Inulin and oligofructose improve sensory quality and increase the probiotic viable count in potentially synbiotic petit-suisse cheese

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LWT 41 (2008) 1037–1046 www.elsevier.com/locate/lwt

Inulin and oligofructose improve sensory quality and increase the probiotic viable count in potentially synbiotic petit-suisse cheese Haı´ ssa R. Cardarellia, Fla´via C.A. Buritia, Inar A. Castrob, Susana M.I. Saada, a

Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 580, B16, 05508-000 Sa˜o Paulo, SP, Brazil b Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 580, B14, 05508-000 Sa˜o Paulo, SP, Brazil Received 23 January 2007; received in revised form 28 June 2007; accepted 3 July 2007

Abstract The influence of inulin, oligofructose and oligosaccharides from honey, combined in different proportions, on the consumers’ sensory acceptance, probiotic viable count and fructan content of novel potentially synbiotic petit-suisse cheeses was investigated. Probiotic populations varied from 7.20 up to 7.69 log cfu g1 (Bifidobacterium animalis subsp. lactis) and from 6.08 up to 6.99 log cfu g1 (Lactobacillus acidophilus). The highest fructan contents were achieved by the cheese trials containing oligofructose and/or inulin (above 8.90 g 100 g1). The control trial showed the lowest mean acceptance (6.63) after 28 days of refrigerated storage, whereas the highest acceptance (7.43) was observed for the trial containing 10 g 100 g1 oligofructose. Acceptance increased significantly during storage (Po0.05) only for cheeses supplemented with oligofructose and/or inulin. Cheeses containing honey did not perform well enough compared to the cheeses with addition of inulin and/or oligofructose, and the best synbiotic petit-suisse cheese considering sensory and technological functional features was that containing oligofructose and inulin combined, therefore encouraging the commercial product use. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Synbiotic; Petit-suisse cheese; Probiotics; Prebiotics; Sensory evaluation

1. Introduction As nutrition is moving towards the use of foods to promote better health and well-being, functional foods become more important for consumers. Probiotic and prebiotic foods, functional foods which are less familiar to consumers, have little known about their acceptance (Luckow & Delahunty, 2004). They may be combined in a food product, called synbiotic (Holzapfel & Schillinger, 2002), when in addition to directly introducing live beneficial bacteria to the colon, there is an increase in the number of beneficial Bifidobacterium and Lactobacillus species in the intestinal microbiota through the use of a prebiotic—‘a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in Corresponding author. Tel.: +55 11 30912378; fax: +55 11 3815 6386.

E-mail address: [email protected] (S.M.I. Saad).

the gastrointestinal microbiota that confers benefits upon host wellbeing and health’ (Gibson, Probert, Van Loo, Rastall, & Roberfroid, 2004). Inulin and oligofructose, non-digestible fermentable fructans, are amongst the most studied and well established prebiotics (Gibson et al., 2004) while oligosaccharides from honey may serve as prebiotic agents and also suppress potentially deleterious bacteria among the gastrointestinal microbiota (Kajiwara, Gandhi, & Ustunol, 2002; Shamala, Jyothi, & Saibaba, 2000). Moreover, Bifidobacterium and Lactobacillus species are widespread applied probiotics, particularly within fermented dairy products like cheese (Boylston, Vinderola, Ghoddusi, & Reinheimer, 2004; Buriti, Rocha, Assis, & Saad, 2005a; Maruyama, Cardarelli, Buriti, & Saad, 2006). Quarg or quark cheese, originally from Eastern and Central Europe, is characterised as a white skimmed soft curd, unripened, with weak acidic taste, and may be used to

0023-6438/$34.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.07.001

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make various types of cheese, depending on the other ingredients added (e.g. flavoured cheese or petit-suisse cheese) (Morgado & Branda˜o, 1998). In Brazil, the petitsuisse cheese is consumed as a pleasantly accepted dessert designed to target children consumers (Veiga, Cunha, Viotto, & Petenate, 2000). Data about synbiotic quark or petit-suisse cheeses are not existent and the development of such a novel functional dairy product combining pro- and prebiotic ingredients may be a good alternative of healthy food. Additionally, the sensory impact of probiotic and prebiotic ingredients incorporated into cheeses has also not been very extensively studied. However, it has been assumed that products containing these ingredients have different taste profiles, when compared to conventional, non-functional products (Mattila-Sandholm et al., 1999). Studies have shown that taste is the primary factor in food selection, followed by health considerations (Tepper & Trail, 1998; Tuorila & Cardello, 2002). Consumers emphasise sensory experiences during consumption (e.g., appearance, texture, aroma and taste) with the pleasure derived from consumption as an important motivator in eating (Westenhoefer & Pudel, 1993). The possibility of producing a petit-suisse cheese capable of presenting a potentially synbiotic effect, due to the incorporation of the probiotics Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and the prebiotics inulin, oligofructose or oligosaccharides from honey is indeed promising. Furthermore, the consumers’ opinion is of great importance when developing a new food product and consumer liking is the key to placing a product successfully on the market (Bolenz, Thiessenhusen, & Scha¨pe, 2003). Keeping in mind the importance of sensory features for new food products, the influence of the prebiotic ingredients inulin, oligofructose and oligosaccharides from honey on sensory acceptance, probiotic viable count and fructan contents of potentially probiotic petit-suisse cheese during storage of the product at 471 1C for up to 28 days was investigated. 2. Materials and methods 2.1. Petit-suisse manufacture Eight pilot-scale petit-suisse cheese-making trials (seven trials plus one control, denoted T1–T7 and T8, respectively) were performed, all of them using Streptococcus thermophilus (TA 040, Danisco, Dange´, France) as starter culture, the potentially probiotic cultures of L. acidophilus (Lac4, Ezal Dried, Danisco), and of B. animalis subsp. lactis (BL04, FloraFit, Danisco) plus different combinations of three prebiotic ingredients: the prebiotic fibre inulin (BeneoTM ST, Orafti, Oreye, Belgium); the prebiotic fibre oligofructose (BeneoTM P95, Orafti) and oligosaccharides from honey (eucalyptus honey, Biosciences Institute-University of Sa˜o Paulo, Sa˜o Paulo, Brazil) during the production of cheeses. The ingredients used

for the production of the cheese formulations are described in Table 1. Cheese-base (quark cheese) for subsequent mixture with the other ingredients described in Table 1 was manufactured in 10 L vats from commercial pasteurised skimmed milk (Salute, Descalvado, Brazil; high temperature short time [HTST]) heated to 37–38 1C, after which addition of cultures proceeded. All cultures employed were freeze-dried commercial cultures for direct vat inoculation (DVS) and they were added at 30 mg L1 (starter culture and L. acidophilus) and at 20 mg L1 (B. animalis subsp. lactis). Calcium chloride (0.25 g L1) was also added in all trials. In the next step, all vats were allowed to set at 38 1C. As soon as pH reached values of about 6.3–6.4, commercial rennet Ha-la (88–92% bovine pepsin+8.0–12.5% bovine chymosin; Christian Hansen, Valinhos, Brazil, 50 mg L1) was added to the cheese-milk, which was allowed to set again until a curd was formed and the pH reached values of about 5.5–5.7. The gel was gently cut into cubes (20  20  20 mm), placed in sterilised cotton cheesecloth, and allowed to drain at 15 1C for 15 h. After draining, the cheese-base was placed in sterilised beakers, covered with a PVC film and stored at 471 1C, until the homogenisation stage (Universal Mixer Machine, model UMM/SK-12 pilot-scale, 12 kg capacity, Geiger, Pinhais, Brazil, 20 1C/ 1 atm) would take place, after the addition of the other ingredients: commercial sucrose (Unia˜o, Limeira, Brazil); commercial sterilised milk cream (25% lipids, Nestle´, Arac- atuba, Brazil); natural colouring agent (Plury Quı´ mica, Diadema, Brazil); artificial strawberry flavour (Mylner, Sa˜o Paulo, Brazil); pasteurised strawberry whole pulp (Maisa, Mossoro´, Brazil); stabilisers and emulsifiers xanthan gum (Rhodigel 80, Rhodia, Melle, France), powdered unflavoured jelly (Oetker, Amparo, Brazil) and the prebiotics (Table 1). The quantities of the ingredients added were determined according to the limits established by the Brazilian regulatory standards for petitsuisse cheese (moisture 455 g 100 g1, non-dairy ingredients p30 g 100 g1, dairy protein X6 g 100 g1) in the ready-to-eat product (Brasil, 2000). During homogenisation, a smooth and homogeneous cream was formed. Table 1 Ingredients used for the production of the petit-suisse cheese trials studied (trials T1–T8 according to the experimental design described in Table 2) Ingredients (g 100 g1) 1. 2. 3. 4. 5. 6. 7. 8. 9.

Quark cheese (cheese-base) Strawberry pulp Sucrose Cream milk Unflavoured powdered jelly Xanthan gum Natural colouring agent Artificial strawberry flavour Prebiotic mixture

Total

T1–T7

T8 (control)

57.510 9.900 9.000 12.600 0.450 0.450 0.054 0.034 10.000

63.900 11.000 10.000 14.000 0.500 0.500 0.060 0.040 0.000

100.000

100.000

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After homogenisation, cheeses were packaged in individual plastic pots, each one containing 35 g of cheese, sealed with an aluminium cover, and stored under refrigeration (471 1C) for up to 28 days. 2.2. Experimental design Eight trials were prepared according to Table 2, using a centroid simplex design (Box & Draper, 1987), changing the oligofructose, inulin and oligosaccharides from honey proportions in the cheeses. 2.3. Physical–chemical compositional analysis and fructan content Moisture, ash, fat and protein contents for all trials of petit-suisse cheese studied were determined after one day of storage at 471 1C on triplicate freeze-dried and grated samples. Moisture content was determined from 5 g samples by oven drying at 70 1C under vacuum (Marconi MA030112, Piracicaba, Brazil) for 24 h. Ash was determined gravimetrically by heating the 2 g sample at 550 1C, until completely ashed (muffle furnace, mod. 1207, Forlabo, Sa˜o Paulo, Brazil) for 5 up to 6 h. Protein was estimated by measuring the nitrogen content of cheeses by Kjeldahl method and multiplying by a conversion factor (6.38), after drying 5 g of cheese samples. Fat was determined by extraction of lipids with ethyl ether, using the Soxhlet device. Analytical procedures followed the appropriate standard methods (Instituto Adolfo Lutz, 1985). The fructan contents were determined for petit-suisse cheeses T1–T7, employing the ion-exchange chromatographic method, based on 997.08 AOAC method, described by Hoebregs (1997). The fructan content in BeneoTM ST fibre (standard) was also determined simultaneously with the samples of petit-suisse cheeses. The enzyme Fructozymes L (Novozymes, Bagsvaerd, Denmark) was employed for enzymatic hydrolysis of samples. Table 2 Experimental design ‘‘centroid simplex’’ employed in the present study Trials

T1 T2 T3 T4 T5 T6 T7 T8

Proportion of each ingredient in the mixture (x1, x2, x3)

Quantities of each ingredient (g) in 100 g of petit-suisse cheesea Oligofructose (x1)

Inulin (x2)

Honey (x3)

(1,0,0) (0,1,0) (0,0,1) (1/2, 1/2, 0) (1/2, 0, 1/2) (0, 1/2, 1/2) (1/3, 1/3, 1/3) (0, 0, 0)

10 0 0 5 5 0 3.33 –

0 10 0 5 0 5 3.33 –

0 0 10 0 5 5 3.33 –

– ¼ ingredients not added (T8—control trial). a 10 g of the total prebiotic mixture was added to the other ingredients used in the petit-suisse cheese formulations.

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For this purpose, a Dionexs HPLC System chromatograph, equipped with an automatic injector A550, a pump EGP40, a pulsed electrochemical detector ED40, and a Carbopac PA1 column with a guard column (Dionex Corporation, Sunnyvale, USA) was used. The gradient employed started with 40% H2O and 60% NaOH 0.018 mol L1, reaching 90% NaOH 0.018 mol L1 after 15 min and 100% 0.018 mol L1 after 25 min. The volume of injections was 25 mL. Determinations were carried out after one and 28 days of storage at 471 1C on triplicate samples. The pH values of cheeses were determined on triplicate samples with a pH meter Analyser Model 300M (Analyser, Sa˜o Paulo, Brazil) equipped with a penetration electrode model 2AO4 GF (Analyser) after 1, 7, 14, 21 and 28 days of storage at 471 1C. 2.4. Microbiological analysis Counts of L. acidophilus, B. animalis subsp. lactis and S. thermophilus were monitored during the storage period (1–28 days) for all trials. For microbiological analysis, 25 g portions of duplicate cheese samples were blended with 225 mL of 0.1 g 100 mL1 peptone water in a Bag Mixer 400 (Interscience, St. Nom, France) and submitted to serial dilutions with the same diluent. L. acidophilus was counted by pour-plating 1 mL of each dilution in DeMan-Rogosa-Sharpe agar (MRS agar) modified by the substitution of glucose for maltose as the main carbohydrate source, as described by the International Dairy Federation (IDF Standard 306, 1995), after 3 days of anaerobic incubation (Anaerobic System Anaerogen, Oxoid, Basingstoke, UK) at 37 1C. B. animalis subsp. lactis was counted by pour-plating 1 mL of each dilution in DeMan-Rogosa-Sharpe agar (MRS agar, Oxoid) to which sodium propionate (0.3 g 100 mL1) and lithium chloride (0.2 g 100 mL1) solutions were added, after 3 days of anaerobic incubation (Anaerobic System Anaerogen, Oxoid) at 37 1C (Vinderola & Reinheimer, 1999). S. thermophilus was counted by pour-plating 1 mL of each dilution in M17 agar (Oxoid) containing 10 g 100 mL1 lactose, followed by incubation at 37 1C (Richter & Vedamuthu, 2001), for 48 h. 2.5. Sensory analysis The sensory evaluation was conducted under controlled conditions, in individual booths. According to the experimental design (Table 2), 8 trials were evaluated during the storage time (7, 14, 21 and 28 days). A balanced incomplete block design (t ¼ 8, l ¼ 1, k ¼ 2, r ¼ 7) was applied in this study (Hinkelmann & Kempthorne, 1994). By this procedure, the 8 samples (t ¼ 8) were divided in 28 blocks and each sample was presented in 7 blocks (r ¼ 7). All 28 blocks, containing 2 samples (k ¼ 2) presented in both positions (AB and BA), were repeated 5 times for each storage period (28  5  4 ¼ 560 assays).

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using non-parametric analysis of variance and Friedman test and LSD rank for the identification of contrasts (Po0.05). The sensory attributes proportion (taste, appearance, texture and aroma) was compared using X2 test. T-test was applied to compare microbial populations one another and also fructan contents one another at different storage periods (after 1 and 28 days of storage) for each trial. Data were analysed using the STATISTICA 7.0 software (Statsoft, Tulsa, USA).

A total of 560 panellists were recruited from students and staff of the University of Sa˜o Paulo via internal advertisements. Each panellist was asked to taste no more than two samples of different trials. Some panellists were present in more than one session because they were available during the experimental period. Panellists ranged in age between 18 and 60 years old, and 55.2% were female. All the panellists were healthy and accepted to taste the samples before the tests, attesting they were fond of the type of product being tested, had petit-suisse cheese consuming habits and did not have any allergy or intolerance problems consuming any of the ingredients present in the cheeses. The study was approved by the University of Sa˜o Paulo-Faculty of Pharmaceutical Sciences Ethics Research Committee. The product samples were served in blind plastic pots (identification consisted only of a three digit random code number), containing a 20 g serving sample. The samples were stored in a refrigerator (471 1C) and directly served from there. The panellists were asked to visually examine, taste the cheese samples and express their opinion by a nine-point hedonic scale, where ‘‘one’’ represented ‘‘dislike extremely’’, ‘‘five’’, ‘‘neither like nor dislike’’ and ‘‘nine’’, ‘‘like extremely’’ (Stone & Sidel, 1993). The panellists were also asked to answer what attribute they liked most and least.

3. Results and discussion 3.1. Physical–chemical composition and fructan content The variations in chemical composition observed for the petit-suisse cheese trials are directly related to the differences in formulation between the various test trials and the control cheese (T8), especially in moisture and total carbohydrate contents, including fructan content (Table 3). The fructan addition to each trial was first established as a function of the expected real human consumption of petit-suisse cheese, at least 100 g day1. The purpose was to enrich a potentially probiotic petit-suisse cheese with the prebiotic ingredients inulin, oligofructose and/or oligosaccharides from honey, thus providing a food with added benefits for human health (potentially synbiotic product). Individual cheese trials presented distinct fructan contents (Po0.05) (Table 4). Maximum fructans contents were obtained for trials T1, T2 and T4 (above 8.90 g 100 g1). For the other cheeses, the fructan contents were above 4.80 g 100 g1, except for cheese T3 (0.43–0.47 g 100 g1). There is no consensus between the researchers considering the minimum effective fructan content for promoting better probiotics viability and, therefore, fulfilling the prebiotic concept. Significant changes in the human faecal microbiota led to the conclusion that chicory inulin and oligofructose (3–15 g day1 for a number of weeks) are prebiotic (Gibson, 1999; Rao, 1999; Roberfroid, Van Loo, & Gibson, 1998). Accordingly, Coussement (1999) recommended up to 10 g inulin per food portion. Moreover, 4–5 g day1 of fructans are considered efficient to stimulate bifidobacterial multiplication (Manning & Gibson, 2004; Rao, 2001; Roberfroid, 1999). Based on these studies, we

2.6. Statistical analysis The effect of the different trials of petit-suisse cheese observed for each time (7, 14, 21 and 28 days of storage) was assessed using ANOVA followed by post-hoc Tukey test, when a homogenous variance was observed, according to the Levene test. Results showing non-homogenous variance were analysed using non-parametric analysis of variance, multiple comparisons based on Kruskal–Wallis test. A value a of 0.05 was adopted in this study. The effect of the different storage periods (7, 14, 21 and 28 days for sensory analysis and also 1 day for the determination of pH) for each trial was assessed using Repeated Measures Analysis of Variance, followed by Tukey test for results with homogenous variance (Po0.05). Results showing non-homogenous variance were analysed

Table 3 Chemical composition of the petit-suisse cheese trials studieda (see Table 2 for description of trials T1–T8) g 100 g1

Cheeses T1

Ash Fat Protein Total carbohydratesb Moisture a

0.81 3.72 9.21 24.72 61.54

T2 [0.01] [0.05] [0.01] [0.13] [0.28]

0.80 3.67 8.89 24.23 62.41

T3 [0.03] [0.03] [0.13] [0.25] [0.10]

0.90 3.49 9.24 24.82 61.65

T4 [0.01] [0.11] [0.13] [0.06] [0.14]

Mean [SD] (n ¼ 3). Values obtained by difference [100(ash+fat+protein+moisture)].

b

0.80 3.73 9.17 24.10 62.20

T5 [0.01] [0.02] [0.02] [0.21] [0.19]

0.87 3.54 9.29 24.58 61.72

T6 [0.02] [0.08] [0.10] [0.29] [0.20]

0.83 3.48 9.31 24.40 61.98

T7 [0.01] [0.04] [0.11] [0.31] [0.13]

0.83 3.54 9.55 25.18 60.90

T8 [0.01] [0.06] [0.30] [0.41] [0.08]

0.90 3.83 9.93 18.53 66.81

[0.03] [0.02] [0.03] [0.00] [0.21]

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conclude that all trials evaluated in this study may be considered prebiotic, except for T3, containing only 0.47 g 100 g1 of petit-suisse cheese, which is due to the low oligosaccharides proportion of honey (4–5% according to Kajiwara et al., 2002). Concerning the achievement of high fructan contents, which may promote intensified prebiotic effects, the trials involving inulin and oligofructose, either alone or combined (T1, T2 and T4) were observed to be the most promising ones. A reduction in pH values, commonly observed in cheeses and other fermented dairy products, is a natural process caused by the continuous production of lactic acid and other organic acids from lactose fermentation by the starter and the probiotic cultures (Buriti, Rocha, & Saad, 2005b; Goncu & Alpkent, 2005; Kristo, Biliaderis, & Tzanetakis, 2003) and may reflect over sensory and technological features of the product (Fox, Guinee, Cogan, & McSweeney, 2000; Shah, 2000). Accordingly, in the present study

Table 4 Fructan contents of the different petit-suisse cheese trials studied during storage at 471 1C1 (see Table 2 for description of trials T1–T8) Cheese trials

Times of storage (days) 1

T1 T2 T3 T4 T5 T6 T7

9.05 9.20 0.47 9.09 4.97 4.99 6.52

28 dA

9.00 8.95 0.43 8.93 4.95 4.80 6.52

[0.04] [0.02]dB [0.02]aA [0.07]dB [0.02]bA [0.04]bB [0.05]cA

[0.09]dA [0.05]dA [0.08]aA [0.01]dA [0.04]bA [0.08]bA [0.05]cA

A,B Data that share a common capital letter in the same row do not differ significantly from one another during the storage times for the same trial (P40.05), a,bData that share a common lowercase letter in the same column do not differ significantly between trials for the same day (P40.05). 1 Mean [SD] g 100 g1 (n ¼ 3).

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the pH decreased significantly during storage for all trials (Po0.05) and the lowest pH values were obtained for T8 (control) (Fig. 1). The different trials showed significant differences for each storage period (Po0.05), but no specific behaviour could be established for trials, except for T8, which always presented the lowest values. The main reason for that is the difference in T8 formulation, which did not contain prebiotics. Proportionally more cheesebase (quark), and therefore more lactose to be degraded into organic acids, was present in T8 (Table 1), leading to lower pH values during the storage period (titratable acidity was also higher for T8 – data not shown). 3.2. Viable counts of microorganisms The microbial populations obtained for all trials are shown in Table 5. The populations of the starter S. thermophilus did not differ between trials after the first day of storage, while some significant variation occurred at 28 days (Po0.05). The high counts during the whole storage period, not less than 9.56 mean log cfu g1 (Table 5), however, confirm that we can disregard the variation found and strongly consider the viable counts found as excellent. These good viable counts associated with the presence of the probiotic microorganisms lead to the reductions in pH values along storage. The probiotic population decreased during storage, both for L. acidophilus and for B. animalis subsp. lactis. Nevertheless, decrease in counts during storage (between 1 and 28 days of storage), considering the same trial, was never above 0.74 and 0.37 log cfu g1, respectively, for L. acidophilus and B. animalis subsp. lactis (Table 5). Moreover, populations were above 106–107 cfu g1 during the whole shelf life of the product, the minimum counts suggested by several authors to produce beneficial health effects on the gut, representing 108–109 cfu 100 g1 of daily product consumption (Hoier et al., 1999; Vinderola, Prosello, Ghiberto, & Reinheimer, 2000; Vinderola &

4.55 4.50

pH

4.45 4.40 4.35 4.30 4.25 0

5

10

15

20

25

30

storage time (days) T1

T2

T3

T4

T5

T6

T7

T8

Fig. 1. Changes in pH during storage time at 471 1C of the petit-suisse cheese-trials studied (see Table 2 for description of trials T1–T8).

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Table 5 Viability of Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis of the different petit-suisse cheese trials studied during storage at 471 1C1 (see Table 2 for description of trials T1–T8) Microorganism

Days

Cheese trials T1

T2

T3

T4

T5

T6

T7

T8

Streptococcus thermophilus

1 28

9.74 [0.15]aA 9.56 [0.01]aA

9.81 [0.13]aA 9.61 [0.03]aA

9.69 [0.05]aA 9.81 [0.02]bcA

9.68 [0.03]aA 9.62 [0.03]aA

9.79 [0.03]aA 9.71 [0.03]bA

9.69 [0.08]aA 9.75 [0.06]bA

9.81 [0.07]aA 9.87 [0.03]cA

9.82 [0.09]aB 9.64 [0.08]abA

Lactobacillus acidophilus

1 28

6.99 [0.06]bB 6.25 [0.18]abA

6.99 [0.08]bB 6.62 [0.05]cA

6.49 [0.16]aB 6.33 [0.18]abcA

6.85 [0.16]bB 6.33 [0.05]abcA

6.83 [0.09]bB 6.53 [0.05]bcA

6.76 [0.04]bB 6.58 [0.10]cA

6.74 [0.01]abB 6.17 [0.13]aA

6.81 [0.08]bB 6.08 [0.07]aA

Bifidobacterium animalis subsp. lactis

1 28

7.60 [0.09]bB 7.34 [0.10]abA

7.69 [0.05]bB 7.35 [0.03]bA

7.44 [0.04]aB 7.21 [0.04]aA

7.69 [0.04]bB 7.37 [0.05]bA

7.44 [0.04]aB 7.25 [0.04]abA

7.40 [0.03]aB 7.26 [0.02]abA

7.60 [0.05]bB 7.31 [0.02]abA

7.69 [0.04]bB 7.32 [0.03]abA

A,B For each microorganism, data that share a common capital letter in the same column do not differ significantly from one another during the storage times for the same trial (P40.05), a,bData that share a common lowercase letter in the same row do not differ significantly between trials for the same day (P40.05). 1 Mean [SD] log cfu g1 (n ¼ 3).

Table 6 Overall acceptability scores obtained for the potentially synbiotic petit-suisse cheese trials studied1 (see Table 2 for description of trials T1–T8) Trials

Overall mean for each trial

T1 T2 T3 T4 T5 T6 T7 T8

6.97 6.53 6.42 6.99 6.79 6.71 6.66 6.39

Overall mean by time

[0.97] [0.98] [1.25] [1.22] [1.38] [1.56] [1.45] [1.23]

Times of storage (days)

7 6.66 6.20 6.29 6.54 6.49 6.26 6.23 6.17

[1.16] [1.13] [1.62] [1.56] [1.15] [1.77] [1.37] [1.07]

aA aA aA aA aA aA aA aA

6.35 [1.37]

14 6.83 6.37 6.14 6.74 6.71 6.60 6.66 6.23

[1.07] [1.06] [1.40] [1.34] [1.58] [1.52] [1.76] [1.42]

6.54 [1.41]

aA aA aA aA aA aA aA aA

21 6.97 6.54 6.49 7.31 6.97 6.83 6.74 6.54

[0.89] [0.78] [1.12] [0.87] [1.36] [1.71] [1.40] [1.15]

6.80 [1.21]

aA aA aA aB aA aA aA Aa

28 7.43 7.00 6.77 7.34 7.00 7.14 7.03 6.63

[0.56] [0.69] [0.55] [0.80] [1.39] [1.06] [1.12] [1.26]

cB abcB abA cB abcA bcA abcA aA

7.04 [1.00]

A,B Data that share a common capital letter in the same row do not differ significantly from one another during the storage times for the same trial (P40.05), a,bData that share a common lowercase letter in the same column do not differ significantly between trials (P40.05). 1 Mean [SD] (n ¼ 35).

Reinheimer, 2000). Other authors also reported satisfactory probiotic viability when producing probiotic fresh cheeses (Buriti et al., 2005a; Gomes & Malcata, 1999; Vinderola et al., 2000), corroborating the use of fresh cheeses like petit-suisse cheese as vehicles for probiotics. Besides the satisfactory probiotic viable counts, a protective behaviour by the prebiotics added to the cheese trials tested was expected. In fact, the fructan-type prebiotics inulin and oligofructose may also aid survival of probiotic organisms during processing and storage of dairy products, particularly increasing or, at least, retaining the viability of Bifidobacterium spp. and of L. acidophilus (Bruno, Lankaputhra, & Shah, 2002; Capela, Hay, & Shah, 2006; O¨zer, Akin, & O¨zer, 2005; Shin, Lee, Pestka, & Ustunol, 2000). Additionally, under in vitro conditions the L. acidophilus and Lactobacillus plantarum counts were reported to increase 10–100 fold in the presence of 1% honey (in the culture medium) compared with sucrose (Shamala et al., 2000). Conversely, in the present study the

presence of prebiotics in T1–T7 lead to a slight or absent reduction in loss of viable counts of B. animalis subsp. lactis and L. acidophilus, compared to the control trial during storage (1–28 days). 3.3. Sensory acceptance The objective of most quantitative consumer research conducted in support of product development is to determine consumers’ affective reaction to new or revised products. The level of consumer acceptance is often assessed by asking consumers to rate how much they like a product overall, using a nine-point hedonic scale (Popper, Rosenstock, Schraidt, & Kroll, 2004). It is known that affective tests (like acceptability tests) help to answer whether the product has commercial potential, especially when consumers are used as panellists. The sensory acceptance results of the present study are shown in Table 6. The acceptance results ranged from ‘‘like

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In the present study, however, differences in the pH decrease of petit-suisse cheeses were not so pronounced (Fig. 1). Nevertheless, the lowest pH values during storage were found for the lowest accepted cheese trial (T8control). It can be speculated that the supplementation of cheeses with prebiotics (T1–T7) might have interfered with the sensory perception of acidity, although not in a significant way. Finally, T1 (only with oligofructose) also provided the most consistent judgements (lowest standard deviation) besides being the most preferred trial. Similarly, in a study with hard and semi-hard cheese, Hersleth, Ueland, Allain, and Naes (2005) observed that the samples which provided the most consistent judgements were the most preferred ones. In the present study, acceptance changed significantly during storage only for the trials containing 10% oligofructose (T1), 10% inulin (T2) and 5% of oligofructose plus 5% of inulin (T4). T1 and T2 showed significantly higher acceptance after 28 days of storage than after 7, 14 and 21 days (Po0.05). Interestingly, T4 was more accepted after 21 and 28 days of storage than after 7 and 14 days. Possibly, the combination of inulin and oligofructose in petit-suisse cheese T4 lead to the best acceptance at 21 days. When oligofructose and inulin were used alone, respectively, in cheeses T1 and T2, the best acceptance was only observed after 28 days. The highest mean score was obtained with T1 (10% oligofructose) reaching 7.43 after 28 days of storage. Hence, cheeses containing oligofructose and/or inulin (T1, T2 and T4), which corresponded to the highest fructan contents cheese trials, were the best accepted as the end of shelf-life got close. The behaviour of acceptance for all petit-suisse cheese trials may be also related to the subjectivity of hedonic tests. Actually, there is a great amount of subjectivity in sensory evaluation when using consumers as panellists. This might explain the differences in sensory acceptance

slightly’’ to ‘‘like moderately’’. A significant difference between trials (Po0.05) was observed at 28 days of refrigerated storage. The lowest acceptance was found for T8 (control), and the highest individual mean acceptance was obtained for T1 (10% oligofructose). T3 did not perform well, and it was the second least accepted cheese. Possibly, the presence of eucalyptus honey contributed to the unfavourable performance of cheese T3. However, when honey was associated with inulin in T6, this cheese was the third most accepted one, after 28 days of storage. The presence of inulin in T6 certainly contributed for this good performance, probably conferring favourable taste and texture features to the cheese (Fig. 2). A less intense effect was observed at 28 days when honey was associated with oligofructose in cheese T5, but still resulted in an increased acceptance, when compared with the product containing only honey (cheese T3). In fact, Buriti (2005) reported that when inulin was present in fresh cream cheese with Lactobacillus paracasei, this cheese behaved similarly to control cheese (without inulin and L. paracasei), regarding preference. According to the author, the presence of inulin decreased the perception of residual taste produced by L. paracasei and improved cheese texture. On the other hand, Aragon-Alegro, Alegro, Cardarelli, Chiu, and Saad (2007) reported no interference of inulin addition in the sensory preference of chocolate mousse with L. paracasei. The oligofructose properties mentioned by Roberfroid (2005), including rounder mouthfeel and sustenance of flavour with reduced aftertaste, probably were responsible for the T5 improved features. Another possible explanation for these acceptance results may be the acidity of each formulation, represented by the modifications in the pH during the storage period. Buriti et al. (2005a) reported that the significantly higher pH in one of the trials of potentially probiotic ‘Minas’ fresh cheese studied by the authors was related to a better preference (though not significant) in sensory evaluation.

1000 900

Frequencies

800

348

530

700 600 500 400 300

597 471

200

174

100

19

67

0 Taste

63

Appearance

Texture

Flavour

Attributes preferred

not preferred

Fig. 2. Frequencies for preferred and not preferred attributes mentioned by the consumers considering all storage periods and trials of petit-suisse cheeses studied (see Table 2 for description of trials T1–T8). The numbers in the bars correspond to the times that a particular attribute is cited.

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during the storage period evaluated (Table 6). Moreover, the consumers’ familiarity with the product over the time despite the degree of novelty in using prebiotic ingredients might have interfered as reported previously (Hersleth et al., 2005; Ko¨ster, 2003). This happened particularly when honey was present, once all formulations with eucalyptus honey in the composition (T3, T5, T6 and T7) had no significant increase on acceptance during storage. It seems that the consumers became more familiar and capable of identifying the taste of eucalyptus honey in T3, T5, T6 and T7. In fact, Bastos, Franco, da Silva, Janzantti, and Marques (2002) verified through quantitative descriptive analysis method that eucalyptus honey, similar to which was used in this study for the production of petit-suisse cheese trials T3, T5, T6 and T7, was sensorially described as ‘‘burnt’’ and having ‘‘after-taste’’. The authors also verified that nonanal and nonanol compounds were the important contributors for these characterizations of eucalyptus honey. 3.4. Sensory attributes comparisons Product developers not only need to know the degree of overall liking, but also what people like and dislike about a specific product, and how these attributes might be changed to increase acceptance. For this reason, studies often include questions about the product attributes that are likely to determine the level of overall liking, and the questions often concern the sensory properties of the food such as its flavour and texture (Popper et al., 2004). But there is some concern that such questions may be a source of bias (Stone & Sidel, 1993). In the present study, considering the preferred and not preferred attributes, the panellists mainly mentioned two attributes: taste and texture (Fig. 2). Considering the overall means between the four storage periods (7, 14, 21 and 28 days) and all of the eight trials, taste and texture showed significant differences between frequencies for preferred and not preferred samples (Po0.01). Taste was almost always the preferred attribute, except for the control trial (T8). The T1, T3 and T5 cheeses were most frequently mentioned as having the preferred taste (data not shown), although no research was carried out in order to elucidate the reason for this. When monitoring acacia honey addition to yogurt, Varga (2006) concluded that at a concentration of approximately 3.0% (w/v) honey highly improved the sensory quality of the finished product, conferring an optimum sweetness to the product. The opposite was observed for the yogurt formulations containing 1.0% (w/ v) and 5.0 (w/v), which presented, respectively, weak and too strong flavour. In the present study, it might be speculated that honey sweetness also influenced the consumer’s opinions about the different petit-suisse cheeses, and those consumers who actually prefer quite sweet products mentioned T3 and T5 as those cheeses with preferred taste. Complementary, oligofructose is expected

to interfere in taste, reducing after-taste or modifying sweet profile (Kaur & Gupta, 2002) and also enhancing fruit flavours (Roberfroid, 2005), therefore explaining the taste preference observed in petit-suisse cheese T1. In fact, taste is of crucial importance for functional foods and, counting on consumer willingness to compromise on the taste of functional foods for health is a highly speculative and risky strategic option (Verbeke, 2006). The texture attribute was the one given the lowest scores by the consumers in the present study. Furthermore, texture attribute results were non-conclusive as to the degree of consumers’ preference, thereby more studies might be suggested. 4. Conclusions The potentially synbiotic petit-suisse cheeses obtained in the present study turned out to be feasible vehicles for probiotic and prebiotic ingredients. Cheeses T1 and T4, both with oligofructose and/or inulin and without honey, showed to be excellent in terms of prebiotic potential, good viable counts of L. acidophilus and B. animalis subsp. lactis and sensory quality. Cheese T3 with eucalyptus honey had the worst performance in terms of fructan content, viable count of B. animalis subsp. lactis and acceptance among the petit-suisse cheeses studied, thus being considered inadequate. As for cheeses containing eucalyptus honey in a smaller proportion (T5, T6 and T7), an improvement of their acceptances during storage might be obtained by the substitution of eucalyptus honey by honey from a flora other than eucalyptus, like orange, for instance, once they also presented satisfactory probiotic viable counts and potential prebiotic effect. Therefore, the petit-suisse cheese T4 (supplemented with both inulin and oligofructose combined) seems to be the most promising concerning the simultaneous achievement of high fructan contents and good viable counts of L. acidophilus and B. animalis subsp. lactis and sensory acceptance. Moreover, the extensive and widespread evidence to support prebiotic efficacy for inulin and oligofructose (Gibson et al., 2004), which are selectively metabolised at different portions of the large intestine (Roberfroid, 2005), is an additional advantage in improving the activities of beneficial intestinal microorganisms and promising for future research projects to be designed to confirm the in vivo effectiveness of the product. Acknowledgements We wish to thank Fundac- a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) (Projects 03/13748-1, 02/14185-8 and 04/13597-6) and Coordenac- a˜o de Aperfeic- oamento de Pessoal de Nı´ vel Superior (CAPES), for financial support. We gratefully acknowledge Prof. Dr. Tullia Maria Clara Caterina Filisetti, Prof. Dr. Beatriz Rosana Cordenunsi and Tania Misuzu Shiga from the University of Sa˜o Paulo—Department of Food and Experimental Nutrition—Faculty of Pharmaceutical

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