Chitosan/whey protein lm as active coating to extend Ricotta cheese shelf-life

Prospero Di Pierro
a
, Angela Sorrentino
a
, Loredana Mariniello
a
, Concetta Valeria L. Giosafatto
a, b
,
Raffaele Porta
a,
*
a
Dipartimento di Scienza degli Alimenti, Universit di Napoli Federico II, Parco Gussone, 80055 Portici, Napoli, Italy
b
Institute of Food Research, Norwich Research Park, Colney, NR4 7UA Norwich, UK
a r t i c l e i n f o
Article history:
Received 4 August 2010
Received in revised form
16 November 2010
Accepted 23 November 2010
Keywords:
Ricotta cheese
Shelf-life
Edible lm coating
Chitosan
Whey protein
a b s t r a c t
Shelf-life extension of Ricotta cheese coated with a chitosan/whey protein edible lm and stored under
modied atmosphere at 4

C was evaluated. The chitosan/whey proteinlmhad 35%and 21%lower oxygen

and carbon dioxide permeability, respectively, and about three times higher water vapor permeability than
lm prepared with chitosan alone. Over a 30-day storage period, no differences in the pH of control and
coated Ricotta cheeses were observed. While the titratable acidity of the control increased linearly during
the rst two weeks and remained constant for the rest of the storage period, the corresponding values for
coated Ricotta cheese did not change signicantly during the rst 21 days and reached the acidity level
(0.34 0.02 milliequivalent/100 g of analyzed sample) of the control only on day 30. The viable numbers of
lactic acid bacteria and mesophilic and psychrotrophic microorganisms were signicantly lower (p <0.05)
in the chitosan/whey protein coated cheese, compared to the control, at each storage time. Our ndings
suggest a potential utility of chitosan/whey protein coatings to extend fresh dairy product shelf-life.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Ricotta cheese is an unripened, creamy dairy product obtained
by heat-induced coagulation of whey protein (WP). It is highly
perishable and has a limited shelf-life even under refrigeration due
to textural changes and mold growth. Recently, modied atmo-
sphere packaging (MAP) has beenproposed for packaging of Ricotta
(Del Nobile, Conte, Incoronato, & Panza, 2009). The current study
investigated if an edible chitosan/whey protein (CWP) lm coating
would be able to delay the appearance of an acid taste and
microbial growth of Ricotta cheese packaged under MAP.
Chitosan [poly-a-(1-4)-2-amino-2-deoxy-D-glucose] is a hydro-
philic biopolymer obtained by alkaline N-deacetylation of chitin
(Kurita, 2001) that possesses broad-spectrum antimicrobial prop-
erties against fungi, bacteria and viruses (Coma et al., 2002; Liu,
Guan, Yang, Li, & Yao, 2001; Rabea, Badawy, Stevens, Smagghe, &
Steurbaut, 2003; Rhoades & Roller, 2000). Approval of chitosan as
food contact material in Europe is pending (Fernandez, Cava, Ocio,
& Lagaron, 2008). Chitosan exhibits its antimicrobial and other
desirable properties, such as decreased transpiration losses (El
Ghaouth, Arul, & Pannampalam, 1991; Jiang & Li, 2001) and
delayed ripening of fruits and vegetables (El Ghaouth,
Pannampalam, Castaigne, & Arul, 1992; Jiang & Li, 2001), in chito-
san-only lms (Butler, Vergano, Testin, Bunn, & Wiles, 1996; Chen &
Hwa, 1996; Kittur, Kumar, & Tharanathan, 1998; Kumar, Bristow,
Smith, & Payne, 2000) and in combination with other polymers
such as pectin (Hoagland & Parris, 1996), methylcellulose (Chen,
Yeh, & Chiang, 1996) or proteins (Jia, Fang, & Yao, 2009; Sztuka &
Ko1odziejska, 2008). Recently, we reported the preparation of
both chitosan/WP (CWP) (Di Pierro, Chico, Villalonga, Mariniello,
Damiao, Masi et al., 2006) and chitosan/ovalbumin lms (Di
Pierro et al., 2007). These lms showed good visual characteris-
tics, and they are likely edible since they are easily degraded by
serine proteases.
In the present study, fresh Ricotta cheese was coated with
CWP lm and stored under 40% CO
2
/60% N
2
MAP conditions to
reduce microbial growth (Dermiki, Ntzimani, Badeka, Savvaidis, &
Kontominas, 2008). Cheese microbiological and physicochemical
changes throughout a 30-days storage period were monitored to
determine if the lms could lead to an extension of shelf-life.
2. Materials and methods
2.1. Materials
Chitosan, prepared as described by Baxter, Dillon, Taylor, and
Roberts (1992) with a degree of N-acetylation 9.0%, was a gift
Abbreviations: CWP, chitosan/whey protein; MAP, modied atmosphere pack-
aging; WP, whey protein.
* Corresponding author. Tel./fax: 39 0812539473.
E-mail address: raffaele.porta@unina.it (R. Porta).
Contents lists available at ScienceDirect
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doi:10.1016/j.lwt.2010.11.031
LWT - Food Science and Technology 44 (2011) 2324e2327
from Professor R. A. A. Muzzarelli (University of Ancona, Italy).
Spray-dried whey from bovine milk (product no. W1500, lot no.
81K0279), containing 11% w/w proteins (biuret) and 65% w/w
lactose, was purchased from Sigma Chemical Co. (St. Louis, MO).
Buffered Peptone Water (BPW), Plate Count Agar (PCA) and de
Man Rogosa Sharpe Agar (MRS) media for microbiological analyses
were from Merck (Darmstadt, Germany). All other chemicals were
analytical grade.
2.2. Preparation of CWP lm forming solution
A CWP lm forming solution, containing 0.8% w/v chitosan and
2.4% w/v lyophilized milk whey (11% WP), was prepared by mixing
of chitosan dissolved in 0.1 N HCl and lyophilized milk whey dis-
solved in distilled water under continuous stirring. The pH of the
chitosan-whey solution was adjusted to pH 5.0 with 0.1 N HCl, and
the solution was lter-sterilized through a Sterilcup ltration
system (0.45 mm cut off, Millipore, Bedford, MA, USA).
2.3. Film formation and characterization
Films were prepared by casting 32.5 mL of either chitosan (0.8%
w/v) or CWP (0.8% w/v chitosan and 2.4% w/v lyophilized milk
whey) solutions into polystyrene Petri dishes (60 15 mm). The
solutions were previously de-aerated under vacuum in order to
prevent small bubbles which could form pinholes in the nished
lms, and then transferred into Petri dishes to be dried at 50

C
overnight under air circulation. The lms were peeled from the
Petri dishes and stored at 20

C in a desiccator (50% RH). Film
thickness was measured using a micrometer model HO62 with
sensitivity of 2 mm (Metrocontrol Srl, Casoria (NA), Italy). Film
water vapor permeability (WVP) was evaluated by gravimetric test
(Di Pierro et al., 2010) according to ASTME96 (ASTM, 1993). Oxygen
and carbon dioxide barrier properties were examined at 25

C and
50% relative humidity by using a Multiperm Oxygen and Carbon
Dioxide Analyzer (ExtraSolution s.r.l., Pisa, Italy).
2.4. Ricotta cheese packaging
Ricotta cheese was obtained from a local dairy manufacturer
(Casa Madaio s.r.l., Castelcivita, Salerno, Italy) both in June and in
September 2009 and three different lots of production were sub-
jected to two different shelf-life experiments. One set of Ricotta
cheese samples, supplied within 4 h from their production in
a traditional polypropylene thermoformed colander (net weight
approximately 100 g), was dipped 30 s into the sterile CWP lm
forming solution. Excess solution was allowed to drain from the
surface of the samples for 30 s before they were placed into plastic
trays and packaged under modied atmosphere. Both dipped and
undipped (control) cheeses were placed into plastic trays and
packaged under modied atmosphere (40% CO
2
/60% N
2
mixture),
as described by Dermiki et al. (Dermiki, Ntzirnani, Badeka,
Savvaidis & Kontominas, 2008), by using an OS 600 INOX VGP
(Ormad macchine s.n.c., Terlizzi, Bari, Italy) and nally stored at
4 0.5

C. After 7, 14, 21 and 30 days of storage, duplicate samples
(5e10 g) from both CWP-coated and uncoated (control) Ricotta
cheeses were taken with pre-sterilized cork-borers.
2.5. pH measurement
Samples of Ricotta cheese (10 g each) were homogenized, at the
maximumspeedfor 5 min, in100 mL of distilledwater withanUltra-
Turrax T8 homogenizer (IKA-Werke GmbH, Staufen, Germany). After
stirring for 15 min, the homogenates were centrifuged at 3000 g
for 10 min andthe obtained supernatants were ltered through both
cotton lint and paper lter.
Measurement of pH was carried out using a digital pH meter,
model 211 (Hanna instruments, PBI International), according to
AOAC procedures (AOAC, 1990).
2.6. Titratable acidity
Samples of 10 g of Ricotta cheese were added to 50 mL of
distilled water and homogenized for 5 min at the maximum speed
with an Ultra-Turrax T8 homogenizer. The homogenates were rst
heated, under stirring, until a temperature of 40

C was reached
and then diluted to the nal volume of 150 mL with distilled
water. After centrifugation at 3000 g for 10 min, the superna-
tants were ltered through both cotton lint and paper lter. Then,
5 drops of phenolphthalein (1% in ethanol) were added to 25 mL
of ltered supernatant and the titratable acidity (TA) was deter-
mined by addition of 0.1 N NaOH until the solution became pink.
The TA, expressed as milliequivalent/100 g, was calculated as
follow:
TA a b 100=c
where a and b correspond to the concentration and the volume of
titrant solution, respectively, and c refers to the grams of analyzed
sample.
2.7. Microbiological analysis
Samples of Ricotta cheese (5 g each) were aseptically transferred
into sterile plastic falcon tubes and homogenized in 45 mL of 0.1%
(w/v) sterile BPW for 5 min, at room temperature and at the
maximum speed, by an Ultra-Turrax T8 homogenizer. The volume
was adjusted to 50 mL and then serial decimal dilutions, prepared
with 0.1% (w/v) sterile BPW, were inoculated in duplicate on growth
media for the estimation of aerobic plate counts (APC). The micro-
biological analyses were carried out according to the pour plate
method (APHA, 1992).
Both mesophilic and psychrotrophic microorganisms were
enumerated on PCA plates which were separately incubated under
different experimental conditions (at 30

C for 48 h for the meso-
philic microora count; at 12

C for 4e5 days for the psychrotrophic

determination). Lactic acid bacteria were determined on MRS
plates according to the pour plate method, with overlay, after
incubation at 30

C for 2e3 days.
2.8. Sensory evaluation
A simple, unstructured sensory evaluation was performed by
seven trained individuals every time the samples were submitted
to microbiological analyses. Colour, avour, odour and texture were
the selected parameters. The tasters evaluated the different
parameters by using a hedonistic scale from 0 to 4 (0 unaccept-
able; 1 poor; 2 fair; 3 good; 4 very good).
Table 1
Barrier properties of lms obtained by casting chitosan or chitosan/whey protein
solution.
Film Thickness
(mm)
Permeability
(g mm m
2
day
2
kPa
2
)
O
2
CO
2
Water vapor
Chitosan 55.3 0.4 32.8 0.7 22.5 0.9 0.4 0.3
Chitosan/whey protein 61.5 0.3 21.3 0.9 17.7 1.2 1.3 0.2
The results are expressed as means of ten samples SD. Further experimental
details are given in the text.
P. Di Pierro et al. / LWT - Food Science and Technology 44 (2011) 2324e2327 2325
2.9. Statistical analysis
JMP software 5.0 (SAS Campus Drive, Building S, Cary, NC, USA)
was used for all statistical evaluations. Data were subjected to
analysis of variance and statistical differences at a given sampling
time were determined using the TukeyeKramer HSD test. Differ-
ences were considered to be signicant at p < 0.05.
3. Results and discussion
We hypothesized that CWP lm would be useful as coating
material for Ricotta cheese since it could combine the antimicrobial
activity of chitosan with desirable oxygen, carbon dioxide and
water vapor permeability (Table 1). The coating would allowfor the
movement of water vapor across the lm, thus preventing water
condensation and, as a consequence, microbial spoilage. The pH
values of control and CWP-coated Ricotta cheeses during storage
are shown in Fig. 1. The pH values decreased, after 7 days of storage
likely because of lactic acid synthesis by Lactobacillus spp. (Dermiki
et al., 2008; Whitley, Muir, & Waites, 2000) and acidic amino acids
and free fatty acids formation due to proteolysis and lipolysis. In
addition, dissolution of some of the CO
2
in the modied atmo-
sphere (approximately 15% during the rst 5 days of storage) might
have contributed to the pH decrease (Daniels, Krishnamurthi, &
Rizvi, 1985; Farber, 1991; Gonzalez-Fandos, Sanz, & Olarte, 2000;
Olarte, Gonzalez-Fandos, Gimenez, Sanz, & Portu, 2002), but it
also could have provided some buffering effect (Salaun, Mietton, &
Gaucheron, 2005). The pHvalues remained relatively constant until
day 30 (Fig. 1).
The TA showed a different trend in CWP-coated than in
uncoated cheese (Fig. 2). In the control sample, it increased linearly
during the rst two weeks and then remained stable, whereas it did
not change signicantly in CWP-coated cheese until day 21 and
then reached the same level as measured for the control sample on
day 30. The increase in TA was likely caused by organic acid
production primarily by lactic acid producing bacteria which
increased during storage (Fig. 3-C). The CWP-coated cheeses
showed only a slight increase in viable lactic acid bacteria during
the rst three weeks which could explain the constant TA values
6.2
6.3
6.4
6.5
6.6
6.7
6.8
0 5 10 15 20 25 30
Storage time (days)
p
H
Fig. 1. Effect of CWP coating on the evolution of pH during Ricotta cheese storage at
4

C under modied atmosphere (40% CO
2
/60% N
2
). C, uncoated samples (control);
B, CWP-coated samples. Data are the means SD of two different experiments run on
different occasions (June and September, see Materials and methods).
0.20
0.25
0.30
0.35
0.40
0 5 10 15 20 25 30
Storage time (days)
T
A

(
m
e
q
/
1
0
0
g
)
Fig. 2. Effect of CWP coating on titratable acidity (TA) during Ricotta cheese storage at
4

C under modied atmosphere (40% CO
2
/60% N
2
). C, uncoated samples (control);
B, CWP-coated samples. Data are the means SD of two different experiments run on
different occasions (June and September, see Materials and methods).
)
g
/
u
f
c
g
o
l
(
s
m
s
i
n
a
g
r
o
o
r
c
i
M
B
C
Storage time (days)
5.0
5.5
6.0
6.5
7.0
7.5
8.0
0 5 10 15 20 25 30
A
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
0 5 10 15 20 25 30
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
0 5 10 15 20 25 30
Fig. 3. Effect of CWP coating on the development of mesophilic (A), psychrotrophic (B)
and lactic acid (C) bacteria during Ricotta cheese storage at 4

C under modied
atmosphere (40% CO
2
/60% N
2
). C, uncoated samples (control); B, CWP-coated
samples. Data are the means SD of two different experiments run on different
occasions (June and September, see Materials and methods).
P. Di Pierro et al. / LWT - Food Science and Technology 44 (2011) 2324e2327 2326
during the rst 21 days (Fig. 2). As the number of lactic acid bacteria
increased (Fig. 3-C), the TA also increased (Fig. 2). In both samples
maximum TA was observed when the lactic acid bacteria were in
the range of 5e6 log cfu/g and this occurred at day 14 for the
control and at day 30 for the CWP-coated cheese.
The viable numbers of mesophilic and psychrotrophic micro-
organisms were signicantly lower (p < 0.05) in CWP-coated than
in control samples (Fig. 3-A and -B). In fact, the microbiological
limit of acceptability, 7 log cfu/g (ICMSF, 1986), was reached in
control samples within 7 days for mesophilic bacteria (Fig. 3-A),
between days 7 and 14 for psychrotrophs (Fig. 3-B), and between
days 14 and 21 for lactic acid bacteria (Fig. 3-C). In contrast, the limit
was never reached in CWP-coated Ricotta cheeses. Thus, CWP lm
might be an effective coating to extend Ricotta cheese shelf-life.
No differences in visual appearance, texture, avor and odor
between uncoated and CWP lm-coated Ricotta cheese samples
were detected during sensory evaluation (data not shown).
Furthermore, CWP-coated Ricotta cheese maintained its texture
better than the control cheese.
4. Conclusions
Coating of Ricotta surface with a CWP edible lm reduced
growth of microbial contaminants and extended the shelf-life of
the product packed under modied atmosphere. The coating
delayed the development of undesirable acidity, better maintained
the texture and did not seem to modify sensory characteristics. It is
possible that the benets derived fromthe biopolymers can also be
realized with other dairy products.
Acknowledgements
The authors would like to thank Mrs. Maria Fenderico for her
skilful technical assistance. This research was supported by a grant
to R.P. from POR 2000e2006 Regione Campania Asse prioritario di
riferimento 3 e Risorse Umane- Misura 3.16 Promozione della
Ricerca e del Trasferimento Tecnologico nei settori connessi alla
crescita ed allo sviluppo sostenibile del sistema Campania.
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