1 Introduction
Edible coatings have long been known to protect perishable food product from
deterioration by retarding dehydration, suppressing respiration, improving textural
quality, helping to retain volatile flavor compounds and reducing microbial growth
(Mauer et al., 2000; Yang and Paulson, 2000; Peressini et al., 2003; Han et al.,
2004). Also, they can be used as a vehicle for incorporating functional ingredients,
such as antioxidants, flavor, colors, antimicrobial agents and nutraceuticals (Kester
and Fennema, 1986; Guilbert et al., 1997; Bifani et al., 2006).
Antimicrobial edible films and coatings are used for improving shelf life of food
products without impairing consumer acceptability (Baker et al., 1994). They are not
designed for totally replace traditional packaging, and might be used as a stress
factor in minimally processed foods in order to prevent surface contamination while
providing a gradual release of the antimicrobial.
Another application of edible films or coatings, is as barrier to lipid absorption by
food during deep fat frying. Oil uptake in fried foods has become a health concern;
high consumption of lipids has been related to obesity and other health problems
such as coronary heart disease. Reducing fat content of fried foods by application of
coatings is an alternative solution to comply with both health concerns and consumer
preferences. Food coatings may become a good alternative to reduce oil uptake
during frying. The effectiveness of a coating is determined by its mechanical and
barrier properties, which depend on its composition and microstructure, and on the
characteristics of the substrate.
Some fried products may contain fat up to 50% of the total weight (Pinthus et al.,
1993). Some of these lipids were not in the food before frying. For example, lipid
content of french fries increases from 0.2% to 14 %, lipid content may reach 40% in
potato chips; raw fish with 1.4% reach 18% fat after frying (Smithet al., 1985;
Mackinson et al., 1987). Several hydrocolloids with thermal gelling or thickening
properties, like proteins and carbohydrates, have been tested to reduce oil and water
migration (Debeaufort and Voilley, 1997; Williams and Mittal, 1999).
225
Thus, the objective of this work is to present and discuss some potentiality or
applications of edible films or coating and to examine its possible uses to improve
the quality and lengthen the shelf life of foods.
12.2. Use of a Tapioca Starch Edible Film

Containing Potassium Sorbate to Extend
the Shelf Life of Minimally Processed Pumpkin
Starch is a natural biopolymer very commonly used to constitute film matrices
(Durango et al., 2005). In particular, tapioca starch is a promising alternative due to
its low price in the world market when compared to starches from other sources
(FAO, 2004). These film matrices are commonly used with additives that provide an
extra protection against surface contamination of some microorganisms. Sorbic acid
and its potassium salt (sorbates) are considered GRAS (generally regarded as safe)
additives and are active against yeasts, molds and many bacteria (Sofos, 1989).
Addition of potassium sorbate to edible films has been proposed as a way of
minimizing surface microbial contamination (Cagri et al., 2001).
Pumpkin is a seasonal crop used as human food. It covers a wide number of species
of the family Cucurbitaceae, most of them with an economic potential due to its low
price in the word market and to the fact that they are a good source of nutrients,
especially β-carotene (Hels et al., 2004). Therefore, in this work the possibility of
controlling microbial growth and lengthening shelf life of minimally processed
pumpkin is studied.
12.2.1 Film preparation and application on minimally processed

pumpkin
The film was prepared using a mixture of tapioca starch, glycerol, sorbates and
water (5.0:2.5:0.15:92.35 in weight). Starch was gelatinized by heating on a hot plate
with magnetic stirrer with at a constant rate of 1.8°C/min for approximately 30 min.
After gelatinization, films were cast over glass plates and dried at 50ºC for two h.
The drying process was completed in a chamber (Velp, Italy) at 25ºC. Once
constituted, films were peeled off from glass plates. Before using to wrap pumpkin
samples were conditioned at 25°C, over a saturated solution of NaCl (water activity,
aW = 0.755) for 4 days.
Pumpkin (Curcumis moschata, Duch.) was purchased at a local supermarket, then
was washed and cut into cylinders of 16 mm length and 23 mm diameter employing
a metallic cork borer. The pumpkin was then cooked in water vapor at 100°C for 10
min and rapidly cooled with water, drained and introduced in a glucose osmotic
solution of 0.895 aw acidified to 3.0 with citric acid and containing 0.02 % (w/w) of
vanillin (ratio pumpkin cylinders:solution, 2:5). The system was stored at 8°C for 4
days; pumpkin cylinders reached a 0.938 aw and 4.4 pH. Portions constituted by four
pumpkin cylinders were wrapped with film and packed into gas impermeable bags
(PET/Al/PE) which were sealed and stored at 25°C for 21 days. Pumpkin cylinders
without edible film wrapping, packed as previously stated, were also stored as
controls for comparison purposes.
226
12.2.2 Microbial and Chemical Analyses
To evaluate the effect of the potassium sorbate wrapping on the development of food
microbiota, microbiological analyses of aerobic mesophilic and lactic acid bacteria,
yeast and molds, were carried out on surface of film and in the pumpkin tissue after
removing the film. Analyses were performed according to the methodology
described by the Compendium of Methods for the Microbiological Examination of
Foods (Vanderzant and Splittstoesser, 1992). Non-coated samples were used as
controls. Three bags from each treatment were sampled at time zero and after 21
days of storage at 25°C.
The evolution of pH and water activity (aw) in the film and in the pumpkin after
removing the film was determined. Weight loss of samples was also determined.
Potassium sorbate concentration in the film and in the pumpkin after removing the
film was evaluated according to the AOAC method (1990) which includes steam
distillation of the preservative followed by oxidation to malonaldehyde and
measurement at 532 nm of the pigment formed between malonaldehyde and
thiobarbituric acid.
All determinations were performed in triplicate and means are reported. A “t”
test was performed to determine significant differences between parameters
evaluated (α= 0.05).
12.2.3 Storage tests

The level of aerobic mesophiles and lactic acid bacteria and yeasts and molds, in the
surface of the film-wrapped pumpkin cylinders was below 42 CFU/cm2 at time zero
and after 21 days of storage, showing that the film carried a low microbial load and
demonstrating that the film used is an efficient barrier to prevent external
contamination.
Figure 12.1 shows the evolution of native flora in the pumpkin after removing
the film. It can be seen that levels of mesophilic aerobes after storage decreased 2 log
cycles. In the case of yeasts and molds and lactic acid bacteria, its initial populations
in pumpkin were approximately 150 CFU/g; after storage, the microorganism’s
counts decreased significantly in all cases. It must be mentioned that for control
samples the level of mesophilic aerobes after storage was similar to the initial level.
However, samples had an unacceptable appearance; the surface was spoiled by
molds and the tissue had an aqueous aspect. This behaviour was related to the high
counts of yeast, molds and lactic acid bacteria (Fig. 12.1).
The pH of the product decreased to 3.3 in controls (Table 12.1) as the result of
lactic acid bacteria metabolism. Moreover, aw increased to 0.951, probably as a
consequence of changes induced by metabolic activity of microbial flora. Otherwise,
the pH of the pumpkin wrapped with the film reached a value of 5.1 and aw
decreased to 0.918 because of the contact with edible films of pH 6.3 and aw of
0.753.
These results demonstrated that depression of aw to 0.938, adjustment of
pH to 4.4 with citric acid, together with the use of an impermeable packaging
were not enough to preserve pumpkin, and demonstrated the need for an
additional stress factor, such as the addition of a preservative agent that could
be provided supported in an edible film, as is proposed in this work.
227
Figure 12.1: Evolution of native flora in samples of processed pumpkin. Bars followed by the
same letter are not significantly different (P> 0.05)
Table 12.1: pH and water activity of pumpkin samples

Initial 21 days
Control Treatment
pH 4.4 ± 0.1 3.3 ± 0.3 5.1 ± 0.1
aw 0.938 ± 0.004 0.951 ± 0.002 0.918 ± 0.003
Reported values are the mean of three measurements. Standard deviations are also shown.
Weight loss for wrapped pumpkins at the end of the experiment was 15.1±1.8%.
This behavior can be attributed, at least partially, to water transfer from pumpkin to
film since higher water content and aw value (0.902) was noted for edible films used.
Weight loss reported here is similar to the values reported by Garcia et al. (1998) for
starch-coated strawberries. For control samples, weight loss could not be determined
because of the high degree of tissue spoilage.
Antimicrobial migration from film to pumpkin was verified by measurement of
potassium sorbate content in wrapped pumpkin cylinders. Concentration observed
was 2588±85 ppm after 21 days, which represent, approximately, a 40% of total
potassium sorbate contained initially in the film (15000 ppm). It must be mentioned
that the content of the preservative in the film after storage was 7140±770 ppm,
suggesting that part of the potassium sorbate initially contained in the film was lost
throughout storage, probably as a result of its oxidation. It is well documented that
potassium sorbate suffers autoxidative degradation in model systems and in food
products, and that the magnitude of this reaction depends on system composition
(Gerschenson and Campos, 1995).
In relation to physico-chemical changes mentioned, the edible film formulation
proposed must be modified in order to avoid the increase in pH in order to minimize
228
weight losses and to ensure sorbate content in the pumpkin is in accordance with
maximum values allowed by food legislation of the country of application.
Results reported here demonstrated that the use of tapioca-starch edible films
containing potassium sorbate to wrap pumpkin cylinders can act as a physical barrier
to exclude the entrance of microorganisms, provide a source of preservative that can
prevent microbial growth at the surface and, at the same time, control spoilage flora
in the pumpkin tissue, since part of the antimicrobial was released to the food.
12.3 Edible Cellulose Derivative Coatings

to Reduce Oil Uptake in Fried Products
The application of edible coatings at the surface of the foods is an interesting
technique that has not been studied extensively. Mallikarjunan et al. (1997), working
with mashed potato balls, reported a reduction, compared to uncoated balls, of
14.9%; 21.9% and 31.1% in moisture loss and of 59.0%; 61.4% and 83.6% in fat
uptake for samples coated with corn zein, hydroxypropylmethylcellulose (HPMC)
and methylcellulose (MC) films, respectively. Williams and Mittal (1999) also found
that MC films showed the best barrier properties, reducing fat uptake more than
hydroxypropylcellulose (HPC) and gellan gum films applied to a pastry mix.
Cellulose derivatives, including MC and HPMC exhibit thermal-gelation. When
suspensions are heated they form a gel that reverts to below the gelation temperature,
and the original suspension viscosity is recovered (Grover, 1993). These cellulose
derivatives reduce oil absorption through film formation at temperatures above their
incipient gelation temperature, or they reinforce the natural barriers properties of
starch and proteins, especially when they are added in dry form (Meyers, 1990).
The objective of this study was to apply coatings based on cellulose derivatives
and plasticizer in order to reduce oil uptake in fried potato strips and pastry products,
optimizing their formulations.
12.3.1 Preparation of Suspension of edible coating

Cellulose derivatives (Methocel), K100LV and E15LV (two different
hydroxypropylmethylcelluloses, HPMC) and A4M (methylcellulose, MC), were
provided by Dow Chemical (USA). Concentrations of aqueous cellulose derivative
suspensions of 1% and 2% were tested to select appropriate formulations for coating
applications. Plasticizer effect of sorbitol on the selected cellulose derivative
formulations was analyzed. Sorbitol (Merck, USA) concentrations assayed in the
suspensions were 0.25; 0.50; 0.75 and 1 % (w/w) (Garcia et al., 2002).
Rheological characterization of the cellulose derivative suspensions was performed
in a Haake RV2 (Haake, Germany) rotational viscometer, at controlled constant
temperature (20°C). A NV type sensor system of coaxial cylinders was used for all the
measurements. Rheological curves were obtained after a stabilization time of 3 minutes
at 20°C. Shear stress was determined as a function of shear rate between 0 and 1382 s-1
with the following program: 3 minutes to attain the maximum shear rate, 1 minute at the
maximum shear rate and 3 minutes to attain 0 shear rate. Apparent viscosities were
calculated at 64 rpm (345.6 s-1) and 128 rpm (691.2 s-1). Apparent viscosities of 1%
229
A4M at 20°C were 138.1 ± 5.2 and 138.1 ± 5.2 mPa.s at shear rates of 345.6 and 691.2
s-1 respectively. These values were significantly higher than the apparent viscosities of
2% K100LV and E15LV solutions
According to manufacturer description, A4M is a modified cellulose that
contains only methyl groups as substituents, allowing stronger interactions between
hydrocolloid chains; thus, it should yield high viscosity solutions even at
concentrations of 1 %. K100LV and E15LV are hydroxyl propylmethyl derivatives
of celluloses containing both substituents: methyl and hydroxypropyl groups. In
these cases, the interaction between chains may be hindered by steric impediment.
Thus, K100LV should yield medium viscosity solutions and E15LV with higher
percentage of hydroxypropyl groups, and more steric impediments should yield
lower viscosity solutions.
Thermal gelation was also analyzed; clouding point was 70ºC for 1%A4M, 80ºC
for 2% K100lLV and 60ºC for 2%E15LV al. Similar results were found by
Mallikarjunan et al. (1997). According to Grover (1993), thermal gelation is
produced because thermal treatments induced perturbations in the hydration layers of
the macromolecules, leading to an increase in the hydrofobic interpolymeric
interactions. Thermal gelation temperature increased with sorbitol concentration; it
was attributed to a more difficult hydrocolloid chain association.
12.3.2. Frying tests

Frying tests were performed with potato strips (0.7×0.7×5 cm) and commercial dough
discs of wheat flour (Pillsbury, Argentina), 3.7cm in diameter and 0.3 cm high.
Samples were dipped in the coating suspensions for 10 sec and immediately fried.
Coated and uncoated (control) samples were fried in a controlled temperature
deep-fat fryer filled with 1.5 L of commercial sunflower oil. Oil composition was
99.93% lipids, with 25.71% mono-unsaturated and 64.29% poly-unsaturated fatty
acids. Used oil was replaced by fresh oil after four frying batches. In each batch six
potato samples or four dough discs were fried. A variety of constant frying
temperatures was tested to select the working frying conditions according to sample
characteristics. These temperatures ranged between 150±0.5ºC and 170±0.5ºC for
dough and between 170±0.5°C and 190±0.5°C for potatoes; frying times ranged
between 2 and 5 minutes in both cases. Optimum time-temperature frying conditions
were determined by both instrumental color and sensory analysis; a non trained panel
judged color, flavor, texture and overall appearance (Garcia et. al. 2002, 2004).
Colorimetric measurements were carried out with a Minolta colorimeter CR 300
Series (Japan). The Hunter scale was used, and lightness (L) and chromaticity
parameters a* (red – green) and b* (yellow – blue) were measured. Samples were
analyzed in triplicate, recording four measurements for each sample.
Instrumental surface color showed no significant differences (P>0.05) between
fried potato strips processed 5 min at 170°C or 4 min at 180°C or for dough discs
processed between 2.5 min at 160°C and 3 min at 150°C. However, sensory analysis
(color, flavor, texture and overall appearance) determined that 4 min at 180±0.5°C
for french fried potatoes and 3 min at 150±0.5°C for dough discs were the best frying
conditions. Accordingly, these parameters were selected as operative conditions for
further determinations.
230
Water content (WC) was determined by measuring the weight loss of fried
products upon drying in an oven at 110°C until constant weight. Relative variation of
water retention % (WR) in the coated product relative to the uncoated one was
calculated as follows:
WCcoated
WR = 1 × 100 ( 12.1 )
WCuncoated
Lipid content (LC) of fried products was determined on dried samples using a
combined technique of successive batch and semi-continuous Soxhlet extractions.
The first batch extraction was performed with petroleum ether:ethylic ether (1:1)
followed by a Soxhlet extraction with the same mixture and another Soxhlet
extraction with n-hexane. Oil uptake relative variation % (OU) in the coated product
relative to the uncoated one was calculated as follows:
LCcoated
OU = 1 × 100 ( 12.2 )
LCuncoated
For each coating formulation, results were obtained using all samples from two
different batches.
The cellulose derivative was selected from experiments done with fried potato
strips. Water and oil contents of the control sample were 62.62±0.92 g water/100g
(dry basis) and 4.43±0.74 g oil/100g (dry basis), respectively. Coatings with
cellulose derivatives without plasticizer applied on potato strips reduced oil uptake
and led to a higher retention of moisture content during deep fat frying (Table 12.2).
As water content in the coated product was higher than in the uncoated product, the
WR was always positive and higher for MC than for the HPMC coatings. The oil
uptake was always negative because the lipid content of the coated sample was lower
than the uncoated one (Garcia et al., 2002).
Table 12.2. Characterization of fried potato strips coated with different formulations.
Relative variation of water
Coating formulation (without Relative variation of oil uptake
retention (WR) % (Eq.
plasticizer) (OU) % (Eq. 12.2)
12.1)
Control - -
1% MC (A4M) -15.53±0.79 2.14±0.49
2% K100LV -6.30±0.21 0.49±0.21
2% E15LV -7.60±0.74 1.00±0.15
Value ± standard deviation
MC coating was selected because it showed the highest oil uptake reduction
compared to HPMC coatings. This could be attributed to the differences in
suspension viscosities, which is related with the covering capacity of coatings HPMC
coatings did not show noticeable differences with regard to oil uptake; the presence
of hydroxypropyl groups may limit film-forming capacity through steric hindrances.
Mallikarjunan et al. (1997) working with potato balls, found that MC coatings have
better moisture barrier performance than HPMC coatings, due to the lower
231
hydrophilic character of MC. In addition, Holownia et al. (2000) reported that MC
and HPMC films applied on chicken strips decreased oil uptake and the degradation
of frying oil, thus extending the useful life of the product..
Coating presence significantly (P<0.05) modified surface color parameters of
potato slices. Chromaticity parameter a* of samples with different coatings and the
uncoated samples were significantly different (P<0.05). Even though instrumental
color parameters showed differences, all samples were accepted by the non trained
panel. Sensory analysis and L values showed that control samples were darker and
more opaque than coated samples.
12.3.3 Effect of Sorbitol Addition to the Coating

Formulation on Oil Absorption and Water Retention
The initial water and oil contents of potato strips before frying were previously
reported. For dough discs, the initial water content before frying was
35.72±0.64g/100g (dry basis) and the lipid content was 4.00±0.10g/100g (dry basis).
After frying the oil content of dough samples without coating (control) was
18.42±1.77 oil/100g (dry basis) and the water content was 17.12±1.43 g water/100g
(dry basis). For potato samples without coating (control), values were 3.49±0.21
oil/100g (dry basis) and 63.97±2.54 g water/100g (dry basis), respectively.
Table 12.3 shows values obtained for the relative decrease of oil uptake and
increase of water retention of fried dough and potato strips coated with MC
formulations.
Table 3. Relative variation of oil uptake and water retention of fried dough discs and potato strips
coated with methylcellulose (MC) and sorbitol (S) formulations.
Sample Formulation Relative decrease of Relative increase of
oil uptake (OU), water retention (WR),
(%)(eq. 2) (%), (eq. 1)
Dough discs Control (without coating)1 0 0
1% MC 14.20±0.143 10.34±0.09
1% MC + 0.25% S 27.53±0.09 27.93±0.16
1% MC + 0.5% S 25.65±0.07 23.68±0.11
1% MC + 0.75% S 35.23±0.06 25.75±0.12
1% MC + 1% S 29.03±0.08 22.87±0.14
Potato strips Control (without coating)2 0 0
1% MC 15.53±0.23 2.14±0.08
1% MC + 0.25% S 26.60±0.18 5.61±0.03
1% MC + 0.5% S 40.62±0.13 6.31±0.04
1% MC + 0.75% S 23.69±0.15 6.98±0.04
1% MC + 1% S 15.58±0.27 7.65±0.03
1-Oil content of dough control sample = 18.42±1.77oil/100g (dry basis); water content = 17.12±1.43 g
water/100g (dry basis).
2-Oil content of potato control sample = 3.49±0.21oil/100g (dry basis); water content = 63.97±2.54 g
water/100g (dry basis).
3-Value ± standard deviation.
MC coating treatment significantly (P<0.05) reduced oil uptake and increased

water retention of both fried potato and dough samples compared to uncoated
samples. Similar results were obtained by other researchers who applied cellulose
232
derivative coatings on different products (Mallikarjunan et al., 1997; Balasubramaniam
et al., 1997; Holownia et al., 2000).
Sorbitol addition improved barrier properties of coatings by decreasing oil
content and increasing moisture retention compared to control and coated samples
without plasticizer. ANOVA analysis (P<0.05) showed that the most effective
sorbitol concentrations to reduce oil uptake were 0.5% for potato strips and 0.75%
for dough discs (Table 12.2). Considering that the plasticizer increases the elasticity
of the coating (Donhowe and Fennema, 1993) and that the dough discs expanded
during the frying process, a higher sorbitol concentration was required for dough
samples. Similarly, Rayner et al. (2000) reported that the performance of soy protein
film applied on dough discs increased by the addition of glycerin as plasticizer,
reducing the food fat uptake.
Dough discs coated with MC and 0.75% sorbitol showed a 35.2% reduction of
oil absorption and a 25.7% increase of moisture retention compared to control
samples. For potato strips coated with MC and 0.5% sorbitol, oil absorption
decreased 40.6% and moisture retention increased 6.3% compared to control
samples. Pinthus et al. (1993) reported that the addition of methylcellulose in dough
formulation significantly reduced the oil uptake of doughnuts in the case of dry
addition.
Coating integrity was analyzed by stereomicroscopy observation (Leitz Ortholux
II, Germany), staining sample surfaces with toluidine blue. Scanning electron
microscopy (SEM) of coated and uncoated samples was performed with a JEOL
JSMP 100 scanning electron microscope (Japan). Coated pieces were mounted on
bronze stubs using double-sided tape and then coated with a layer of gold (40-50
nm). All samples were examined using an accelerating voltage of 5 kV.
Staining with toluidine blue only helped to visualize coated dough samples under
light microscopy; the similar chemical structures of the potato matrix and cellulose
derivatives did not allow to effective differentiation of the coating in french fries.
Fried samples with unplasticized coatings showed cracks that may reduce barrier
properties of the coating. Mallikarjunan et al. (1997) attributed the reduction in oil
uptake and moisture loss to the formation of a protective layer on the surface of the
samples during the initial stages of frying due to thermal gelation above 60°C. This
protective layer inhibits the transfer of moisture and fat between the sample and the
frying medium. Coating integrity is an important factor, because the presence of cracks
may reduce barrier properties of coatings and may limit coating applications (Donhowe
& Fennema, 1993). In the case of frying applications coating integrity also depends on
the substrate. While potato fries almost maintained their shape and volume after frying,
dough discs increased their volume and modified the shape. Observations by both SEM
and stereomicroscope of stained dough discs showed cracks in the coatings without
plasticizer, evidencing the fragility of the coating structure.
Addition of plasticizer (sorbitol) to MC coatings was necessary to achieve
coating integrity (Fig. 12.2). A similar trend was observed by Rayner et al. (2000),
working on deep-fat fried potato discs coated with plasticized soy protein films.
Formation of a uniform coating on the surface of the sample is essential to limit mass
transfer during the frying process (Huse et al., 1998).
MC is characterized by its thermogelation properties, i.e., the gel formed upon
heating above 60°C (Meyers, 1990; Grover, 1993; García et al. 2002). The fact that a
layer corresponding to the coating was seen after frying indicated that dehydration
also took place on the coating. Absorption of oil on the surface of the fried product
occurs when samples are removed from the frying medium; oil that remains on the
233
piece surface enters into the product (Ufheil and Escher, 1996; Aguilera and Gloria,
1997; Aguilera and Gloria-Hernandez, 2000; Mellema, 2003). According to these
authors, oil does not invade the sample itself, so no oil uptake occurs during frying.
Conditions at which potato slices are removed from the frying oil seem decisive for
the uptake of oil; this is related to adhesion of oil to the surface and draining
phenomena. Two main mechanisms are proposed to explain oil uptake: condensation
and capillary mechanisms; in both cases, oil penetrates through the pores to the
inside the product (Mellema, 2003). Thermogelling of the coating could lead to a
stronger barrier that would limit oil uptake after that frying process.
coating
Fig. 12.2: SEM Micrograph of fried dough disc coated with 1% methylcellulose (MC) with
0.75% sorbitol. Magnification: 100μm between marks.
The breaking force of samples was measured by puncture test using a texture
analyzer TA.XT2i – Stable Micro Systems (Haslemere, Surrey, UK) with a 5 kg cell.
Samples were punctured with a cylindrical plunger (2mm diameter) at 0.5mm/sec.
Maximum force at rupture was determined from the force-deformation curves. At
least 10 samples were measured in each assay. Samples were allowed to reach room
temperature before performing the tests.
Table 12.4 shows breaking force results for dough discs and potato strips with
and without coatings. Coating did not significantly (P>0.05) modify texture of fried
products. Instrumental results of breaking force agree with sensory analysis data.
Similar results were obtained by Rayner et al. (2000), working on deep-fat fried
potato discs coated with plasticized soy protein films. Besides, Funamiet al. (1999)
working on doughnuts, found that MC addition had no effect on breaking stress of
the samples.
After frying, dough discs showed a volume increase that was quantified by the
linseed method. Samples coated with MC and sorbitol showed a significantly
(P<0.05) lower volume increase than uncoated samples. Samples coated with MC
solutions plasticized with 0.5% sorbitol showed a higher density value (0.95 ± 0.05
g/mL) than the uncoated samples (0.70 ± 0.05 g/mL). With regard to volume,
samples coated with MC without plasticizer did not differ significantly (P>0.05) than
those of uncoated ones. This could be attributed to the more elastic layer of coating,
which limits the development of the dough network (Normen et al., 1998).
234
Color of potato strips did not show significant differences (P>0.05) between
uncoated samples and those with unplasticized coatings (Table 12.4). However, for
dough discs, significant differences (P<0.05) in lightness and chromaticity a*
parameter were found between the uncoated samples and those with unplasticized
coatings. In any case, all samples were accepted by the sensory panel. Sorbitol
addition showed a highly significant (P<0.01) effect on chromaticity parameters a*
and b* for both potato and dough fried samples. In the case of lightness, the presence
of sorbitol was significant (P<0.01) only for potato strips (Table 12.4). Samples
coated with MC and sorbitol showed a lighter color which could be associated to a
lower cooking time, even though all samples were cooked for the same time period.
The panelists did not detect flavor or texture differences between coated and control
samples.
Table 12.4: Instrumental texture and color parameters of fried dough discs and potato strips.
Sample Formulation Breaking Surface color (Hunter units)
force (N) L a* b*
Dough Control 6.69 ± 2.971 54.83 ± 3.85 -2.11 ± 2.54 33.67 ± 2.33
discs 1% MC2 4.81 ± 1.53 61.68 ± 3.34 -7.15 ± 1.85 31.62 ± 2.31
1% MC + 0.75% S3 5.33 ± 1.58 62.58 ± 1.62 -10.03 ± 0.62 27.77 ± 1.78
Potato Control 2.57 ± 0.64 51.09 ± 3.62 6.21 ± 2.39 36.22 ± 1.68
strips 1% MC 2.77 ± 0.44 52.73 ± 4.08 4.32 ± 2.58 34.92 ± 1.86
1% MC + 0.5% S 2.66 ± 0.30 46.91 ± 3.58 8.86 ± 1.78 33.29 ± 3.29
1 Value ± standard deviation
2 MC = methylcellulose
3 S = sorbitol.
French fries and fried dough discs coated with the selected formulations were
evaluated by a non-trained sensory panel of 22 members. Two triangle tests were
performed to determine if consumers could distinguish between coated and uncoated
samples. Each sample was randomly numbered and presented to the panel member
with the instructions to pick the two equal samples out of three. A hedonic panel was
also performed with the same panelists, who evaluated texture, color, flavor and
overall characteristics. A hedonic 4-point scale was used, 1= dislike, 2= acceptable,
3= like and 4= like very much.
The triangular test showed no significant differences (P>0.05) between samples
coated with MC and sorbitol and the uncoated controls for both fried products. Panelists
did not reject any sample; in all cases, scores were higher than 2 (acceptable). Texture
was the lowest scored parameter (2.2) with regard to color and flavor; however texture
scores of coated samples were similar to control ones. Flavor scores of coated samples
were the highest and similar to the control values, these results were of great
significance because no off flavors were detected considering the presence of the
coating. Mallikarjunan et al. (1997) reported that HPMC coatings improved sensory
attributes of fried chicken nuggets and marinated chicken strips, even more when
HPMC was incorporated into the breading mix.
In conclusion, methylcellulose (MC) and hydroxypropylmethylcellulose
(HPMC) were used in coating formulations to reduce oil uptake in deep-fat frying
potato strips and dough discs. MC coatings were more effective in reducing oil
uptake than HPMC coatings, and the cellulose derivative was selected for coating
formulations. The addition of a plasticizer (sorbitol) was necessary to maintain
coating integrity and improve barrier properties. The most effective coating
235
formulations were 1% MC and 0.75% sorbitol for dough discs and 1% MC and 0.5%
sorbitol for potato strips. For these formulations, oil uptake reduction was 35.2% and
40.6% for dough discs and potato slices, respectively, compared to the uncoated
samples, and the increase in water content of the same products was 25.7% and
6.3%, respectively.
All samples were accepted by the panelists, although color differences between
coated and uncoated samples were detected by instrumental analysis. Coating
application did not modify texture characteristics of fried samples. This is a
favorable result since our goal was to incorporate the coating to decrease oil uptake
without having a significant impact on the sensory characteristics of the fried
products.
12.4 Incorporation of Natural Antioxidants

on Carboxy-methylcellulose Based Edible Films
Traditionally, the hot water extract of leaves from a plant native to the south of
Chile, known as “murta” or “murtilla” (Ugni molinae Turcz), is highly valued as a
folk medicine by the indigenous Chilean Mapuche for its physiological benefits,
mainly for its kidney-protection effect (Montenegro, 2002). Due to this interest, the
germoplasm of this plant has been collected and characterised (Seguel et al., 2000).
Methanolic, ethanolic and water extracts of the leaves were found to be high in
polyphenols content and antioxidant abilities, as measured through DPPH and
TBARS (Rubilar et al., 2006). Finally, through reverse phase HPLC-MS analysis of
the aqueous extracts, it was shown that the main difference between the polyphenol
content of the two ecotypes studied, Pumalal Soloyo Grande (SG) and Soloyo Chico
(SC), is the flavonol derivatives myricetin dirhamnoside, myricetin glucoside or
myricetin galactoside and quercetin dirhamnoside (Bifani et al., 2006).
The use of edible films prepared with the aqueous extracts of murta leaves is an
interesting alterative to give additional antioxidant properties to the films and
therefore improve the coatings. But the presence of polyphenols may affect the
polymeric matrix, changing some physical properties of the films; there is a need to
study their effect on rheological characteristics of the film forming solutions and on
physical characteristics of the edible films.
12.4.1 Films Production

Using 2 g L-1 of carboxymethylcellulose as structural basis of the film forming
solutions, the addition of the leaves extracts of each murta ecotype were done
without dilution (SC-100 and SG-100), mixing 50% extract with 50% water (SC-50
and SG-50), and 100% distilled water (Control); 0.4 g L-1 of glycerol and 0.5 g L-1 of
sunflower oil were always added as plasticizers (Table 12.5).
The CMC solutions behave as pseudoplastic fluids and can be described through
the Power Law Model. SG samples always show a higher resistance to the
deformation than the SC samples. For both ecotypes, the consistency coefficient K
decrease when the extract concentration in the sample was increased, being one order
of magnitude higher for SG than for SC samples. The flow behavior index n for the
236
SG samples was always higher than for the SC ecotype; it increases with the
concentration, for both samples, but it is always lower than 1, corroborating the
pseudoplastic behavior of the CMC solution (Vais et al. 2002). Both the shear
storage modulus G’ (or elastic modulus) and the shear loss modulus G” (or viscous
modulus) in the lineal range of viscoelasticity were performed to evaluate the
viscoelastic behavior of the filmogenic solutions.
Table 12.5. Composition of forming solution of carboxymethylcellulose-based edible film

with murta (Ugni molinae Turcz) leave extract (Bifani et al. 2006).
Extract con-
Sample centration CMC Glycerol Sun flower Water Soloyo Chico SoloyoGrande
[g/100g] [g] [mL] oil [mL] [mL] extract [mL] extract [mL]
SC50 48.57 2.000 0.40 0.50 0.00 100.00 0.00
SC100 97.18 2.000 0.40 0.50 50.00 50.00 0.00
SG50 48.57 2.000 0.40 0.50 0.00 0.00 100.00
SG100 97.18 2.000 0.40 0.50 50.00 0.00 50.00
Control 0.00 2.000 0.40 0.50 100.00 0.00 0.00
The shear storage modulus (G’) and also the shear loss modulus were higher for
the SG samples than for the SC samples. In all of the analyzed conditions, the results
are in the so-called initial region of low frequency or diluted state, where G” is
higher than G’, indicating that the samples were more viscous than elastic. The
chains are not more imbricated on these frequencies, and therefore, the solution can
flow more easily. The gel point, where values of G’ and G” are the same, were
observed only for SG samples, with a displacement to the left; therefore the addition
of murta leaves extracts of SG let to higher molecular weight of the solution (De
Matos, 1997) (Table 12.6).
12.4.2 Films characteristics

Using dynamic mechanical analysis (DMA), viscoelastic properties of the films were
studied. The shear loss modulus G” was always smaller than the shear storage
modulus G’; this is a characteristic of physical gels, and means that gels with most of
the time behave as solids, instead behave as liquids (Paschoalick et al., 2003).
Water vapor permeability (WVP), measured by the method described in Mali et
al. (2002), was significantly lower (p≤ 0.05) for SG films than control and SC films,
without any significant difference (p>0.05) between WVP for control and SC films
(Table 12.7). Carbon dioxide (CO2) and oxygen (O2) permeabilities of the films were
assessed by the accumulation method (Garcia et al., 2000). Carbon dioxide
permeability of SG films is significantly higher (p≤ 0.05) than control and SC films;
oxygen permeability of SG films was significantly lower (p≤ 0.05) than SC films,
and both films showed significantly lower (p≤ 0.05) oxygen permeability than
control (Bifani et al. 2006).
237
Table 12.6. Rheological properties of film- forming solution of carboxymethylcellulose-
based edible film with murta (Ugni molinae Turcz) leave extract a
Extract concentration Consistency Flow behavior Elastic modulus Viscous modulus
Sample [g/100g] coefficient K Index n G’ [Pa] G” [Pa]
SC50 48.57 6.87 0.56 6.50 – 7.56 15.04 – 17.00
SC100 97.18 2.66 0.67 2.30 – 2.60 7.97 – 8.97
SG50 48.57 28.72 0.35 36.86 – 43.05 40.21 – 47.18
SG100 97.18 23.89 0.41 31.77 – 34.63 38.69 – 39.94

a
Means value of two replicates
In conclusion, the addition of aqueous murta leave extract with different

concentration of same polyphenols to CMC based film forming solution, not only
affects physical properties of this solutions but also affect physical properties of
formed films, in particular important physical properties as water vapor and gases
(CO2 and O2) permeability. It is necessary to know the behavior of CMC chains with
polyphenols present in the water extract of murta leaves that have different
distribution of components to predict physical properties of edible films. This
conclusion is not only valid for CMC based films added with murta extract, but for
the addition of any natural extract to any carbohydrate or protein-based films.
TABLE 12.7.- Water vapor and gases permeability of CMC based edible films with murta
leave extract (Bifani et al. 2006).
Water vapor permeability x CO2 permeability x 1010 O2 permeability x 1010
Extract con- 1011 [g m-1 s-1 Pa-1] [cm3 m-1 s-1 Pa-1] [cm3 m-1 s-1 Pa-1]
centration
Soloyo Soloyo Soloyo Soloyo Soloyo Soloyo
[g/100g]
Grande Chico Grande Chico Grande Chico
7.144 7.144 4.092 4.092 8.096 8.096
0 ± 0.130a ± 0.130 ± 0.095 ± 0.095 ± 0.077 ± 0.077
Aa b Aa Aa Aa Aa Aa
6.738 7.200 7.485 3.914 3.260 2.344
48.57 ± 0.032 ± 0.038 ± 0.002 ± 0.486 ± 0.035 ± 0.011
Bb Aa Bb Aa Ab Cb
5.655 7.188 6.850 3.551 4.382 5.941
97.18 ± 0.049 ± 0.064 ± 0.007 ± 0.093 ± 0.078 ± 0.141
Cc Aa Cc Aa Ac Bc
a
Means value ± standard deviations of two replicates
b
For each property, means in same row with different capital letters are significantly different (p≤ 0.05);
means in same column with different small letters are significantly different (p≤ 0.05)
Acknowledgements To CYTED (Project XI.20). The authors acknowledge the

financial support from Agencia Nacional de Promoción Científica y Tecnológica
(ANPCyT), Universidad de Buenos Aires (UBA), Universidad de La Plata (UNLP),
Consejo Nacional de Investigaciones Científicas y Técnicas de la República
Argentina (CONICET) and Fundación Antorchas; Universidad de La Frontera
(Temuco, Chile). To Fapesp and CNPq (Brazil).
238
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UNIVERSIDAD DE LA FRONTERA

Facultad de Ingeniería, Ciencias y Administración

Development of edible coatings and films based on

Andrea Cristina Silva Weiss

Reviewers: Dr. Carmen Gómez Guillén

Professor Dr. Carolina Shene de Vidts

Professor Dr. Ma Cristina Diez Jerez

Professor Dr. Erick Scheuermann Salinas

Dr. Paulo José do Amaral Sobral

Dr. Francisco Matus Baeza Prof. Mg. Valerio Bifani Cosentini

Dra. Carmen Gómez Guillén

Dirección de Postgrado Prof. Dra. Carolina Shene de Vidts

Prof. Dra. Ma Cristina Diez Jerez

Prof. Dr. Erick Sheuermann Salinas

Dr. Paulo José do Amaral Sobral

El desarrollo de esta tesis fue posible principalmente gracias al financiamiento de la Comisión

Al Programa de Doctorado en Ciencias de Recursos Naturales y a la Dirección de Postgrado de la

Al proyecto INNOVA-CORFO 06CN12PAT-57 del profesor Valerio Bifani, que permitió

A la comisión FULBRIGHT por haberme otorgado la beca de pasantía doctoral, Fulbright

Agradezco a los miembros de la comisión evaluadora de tesis de la Universidad de La Frontera,

Quiero agradecer profundamente a mi familia, especialmente a mi papá, Benjamín Silva

ISI publications: Published

Other publications: Published

ISI publications: In preparation

Ultrasound-assisted extraction allows a higher recovery of phenolic compounds from murtilla

In this investigation, a viscosity between 3 and 11 Pa s at 25 °C proved to be adequate for

G' : Storage or elastic modulus [Pa] δ : Phase angle [ º ]

G'' : Loss or viscous modulus [Pa] γ& : Shear rate [ s-1]

G* : Complex modulus [Pa] η : Apparent viscosity [Pas]

K : Consistency index [Pa s] η* : Complex viscosity [Pas]

n : Flow index [-] τ : Shear stress [Pa ]

tg : Gel point time [min] ω : Angular frequency [rad/s]

1.1. General introduction

Leaf source Scientific name TPC 1 I.O.AS.A 2 IC 50 % 3 IC 50 % 3

Figure 1.4 Murta or murtilla (Ugni molinae Turcz) shrub,

Figure 1.5 Chemical structures of some polyphenols found in aqueous extract

Phenolic compounds Formule m/z RT (min)

Extraction solvent: 1 ethanol : water 1:1 (v/v) (García, 2007), 2

Rate, Time F (kHz), A (%),

• The efficiency of the phenolic compounds recovery by ultrasound-assisted extraction (UAE)

• Polyphenol-rich extract from murtilla leaves (PEML), incorporated in biopolymer matrices

1.3 General objective

1.4 Specific objectives

• To determine the parameter combination of ultrasound-assisted extraction (UAE); ultrasonic

• To evaluate the changes on the physical stability of film-forming solutions as well as

diagrams, mechanical and permeation properties. Carbohydrate Polymers, 37(4), 371-382.

Research and Technology, 228(3), 467-475.

A. Silva‒Weissa, ∗, V. Bifanib, M. Ihlb, P.J.A. Sobralc, M.C. Gómez‒Guillénd

Keywords: Biopolymers, Bioactive compounds, Plant-based products, Vitamins, Interactions

2.2 Edible films and coatings: concept, formulation and requirements

2.3 Interactions among additives, film and food

reveals the importance of electrostatic interactions, in which negatively charged chain

2.4.1 Synthetic versus natural antioxidants

2.4.2 Antioxidant mechanisms to prevent lipid oxidation

2.4.3 Antioxidant plant extract and bioactive compounds as antimicrobial agents

2.5.1 Browning mechanism

Figure 2.4 General scheme of browning reactions in foods

2.5.2 Browning in fruits and vegetables

2.5.3 Browning inhibition

2.5.4 Ascorbic acid and acidulants as inhibitors of browning

2.5.5 Polyphenols: inhibitors of browning?

diffusion of oxygen through membranes or films by diminishing their permeability (Pokomý,

2.5.6 Packaging, films or coating applications to prevent browning