Accepted Manuscript

Storage stability of prebiotic fermented milk obtained from permeate resulting of the microfiltration process Janaina Debon, Elane Schwinden Prudncio, Jos Carlos Cunha Petrus, Carlise Beddin Fritzen-Freire, Carmen M.O. Mller, Renata D. de M. Castanho Amboni, Cleide Rosana Werneck Vieira PII: DOI: Reference: To appear in: S0023-6438(11)00429-4 10.1016/j.lwt.2011.12.029 YFSTL 2941 LWT - Food Science and Technology

Received Date: 25 July 2011 Revised Date: 22 December 2011 Accepted Date: 28 December 2011

Please cite this article as: Debon, J., Prudncio, E.S., Cunha Petrus, C., Fritzen-Freire, C.B., Mller, C.M., de M. Castanho Amboni, R.D., Werneck Vieira, C.R., Storage stability of prebiotic fermented milk obtained from permeate resulting of the microfiltration process, LWT - Food Science and Technology (2012), doi: 10.1016/j.lwt.2011.12.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Abstract Janaina Debona, Elane Schwinden Prudnciob*, Jos Carlos Cunha Petrusa, Carlise Beddin Fritzen-Freireb, Carmen M. O. Mllera, Renata D. de M. Castanho Ambonib & Cleide Rosana Werneck Vieirab
a

Storage stability of prebiotic fermented milk obtained from permeate resulting of the microfiltration process

Departament of Chemistry and Food Engineering, Technology Center, Federal

University of Santa Catarina, Trindade, 88040-970, Florianpolis, SC, Brazil.

b

Department of Food Science and Technology, Center of Agricultural Science, Federal

University of Santa Catarina, Rod. Admar Gonzaga, 1346, Itacorubi, 88034-001, Florianpolis, SC, Brazil

* Corresponding author. Tel: (55) 48 37215366: Fax: (55) (48) 37219943 E-mail: elane@cca.ufsc.br

The permeate, obtained from the best microfiltration process, was employed in the preparation of fermented milks, without inulin (control) and with 5 g 100g-1 inulin (prebiotic), stored at 5 1 C for 28 days. It could be verified that the storage period and addition of inulin increased the total solids and carbohydrate contents, the caloric

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value and the acidity, and decreased the pH. The addition of inulin resulted in a fermented product with a lower syneresis index, and greater firmness and cohesiveness. The inulin employed resulted in a product with a greater tendency toward a greenish coloration. Keywords: Microfiltration; permeate; fermented milk; prebiotic; inulin.

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2 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Currently, separation processes which employ membranes, such as 1. Introduction

microfiltration (MF), are widely used and mainly by the dairy industry. Among the various applications of MF, is the removal of bacteria (Pafylias, Cheryan, Mehaia, &

Saglam, 1996; Lawrence, Kentish, OConnor, Barber, & Stevens, 2008). Microfiltration produces a permeate, which is the liquid that passes through the membrane (Saboya &

Maubois, 2000) and can be used to prepare dairy products that have distinct properties (Lawrence et al., 2008) and that are thus well accepted by the market (Saboya & Mabouis, 2000). However, the composition and the quality of the permeate depend on factors such as the operational parameters employed during the process, as well the pore size of membrane. Pafylias et al. (1996) reported that larger pore of microfiltration membranes of about 1.4 m pore size, can achieve the right balance between rejection of other milk components, such as the protein, lactose and ash component. In comparison with the conventional thermal treatments of milk, MF has the advantages of requiring lower temperatures, and thus maintaining the structure of casein, which results in a raw material with fewer functional and nutritional modifications (Lawrence et al., 2008). Dairy products, such as cheese and fermented milk, have been successfully prepared by using microfiltration (Saboya & Maubois,

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2000). However, few studies, as that by Debon, Prudncio, and Petrus (2010), have used a functional ingredient, such as inulin, to obtain previously microfiltered fermented milk.

Inulin, which is a fiber that can be extracted from chicory root, for example, and is classified as a prebiotic food ingredient, offers both nutritional and technological benefits when added to the dairy products (Gibson & Fuller, 2000). Studies carried out

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3 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 2.1. Materials 2. Materials and methods by Gibson, Beatty, Wang, and Cummings (1995) showed that regular ingestion of inulin is beneficial to the microbiota of the human intestine. From a technological point of view, inulin can be used as a substitute for fat in milk products (Paseephol, Small, & Sherkat, 2008; Debon et al., 2010) since it forms microcrystals in the presence of milk, resulting in products with a fine and creamy texture (Kaur & Gupta, 2002).

The objective of this study was to use MF to replace conventional thermal treatments of milk to obtain a permeate that is microbiologically and physico-

chemically acceptable for the production of fermented milks with or without the addition of inulin. The products were evaluated for their physico-chemical properties, syneresis index, instrumental texture profile analysis, and color parameters during storage for 28 days at 5 1 C.

Raw skimmed milk, thermophilic milk culture (YC-X11 Yo Flex, Chr. Hansen) composed of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. Thermophilus, saccharose, and inulin (HP-Gel-Beneo, Orafti, Oreye,

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Belgium) were used in the production of fermented milk. All the reagents used were of analytical grade (P.A.).

2.2. Microfiltration process

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4 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 The fermented milks, which were manufactured from the permeate of the best microfiltration process, were produced by using a methodology adapted from the one
VRF = J= VP AP .t

The raw skimmed milk was microfiltered in a pilot unit using an organic poly(imide) membrane (MF-1, PAM Membranas Seletivas, Rio de Janeiro, Brazil), hollow fiber type, with an average pore size of 1.4 m and 0.4 m2 of useful filtering area. In this stage, five experiments were carried out using the following transmembrane pressures and tangential velocities: 100 kPa and 0.8 m s-1, 300 kPa and 0.8 m s-1, 100

kPa and 1.4 m s-1, 300 kPa and 1.4 m s-1, and 200 kPa and 1.2 m s-1. The best microfiltration process for the raw skimmed milk, i.e., with higher permeate flux (J) (L

one selected for the production of the fermented milks. The temperature used in the experiments was 45 1 C. All the experiments were carried out in duplicate. During microfiltration, the J was calculated every five minutes according to Eq. 1.

(L h-1 m-2)

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h-1 m-2), volumetric reduction factor (VRF) and protein content (g 100g-1) VRF, was the

where Vp is the permeate volume collected during the time interval t (h) and Ap (m2) is the membrane surface area of permeation.

The volumetric reduction factor (VRF) was determined as follows (Eq. 2):
initial milk volume (L ) retentate volume ( final ) (L )

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5 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 2.4. Microbiological analysis developed by Almeida, Bonassi, and Roa (2001). The permeate was used to produce two fermented milks, denominated as control (without inulin) and prebiotic (with 5 g 100g-1 of inulin). The permeate samples with and without inulin, at 42 1 C, were both added with 8 g 100g-1 saccharose and the thermophilic milk culture for the fermentation stage. The fermentation was stopped when pH reached between 4.5 and 4.7, and the fermented milks were cooled at 10 1 C, being gently stirred, and then conditioned in

plastic flasks and stored at 5 1 C. The physico-chemical composition, syneresis index, instrumental texture profile analysis (TPA), and color parameters were determined every 7 days for 28 days of storage at 5 1 C. All these evaluations were carried out in triplicate.

Samples of the raw skimmed milk and of the permeate were submitted to mesophilic and psychrophilic bacteria counts, using the values of CFU (Colony Forming Units) mL-1, following the methodology of APHA (2001). The mesophilic and psychrophilic bacteria counts were carried out on the samples stored at 32 C for 48 h and at 7 C for 10 days, respectively.

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2.5. Physico-chemical analysis

The raw skimmed milk, the permeate, and the fermented milks (control and

prebiotic) were analyzed for content of total solids (g 100g-1), through the drying of the samples until reaching constant weight (method 925.23), total proteins (g 100g-1) by the Kjeldahl method (N x 6.38) (method 991.20) and ash (g 100g-1) through a gravimetric

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6 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 method (method 945.46). The lipid content (g 100g-1) of the raw skimmed milk and of the permeate were determined through the Gerber method, while the lipid content of the fermented milks were determined according to the Monjonnier method (method 989.05) (AOAC, 2005). The value for total carbohydrate was obtained by difference. The caloric value for the fermented milks were calculated from the conversion factors,

which were 4.0 kcal g-1 for proteins, 4.0 kcal g-1 for carbohydrates and 9.0 kcal g-1 for lipids. The acidity (% lactic acid) of the fermented milks was determined according to

the methodology described by IAL (2005). The measurements of pH were carried out with a pH meter (MP220, Metler-Toledo, Greinfensee, Switzerland).

2.6. Percent recovered of the permeate components (% Rc)

The percent recovered of the permeate components (% Rc), which indicates how much of a component passes through the membrane in a filtration process, was calculated only for the permeate obtained with the selected variables of the MF process, through Eq. 3.

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2.7. Syneresis index

The syneresis indexes of the two fermented milk samples (control and prebiotic)

were determined according to the method proposed by Farnsworth, Li, Hendricks, and Guo (2006), with modifications. The samples (15 g) were centrifuged at 350 g in a refrigerated centrifuge (5 1 C) (Jaetzki K24, Jena, Germany) for 10 min. The

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7 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 2.8. Instrumental texture profile analysis (TPA) Syneresis (% ) = Supernatant (g) x 100 Fermented milk ( g ) (4) supernatant was collected and weighed, and the syneresis index was calculated through Eq. 4.

The textural properties of the fermented milks were measured using a texturometer model TA-XT2 (Stable Micro System, Texture Expert, Surrey, UK), operating with the Texture Expert software program. The double compression analysis was carried out using a 25 mm-diameter acrylic probe (P25/L). The analysis was performed in a 50 mL aluminum capsule with the samples at a temperature of 5 1 C. The test velocity, the time, and the distance were equal at 2.0 mm.s-1; 5.0 s, and 5.0 mm, respectively. From the TPA curve, the texture parameters obtained were firmness, gumminess, and adhesiveness. Firmness is defined by peak force during the first compression cycle, whereas gumminess is the product of the firmness and the cohesiveness, and adhesiveness is the negative area under the curve obtained between the cycles.

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2.9. Color measurements

The measurements of the color parameters of the fermented milks were carried

out with a previously calibrated colorimeter Minolta Chroma Meter CR-400 (Minolta, Japan), adjusted to operate with a D65 illuminant and an observation angle of 10. Luminosity (L*), red color intensity (a*) and yellow color intensity (b*) were measured.

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8 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 3.1. Microfiltration The data were expressed as means and standard deviation. One-way analyses of
variance (ANOVA) and Tuckeys studentized range (5 g 100g-1 significance) were carried

2.10. Statistical Analysis

out to test any significant differences between the results. The data were obtained using the software STATISTICA version 6.0 (2001) (StatSoft Inc., Tulsa, OK, USA).

3. Results and discussion

There were small variations in the behavior of the permeate flux (J) as a function of time for all of the experiments, as shown in Fig. 1. A similar behavior was observed by Beolchini, Veglio, and Barba (2004) and Akbache et al. (2009) during the first 30 minutes of microfiltration of skimmed sheeps milk (with 0.1 g 100g-1 of lipids) using tubular ceramic membrane, and in the ultrafiltration of whey, using a hollow fiber membrane. According to Nbrega, Borges, and Habert (2005), in the tangential flow there usually is an initial drop in the J values, which later stabilize during the process. In

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this present study, a positive variation in the J was noted, mainly according as the increase in the transmembrane pressure. It was also possible observed that the J of experiment 4 (300 kPa, 1.4 m s-1) was lower than experiment 2 (300 kPa, 0.8 m s-1).

According to Mourouzidis-Mourozidis and Karabelas (2006) this effect is more pronounced at higher filtration pressure (300 kPa). These authors observed that aggregates of measured diameter less than the mean pore size, can infiltrate the internal

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9 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 membrane structure, possibly blocking some passages, moreover we should also take into consideration the aggregates formed. Thus, during filtration, the deposition of casein micelles, the size of which is close to 200 nm (much higher than the size of other skimmed milk constituents, smaller than 4 nm), could lead to the formation of a porous layer becoming a host network for small components (Rabiller-Baudry, Gesan-Guiziou,

Roldan-Calbo, Beaulieu, & Michel, 2005). Mourouzidis-Mourozidis and Karabelas (2006) related that aggregates higher than 1 m can effectively block the pores of

membrane. It is reasonable to assume that protein aggregates can be firmly packed inside membrane pores, also contributing to the decrease of protein content in the permeate when compared with raw skimmed milk. Hwang, Hsu and Tung (2006) cited that an increase in filtration pressure led to a decrease in cake porosity, but to an increase in the mass and average filtration resistance of the cake. However, the mass and porosity of the cake decreased, but the average filtration resistance increased with the increase of the velocity, as observed in present work.

The results for the parameters of the five experiments employed in the MF are shown in Table 1. The best microfiltration process for the raw skimmed milk was that of Experiment 2, where a transmembrane pressure of 300 kPa and tangential velocity of 0.8 m s-1 were employed and whose VRF, J, and protein content were higher. According to Nbrega et al. (2005), separation processes with membranes that use a pressure

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gradient as the driving force will result, within a certain range, in a J directly proportional to the pressure gradient employed. Similar VRF values were detected by Ozer, Robinson, Grandison, and Bell (1998) for cows milk. The J value of best the microfiltration was higher than that obtained by Krstic, Teric, Caric, and Milanovic (2002) (35 L h-1 m-2), who employed a 0.1 m membrane and pressure of 100 kPa. The

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10 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 3.2. Microbiological analysis difference in these values may be related to the average membrane pore diameter and the pressure employed.

The raw milk and the permeate showed mesophilic counts of 4 log CFU mL-1

and 3 log CFU mL-1, respectively. The psychrophilic count for the raw milk was 3 log

Prieto, and Chen (2006) and Lawrence et al. (2008) verified that MF can be used in the separation of microorganisms, and thus reduce the nutrient damage caused by high temperature. According to Tomasula et al. (2011), in practical the use of MF has the potential to serve as a nonthermal intervention to high-temperature/short-time pasteurization to ensure the safety and quality of milk, because it showed efficient removal of the native microorganisms, as a transmission of the casein proteins to permeate. Comparing MF and pasteurization, Walkling-Ribeiro, Rodrguez-Gonzalez, Jayaram and Griffiths (2011) observed a similar reduction of the native microorganism in milk. Pafylias et al. (1996) and Saboya and Maubois (2000) stated that the bacterial decrease in the MF of milk is from 4 to 5 log CFU mL-1, such decrease could be even lower, according to the findings of Trouv et al. (1991), because of a lower bacterial

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contamination initially detected in the raw milk. The bacterial counts (mesophilic and psychrophilic) in the permeate show that it

can be employed in the production of a prebiotic fermented milk, since Angelidis et al. (2006) noted that a count above 6 log CFU mL-1 is indicative of utilization of low-

quality raw material.

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CFU mL-1, whereas for the permeate it was 2 log CFU mL-1. Bansal, Al-Ali, Mercad-

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11 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 The MF of the milk resulted in a permeate with lower contents of total solids (TS) (p < 0.05), proteins, and lipids (Table 2), when compared with the raw skimmed milk. Thus, the reduction in the TS content could be related with the reduction in the lipid and protein contents. 3.3. Physico-chemical analysis

The result obtained for the % Rc of the total proteins, approximately 96 %, is in

agreement with that of the study carried out by Mourouzidis-Mourouzis and Karabelas (2006) on whey. Such authors used a tubular ceramic MF membrane, with a pore

%. These observations are also in agreement with those in previous studies carried out by Guell and Davis (1996) in the tangential MF process, where the retention of proteins was below 15 %. In this present study, the % Rc obtained at the end of the MF process showed that 73 % of the lipids initially contained in the raw skimmed milk were transferred to the permeate.

Through the physico-chemical analysis of the permeate it is possible to note that the pH and acidity values (Table 2) were not affected by the MF. The physico-chemical composition of the permeate was similar to that of the skimmed milk used by Sivieri and Oliveira (2002) in the preparation of lactic beverage with fat substitutes, where the

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pH value was 6.65 and the acidity was 0.197 % lactic acid, whereas the lipid and TS contents were 0.40 g 100g-1 and 8.56 g 100g-1, respectively. The pH and acidity values were also in agreement with those obtained for the pasteurized milks used by Sivieri and Oliveira (2002) (pH = 6.68 and acidity = 0.193 % lactic acid) and by Cunha, Castro, Barreto, Benedet, and Prudncio (2008) (pH = 6.69 and acidity = 0.173 % lactic acid) in the preparation of fermented milks.

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diameter of 0.8 m, where the percent permeability of the proteins was greater than 96

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12 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 Table 3 shows the results of the physico-chemical composition for the two fermented milks (control and prebiotic) during the 28 days of storage. As expected, the addition of inulin increased the TS content, reflecting also in an increase in the total carbohydrates and, consequently, in the caloric value (p < 0.05). During storage the control and prebiotic fermented milk showed an increase (p < 0.05) in the TS content due to moisture loss, which can be attributed to the form of storage employed, i.e., the

use of plastic packaging without a perfect sealing system. For the prebiotic fermented milk, the TS values were similar to those found by Penna, Sivieri, and Oliveira (2001) (19.01 g 100g-1 to 21.71 g 100g-1) for commercial lactic beverage.

The storage period did not influence on (p > 0.05) the protein or lipid contents, neither in the control nor in the prebiotic fermented milk. When the protein and lipid contents of the control and prebiotic fermented milks are compared, it is possible to note (Table 3) that there were no differences (p < 0.05) on days 1 and 28 of storage. The storage period and the addition of inulin did not influence on the ash contents of the fermented milks evaluated (p > 0.05). Moreover, the addition of a prebiotic did not change the ash content in a study carried out by Thamer and Penna (2006) (0.61 g 100g-1), Castro, Cunha, Barreto, Amboni, and Prudncio (2008) (0.66 g 100g-1) and Cunha et al. (2008) (0.65 g 100g-1) in lactic beverages. The fermented milks prepared in this present study showed a decrease (p < 0.05)

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in the pH values on days 1 and 21 for the control and on days 1 and 28 for the prebiotic (Fig. 2). When evaluated on the same day of storage, the pH values for the fermented milks were not different (p > 0.05). A similar behavior was noted by Fuchs, Tanamati, Antonioli, Gasparello, and Doneda (2006) in yogurt containing 5 g 100g-1 oligofructose and 1 g 100g-1 inulin, where the pH values remained around 4.33 and 4.20 on days 1

and 28 of storage, respectively.

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13 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 3.4. Syneresis index For the control fermented milk, the difference in acidity (p < 0.05) was verified from day 7 on, while for the prebiotic fermented milk the increase (p < 0.05) was verified only from day 21 on. However, the acidity remained constant (p > 0.05) until day 28 of storage (Fig. 3). According to Kailasapathy (2006), the decrease in the pH values can occur due to the post-acidification, which is detected in fermented milks stored at temperatures between 0 C and 5 C.

The acidity values were higher (p < 0.05) for the prebiotic fermented milk when

compared with the control, on days 1 and 7 of storage. However, these values were higher than those found by Fuchs et al. (2006) (1.76 %) for probiotic yogurts supplemented with prebiotics.

The syneresis index of fermented milks (control and prebiotic) increased (p < 0.05) on day 7, however, it remained constant (p > 0.05) until day 28 of storage (Fig. 4). As expected, the use of inulin in the preparation of the prebiotic fermented milk contributed to the obtainment of a product with a lower syneresis index (p < 0.05). This decrease may be related to TS present in the prebiotic fermented milk because, according to Lucey (2001), a greater content of TS leads to a lower syneresis index.

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Neven (2001) and Kaur and Gupta (2002) stated that the main use of inulin in fermented milks can be as a substitute for fat. However, Gauche, Tomazi, Barreto, Ogliari, and Bordignon-Luiz (2009) noted a

lower syneresis (22.93 %) index for yogurts, prepared from pasteurized milk, than those verified for the fermented milks produced in this present study. It is important to note that this difference may be associated with the thermal treatment initially applied to the

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14 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 Table 5 shows the parameters L*, a*, and b* for the fermented milks (control and prebiotic) during storage period. On the same day of storage, it was possible to note 3.5. Instrumental texture profile analysis (TPA) milk. These fermented milks (control and prebiotic) were produced from the permeate without undergoing any thermal treatment. According to Varnan and Sutherland (1994) thermal treatments, as the pasteurization employed by Gauche et al. (2009), result in the association of whey proteins with caseins, thus generating a more stable gel with a decrease in syneresis.

No alterations in the firmness, gumminess, or adhesiveness were observed in the control and prebiotic fermented milks during the storage period (Table 4). Also, it was possible to verify that the use of inulin contributed to the obtainment of a prebiotic fermented milk with greater firmness (p < 0.05) than that of the control product. Castro et al. (2008) also verified that the addition of oligofructose at the proportion of 5 g 100g-1 in fermented lactic beverages increased the firmness of the product. Inulin has also contributed to prebiotic fermented milks with higher values (p < 0.05) for adhesiveness on days 1 and 7 of storage. El-Nagar, Clowes, Tudoric, and Kuri (2002) noted an increase in adhesiveness when inulin was used in frozen yogurt and these authors attributed this increase to a higher gel viscosity resulting from this process. This statement is in agreement with that of Neven (2001), who defined inulin

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as a substitute for fat in milk derivatives, contributing to the increase in adhesiveness.

3.6. Color measurements

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15 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 that for the fermented milks only the parameter a* differed (p < 0.05), whereas the parameters L* and b* remained practically unaltered (p > 0.05). The values obtained for the parameter a* indicate that the control fermented milk tends towards a slightly more greenish color than the prebiotic fermented milk, i.e., with the addition of inulin, a reducing sugar. The decrease in the greenish color and the increase in the reddish color may be attributed to the inulin employed associated with the lower moisture content and fermentation temperature. According to Dattatreya and Rankin (2006), factors as the reducing carbohydrates content and the temperature may contribute to maillard reaction, resulting in change in color of dairy products. The results obtained in this present study are in accordance with those reported by Castro et al. (2008), where the supplementation of fermented milk with a prebiotic of white color did not affect the parameters L* and b*.

Moreover, the storage of the fermented milks for 28 days did not influence (p > 0.05) on the parameters L* and a*. However, after 14 days of storage it was possible to verify an increase (p < 0.05) in the yellow coloration (b* values) of the prebiotic fermented milk. This occurrence may be related to the addition of the prebiotic, possibly attributable for the maillard reaction. According to Dattatreya and Rankin (2006), the increase in the parameter b* is related to the intermediate phase of the maillard reaction, where a greater production of yellow compounds occurs, thus confirming the instability

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of the b* values obtained. In relation to the parameters L* and a* during storage, a similar behavior was observed by Dello Staffolo, Bertola, Martino and Bevilacqua (2004) in yogurts added with inulin and stored for 21 days. Therefore, the use of microfiltration as a replacement for the thermal treatment of pasteurization in the preparation of the fermented milks (control and prebiotic) may be attributable for the lower values obtained to parameters a* and b*.

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16 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 The best microfiltration process was when a pressure of 300 kPa and a velocity of 0.8 m s-1 were used, resulting in an average permeate flux of 41.27 L h-1 m-2, a

4. Conclusion

volumetric reduction factor of 4.24, and a protein content of 2.59 g 100g-1. The

permeate showed a reduction in the mesophilic and psychrophilic bacterial counts and in total solids, protein, and lipid contents when compared with the raw skimmed milk. Neither the storage period (28 days) nor addition of inulin affected the lipid, protein, or ash contents of the prebiotic fermented milk. As happened with the control fermented milk, the storage period of 28 days also led to an increase in the total solids, carbohydrate content, caloric value, and of acidity, which consequently decreased the pH of the prebiotic fermented milk.

The addition of inulin resulted in a fermented milk with a lower syneresis index and greater firmness and cohesiveness. There were no modifications in firmness, gumminess, or cohesiveness during the 28 days of storage. The addition of inulin caused the fermented milk to show a lower tendency towards a green coloration, whereas the storage period of the prebiotic fermented milk showed a greater tendency towards a yellow coloration.

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Finally, the results showed that microfiltration can be considered as a good

alternative to obtain a permeate from microfiltration with appropriate characteristics to be employed in obtaining of prebiotic fermented milk with storage stability.

Acknowledgements
The authors wish to thank to Beneo Orafti and Victoria Alimentos Ltda.

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17 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 Akbache, A., Lamiot, ., Moroni, O., Turgeon, S., Gauthier, S. F., & Pouliot, Y. (2009). Use of membrane processing to concentrate TGF-2 and IGF-I from bovine milk and whey. Journal of Membrane Science, 326, 435-440.

References

Almeida, K.E., Bonassi, I.A., & Roa, R.O. (2001). Physical and chemical characteristics of fermented dairy beverages prepared with whey of minas cheese. Cincia e Tecnologia de Alimentos, 21, 187-192.

American Public Health Association (APHA) (2001). Compendium of Methods of the Microbiological Examination of Foods, 4th edn. Washington, DC: APHA. Angelidis, A.S., Chronis, E.N., Papageorgiou, D.K., Kazakis, I.I., Arsenoglou, K.C., & Stathopoulos, G.A. (2006). Non-lactic acid, contaminating microbial flora in readyto-eat foods: A potential food-quality index. Food microbiology, 23, 95-100. Association of Official Analytical Chemists (AOAC) (2005). Official methods of analysis of the association analytical chemists, 18th edn. Maryland, DC: AOAC. Bansal, B., Al-Ali, R., Mercad-Prieto, R., & Chen, X.D. (2006). Rinsing and cleaning -lactalbumin fouled MF membranes. Separation and Purification Technology, 80, 257-266.

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Beolchini, B., Veglio, F., & Barba, D. (2004). Microfiltration of bovine and ovine milk for the reduction of microbial content in a tubular membrane: a preliminary investigation. Desalination, 161, 251-258.

Castro, F.P., Cunha, T.M., Barreto, P.L.M., Amboni, R.D.M.C., & Prudncio, E. S. (2008). Effect of oligofructose incorporation on the properties of fermented probiotic lactic beverages. International Journal of Dairy Technology, 62, 82-74.

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18 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 Cunha, T.M., Castro, F.P., Barreto, P.L.M., Benedet, H.D., & Prudncio, E.S. (2008). Physico-chemical, microbiological and rheological behavior of fermented lactic beverage and fermented milk added of probiotics. Semina: Cincias Agrrias, 29, 103-116. Dattatreya, A., & Rankin, S. A. (2006). Moderately acidic pH potentiates browning of sweet whey powder. International Dairy Journal, 16, 822-828.

Debon, J., Prudncio, E.S., & Petrus, J.C.C. (2010). Rheological and physico-chemical

characterization of prebiotic microfiltered fermented milk. Journal of Food Engineering, 99, 128135

Dello Staffolo, M., Bertola, N., Martino, M., & Bevilacqua, A. (2004). Influence of dietary fiber addition on sensory and rheological properties of yogurt. International Dairy Journal, 14, 263-268.

El-Nagar, G., Clowes, G., Tudoric, C.M., & Kuri, V. (2002). Rheological quality and stability of yog-ice cream with added inulin. International Journal Dairy Technology, 55, 89-93.

Farnsworth, J.P., Li, J., Hendricks, G.M.. & Guo, M.R. (2006). Effects of Transglutaminase treatment on functional properties and probiotic cultures survivability of goat milk yogurt. Small Ruminant Research, 65, 113-121. Fuchs, R.H.B., Tanamati, A.A.C., Antonioli, C.M., Gasparello, E.A., & Doneda, I.

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(2006). Utilization of Lactobacillus casei and cultura starter in obtaining of yogurt

supplemented with inulin and oligofructose. Boletim CEPPA, 24, 83-98.

Gauche, C., Tomazi, T., Barreto, P.L.M., Ogliari, P.J., & Bordignon-Luiz, M.T. (2009). Physical properties of yogurt manufactured whit whey and transglutaminase. LWT Food Science and Technology, 42, 239-243.

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19 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 Gibson, G.R., Beatty, E.R., Wang, X., & Cummings, J. H. (1995). Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology, 108, 975-982. Gibson, G.R., & Fuller, R. (2000). Aspects of in vitro and in vivo research approaches directed toward identifying probiotics and prebiotics for human use. Journal of Nutrition, 130, 391S-5S.

Guell, C., & Davis, R.H. (1996). Membrane fouling during microfiltration of protein mixtures. Journal of Membrane Science, 119, 269-284.

Hwang, K.J., Hsu, Y.L., & Tung, K.L. (2006). Effect of particle size on the performance of cross-flow microfiltration. Advanced Powder Technology, 17, 189206.

Instituto Adolfo Lutz (IAL) (2005). Normas Analticas do Instituto Adolfo Lutz, 4th edn. So Paulo, DC: IAL.

Kailasapathy, K. (2006). Survival of free and encapsulated probiotic bacteria and their effect on the sensory properties of yoghurt. LWT-Food Science and Technology, 39, 1221-1227.

Kaur, N., & Gupta, A.K. (2002). Applications of inulin and oligofructose in health and nutrition. Journal of Biosciences, 27, 703-714. Krstic, D.M., Teric, M.N., Caric, M.D., & Milanovic, S.D. (2002). The effects of

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turbulence promoter on cross-flow microfiltration of skim milk. Journal of

Membrane Science, 208, 303-314.

Lawrence, N.D., Kentish, S.E., OConnor, A.J., Barber, A.R., & Stevens, G.W. (2008). Microfiltration of skim milk using polymeric membranes for casein concentrate manufacture. Separation and Purification Technology, 60, 127-244.

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20 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 Lucey, J.A. (2001). The relationship between rheological parameters and whey separation in milk gels. Food Hydrocolloids, 15, 603-608. Mourouzidis-Mourouzis, S.A., & Karabelas, A.J. (2006). Whey protein fouling of microfiltration ceramic membranes-pressure effects. Journal of Membrane Science, 282, 124-132.

Neven, E. (2001). Inulin and oligofructose - multifunctional ingredients for the development of dairy products. Revista Leite e Derivados, 11, 32-37.

Nbrega, R., Borges, C.P., & Habert, A.C. (2005). Membrane separation processes. In: Purification of biotech products (edited by A. P. Junior and B. V. Kilikian). So Paulo, SP: Manole.

Ozer, B.H., Robinson, R.K., Grandison, A.S., & Bell, A.E. (1998). Gelation properties of Milk concentrated by different techniques. International Dairy Journal, 8, 793799.

Pafylias, I., Cheryan, M., Mehaia, M.A., & Saglam, N. (1996). Microfiltration of Milk with ceramic membranes. Food Research International, 29, 141-146. Paseephol, T., Small, D.M., & Sherkat, F. (2008). Rheology and texture of set yogurt as affected by inulin addition. Journal of Texture Studies, 39, 617-634. Penna, A.L.B., Sivieri, K., & Oliveira, M.N. (2001). Relation between quality and rheological properties of lactic beverages. Journal of Food Engineering, 49, 7-13.

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Rabiller-Baudry, M., Gesan-Guiziou, G., Roldan-Calbo, D., Beaulieu, S., & Michel F. (2005). Limiting flux in skimmed milk ultrafiltration: impact of electrostatic repulsion due to casein micelles. Desalination, 175, 49-59.

Saboya, L.V., & Maubois, J.L. (2000). Current developments of microfiltration technology in the dairy industry. Lait, 80, 541-553.

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21 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 Sivieri, K., & Oliveira, M.N. (2002). Evaluation of shelf life of lactic beverages prepared with fat replacers (Litesse and Dairy-Lo). Cincia e Tecnologia de Alimentos, 22, 24-31. Statsoft Inc. (2001). STATISTICA (data analysis system) version 6.0. Tulsa, OK: StatSoft Inc.

Thamer, K.G., & Penna, A.L.B. (2006). Characterization of functional lactic beverage fermented by probiotics and added of prebiotic. Cincia e Tecnologia de Alimentos, 26, 589-595.

Tomasula, P.M., Mukhopadhyay, S., Datta, N., Porto-Fett et al. (2011). Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk. Journal Dairy Science, 94, 4277-4291.

Trouv, E., Maubois, J.L., Piof, M.M.N., Fauquant, J., Rouault, A., Tabard, J., et al. (1991). Retention de differentes espces microbiennes lors de lpuration du lait par microfiltration en flux tangentiel. Lait, 71, l-13.

Varnan, A.H., & Sutherland, J.P. (1994). Milk and dairy products: technology, chemistry and microbiology. Zaragoza: Acribia. Walkling-Ribeiro, M., Rodrguez-Gonzalez, O., Jayaram, S., & Griffiths, M.W. (2011). Microbial inactivation and shelf life comparison of cold hurdle processing with pulsed electric fields and microfiltration, and conventional thermal pasteurisation in

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skim milk. International Journal of Food Microbiology, 144, 379-386.

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- Storage stability of prebiotic fermented milk obtained from permeate. - Evaluation of the permeate obtained from the microfiltration process. - Preparation of prebiotic fermented milk obtained from permeate. - Evaluation of fermented milks without and with the addition of inulin. - The use of microfiltration to replace conventional thermal milk treatments.

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22 521 522 523 524 Table 1 Results of permeate flux (J), volumetric reduction factor (VRF) and protein content of the permeate during the microfiltration process with different pressure and tangential velocity EXPERIMENT J (L h-1 m-2) 1 2 3 4 5 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 VRF PROTEIN CONTENT (g 100g-1) 1.68 0.01 2.59 0.01 1.67 0.01 2.46 0.00 2.10 0.00

16.79 0.01 2.26 0.01 41.27 0.01 4.24 0.01 15.93 0.01 2.55 0.01 29.41 0.01 2.58 0.01 24.72 0.02 3.41 0.01

Results expressed as mean standard deviation (n = 2)

Experiment 1: Pressure = 1 bar and tangential velocity = 0.8 m s-1, during 30 minutes ; experiment 2 = 3 bar and 0.8 m s-1, during 25 minutes; experiment 3: 1 bar and 1.4 m s-1, during 25 minutes; experiment 4: 3 bar and 1.4 m s-1, during 25 minutes; experiment 5: 2 bar and 1.2 m s-1, during 30 minutes

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23 541 542 543 Table 2 Physico-chemical composition of the raw skimmed milk and of the permeate obtained of microfiltration process with pressure of 3 bar and tangential velocity of 0.8 m s-1 RAW SKIMMED MILK Proteins (g 100g-1) Lipids (g 100g-1) Total solids (TS) (g 100g-1) Ash (g 100g-1) Carbohydrates (g 100g-1) pH Acidity (% lactic acid) 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 3.27a 0.01 0.55a 0.07 8.80a 0.02 0.74a 0.01 4.23a 0.04 6.66a 0.00 PERMEATE

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0.195a 0.007

Results expressed as mean standard deviation (n = 3)

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Within a line, different superscript lowercase letters denote significant differences (p < 0.05) between

the samples (raw skimmed milk and permeate)

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3.13b 0.01 0.40b 0.01 8.41b 0.03 0.72a 0.01 4.15a 0.01 6.67a 0.00 0.185a 0.007

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24

FERMENTED MILK

DAY

TOTAL SOLIDS (g 100g-1)

PROTEINS (g 100g-1) 2.78Aa 0.02 2.82Aa 0.02 2.84Aa 0.02 2.84Aa 0.03 2.85Aa 0.03 2.72Aa 0.03 2.72Ba 0.03 2.69Ba 0.03

LIPIDS (g 100g-1) 0.34Aa 0.03 0.35Aa 0.02 0.36Aa 0.02 0.37Aa 0.03 0.37Aa 0.02 0.30Aa 0.03 0.33Aa 0.01 0.33Aa 0.04

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ASH (g 100g-1)

559 560

Table 3 Composition of the control (without inulin) and prebiotic fermented milk (with inulin) during 28 days of storage at 5 1 C
CARBOHYDRATES (g 100g-1) 10.75Aa 0.04 10.89Ab 0.03 10.89Ab 0.03 11.34Ac 0.02 11.34Ac 0.04 14.71Ba 0.03 14.76Ba 0.04 14.78Ba 0.04 15.26Bb 0.04 15.25Bb 0.03 CALORIC VALUE (kcal g-1) 57.44Aa 0.23 57.99Aa 0.18 58.16Aa 0.01 60.05Ab 0.48 60.09Ab 0.18 72.42Ba 0.03 72.89Ba 0.54 72.85Ba 0.03 75.06Bb 0.19 75.15Bb 0.07

1 7 CONTROL 14 21 28 1 7 PREBIOTIC 14 21 28

14.50Aa 0.03 14.69Ab 0.03 14.71Ab 0.04 15.20Ac 0.04 15.22Ac 0.03 18.39Ba 0.08 18.49Ba 0.05 18.47Ba 0.03 19.01Bb 0.06 19.04Bb 0.04

0.63Aa 0.03 0.63Aa 0.01 0.62Aa 0.04 0.65Aa 0.03 0.66Aa 0.04 0.66Aa 0.03 0.68Aa 0.03 0.67Aa 0.04 0.67Aa 0.02 0.69Aa 0.01

2.74Aa 0.03 2.75Aa 0.01

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561 562 563 564

Results expressed as mean standard deviation (n = 3)

A,B,C

Within a column, different superscript uppercase letters denote significant differences (p < 0.05) among the different fermented milk (without and with inulin) for the

same storage period

a,b,c

Within a column, different superscript lowercase letters denote significant differences (p < 0.05) among the different storage day, for each sample

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0.34Aa 0.03 0.35Aa 0.01

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25 565 566 567 Table 4 Results of the instrumental texture profile (TPA) analysis of the control (without inulin) and prebiotic fermented milk (with inulin) during 28 days of storage at 5 1 C
FERMENTED MILK 1 7 CONTROL 14 21 28 1 7 PREBIOTIC 14 21 27.13Aa 1.71 30.10Ab 0.62 24.40Aa 0.85 26.50Aa 0.69 25.75Aa 0.07 36.10Ba 1.30 9.92Aab 0.37 10.40Ab 0.39 8.92Aa 0.69

9.87Aab 0.27 9.21Aab 0.42

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10.83Aa 0.47 10.05Aa 1.44 10.29Aa 0.10 10.29Aa 0.51 10.16Aa 0.82

35.20Bba 1.84 31.50Bb 1.27

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32.87Bba 1.23

34.65Bba 1.48

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568 569 570 571 572 573 574 575 576 577

Results expressed as mean standard deviation (n = 3)

A,B,C

Within a column, different superscript uppercase letters denote significant differences (p < 0.05)

among the different fermented milk (without and with inulin) for the same storage period
a,b,c

Within a column, different superscript lowercase letters denote significant differences (p < 0.05)

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among the different storage day, for each sample

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- 27.05Aa 2.29 - 26.99Aa 1.20 - 41.87Ab 8.26 - 31.84Aab 2.43 - 38.56Aab 8.57 - 48.52Ba 1.77 - 43.81Bba 5.26 - 45.60Aba 6.32 - 36.24Ab 1.61 - 42.91Aba 1.41

DAY

FIRMNESS (g)

GUMMINESS (g)

ADHESIVENESS (g.s)

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26 578 579 580 Table 5 Color parameters L*, a* and b* of the control (without inluin) and prebiotic fermented milk (with inulin) samples during 28 days of storage at 5 1 C
L* DAY CONTROL 1 7 14 21 28 48.59Aa1.86 50.12Aa0.51 49.07Aa0.16 49.35Aa1.36 50.09Aa0.92 PREBIOTIC 50.46Aa2.26 51.09Aa1.29 49.82Aa0.48 49.51Aa1.24 50.57Aa0.48 a* CONTROL PREBIOTIC -2.44Aa0.11 -2.12Ba0.02 -2.43Aa0.05 -2.11Ba0.03 -2.45Aa0.01 -2.08Ba0.03 -2.50Aa0.02 -2.10Ba0.01 -2.36Aa0.03 -2.02Ba0.02 b* CONTROL PREBIOTIC 6.12Aa0.14 6.20Aa0.02 6.32Aa0.09 6.09Aa0.06 6.29Aa0.06 5.97Aa0.11 6.11Aa0.02

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581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596

Results expressed as mean standard deviation (n = 3)

A,B

Within a line, for each color parameter evaluated, different superscript uppercase letters denote

significant differences (p < 0.05) among the different fermented milk (without and with inulin) for the same storage period
a,b

Within a column, different superscript lowercase letters denote significant differences (p < 0.05)

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6.24Ab0.13 6.25Bb0.04 6.28Ab0.04

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27 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621

Fig. 1. Average permeate flux (J) of milk submitted to microfiltration in different process conditions.

P = 200 kPa, v = 1.2 m s-1; s-1.

P = 300 kPa, v = 0.8 m s-1;

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P = 100 kPa, v = 0.8 m s-1;

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P = 100kPa, v = 1.4 m s-1; P = 300 kPa, v = 1.4 m

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28 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647

Fig. 2. Results of the average(standard deviation) pH during 28 days of storage (5 1 C), of fermented milks (control and prebiotic). Prebiotic fermented milk.
A,B

Different superscript uppercase letters denote significant differences (p < 0.05) among the different

storage day, for each sample

a,b

Different superscript lowercase letters denote significant differences (p < 0.05) among the different

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Control fermented milk;

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29 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673
A,B

Fig. 3. Results of the average acidity(standard deviation) (% lactic acid), during 28 days of storage (5 1 C), of fermented milks (control and prebiotic). Control fermented milk;

Different superscript uppercase letters denote significant differences (p < 0.05) among the different

storage day, for each sample

a,b

fermented milk (without and with inulin) for the same storage period

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Prebiotic fermented milk.

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30 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699

Fig. 4. Results of the average standard deviation (

milks (control and prebiotic), during 28 days of storage at 5 1 C. Milk; Prebiotic fermented milk.
A,B

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a,b

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fermented milk (without and with inulin) for the same storage period

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) syneresis index of fermented Control fermented

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