Multilizer PDF Translator Free version - translation is limited to ~ 3 pages per translation.

Manuscrito aceptado

Efecto de la deshidratación osmótica de las aceitunas como tratamiento de prefermentación y sustitución

parcial del cloruro de sodio por glutamato monosódico en el perfil de fermentación de las aceitunas
negras naturales Kalamata

Stamatoula Bonatsou, Vasilis Iliopoulos, Athanasios Mallouchos, Eleni Gogou,

Vasiliki Oikonomopoulou, Magdalini Krokida, Petros Taoukis, Efstathios Z. Panagou
Pii: S0740-0020(16)30729-8
Doi: 10.1016/j.fm.2016.11.001
Referencia: YFMIC 2662

Para aparecer en: Microbiología alimentaria

Fecha de recepción: 4 de septiembre de 2016

Fecha revisada: 28 de octubre de 2016

Fecha aceptada: 1 de noviembre de 2016

Por favor, cite este artículo como: Bonatsou, S., Iliopoulos, V., Mallouchos, A., Gogou, E., Oikonomopoulou, V., Krokida, M.,
Taoukis, P., Panagou, E.Z., Efecto de la deshidratación osmótica de las aceitunas como tratamiento de prefermentación y
sustitución parcial de cloruro de sodio por monosodium glutamato en el perfil de fermentación de aceitunas negras naturales de
Kalamata,
Microbiología alimentaria (2016), doi: 10.1016/j.fm.2016.11.001.

Este es un archivo PDF de un manuscrito sin editar que ha sido aceptado para su publicación. Como un servicio para
nuestros clientes estamos proporcionando esta versión temprana del manuscrito. El manuscrito se someterá a la edición,
tipografía y revisión de la prueba resultante antes de que se publique en su forma final. Tenga en cuenta que durante el proceso
de producción se pueden descubrir errores que podrían afectar al contenido, y todas las exenciones de responsabilidad legales
que se aplican a la revista pertenecen.

Multilizer PDF Translator Free version - translation is limited to ~ 3 pages per translation.
Multilizer PDF Translator Free version - translation is limited to ~ 3 pages per translation.

MANUSCRIPT ACEPTADO

1 Efecto de la deshidratacin osmtica de las aceitunas como tratamiento pre-fermentacin y

sustitucin parcial del cloruro de sodio por glutamato monosdico en el 2

perfil de fermentacin de las aceitunas negras naturales Kalamata 3

T
5 Stamatoula Bonatsou Un
, Vasilis Iliopoulos ,Un
Athanasios Mallouchos , BEleni Gogou , Vasiliki
C

ip
6 Oikonomopoulou D, Magdalini Krokida ,DPetros Taoukis , Efstathios
C
Z. Panagou a,*

Cr
7

Un
8 Laboratorio de Microbiologa y Biotecnologa de Alimentos, Departamento de Ciencia de los Alimentos y

9
us
Nutricin, Universidad Agrcola de Atenas, Iera Odos 75, 11855 Atenas, Grecia
an
B Laboratorio
10 de Qumica y AnÆlisis de Alimentos, Departamento de Ciencia de los Alimentos y Nutricin Humana,

11 Universidad Agrcola de Atenas, Iera Odos 75, 11855 Atenas, Grecia

M

C
12 Laboratorio de Qumica y Tecnologa alimentaria, Escuela de Ingeniera Qumica,
ed

13 Universidad TØcnica de Atenas, Campus Zografou, 5 Iroon Polytechneiou, Atenas, 15780, Grecia
PT

D
14 Laboratorio de AnÆlisis y Diseæo de Procesos, Escuela de Ingeniera Qumica,

15 Universidad de Atenas, Campus Zografou, 5 Iroon Polytechneiou, Atenas, 15780, Grecia

CE

16

17
AC

18

19

20

21 * Autor correspondiente: stathispanagou@aua.gr (E.Z. Panagou)

Multilizer PDF Translator Free version - translation is limited to ~ 3 pages per translation.
Multilizer PDF Translator Free version - translation is limited to ~ 3 pages per translation.

MANUSCRIPT ACEPTADO
22 Abstracto

23 Este estudio examinó el efecto de la deshidratación osmótica de las aceitunas negras naturales de Kalamata

24 tratamiento de prefermentación en combinación con sustitución parcial de NaCl por monosodio

25 glutamato (MSG) en el perfil de fermentación de las aceitunas. Se llevó a cabo la deshidratación osmótica

26 sumergiendo las aceitunas en un 70% (p/p) de jarabe de glucosa durante la noche a temperatura ambiente. Más

T
27 en, tres mezclas diferentes de NaCl y MSG con/sin deshidratación osmótica previa de

ip
28 se investigaron las aceitunas, a saber: i) 6,65% NaCl 0,35% MSG (5% de sustitución), (ii) 6,30%

29 NaCl 0.70% MSG (10% sustitución), (iii) 5.95% NaCl 1.05% MSG (15% sustitución),

Cr
30 y (iv) 7% NaCl sin deshidratación osmótica (tratamiento de control). Cambios en el microbiano

31 (bacterias del ácido láctico [LAB], levaduras, Enterobacteriaceae ), pH, acidez valorable,

32 us
ácidos orgánicos, azœcares y compuestos volátiles en la salmuera se analizaron durante un período de 4
an
33 Meses. El producto final fue sometido a análisis sensoriales y al contenido de MSG en aceitunas

fue determinado. Los resultados demostraron que la deshidratación osmótica de las aceitunas antes de
M

34

35 a los vigorosos procesos de ácido láctico segœn lo indicado por los valores obtenidos de pH (3.7-4.1) y

36 acidez (0,7-0,8 %) independientemente de la cantidad de MSG utilizado. Sin embargo, en

ed

37 aceitunas deshidratadas, el nivel de sustitución más alto de MSG dio lugar a un pH final (4,5) que fue
PT

38 más allá de las especificaciones para este tipo de aceitunas. MSG se degradó en las salmueras siendo casi

39 completamente convertido en ácido aminobutírico (GABA) al final de la fermentación. Por œltimo, el

CE

40 evaluación sensorial de aceitunas fermentadas con/sin deshidratación osmótica y a todos los niveles de
AC

41 MSG no mostró ninguna desviación en comparación con el tratamiento de control.

42

43 Palabras clave: aceitunas negras, fermentacin, glutamato monosdico, deshidratacin osmtica

44

Multilizer PDF Translator Free version - translation is limited to ~ 3 pages per translation.
 ACCEPTED MANUSCRIPT
45 1. Introduction

46 Natural black olives in brine are a major trade preparation of table olives in the

47 international market together with Spanish-style green olives and Californian-style black

48 oxidized olives (Sánchez Gómez et al., 2006). According to their relevance in the

49 international market, Kalamata and Conservolea are the two most economically important

PT
50 varieties for the production of natural black olives in Greece. Based on a recent estimation of

RI
51 the International Olive Council (IOC, 2016), the production of table olives in Greece

52 amounted to 231,000 tons for the season 2014-2015 from which almost 50% was processed as

SC
53 natural black olives. It must be noted that Conservolea was the most wide spread variety

54 corresponding to at least 80-85% of annual Greek table olive production (Garrido-Fernández

55
U
et al., 1997). However, this trend has changed recently and the cultivation of Kalamata variety
AN
56 is rapidly expanding in the country due to the exceptional technological and sensory
M

57 characteristics of the final fermented product and the high demand in domestic and

58 international markets (DOEPEL, 2016). Greek-style processing involves direct brining of

D

59 olives in 8-10% salt, without any prior chemical pre-treatment for debittering, where olives
TE

60 are subjected to spontaneous fermentation by a mixed microbiota of Gram negative bacteria,

61 LAB and yeasts (Brenes, 2004). The main advantages of the process are low energy
EP

62 consumption and simplicity, providing a product with minimum input of chemicals. However,

63 the long processing time (8-10 months) required for the fermentation to be completed could
C

64 be considered a major drawback (Garrido-Fernández et al., 1997).

AC

65 Monosodium glutamate (MSG) is the sodium salt of glutamic acid, a non-essential amino

66 acid for humans and other mammals, that is used as food additive to enhance the flavor of

67 many foods (Jinap and Hajeb, 2010). The taste of MSG is defined as savory or meaty,

68 whereas in Japan and other Asian countries it is called “umami” which has been defined as a

69 fifth basic taste of Asian foods separate from the four basic tastes of salty, sour, sweet and

3
 ACCEPTED MANUSCRIPT
70 bitter (Hajeb and Jinap, 2015). Apart from its unique taste properties, MSG can improve the

71 palatability of many foods when the salt content is reduced and thus it may increase the

72 acceptability of low-salt foods making easier for people to accept and maintain a low-salt diet

73 (Fernstrom, 2007). In the case of table olives, MSG has been employed in Spanish-style green

74 olives to provide a typical flavor known as “anchovy flavor” which is highly appreciated by

PT
75 consumers. The trade standard applying to table olives of the International Olive Council

RI
76 permits the use of MSG as flavor enhancer in table olive processing at a maximum level of 5

77 g kg-1 net weight (IOC, 2004). Despite its extended use in other foods, there are few research

SC
78 works on the application of MSG in table olives. Spanish researchers (Rejano Navarro and

79 Sánchez Gómez, 1996) studied the effect of MSG on the physicochemical characteristics and

80
U
sensory profile of green table olives from Spain, Greece, Israel and Turkey and reported an
AN
81 increase in pH values due to the addition of MSG, recommending a pasteurization treatment
M

82 to ensure product stability during packing and storage. In another work (De Castro et al.,

83 2014) the stability of MSG in Spanish-style green table olives and pickled cucumbers was
D

84 reported as a function of packing conditions and storage time. Different packing materials
TE

85 (glass container, plastic pouch), thermal treatment (pasteurization) and preservatives

86 (potassium sorbate) were investigated and as far as green olives is concerned the authors
EP

87 reported that pasteurization was necessary to stabilize packed olives and maintain the

88 physicochemical characteristics and MSG content. However, the use of MSG in natural black
C

89 olives has not been reported so far and it would be interesting to investigate the effect of MSG
AC

90 on the microbiological, physicochemical and sensory profile of this trade preparation.

91 Osmotic dehydration is a process based on the partial removal of the water by

92 submerging the plant tissue in a hypertonic solution. During osmotic processing, water flux

93 from the product into the osmotic solution is accompanied by osmotic solute transfer from the

94 solution into the product (Dermesonlouoglou et al., 2008). Other mass transfer process occurs,

4
 ACCEPTED MANUSCRIPT
95 such as leaching of small amounts of product-soluble compounds (sugars, acids, minerals and

96 vitamins) that may affect the sensory and nutritional characteristics of the product. Both the

97 concentration of solution and the nature of the solutes influence the extent and the rate of

98 water removal and solute impregnation (Beristain et al., 1990; Rastogi et al., 2000; Tedjo et

99 al., 2002). Substances used for osmotic dehydration are solutions of sugars, sodium chloride,

PT
100 sorbitol or other substances acceptable for the consumer allowing for the reduction of water

RI
101 activity of the dehydrated material (CiurzyDska et al., 2016). It has been reported that osmotic

102 dehydration pre-treatment decreases enzymatic activity with slight changes in product quality

SC
103 (Giraldo et al., 2003) and in the case of enzymatic browning it can retain or even improve

104 product color (Krokida et al., 2000; Giannakourou and Taoukis, 2003a,b). Moreover,

105
U
dehydration pre-freeze treatments could have a positive effect on the textural and nutritional
AN
106 characteristics of fruits and vegetables during storage (Talens et al., 2002; Giannakourou and
M

107 Taoukis, 2003a; Kucner et al., 2013; CiurzyDska et al., 2016). In the case of table olive

108 processing, green olives were subjected to osmotic dehydration using 70% (w/w) glucose
D

109 syrup as osmotic solution prior to brining (Kalomiri et al., 2015). Olives were subsequently
TE

110 fermented for 3 months in 8% (w/w) NaCl brine followed by packing in low-salt solutions

111 (3%, w/w, NaCl) and pasteurization. The authors reported that osmotic pre-treatment of olives
EP

112 resulted in the development of a final fermented product with enhanced sensory

113 characteristics while lowering the salt intake during fermentation.

C

114 The objective of the present work was to investigate the influence of osmotic dehydration
AC

115 as pre-fermentation treatment in Kalamata natural black olives in combination with partial

116 substitution of sodium chloride by monosodium glutamate in the brines on the

117 microbiological, physicochemical and sensory profile of olives during fermentation.

118

119 2. Materials and methods

5
 ACCEPTED MANUSCRIPT
120 2.1Olive samples and fermentation procedures

121 Kalamata table olives (180 kg) were harvested at their mature stage of ripeness (in

122 November) when the superficial color of the drupes was black with intermediate shades of

123 brown and violet which is appropriate for Greek style processing. Olives were supplied by

124 Konstantopoulos S.A. table olive industry (Katerini, Greece), subjected to quality control at

PT
125 the processor’s facilities and transported to the laboratory with minimal delay (within 24 h).

RI
126 On arrival, olives were divided into two lots. One lot was immersed directly in 20 L plastic

127 vessels containing 13 kg of olives and 8 L of freshly prepared brine. Sodium chloride, which

SC
128 is normally used in brine preparation, was partially substituted by monosodium glutamate

129 (MSG) in concentrations shown in Table 1. The second lot was placed in the same vessels and

130
U
subjected to osmotic dehydration as pre-fermentation treatment by immersing the olives in
AN
131 70% (w/w) glucose syrup (56° Brix) (C*Sweet M1521, FALCON SA, Greece) overnight at
M

132 room temperature. The ratio of olives to osmotic solution was 1:3. At the end of the process,

133 the osmotic solution was discarded and replaced by brine in which NaCl was partially
D

134 substituted by MSG in the same proportions as for non osmotically dehydrated olives (Table
TE

135 1). The details of the osmotic dehydration of Kalamata black olives have been reported

136 elsewhere (Gogou et al., 2014). All treatments were performed in duplicate and fermentation
EP

137 vessels were maintained at room temperature (ca. 20-22°C) for a period of 4 months (127

138 days). The processes were allowed to evolve spontaneously by the autochthonous microbiota
C

139 of olives according to the traditional Greek style processing (Panagou, 2016).
AC

140

141 2.2Microbiological analysis

142 Brine samples were analyzed at regular time intervals (18 sampling points per treatment)

143 to allow for the determination of the population dynamics of the main microbial groups

144 driving fermentation, namely LAB, yeasts, and Enterobacteriaceae. Specifically, brine

6
 ACCEPTED MANUSCRIPT
145 samples (1 mL) were aseptically transferred to 9 mL sterile ¼ Ringer’s solution (LabM, Bury,

146 UK). Decimal dilutions in the same Ringer’s solution were prepared and duplicate 1 or 0.1

147 mL samples of the appropriate dilutions were poured or spread on the following agar media:

148 de Man-Rogosa-Sharp medium (MRS; LabM, Bury, UK) for the enumeration of LAB,

149 supplemented with 0.05% (w/v) cycloheximide (Sigma, St. Luis, Mo.), overlaid with 10 mL

PT
150 of molten medium and incubated at 30ºC for 48-72 h; Rose Bengal Chloramphenicol agar

RI
151 (RBC; LabM, Bury, UK) for the enumeration of yeasts, incubated at 25 ºC for 72 h; Violet

152 Red Bile Glucose agar (VRBGA; Biolife, Milano, Italy) for the enumeration of

SC
153 Enterobacteriaceae counts, overlaid with 10 mL of molten medium and incubated at 37 ”C for

154 24 h. Results were expressed as log values of colony forming units per mL of brine (log

155 CFU/mL).
U
AN
156
M

157 2.3Physicochemical analysis and sensory assessment

158 During fermentation physicochemical analyses were performed in the brine to monitor
D

159 the changes in pH, titratable acidity, organic acids, sugars, and volatile compounds.
TE

160 Combined acidity was also measured in the brines at the end of fermentation. Specifically, pH

161 values were measured using a digital pH-meter (Russel Inc., Boston, MA). Titratable acidity
EP

162 and combined acidity were determined according to the protocols described by Garrido-

163 Fernández et al. (1997). Organic acids and sugars in the brine were analyzed by HPLC as
C

164 reported elsewhere (Bleve et al., 2015). The profile of volatile compounds was analyzed by
AC

165 Gas Chromatography following the static headspace technique (Montaño et al., 1990) with

166 slight modifications (Villegas Vergara et al., 2013). At the end of fermentation, olives from

167 the different processes were analyzed according to Populin et al. (2007) slightly modified to

168 determine the concentration of glutamic acid and ‡-aminobutyric acid (GABA) which is a

169 major end-product of MSG degradation in fermented vegetables (De Castro et al., 2014).

7
 ACCEPTED MANUSCRIPT
170 Specifically, 25 g of olive flesh were added in 50 ml distilled water, homogenized in an Ultra-

171 Turrax T-25 blender (IKA Werke, Staufen, Germany) at room temperature until obtaining a

172 slurry, and filtered through Whatman No. 41 paper. The extract was diluted 1/100 and filtered

173 through PVDF 0.22 m (pore size) filters before derivatization. Derivatives of o-

174 Phthaldialdehyde (OPA) were prepared by adding 80 L of a ready-to-use OPA reagent

PT
175 solution (Sigma-Aldrich) in 80 L aliquot of the diluted extract. HPLC determinations were

RI
176 performed using a C18 Spherisorb ODS2 (5.0 m, 250 x 4.6 mm i.d.; Waters Corporation,

177 Milford, MA) column held at 40°C. Mobile phase was composed of Potassium Phosphate

SC
178 Buffer (20 mM; pH 6.5-6.6) (Eluent A) and Acetonitrile/Methanol/Water (45/45/10 v/v/v)

179 (Eluent B). The gradient elution programme was held at 10% of B for 5 min, ramped at 26%

180
U
of B (24 min) and then at 60% of B (33 min) and held for 7 min with a flow rate of 1.2 ml
AN
181 min-1. The mobile phase returns to original conditions (10% of B) and held until the end of the
M

182 rum (60 min) so that the column is equilibrated for the next run. The HPLC system consisted

183 of a Jasco AS-2055Plus autosampler, a Jasco PU-980 pump, a Jasco LG-980-02 gradient
D

184 mixer, and a Jasco FP-2020Plus fluorescence detector (excitation wavelength 340 nm,
TE

185 emission wavelength 450 nm) (JASCO Inc., Easton, USA).

186 Sensory assessment was undertaken at the end of fermentation by a panel of 10 judges of
EP

187 the Laboratory of Microbiology and Biotechnology of Foods according to the method of

188 sensory analysis of table olives established by the International Olive Council (IOC, 2011).
C

189 Olive samples were presented in the standard tasting glasses recommended by the IOC
AC

190 method for sensory analysis, coded with a 3-digit random number. Olives were initially

191 assessed for the perception of negative sensations (abnormal fermentation, other defects), and

192 then for gustatory (bitter, salty, acid) and kinaesthetic (hardness, fibrousness, crispness)

193 sensations. Results were expressed as the median score provided by the members of the panel

194 together with the robust standard deviation using the special software of the IOC. Sensory

8
 ACCEPTED MANUSCRIPT
195 data were subjected to Kruskal-Wallis analysis of variance using Statgraphics Centurion XV

196 (Statpoint, Inc., Warrenton, Virginia) to define significant differences among the sensory

197 descriptors for the fermentation procedures (P < 0.05).

198

199 2.4Exploratory data analysis

PT
200 Hierarchical cluster analysis (HCA) was performed to explore the unsupervised

RI
201 discrimination of the different fermentation procedures based on the microbiological and

202 physicochemical profiles attained. The input matrix for the analysis consisted of the total

SC
203 areas under the growth/decline curves of LAB, yeasts, and enterobacteria, together with those

204 corresponding to the changes in the profile of organic acids, volatile compounds, sugars, pH,

205
U
and titratable acidity. The calculation of the area under curve for each variable was
AN
206 implemented by integration using the trapezoid method (Billo, 2007). It needs to be noted that
M

207 combined acidity (CA), monosodium glutamate (MSG) and ‡-aminobutyric acid (GABA)

208 concentrations were determined at the end of fermentation and hence only these values were
D

209 taken into account in the input matrix. Prior to analysis data were autoscaled (mean-centered
TE

210 and divided by the standard deviation of each variable) to avoid bias due to differences in

211 scale (van den Berg et al., 2006). HCA was performed based on Euclidean distance as
EP

212 similarity measure and Ward’s linkage as clustering algorithm using Metabo-Analyst 3.0

213 software (Xia et al., 2015) and the results were graphically illustrated as heatmap. Heatmap is
C

214 a two-dimensional visualization technique, initially used in bioinformatics, where data are
AC

215 displayed by colours. The heatmap also re-arranges the rows and columns of the data so that

216 similar rows and similar columns are grouped together with their similarity presented by a

217 dendrogram (Moon et al., 2009).

218

219 3. Results and discussion

9
 ACCEPTED MANUSCRIPT
220 3.1Changes in population dynamics during fermentation

221 The changes in population dynamics of LAB, yeasts, and enterobacteria in processes

222 where olives were treated with/without osmotic dehydration as pre-fermentation treatment

223 and immersed in brines with different levels of substitution of NaCl by MSG are illustrated in

224 Figures 1 and 2. In the control fermentation undertaken in 7% (w/v) NaCl, LAB were the

PT
225 dominant microbial group in population exceeding 6.0 log CFU/mL (Fig. 1a). Specifically, in

RI
226 the beginning of the process the counts of LAB were enumerated at 6.6 log CFU/mL

227 presenting an increase until day 13 reaching 8.0 log CFU/mL. From this time onwards, L‘’

SC
228 counts presented a decreasing trend until the end of the process. The second dominant

229 microbial group was yeasts, the population of which was maintained 4-5 log cycles below

230
U
LAB counts. Yeast growth reached a maximum level after about 30 days at 4.5 log CFU/mL
AN
231 followed by a decline thereafter. However, it needs to be noted that in the last two weeks of
M

232 fermentation, yeasts presented an increase at 3.1 log CFU/mL. This could be attributed to

233 lower competition by LAB towards the end of fermentation due to the decreasing population
D

234 of this microbial group. It has been reported that the interrelationship between LAB and
TE

235 yeasts plays an important role in fermented foods, as in these ecosystems they may compete

236 for the same substrates or synergistically promote the growth of each other (Viljoen, 2006).
EP

237 Finally, enterobacteria were counted at relatively high population of 5.0 log CFU/mL but

238 decreased rapidly until day 13 where they became undetectable in the brines.
C

239 The population dynamics of the same microbial groups in brines supplemented with
AC

240 MSG are illustrated in Fig. 1(b,c,d) for non-osmotically dehydrated olives and Fig. 2(a,b,c)

241 for osmotically dehydrated olives, respectively. In both cases it can be inferred that the

242 evolution of the microbiota was similar to the control fermentation. Specifically, LAB were

243 the dominant species with counts exceeding 7.0 log CFU/mL throughout the process,

244 presenting a decreasing trend after their maximum population was attained, whereas yeasts

10
 ACCEPTED MANUSCRIPT
245 were always maintained at lower populations being the second most important microbial

246 group. It needs to be noted that yeasts reached higher populations within the first 30 days of

247 fermentation in osmotically dehydrated olives (5.5 ± 0.5 CFU/mL on average) compared to

248 non-osmotically dehydrated ones (4.6 ± 0.2 log CFU/mL on average). There was however, a

249 distinct difference in the initial population of enterobacteria in olives subjected to osmotic

PT
250 dehydration prior to fermentation. Specifically, their counts were lower ranging from 3.0-3.5

RI
251 log CFU/mL compared to non-osmotically dehydrated olives where the initial counts were

252 above 5.0 log CFU/mL. This could be attributed to the lower water activity (aw) value of

SC
253 Kalamata black olives after osmotic dehydration (0.866 ± 0.005) that could have possibly

254 affected the survival of enterobacteria on the surface of olives prior to brining. It is known

255
U
that enterobacteria, as Gram negative microorganisms, are less tolerant to lower aw levels
AN
256 compared to Gram positive bacteria (e.g., LAB) (Adams and Moss, 2000), indicating a clear
M

257 benefit of osmotic dehydration as pre-fermentation treatment in reducing the risk of

258 developing unpleasant odors and flavors due to the activity of this microbial group
D

259 (Bevilacqua et al., 2015). Overall, the different levels of NaCl substitution by MSG in the
TE

260 brines combined with osmotic dehydration of the raw olives prior to brining resulted in

261 fermentation profiles that were typical for natural black olive processing as also reported by
EP

262 other researchers (Nisiotou et al., 2010; Bleve et al., 2014, 2015; Tufariello et al., 2015;

263 Grounta et al., 2016) at least as far as the evolution of the microbiota is concerned. At the
C

264 same time, the use of initial NaCl concentration below 8% (w/v) in all treatments resulted in a
AC

265 mixed fermentation by LAB and yeasts that favored the dominance of LAB from the onset of

266 fermentation (Tassou et al., 2002; Hurtado et al., 2012).

267

268 3.2Changes in the biochemical profile during fermentation and sensory assessment

11
 ACCEPTED MANUSCRIPT
269 The changes in pH and titratable acidity in the brines was typical of natural black olive

270 fermentation in all treatments assayed (Fig. 3a, b). Specifically, for non-osmotically

271 dehydrated olives, pH values presented a rapid decrease within the first 13 days of

272 fermentation reaching a plateau thereafter with the exception of the treatment with 15%

273 substitution of NaCl by MSG, where a gradual increase of pH was noticeable after day 71

PT
274 (Fig. 3a). At the end of fermentation, the lowest pH value was observed in the control

RI
275 treatment (3.73±0.09), whereas for the other treatments a gradual increase in pH values was

276 observed according to the substitution percentage of NaCl by MSG, namely 3.90 (±0.09),

SC
277 4.10 (±0.01), and 4.55 (±0.04) for 5, 10, and 15% NaCl substitution by MSG, respectively. It

278 needs to be noted that the last treatment does not meet the specifications of the trade standard

279
U
of the IOC (IOC, 2004) for table olives, as for natural fermentations the maximum limit of pH
AN
280 should be 4.3. For the other treatments, thermal processing (pasteurization) would be
M

281 recommended to ensure the safety and stability of the final product, in case where the increase

282 in pH values beyond the time scale of the experiment (127 days) is above the IOC limit for
D

283 this type of olives. The profile of acidity was also similar presenting a gradual increase until
TE

284 day 40, followed by a steady condition until the end of fermentation reaching final values of

285 0.76-0.81 g of lactic acid/100 mL of brine. An exception was noticeable for the treatment with
EP

286 15% substitution of NaCl by MSG in the brine where a gradual decrease of acidity was

287 observed after day 50, reaching a final value of 0.54 g of lactic acid/100 mL of brine. In the
C

288 case of black olives subjected to osmotic dehydration prior to brining, similar pH profiles
AC

289 were observed (Fig. 3b). Again, a gradual increase in the final pH values was recorded

290 according to the percentage of NaCl substitution by MSG, namely 3.66 (±0.08), 3.84 (±0.06),

291 and 4.07 (±0.04) for 5, 10, and 15% MSG substitution, respectively. However, a remarkable

292 difference was noticeable in the profile of titratable acidity compared to non-osmotically

293 dehydrated olives. Specifically, titratable acidity presented a gradual increase until day 105

12
 ACCEPTED MANUSCRIPT
294 reaching values up to 0.93-0.99 g of lactic acid/100 mL of brine for 5 and 10% NaCl

295 substitution by MSG, whereas for 15% MSG use the respective value of acidity was 0.86 g of

296 lactic acid/100 mL of brine. From this point onwards, a gradual decrease in acidity was

297 evident that was finally maintained between 0.71-0.80 g of lactic acid/100 mL of brine. The

298 decrease in acidity could be associated with the increase in yeast population that occurred

PT
299 after 100 days of fermentation (Fig. 2). This could be attributed to the depletion of

RI
300 fermentable substrates in the brine allowing yeasts to utilize other carbon sources, such as

301 organic acids and thereby decrease the values of titratable acidity (Garrido-Fernández et al.,

SC
302 1997; Viljoen, 2006). Decreased values of titratable acidity due to yeast activity have also

303 been reported by Spanish researchers during post-fermentation storage of Spanish-style green

304
U
Manzanilla olives (Rodríguez-Gómez et al., 2014, 2015). Another interesting observation is
AN
305 that the rapid pH decrease below the value of 4.5 within the first 13-14 days in all
M

306 fermentations coincided with the inhibition of enterobacteria in the brines within the same

307 time period (Figs. 1, 2), minimizing thus the risk of spoilage due to prolonged survival in the
D

308 brines (De Angelis et al., 2015).

TE

309 The changes in the concentration of organic acids with major presence in the brines are

310 illustrated in Fig. 4. Lactic acid was the main metabolic product with considerable presence in
EP

311 the brines as determined by HPLC. It was detected in higher concentrations in treatments in

312 which olives were subjected to osmotic dehydration prior to fermentation, with final values
C

313 ranging from 9.9-10.6 g/L. In the case of non-osmotically dehydrated olives, a similar profile
AC

314 of lactic acid was observed in the brines, but lower concentrations were measured at the end

315 of fermentation ranging from 6.6-7.7 g/L. The obtained values of lactic acid concentration at

316 the end of all fermentation processes are consistent with the results of titratable acidity (Fig.

317 3). A continuous increase of acetic acid was also observed from the onset of fermentation in

318 all treatments. Higher concentrations of acetic acid were detected in the brines of olives

13
 ACCEPTED MANUSCRIPT
319 treated with osmotic dehydration before fermentation with amounts reaching 3.5-3.7 g/L at

320 the end of the process, whereas lower concentrations were detected in the brines of non-

321 osmotically dehydrated olives (1.7-2.3 g/L). However, no clear trend could be established for

322 the different substitution levels of NaCl by MSG in the brines. The high concentrations of

323 acetic acid could be attributed to a shift from homo- to hetero-fermentative metabolism of

PT
324 bacteria under conditions of environmental stress (e.g., oxygen availability, nutrient

RI
325 limitation, salt content, pH values) (Bobillo and Marshall, 1991). Malic, tartaric, citric, and

326 succinic acids were also detected in the brines but their concentrations were low with no

SC
327 marked differences among fermentations, with the exception of succinic acid that presented

328 higher concentrations in the brines of olives treated with osmotic dehydration prior to

329
U
fermentation (Supplementary Fig. 1). Succinic acid is an intermediate in the citric acid cycle
AN
330 and serves as an electron donor in the electron transport system (Pratt and Cornely, 2004).
M

331 Thus, the increased concentration of succinic acid in osmotically dehydrated olives prior to

332 fermentation could be attributed to the higher concentration of citric acid in these olives
D

333 compared to non osmotically treated samples. The observed profile of organic acids in all
TE

334 fermentations was typical for both green and black olive processing and is in good agreement

335 with recently published reports (Cortés-Delgado et al., 2016; Bleve et al., 2014, 2015;
EP

336 Panagou et al., 2008).

337 Glucose was the main sugar in the brines detected by HPLC presenting a completely
C

338 different profile based on whether olives had been subjected to osmotic dehydration prior to
AC

339 fermentation or not (Fig. 4). Specifically, for non-osmotically dehydrated olives low amounts

340 of glucose were detected throughout fermentation in concentrations not exceeding 0.4 g/L,

341 without any clear pattern among the different levels of NaCl substitution by MSG.

342 Apparently, the high cell densities of LAB from the onset of fermentation in populations

343 exceeding 6.5 log CFU/mL resulted in a vigorous fermentation with rapid depletion of

14
 ACCEPTED MANUSCRIPT
344 glucose in the brines. Our findings are in line with a recently published report on black olive

345 processing (Tufariello et al., 2015) where low levels of glucose were detected in the brines of

346 Kalamata olives after 90 days of spontaneous fermentation. A different profile of glucose in

347 the brines was obtained when olives were subjected to osmotic dehydration prior to

348 fermentation. In this case, glucose concentration showed a rapid increase within the first 4

PT
349 days of fermentation with amounts ranging from 4.5 to 5.6 g/L. From this point, a rapid

RI
350 decrease was evident until day 27 where no marked differences in glucose concentration

351 could be established among the different treatments. The increased concentration of glucose

SC
352 in the brines could be attributed to the pre-fermentation treatment of olives since osmotic

353 dehydration was undertaken by immersing the drupes in 70% (w/w) glucose syrup overnight

354
U
prior to brining. It is well known that osmotic dehydration is a process based on partial
AN
355 removal of the water by submerging the plant tissue in a hypertonic solution of an osmotic
M

356 substance (Dermensonlouoglu et al., 2008). In the case of table olives, during osmotic

357 dehydration water flowed from the product (raw olives) into the osmotic solution, while
D

358 osmotic solute (glucose) was transferred from the solution into the product. Subsequently,
TE

359 when osmotically dehydrated olives were placed into the brine for fermentation, glucose was

360 gradually released from the olives reinforcing the brine with fermentable substrate. This could
EP

361 also justify the higher values of titratable acidity attained in the fermentation of osmotically-

362 dehydrated olives, as well as the lactic and acetic acid concentrations detected in the
C

363 fermentation procedures.

AC

364 The volatile compounds with major presence in the brines were ethanol and methanol

365 (Fig. 5). For non-osmotically dehydrated olives, ethanol increased rapidly within the first 14

366 days reaching a value of ca. 3,000 g/L. A slight decrease was observed until the end of

367 fermentation where ethanol concentration was maintained at 2,500 g/L without presenting a

368 clear pattern for the different substitution levels of NaCl by MSG. For osmotically dehydrated

15
 ACCEPTED MANUSCRIPT
369 olives, higher levels of ethanol were generally observed throughout fermentation with final

370 values ranging from 3,000 to 3,300 g/L. The higher level of ethanol in osmotically dehydrated

371 olives prior to brining could be attributed to the higher activity of yeasts in the brines due to

372 the increased concentration of glucose in these treatments (Fig. 4). Ethanol is an important

373 volatile compound for the sensory properties of natural black olives and it has been defined as

PT
374 a precursor of ethyl esters which are responsible for the fruity notes of fermented olives

RI
375 (Sabatini and Marsilio, 2008). Considerable amounts of methanol were also detected

376 presenting a similar pattern with ethanol but in lower concentrations (Fig. 5). Specifically, for

SC
377 non-osmotically dehydrated olives, methanol concentration increased rapidly from ca. 50 g/L

378 at the onset of fermentation to 600-700 g/L at day 14 and it was further maintained between

379
U
500-600 g/L throughout the process without presenting a clear pattern for the different
AN
380 concentrations of MSG. Lower levels for this volatile compound were generally observed for
M

381 osmotically dehydrated olives with amounts ranging from 400-500 g/L. The presence of this

382 compound has been reported by previous researchers in both black and green olive
D

383 fermentations (Montaño et al., 1992; Sabatini et al., 2009; Grounta et al., 2016) and is
TE

384 associated with the activity of pectinolytic enzymes, namely pectin methyl esterase, that

385 degrade pectin on the cell wall of olives (Balatsouras, 1990). Higher alcohols (2-butanol,
EP

386 propanol) were also detected in the brines. Concerning 2-butanol, the treatments with non-

387 osmotically dehydrated olives presented higher amounts of 2-butanol with progressively
C

388 increasing concentrations according to the substitution level of NaCl by MSG. Thus, the
AC

389 amount of this alcohol at the end of fermentation was detected at 80.1, 51.7, and 29.4 g/L for

390 5, 10, and 15% substitution of NaCl by MSG (Fig. 5). In contrast, in the brines of osmotically

391 dehydrated olives, lower concentrations were observed not exceeding 13 g/L throughout the

392 process regardless of MSG substitution level. It needs to be noted that the presence of this

393 compound in the brines has been associated with a typical fermentation in green olives and

16
 ACCEPTED MANUSCRIPT
394 more recently with winery off-odor in inoculated fermentation of green olives (Rodríguez-

395 Gómez et al., 2013), although no such sensory defect was perceived by the sensory analysis of

396 the final fermented product. Higher amounts of propanol were detected in the brines of non-

397 osmotically dehydrated olives compared to osmotically dehydrated ones (Fig. 5). In both

398 cases, the concentration of this compound presented a progressive increase according to the

PT
399 proportion of MSG used in the brines but generally lower concentrations were detected at the

RI
400 end of fermentation in osmotically dehydrated olives. This compound has been associated

401 with alcoholic/musty notes and its presence could be attributed to yeast activity (Sabatini et

SC
402 al., 2009). This alcohol has been previously detected in both Spanish-style green (Sabatini et

403 al., 2009; Blana et al., 2014; Cortés-Delgado et al., 2016) and black olive fermentations

404
U
(Sabatini et al., 2008; Bleve et al., 2015; Grounta et al., 2016). Ethyl acetate and acetaldehyde
AN
405 were also detected in the brines with increasing concentration throughout the course of
M

406 fermentation, without presenting any clear pattern between osmotically and non-osmotically

407 dehydrated olives as well as between the different concentrations of MSG employed
D

408 (Supplementary Fig. 2). Generally, acetaldehyde was maintained in higher amounts (12-20
TE

409 g/L) at the end of fermentation compared to ethyl-acetate (4-7 g/L). These compounds have a

410 major contribution in odor development of fermented Kalamata black olives as reported
EP

411 elsewhere (Sabatini et al., 2008; Bleve et al., 2015). Finally, dimethyl sulfide (DMS) was

412 detected in low amounts (< 2.0 g/L) in treatments with non-osmotically dehydrated olives
C

413 compared to osmotically dehydrated olives where DMS was detected at lower concentrations
AC

414 (< 1.0 g/L), without presenting a clear pattern for the different concentrations of MSG used

415 (Supplementary Fig. 2). This compound has been associated with cabbage-like odor due to

416 microbial degradation of sulfur amino acids (Casaburi et al., 2015). The same compound has

417 been reported in the volatile profile of Spanish-style green olives prepared from different

17
 ACCEPTED MANUSCRIPT
418 olive cultivars (Cortés-Delgado et al., 2016) as well as from natural black olives (Grounta et

419 al., 2016).

420 The values of combined acidity (CA) in the brines at the end of fermentation are

421 illustrated in Fig. 6. For control fermentation, a value of 0.056 (±0.007) N was measured

422 which is in good agreement with previously reported values of CA (0.044 ± 0.005 N) for

PT
423 directly brined olives (López-López et al., 2004). Overall, the values of CA were affected by

RI
424 both the osmotic dehydration pre-treatment of the drupes prior to fermentation and the

425 substitution level of NaCl by MSG. Specifically, in fermentations with non-osmotically

SC
426 dehydrated olives, CA presented a gradual increase according to the level of NaCl substitution

427 by MSG. Thus, the highest CA value (0.133 ± 0.008 N) was detected in 15% MSG

428
U
substitution level. However, statistically significant (P < 0.0001) lower values of CA were
AN
429 measured in the brines of osmotically-dehydrated olives that also presented a progressive
M

430 increase according to the MSG level employed. The amounts of MSG used in the brines

431 resulted in a gradual increase in the profile of pH and this was more pronounced in non-
D

432 osmotically dehydrated olives (Fig. 3a). Thus, the highest level of NaCl substitution by MSG
TE

433 (i.e., 15%) resulted in the highest pH values throughout fermentation, reaching a final pH of

434 4.55 that was beyond specification for this trade preparation of table olives. This is in
EP

435 agreement with a previous work of Spanish researchers (Rejano Navarro and Sánchez Gómez,

436 1996) on the use of MSG in pickled green olive packing, who reported that the addition of
C

437 MSG in the packing brine provoked an increase in pH. To overcome this effect, the use of
AC

438 MSG in the brines should be combined with osmotic dehydration of olives prior to brining

439 where lower pH profiles were obtained.

440 The presence of glutamic acid in the olives and ‡-aminobutyric acid (GABA) as a major

441 degradation product (De Castro et al., 2014) at the end of fermentation is shown in Fig. 7. It

442 can be seen that low levels of glutamic acid were detected in the brines without any marked

18
 ACCEPTED MANUSCRIPT
443 differences among the treatments. In all fermentations, glutamic acid was detected in

444 concentrations below 0.1 g/kg of olives, with the exception of 15% substitution of NaCl by

445 MSG where slightly higher amounts were detected (0.142±0.008 g/kg). Moreover, higher

446 yields of GABA were detected in the olives in increasing concentrations according to the

447 initial substitution level of NaCl by MSG. However, no statistical differences could be

PT
448 established for the effect of osmotic dehydration in GABA concentration within the same

RI
449 substitution level of MSG. Thus, for instance, the amount of GABA in 15% MSG was the

450 same regardless of the pre-treatment of olives with osmotic dehydration prior to fermentation.

SC
451 Our observations about the fate of MSG are in agreement with a recent work (De Castro et al.,

452 2014) in which MSG was used in the packing brine of fermented green olives and pickled

453
U
cucumbers in order to investigate the stability of MSG during long-term storage under
AN
454 different packing conditions. The authors reported extensive (> 75%) degradation of MSG in
M

455 unpasteurized green olives due to the activity of LAB and yeasts with concurrent formation of

456 GABA as a major end-product. In another work (Montaño et al., 2000) a LAB strain of
D

457 Lactobacillus pentosus isolated from green olive fermentation has been shown to utilize
TE

458 glutamic acid in olive brine, although the end products were not identified. It has been

459 reported that GABA has a functional role in depressing the blood pressure in mild
EP

460 hypertensive humans and therefore it could be used as a potential bioactive component to

461 develop novel fermented products with enhanced value (Shekh et al., 2016).
C

462 With regard to the sensory analysis of the olives at the end of fermentation, no off-odours
AC

463 indicating sign of abnormal fermentation such as butyric, putrid fermentation or zapateria

464 were detected by the panelists. Thus, all olives were classified as ‘Extra’ category according

465 to the method of sensory analysis of table olives developed by the IOC. Other defects were

466 not also detected in any fermentation. For the gustatory sensations of saltiness, bitterness, and

467 acidity no statistically significant differences could be established among fermentations. It

19
 ACCEPTED MANUSCRIPT
468 needs to be noted that the values of acidity measured in the brines of osmotically and non-

469 osmotically dehydrated olives presented a different profile (Fig. 3), but this difference in

470 acidity could not be organoleptically perceived. Finally, no significant differences were

471 observed concerning the effect of osmotic dehydration on the kinesthetic sensations of

472 hardness, fibrousness, and crunchiness as all of them received similar scores by the panelists.

PT
473 Overall, the treatment of olives with osmotic dehydration prior to fermentation did not affect

RI
474 the sensory properties of the final fermented product. Moreover, no effect of MSG was

475 evident on the sensory characteristics of the final product as this compound was extensively

SC
476 degraded during fermentation. MSG degradation could result in the development of off-

477 flavours in processed vegetables due to extended conversion of MSG to pyroglutamic acid

478
U
(pGlu). However, in a recent work (De Castro et al., 2014) it was found that the conversion of
AN
479 MSG to pGlu was almost non-existent in both fermented green olives and cucumbers, with
M

480 MSG being almost completely converted to GABA.

481
D

482 3.3Exploratory analysis of fermentation profiles

TE

483 The two-dimensional graphical representation of the data is shown in Fig. 7, where each

484 microbiological and biochemical parameter was represented by a single row of coloured
EP

485 boxes, whereas columns represented different fermentation treatments. Strong and weak

486 similarities between variables and subjects were displayed in various shades of brown and
C

487 blue colours, respectively. The heatmap readily showed discrimination between osmotically
AC

488 and non-osmotically dehydrated olives in two clusters. Moreover, for osmotically dehydrated

489 olives two well-differentiated sub-clusters were evident, one for 5% MSG substitution and the

490 other for 10% and 15% MSG substitution. Similar observations were made for non-

491 osmotically dehydrated olives with two differentiated sub-clusters, one for 5% MSG

492 substitution and control fermentation and the second for 10% and 15% MSG substitution.

20
 ACCEPTED MANUSCRIPT
493 Concerning the discrimination of variables within the different fermentation processes, two

494 blocks of variables could be generally differentiated. The first block (at the top left)

495 corresponding to osmotically dehydrated olives, is positively related to acetic, succinic, citric,

496 malic, and lactic acids, ethanol, acetaldehyde, and acidity. The second block (at the bottom

497 right) corresponding to non-osmotically dehydrated olives is positively related to the

PT
498 indigenous microbiota (LAB, yeasts, enterobacteria), methanol, 2-butanol, propanol, pH, and

RI
499 dimethyl-sulfide. Table olive fermentation is a complex process in which heterogeneous data

500 collected throughout the different sampling times including both microbiological and

SC
501 biochemical data should be assessed by means of multivariate analysis and provide

502 presentation in a clear and graphical way (Berrueta et al., 2007). Cluster analysis is the most

503
U
commonly employed unsupervised pattern recognition technique that relates treatments
AN
504 (fermentations) according to similarity, whereas a two-dimensional presentation of the data in
M

505 the form of heatmap could provide a convenient way to illustrate the profile of the different

506 processes in a single column with different colours. Multivariate analysis is expanding in
D

507 table olive research in the last years with promising results as it can reveal underlying
TE

508 relationships among process variables that could not be perceived by examining each variable

509 separately (Rodríguez-Gómez et al., 2012, 2013; Bleve et al., 2014, 2015; Tufariello et al.,
EP

510 2015; Grounta et al., 2016).

511
C
AC

512 4. Conclusion

513 The obtained results illustrated that the osmotic dehydration of Kalamata black olives

514 prior to brining resulted in vigorous lactic acid fermentation based on the final values of pH

515 (3.7-4.1) and titratable acidity (0.7-0.8 %) regardless of the substitution level of NaCl by

516 MSG. The use of glucose as osmotic solute in pre-fermentation treatment of olives resulted in

517 subsequent enrichment of the brine with fermentable substrates that supported fermentation.

21
 ACCEPTED MANUSCRIPT
518 However, in non-osmotically dehydrated olives, 15% substitution of NaCl by MSG resulted

519 in final pH of 4.5 that was beyond specification for this trade preparation and could not ensure

520 the microbiological stability of the product. Moreover, the use of MSG in non-osmotically

521 dehydrated olives prior to fermentation resulted in higher buffer capacity of the brines

522 compared to osmotically dehydrated olives as indicated by combined acidity measurements.

PT
523 MSG was highly degraded in the brines being almost completely converted to GABA at the

RI
524 end of fermentation. Finally, the sensory characteristics of fermented olives with/without

525 osmotic dehydration and at all levels of MSG employed did not show any significant

SC
526 difference compared to the control treatment. As a next step, large-scale experiments will be

527 implemented in a Greek table olive industry to validate the applicability of osmotic

528
U
dehydration as pre-fermentation treatment in large scale productions taking also into
AN
529 consideration other trade preparations.
M

530

531 Acknowledgments
D

532 This work has been supported by the project ‘Development and adaptation of traditional
TE

533 Greek olive based products to Chinese dietary and culinary preferences-GRECHIN OLIV’

534 (project number 12CHN324), co‐financed by the European Union (European Social Fund –
EP

535 ESF) and Greek national funds through the O.P. “Competitiveness and Entrepreneurship

536 (OPC II)” ROP Macedonia – Thrace, ROP Crete and Aegean Islands, ROP Thessaly –
C
AC

537 Mainland Greece – Epirus, ROP Attica, Framework NSRF 2007-2013.

538

539 References

540 Adams, M.R., Moss, O.M., 2000. Food Microbiology. The Royal Society of Chemistry.

541 Cambridge, UK.

542

22
 ACCEPTED MANUSCRIPT
543 Beristain, C.I., Azuara, E., Cortés, R., Garcia, H.S., 1990. Mass transfer during osmotic

544 dehydration of pineapple rings. Int. J. Food Sci. Technol. 25, 576-582.

545 Berrueta, L.A., Alonso-Salces, R.M., Héberger, K., 2007. Supervised pattern recognition in

546 food analysis. J. Chromatogr. A 1158, 196-214.

547 Bevilacqua, A., de Stefano, F., Augello, S., Pignatiello, S., Sinigaglia, M., Corbo, R.M., 2015.

PT
548 Biotechnological innovations for table olives. Int. J. Food Sci. Nutr. 66, 127-131.

RI
549 Billo, E.J., 2007. Excel for Scientists and Engineers. Wiley & Sons, Inc., New Jersey.

550 Blana, V.A., Grounta, A., Tassou, C.C., Nychas, G.J.E., Panagou, E.Z., 2014. Inoculated

SC
551 fermentation of green olives with potential probiotic Lactobacillus pentosus and

552 Lactobacillus plantarum starter cultures isolated from industrially fermented olives. Food

553 Microbiol. 38, 208-218.

U
AN
554 Bleve, G., Tufariello, M., Durante, M., Perbellini, E., Ramires, F.A., Grieco, F., Cappello,
M

555 M.S., De Domenico, S., Mita, G., Tasioula-Margari, M., Logrieco, A.F., 2014. Physico-

556 chemical and microbiological characterization of spontaneous fermentation of Cellina di

D

557 Nardò and Leccino table olives. Front. Microbiol. 5:570.

TE

558 Bleve, G., Tufariello, M., Durante, M., Grieco, F., Ramires, F.A., Mita, G., Tasioula-Margari,

559 M., Logrieco, A.F., 2015. Physico-chemical characterization of natural fermentation

EP

560 process of Conservolea and Kalamata table olives and development of a protocol for the

561 pre-selection of fermentation starters. Food Microbiol.46, 368-382.

C

562 Bobillo, M., Marshall, V.M., 1991. Effect of salt and culture aeration on lactate and acetate
AC

563 production by Lactobacillus plantarum. Food Microbiol. 8, 153-160.

564 Brenes, M., 2004. Olive fermentation and processing: Scientific and technological challenges.

565 J. Food Sci. 69, FMS33-FMS34.

566 Casaburi, A., Piombino, P., Nychas, G.-J., Villani, F., Ercolini, D., 2015. Bacterial

567 populations and the volatilome associated to meat spoilage. Food Microbiol. 45, 83-102.

23
 ACCEPTED MANUSCRIPT
568 CiurzyDska, A., Kowalska, H., Czajkowska, K., Lenart, A., 2016. Osmotic dehydration in

569 production of sustainable and healthy food. Trends Food Sci. Technol. 50, 186-192.

570 Cortés-Delgado, A., Sánchez, A.H., de Castro, A., López-López, A., Beato, V.M., Montaño,

571 A., 2016. Volatile profile of Spanish-style green table olives prepared from different

572 cultivars grown at different locations. Food Res. Int. 88, 131-142.

PT
573 De Angelis, M., Campanella, D., Cosmai, L., Summo, C., Rizzello, C.G., Caponio, F., 2015.

RI
574 Microbiota and metabolome of un-started and started Greek-type fermentation of Bella di

575 Cerignola table olives. Food Microbiol. 52, 18-30.

SC
576 De Castro, A., Sánchez, A.H., Beato, V.M., Casado, F.J., Montaño, A., 2014. Stability of

577 monosodium glutamate in green table olives and pickled cucumbers as a function of

578
U
packing conditions and storage time. Food Addit. Contam. 31, 1158-1164.
AN
579 Dermesonlouoglou, E.K., Pourgouri, S., Taoukis, P.S., 2008. Kinetic study of the effect of the
M

580 osmotic dehydration pre-treatment to the shelf-life of frozen cucumber. Innov. Food Sci.

581 Emerg. Technol. 9, 542-549.

D

582 Fernstrom, J.D., 2007. Health issues relating to monosodium glutamate use in the diet. In:
TE

583 Kilcast, D., Angus, F. (Eds.), Reducing Salt in Foods. Woodhead Publishing Ltd.,

584 Cambridge, UK, pp. 55-76.

EP

585 Garrido-Fernández, A., Fernández Díez, M.J., Adams, M.R., 1997. Table Olives: Production

586 and Processing. Chapman & Hall, London.

C

587 Giannakourou, M.C., Taoukis, P.S., 2003a. Kinetic modelling of vitamin C loss in frozen
AC

588 green vegetables under variable storage conditions. Food Chem. 83, 33-41.

589 Giannakourou, M.C., Taoukis, P.S., 2003. Stability of dehydrofrozen green peas pretreated

590 with non conventional osmotic agents. J. Food Sci. 68, 2002−2010.

591 Giraldo, G., Talens, P., Fito, P., Chiralt, A., 2003. Influence of sucrose solution concentration

592 on kinetics and yield during osmotic dehydration of mango. J. Food Eng. 58, 33-43.

24
 ACCEPTED MANUSCRIPT
593 Gogou, E., Oikonomopoulou, V., Papadaki, S., Tsimogiannis, D., Krokida, M. Taoukis, P.

594 2014. Pre-dehydration processing of table olives for the development of reduced salt

595 products. IFT Annual Meeting, New Orleans, USA.

596 Grounta, A., Doulgeraki, A.I., Nychas, G.-J.E., Panagou, E.Z., 2016. Biofilm formation on

597 Conservolea natural black olives during single and combined inoculation with a functional

PT
598 Lactobacillus pentosus starter culture. Food Microbiol. 56, 35-44.

RI
599 Hajeb, P., Jinap, S. 2015., Umami taste components and their sources in Asian foods. Crit.

600 Rev. Food Sci. Nutr. 55, 778-791.

SC
601 Hurtado, A., Reguant, C., Bordons, A., Rozès, N., 2012. Lactic acid bacteria from fermented

602 table olives. Food Microbiol. 31, 1-8.

603
U
International Olive Council (IOC), 2004. Trade standard applying to table olives. DOI/OT/Nc
AN
604 no 1, Madrid, Spain.
M

605 International Olive Council (IOC), 2011. Sensory analysis of table olives. Document

606 COI/OT/MO No1/Rev. 2, Madrid, Spain.

D

607 International Olive Council (IOC), 2016. World Table Olives Figures
TE

608 (http://www.internationaloliveoil.org/estaticos/view/132-world-table-olive-figures,

609 assessed on 14 July 2016).

EP

610 Interprofessional Association for Table Olives (DOEPEL), 2016. Table olive varieties

611 (http://doepel.gr/index.php/poikilies-elias, assessed on 25 October 2016) (in Greek).

C

612 Jinap, S., Hajeb, P., 2010. Glutamate. Its applications in food and contribution to health.
AC

613 Appetite 55, 1-10.

614 Kalomiri, F., Alexandraki, M., Karamitsiou, F., Ntzimani, A., Gogou, E., Oikonomopoulou,

615 V., Krokida, M., Taoukis, P., 2015. Effect of osmotic dehydration as a pre-fermentation

616 treatment on the quality and sensory characteristics of green table olives. 29th EFFoST

617 International Conference Proceedings, Athens, Greece, pp. 1416.

25
 ACCEPTED MANUSCRIPT
618 Krokida, M.K., Kiranoudis, C.T., Maroulis, Z.B., Marinos-Kouris, D., 2000. Effect of

619 pretreatment on colour of dehydrated products. Drying Technol. 18, 1239-1250.

620 Kucner, A., Klewicki, R., Sójka, M., 2013. The influence of selected osmotic dehydration and

621 pretreatment parameters on dry matter and polyphenol content in highbush blueberry

622 (Vaccinium corymbosum L.) fruits. Food Bioprocess Technol. 6, 2031-2047.

PT
623 López-López, A., García-García, P., Durán-Quintana, M.C., Garrido-Fernández, A., 2004.

RI
624 Physicochemical and microbiological profile of packed table olives. J. Food Prot. 67,

625 2320-2325.

SC
626 Montaño, A., Sánchez, A.H., Rejano, L., 1990. Rapid quantitative analysis of headspace

627 components of green olive brine. J. Chromatogr. 521, 153–157.

628
U
Montaño, A., de Castro, A., Rejano, L. and Sánchez, A.H., 1992. Analysis of zapatera olives
AN
629 by gas and high-performance liquid chromatography. J. Chromatgr. 594, 259–267.
M

630 Montaño, A., Sánchez, A.H., de Castro, A., 2000. Changes in the amino acid composition of

631 green olive brine due to fermentation by pure culture of bacteria. J. Food Sci. 65, 1022-
D

632 1027.
TE

633 Moon, J.Y., Jung, H.J., Moon, M.H., Chung, B.C., Choi, M.H., 2009. Heat-map visualization

634 of gas chromatography-mass spectrometry based quantitative signatures on steroid

EP

635 metabolism. J. Am. Soc. Mass Spectrom. 20, 1626-1637.

636 Nisiotou, A.A., Chorianopoulos, N., Nychas, G.J.E., Panagou, E.Z., 2010. Yeast
C

637 heterogeneity during spontaneous fermentation of black Conservolea olives in different

AC

638 brine solutions. J. Appl. Microbiol. 108, 396-405.

639 Panagou, E.Z., Schillinger, U., Franz, C.M.A.P., Nychas, G.J.E., 2008. Microbiological and

640 biochemical profile of cv. Conservolea naturally black olives during controlled

641 fermentation with selected strains of lactic acid bacteria. Food Microbiol. 25, 348-358.

26
 ACCEPTED MANUSCRIPT
642 Panagou, E.Z., 2016. Greek table olives processing. In: Hui, Y.H., Özgül Evranuz, E. (Eds.),

643 Handbook of Vegetable Preservation and Processing. CRC Press, Boca Raton, FL, pp.

644 527-554.

645 Rastogi, N.K., Angersbach, A., Niranjan, K., Knorr, D., 2000. Rehydration kinetics of

646 high pressure pretreated and osmotic dehydrated pineapple. J. Food Sci. 65, 838−841.

PT
647 Populin, T., Moret, S., Truant, S., Conte, L.S., 2007. A survey on the presence of free

RI
648 glutamic in foodstuffs, with and without added monosodium glutamate. Food Chem. 104,

649 1712-1717.

SC
650 Pratt, C.W., Cornely, K., 2004. Essential Biochemistry. John Wiley & Sons, Hoboken, NJ,

651 USA.

652
U
Rejano Navarro, L., Sánchez Gómez, A.H., 1996. Utilización de glutamate sódico en el
AN
653 envasado de aceitunas verdes aderezadas. Efecto sobre las características químicas y el
M

654 sabor. Grasas Aceites 47, 255-259.

655 Rodríguez–Gómez, F., Romero-Gil, V., Bautista-Gallego, J., Garrido-Fernandez, A., Arroyo-
D

656 López, F.N., 2012. Multivariate analysis to discriminate yeast strains with technological
TE

657 applications in table olive processing. World J. Microbiol. Biotechnol. 28, 1761-1770.

658 Rodríguez–Gómez, F., Bautista – Gallego, J., Arroyo- López, F.N., Romero-Gill, V.,
EP

659 Jiménez-Díaz, R., Garrido-Fernández, A., García-García, P., 2013. Table olive

660 fermentation with multifunctional Lactobacillus pentosus strains. Food Control 34, 96-105.
C

661 Rodríguez–Gómez, F., López-López, A., Romero-Gill, V., Arroyo- López, F.N., Moreno-
AC

662 Baquero, J.M., Garrido-Fernández, A., García-García, P., 2014. Effect of post-fermentation

663 storage on Spanish-style green Manzanilla olives. LWT-Food Sci. Technol. 57, 789-793.

664 Rodríguez–Gómez, F., Romero-Gill, V., Arroyo- López, F.N., Bautista Gallego, J., García-

665 García, P., Garrido-Fernández, A., 2014. Effect of packaging and storage conditions on

27
 ACCEPTED MANUSCRIPT
666 microbial survival, physicochemical characteristics and colour of non-thermally preserved

667 green Spanish-style Manzanilla olives. LWT-Food Sci. Technol. 63, 367-375.

668 Sánchez Gómez, A.H., Garcia Garcia, P., Rejano Navarro, L., 2006. Elaboration of table

669 olives. Grasas Aceites 57, 86-94.

670 Sabatini, N., Marsilio, V., 2008. Volatile compounds in table olives (Olea europea L.,

PT
671 Nocellara del Belice cultivar). Food Chem. 107, 1522-1528.

RI
672 Sabatini, N., Mucciarella, M.R., Marsilio, V., 2008. Volatile compounds in uninoculated and

673 inoculated table olives with Lactobacillus plantarum(Olea Moresca and Kalamata).

SC
674 LWT-Food Sci. Technol. 41, 2017 – 2022.

675 Sabatini, N., Perri, E., Marsilio, V., 2009. An investigation on molecular partition of aroma

676
U
compounds in fruit matrix and brine medium of fermented table olives. Innov. Food Sci.
AN
677 Emerg. Technol. 10, 621-626.
M

678 Shekh, S.L., Dave, J.M., Vyas, B.R.M., 2016. Characterization of Lactobacillus plantarum

679 strains for functionality, safety and ‡-amino butyric acid production. LWT-Food Sci.
D

680 Technol. 74, 234-241.

TE

681 Talens, P., Martinez-Navarrete, N., Fito, P., Chiralt, A., 2002. Changes in optical and

682 mechanical properties during osmodehydrofreezing of kiwi fruit. Innov. Food Sci. Emerg.
EP

683 Technol. 3, 191−199.

684 Tassou, C.C., Panagou, E.Z., Katsaboxakis, K.Z., 2002. Microbiological and physicochemical
C

685 changes of naturally black olives fermented at different temperatures and NaCl levels in
AC

686 the brines. Food Microbiol. 19, 605-615.

687 Tedjo,W., Taiwo, K.A., Eshtiaghi, M.N., Knorr, D., 2002. Comparison of pretreatment

688 methods on water and solid diffusion kinetics of osmotically dehydrated mangos. J. Food

689 Eng. 53, 133−142.

28
 ACCEPTED MANUSCRIPT
690 Tufariello, M., Durante, M., Ramires, F.A., Grieco, F., Tommasi, L., Perbellini, E., Falco, V.,

691 Tasioula-Margari, M., Logrieco, A.F., Mita, G., Bleve, G., 2015. New process for

692 production of fermented black table olives using selected autochthonous microbial

693 resources. Front. Microbiol. 6, 1007.

694 Van den Berg, R.A., Hoefsloot, H.C.J., Westerhuis, J.A., Smilde, A.K., van der Werf, M.J.,

PT
695 2006. Centering, scaling and transformations: improving the biological information content

RI
696 of metabolomics data. BMC Genomics 7, 142-156.

697 Viljoen, B.C., 2006. Yeast ecological interactions. Yeast-yeast, yeast-bacteria, yeast-fungi

SC
698 interactions and yeasts as biocontrol agents. In: Querol, A., Fleet, G.H. (Eds.), Yeasts in

699 Foods and Beverages. Springer-Verlag, Berlin, pp. 83-110.

700
U
Villegas Vergara, J., Blana, V., Mallouchos, A., Stamatiou, A., Panagou, E.Z., 2013.
AN
701 Evaluating the efficacy of brine acidification as implemented by the Greek table olive
M

702 industry on the fermentation profile of Conservolea green olives. LWT-Food Sci. Technol.

703 53, 113-119.

D

704 Xia, J., Sinelnikov, I.V., Han, B., Wishart, D. S., 2015. MetaboAnalyst 3.0-making
TE

705 metabolomics more meaningful. Nucleic Acids Res., 43, 251–257.

C EP
AC

29
 ACCEPTED MANUSCRIPT
706 Figure captions
707
708 Figure 1. Changes in the population of LAB ( ), yeasts ( ) and enterobacteria ( ) in the
709 brine during Greek-style processing of Kalamata black olives in (a) 7% NaCl brine (control),
710 (b) 6.65% NaCl-0.35 % MSG , (c) 6.30% NaCl-0.70% MSG, and (d) 5.95% NaCl-1.05%
711 MSG. Data points are average values of duplicate fermentations ± standard deviation.
712

PT
713
Figure 2. Changes in the population of LAB ( ), yeasts ( ) and enterobacteria ( ) in the

RI
714 brine during Greek-style processing of Kalamata black olives subjected to osmotic
715 dehydration as pre-fermentation treatment with (a) 6.65% NaCl-0.35 % MSG , (b) 6.30%

SC
716 NaCl-0.70% MSG, and (c) 5.95% NaCl-1.05% MSG . Data points are average values of
717 duplicate fermentations ± standard deviation.
718

U
Figure 3. Evolution of pH (open symbols) and titratable acidity (solid symbols)
AN
during 719
720 Greek-style processing of Kalamata black olives without (a) and with (b) osmotic dehydration
721 of the olives as pre-fermentation treatment. Data points are average values of duplicate
M

722 fermentations ± standard deviation. ( , ) 7% NaCl brine (control); ( , ) 6.65% NaCl-

723 0.35 % MSG; ( , ) 6.30% NaCl-0.70% MSG ; ( , ) 5.95% NaCl-1.05% MSG.
724
D

Figure 4. Changes in lactic acid, acetic acid and glucose concentrations (g/L) in the
TE

brines 725
726 during processing of Kalamata black olives with (open symbols) and without (solid symbols)
727 osmotic dehydration of olives prior to fermentation. ( , ) 6.65% NaCl-0.35 % MSG;
EP

728 ( , ) 6.30% NaCl-0.70% MSG ; ( , ) 5.95% NaCl-1.05% MSG ; ( ) 7% NaCl brine

729 (control). Data points are average values of duplicate fermentations ± standard deviation.
730
C

Figure 5. Changes in ethanol, methanol, 2-butanol and propanol concentrations (mg/L)

AC

in the 731
732 brine during processing of Kalamata black olives with (open symbols) and without (solid
733 symbols) osmotic dehydration of olives prior to fermentation. ( , ) 6.65% NaCl-0.35 %
734 MSG; ( , ) 6.30% NaCl-0.70% MSG ; ( , ) 5.95% NaCl-1.05% MSG ; ( ) 7% NaCl
735 brine (control). Data points are average values of duplicate fermentations ± standard
736 deviation.
737
738 Figure 6. Combined acidity (N) in the brines of Kalamata black olives at the end
of fermentation (127 days). Data are mean values of duplicate fermentations – standard
739

30
 ACCEPTED MANUSCRIPT
740 deviation. Mean values with common lowercase letters shown above the bars are not
741 statistically different at a P value of < 0.05.
742
743
Figure 7. Concentration of glutamic acid and ‡-aminobutyric acid (GABA) in the
flesh of Kalamata black olives at the end offermentation (127 days). Data are mean values of
744
745 duplicate fermentations ± standard deviation. Mean values with common lowercase letters

PT
746 shown above the bars are not statistically different at a P value of < 0.05.
747
748

RI
Figure 8. Hierarchically clustered heatmap of variables (microbiological and
biochemical profile) and objects (different fermentation processes of Kalamata black olives). OD+ and
749

SC
750 OD- indicate osmotically and non-osmotically dehydrated olives prior to fermentation. The
751 number after the fermentation process (1 or 2) indicates the replication of the experiment.
752

U
753 AN
M
D
TE
C EP
AC

31
 ACCEPTED MANUSCRIPT

9.0 9.0

(a) (b)
8.0 8.0

7.0 7.0

6.0 6.0

PT
5.0 5.0

RI
4.0 4.0

SC
3.0 3.0
Population (log CFU/mL)

2.0 2.0

U
AN
1.0 1.0

0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

M
9.0 9.0

(c) (d)

D
8.0 8.0

TE
7.0 7.0

6.0 6.0
EP
5.0 5.0
C

4.0 4.0
AC

3.0 3.0

2.0 2.0

1.0 1.0

0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
32
Fig. 1 Fermentation time (days)
 9.0
(a)
8.0 ACCEPTED MANUSCRIPT
7.0

6.0

5.0

4.0

3.0

PT
2.0

1.0

RI
0.0
0 20 40 60 80 100 120 140

SC
9.0
(b)
8.0

U
Population (log CFU/mL)

7.0 AN
6.0

5.0
M

4.0

3.0
D

2.0
TE

1.0

0.0
0 20 40 60 80 100 120 140
EP

9.0

(c)
8.0
C

7.0
AC

6.0

5.0

4.0

3.0

2.0

1.0

0.0
0 20 40 60 80 100 120 140
Fig. 2
33
Fermentation time (days)
 ACCEPTED MANUSCRIPT
7.0 1.1

6.5 (a) 1

6.0 0.9

0.8
5.5
0.7
5.0

PT
0.6
4.5
0.5
4.0

RI
0.4
3.5
0.3

SC
3.0 0.2

Acidity (%, lactic acid)

2.5 0.1

U
2.0 0
0 20 40 60 80 100 120 140
pH

AN
7.0 1.1

6.5 (b) 1
M

6.0 0.9

0.8
D

5.5
0.7
TE

5.0
0.6
4.5
0.5
EP

4.0
0.4
3.5
0.3
C

3.0 0.2
AC

2.5 0.1

2.0 0
0 20 40 60 80 100 120 140

Fermentation time (days)

Fig. 3

34
 12.0

ACCEPTED MANUSCRIPT
10.0

8.0
Lactic acid (g/L)

6.0

4.0

PT
2.0

RI
0.0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

SC
6.0

5.0

U
AN
4.0
Acetic acid (g/L)

3.0
M

2.0
D

1.0
TE

0.0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
EP

7.0

6.0
C

5.0
AC
Glucose (g/L)

4.0

3.0

2.0

1.0

0.0
Fig. 4 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
35
Fermentation time (days)
 ACCEPTED MANUSCRIPT

4500.0 900.0

4000.0 800.0

3500.0 700.0
Ethanol (mg/L)

Methanol (mg/L)
3000.0 600.0

PT
2500.0 500.0

RI
2000.0 400.0

1500.0 300.0

SC
1000.0 200.0

U
500.0 100.0

AN
0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

M
100.0 275.0

D
90.0 250.0

80.0 225.0

TE
70.0 200.0
2-Butanol (mg/L)

Propanol (mg/L)
175.0
EP
60.0
150.0
50.0
125.0
C

40.0
100.0
AC

30.0
75.0
20.0
50.0
10.0 25.0
0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
36
Fig. 5
 Concentration (g/kg of olives) Combined acidity in the brine (N)

Fig. 6

Fig. 7
a
AC
b

a
C EP
d

b
TE
D
e

c
M
AN
a

a
ACCEPTED MANUSCRIPT

U SC

b
c

RI
c
d

PT

37
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC

Fig. 8

38
 ACCEPTED MANUSCRIPT
Table 1. Experimental treatments of Kalamata black olives subjected to osmotic dehydration

(OD) prior to fermentation and brined in solutions with different levels of NaCl substitution

by monosodium glutamate (MSG).

Fermentation code Pre-fermentation treatment Brine solution

PT
Control none 7% (w/v) NaCl

5% MSG/no OD none 6.65% (w/v) NaCl – 0.35% (w/v) MSG

RI
(5% substitution)

10% MSG/no OD none 6.30% (w/v) NaCl – 0.70% (w/v) MSG

(10% substitution)

SC
15% MSG/no OD none 5.95% (w/v) NaCl – 1.05% (w/v) MSG
(15% substitution)

5% MSG/with OD OD
U 6.65% (w/v) NaCl – 0.35% (w/v) MSG
AN
(5% substitution)

10% MSG/with OD OD 6.30% (w/v) NaCl – 0.70% (w/v) MSG

(10% substitution)
M

15% MSG/with OD OD 5.95% (w/v) NaCl – 1.05% (w/v) MSG

(15% substitution)
D
TE
C EP
AC

39
 ACCEPTED MANUSCRIPT

Table 2. Sensory attributes (median value – standard deviation) of Kalamata natural black olives at the end of fermentation process (127 days).

Sensory Fermentation process

PT
attribute Control 5% MSG/no OD 10% MSG/no OD 15% MSG/no OD 5% MSG/with OD 10% MSG/with OD 15% MSG/with OD

RI
Abnormal 1.0 ± 0.02a 1.0 ± 0.07 a 1.2 ± 0.08 a 1.0 ± 0.03 a 1.0 ± 0.02 a 1.0 ± 0.05 a 1.0 ± 0.04 a

SC
fermentation

U
Other defects 1.0 ± 0.09 a 1.0 ± 0.05 a 1.0 ± 0.11 a 1.0 ± 0.06 a 1.0 ± 0.04 a 1.0 ± 0.06 a 1.0 ± 0.05 a

AN
Salty 4.7 ± 0.65 a 3.0 ± 0.37 a 3.5 ± 0.37 a 3.8 ± 0.77 a 3.7 ± 1.17 a 3.5 ± 0.56 a 3.8 ± 1.14 a

M
Bitter 5.3 ± 1.05 a 6.0 ± 0.68 a 5.2 ± 0.83 a 6.0 ± 0.83 a 7.2 ± 0.22 a 6.4 ± 0.77 a 6.0 ± 0.37 a

D
Acid 3.4 ± 0.77 a 2.5 ± 0.22 a 2.4 ± 0.62 a 3.0 ± 0.71 a 2.6 ± 0.62 a 3.5 ± 0.46 a 3.6 ± 0.40 a

Hardness 4.2 ± 0.31a 4.0 ± 1.05 a

TE
3.3 ± 0.52 a 4.0 ± 1.30 a 4.9 ± 1.33 a 4.0 ± 0.59 a 5.6 ± 0.65 a
EP
Fibrousness 3.2 ± 1.05 a 4.0 ± 1.05 a 4.2 ± 0.77 a 5.0 ± 0.90 a 2.7 ± 1.30 a 4.9 ± 1.14 a 4.1 ± 1.27 a
C

Crunchiness 5.1 ± 0.49 a 5.0 ± 0.90 a 5.0 ± 0.68 a 5.4 ± 0.96 a 4.2 ± 0.65 a 5.0 ± 0.96 a 4.0 ± 1.27 a
AC

Median values with same lowercase letters within the same row are not statistically significant (P>0.05) according to Kruskal-Wallis test.

40
 ACCEPTED MANUSCRIPT
• The effect of osmotic dehydration of olives and use of MSG in the brine was investigated

• Osmotic dehydration of olives resulted in vigorous fermentation based on pH and acidity

• The use of glucose as osmotic solute enriched the brine with fermentable material

• High levels of MSG in the brines resulted in high combined acidity values

• MSG was almost completely converted to GABA at the end of fermentation

PT
• Osmotic dehydration did not affect the sensory attributes of olives

RI
U SC
AN
M
D
TE
C EP
AC