Stability and functionality of synbiotic soy food during shelf-life

Journal of Functional Foods 35 (2017) 134–145

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Stability and functionality of synbiotic soy food during shelf-life Olga Lucía Mondragón-Bernal a,⇑, José Guilherme Lembi Ferreira Alves a, Mariá Andrade Teixeira a, Maria Fernanda Perina Ferreira b, Francisco Maugeri Filho b a

Laboratory of Bioprocess Engineering, Department of Food Science, Federal University of Lavras, Campus Universitário s/n - P.B. 3037, CEP 37200-000 Lavras, MG, Brazil Food Engineering Faculty, Laboratory of Bioprocess Engineering, Department of Food Engineering, University of Campinas, Monteiro Lobato, 80 - UNICAMP - Barão Geraldo, CEP: 13083-862 Campinas, SP, Brazil b

a r t i c l e

i n f o

Article history: Received 31 December 2016 Received in revised form 25 April 2017 Accepted 3 May 2017

Chemical compounds studied in this article: Anthrone (PubChem CID: 7018) Calcium lactate (PubChem CID: 13144) Dextran (PubChem CID: 4125253) Fructooligosaccharides (PubChem CID: 439709) Galactose (PubChem CID: 6036) Glucose (PubChem CID: 5793) Polydextrose (PubChem CID: 71306906) Raffinose (PubChem CID: 439242) Sucrose (PubChem CID: 5988) Stachyose (PubChem CID: 439531)

a b s t r a c t Synbiotic soy foods fermented by lactic acid bacteria suffer pH decrease, viscosity and syneresis changes during storage. Lactobacillus rhamnosus LR32 (LR) has capacity of producing exopolysaccharides (EPS) when inoculated in complex media. A Plackett & Burman experimental design was carried out to investigate the effects of LR (plus standard probiotic mixture: Lactobacillus spp and Bifidobacterium sp), sucrose, soy extract (SE), calcium lactate (CL), fructooligosaccharides (FOS) and polydextrose on the responses: EPS, syneresis, apparent viscosity, rheological behaviour and viable counts during 30 days of shelf-life. EPS producing showed a heteropolysaccharide with molecular mass >39 KDa. CL showed the greatest significant positive effect on the syneresis, while sucrose and polydextrose showed significant negative effects. Sucrose and SE had a significant positive effect in most stability responses. The best tests for the stability study for the synbiotic and fermented soy food were without CL, added with 12% sucrose and 10% SE. Ó 2017 Elsevier Ltd. All rights reserved.

Keywords: Plackett & Burman experimental design Lactobacilli Bifid bacteria Soy extract fermentation Exopolysaccharides Rheology

1. Introduction There has been great progress in the development of the so called probiotic products, which are food supplements containing live micro-organisms that confer a health benefit on the host when administered in adequate amounts (FAO/WHO, 2001). According to the Technical Meeting on Prebiotics promoted by FAO (Pineiro et al., 2008), prebiotics are non-viable food components that confer benefits to host health associated with the modulation of their intestinal microbiota. The positive influence of prebiotic substances, considered as soluble fibres, in intestinal flora has been ⇑ Corresponding author. E-mail addresses: [email protected] (O.L. Mondragón-Bernal), [email protected] (José Guilherme Lembi Ferreira Alves), [email protected] (F. Maugeri Filho). http://dx.doi.org/10.1016/j.jff.2017.05.021 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

tested in several studies, where the utilization of probiotic species in combination with prebiotic substances provides a combined effect called ‘‘synbiotic” (Mattila-Sandholm et al., 2002; Saad, Assis, & Faria, 2011). Then, synbiotic functional foods are those that supply both prebiotics and probiotics and their synergy effects (Mondragón-Bernal, Rodrigues, André-Bolini, & Maugeri, 2011). A functional product must contain between 106 and 108 colonyforming units per gram (CFU/g) for optimum therapeutic effects; however, various international legislations recommend a minimum probiotic population ranging from 108 up to 109 CFU/daily serving portion of the food product (Brasil, 2002; Martinez, Bedani, & Saad, 2015). According to Brasil (1998), in relation to food containing edible fibres, the following nutritional statements can be added: ‘‘source of fibres”, when the content is 3.0 g/100 g in

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case of solid foods and 1.5 g/100 mL for liquids, or, ‘‘high fibre content”, when containing 6.0 g/100 g in case of solids or 3.0 g/100 mL for liquids. Soy products have an excellent nutritional status based on their high protein, essential vitamins, minerals and phytoestrogens content, called isoflavones, primarily as a health promoter for Seniors (Bedani et al., 2015; He & Chen, 2013). Raffinose and stachyose are a-galactosides presents in soybeans and products. These oligosaccharides have prebiotic effect, but are not digested by the human intestine, that does not express the pancreatic a -galactosidase required for hydrolyzing the a-1,6 links of these sugars, that could cause problems such as distending (LeBlanc et al., 2004). Bifidobaterium strains possess high activity a–galactosidase, bgalactosidase and b-2,1-D-fructano-fructanohidrolyse (inulinase), are indicated for biotechnological processes that employ soymilk as substrate and fructooligosaccharides (FOS). Bifidobacteria bioavailable simple sugars for lactobacilli, which in turn, bioprovides peptides for bifid bacterium by protease enzymes going well a symbiotic relationship between strains (Hou, Yu, & Chou, 2000; Mondragón-Bernal, Maugeri-Filho, Alves, & Rodrigues, 2012). Soy fermented products containing prebiotics and probiotics (synbiotic food) after fermentation by lactic bacteria show a decrease in pH, which gives them sensory characteristics such as viscosity and acidity. It is necessary that fermented products conserve their sensory characteristics during shelf-life, but it has been observed that pH continues to decrease, with consequent syneresis and changes in consistency (Mondragón-Bernal et al., 2012). Furthermore, the maintenance of high probiotic’s numbers in functional foods during storage in low temperatures required technological challenge, as protect compounds (Etchepare et al., 2016; Saad et al., 2011). Starter cultures ferment sugars to produce lactic acid, which serves to acidify, preserve and give flavour to the product. They also hydrolyze proteins, changing the product texture, and some of them have the capacity of producing exopolysaccharides (EPS) when fermented in appropriate medium. EPS-producing cultures are capable of improving the textural properties, contributing to the consistency and rheology of the fermented products. Beneficial effects to health have been attributed to a few EPS-producing lactic bacteria (LAB) (Grattepanche, Audet, & Lacroix, 2007; Hamet, Piermaria, & Abraham, 2015; Li et al., 2014). In many cases, polysaccharides released extracellularly by lactic bacteria are advantageous in a variety of fermented food products (Hamet et al., 2015; Sheng, Yu, & Li, 2010). Some studies noticed that instead of using additives as texture improvers, stabilizers, emulsifiers, jellifies or anti-syneresis agents in fermented foods, it may be convenient to use EPS-producing lactic bacteria as starter cultures (Hamet et al., 2015; Li et al., 2014). Exopolysaccharides (EPS) produced by LAB strains, are used in the production of fermented products to improve their rheological properties (Jaiswal, Sharma, Sanodiya, & Bisen, 2014). The EPS polymers can be considered as natural biothickeners because they are produced in situ by the LAB-starters that have General Recognised As Safe (GRAS) status, and for this reason is not considered as food chemical additive (Florencia, 2013). EPS of some strains of LAB contribute a gelatinous texture to fermented products and these polysaccharides are also digestible (Jaiswal et al., 2014). During fermentation of complex media such as soy extract and milk by LAB, the produced lactic acid causes the aggregation of protein particles (globulins), leading to the formation of a fragile gel. The gel in which bacteria cells, sugars and other smaller components are retained is a structure of the highly complex network of proteins and EPS (Goh, Haisman, Archer, & Singh, 2005). EPS producing microorganisms utilize sugars as their carbon and energy source, ammonium salts and amino acids are their source of nitro-

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gen. The synthesis of EPS by microbial cells basically depends on the carbon and nitrogen availability in the culture media (Grattepanche et al., 2007; Jaiswal et al., 2014; Shihata & Shah, 2002). Species with potential for EPS production are Lactococcus lactis spp., Leuconostoc spp., Leuconostoc fructosum, Leuconostoc mesenteroides spp.; Pediococcus acidilactici var and Proponibacterium as Propionibacterium freudenreichii spp. (Mondragón-Bernal et al., 2012). Approximately 30 species of lactobacilli are described as EPS producers (Jaiswal et al., 2014). Among them Lactobacillus rhamnosus ssp. is an EPS producing species with probiotic potential (Champagne, Barrete, Roy, & Rodrigues, 2006; Laneuville & Turgeon, 2014; Macedo, Lacroix, Gardner, & Champagne, 2002). It was observed that L. rhamnosus LR32 showed the best growth in soy extract with high sucrose concentrations (12%) and the best capacity to produce EPS (unpublished results) when compared with several LAB strains. Therefore new studies about the viscous properties and the behaviour of EPS produced by L. rhamnosus on specific soy fermented products are necessaries. The experimental design proposed by Plackett & Burman (P&B) in 1946, ‘‘Screening Design”, based on the factorial design methodology, is a very useful statistical tool for the previous evaluation of a process when one has a great number of variables. As advantages, one has the reduction of the number of tests to be performed, it allows for estimating the main effects and identifying the most relevant variables that must be chosen to make a complete planning, but the number of treatments should be bigger than the number of independent variables and it has the disadvantage of not allowing to optimize the process (Rodrigues & Iemma, 2014). The goal of this study was to determine the conditions to obtain stable functional and physical-chemical characteristics of soybased synbiotic food during 30 days of shelf-life. Therefore, using a P&B experimental design, the effects of the following variables were studied: proportion of Lactobacillus rhamnosus sp., sucrose contents, soy hydro-soluble extract, acidity regulator (calcium lactate), fructooligosaccharides (FOS) and polydextrose in the production of EPS (exopolysaccharides), syneresis reduction, apparent viscosity, rheological parameters and total probiotic counts during shelf-life. A sensory validation of the best prototypes obtained was based on sensory optimization made by Mondragón-Bernal, Rodrigues, Bolini, and Maugeri (2010).

2. Materials and methods 2.1. Raw material Soy extract was used as main ingredient of the fermentation medium (Provesol FB Olvebra Industrial S/A, São Paulo, SP, Brazil) obtained from non-GMO dehulled and defatted white soy flour by an aqueous extraction process, thermal inactivation, formulation whit lecithin and refined soy oil, and spray drying, with properties as good solubility and dispensability, free of chemical additives, lactose, sucrose, casein and gluten, and its composition was: 19 g/100 g of Carbohydrates; 19 g/100 g of Sugars (without lactose); 43.3 g/100 g of Protein; 26 g/100 g of Total Fat; 4 g/100 g of Saturated Fat, 0 g/100 g of Trans Fat; 1 g/100 g of Monounsaturated Fat; 2.3 g/100 g of Polyunsaturated Fat; 0 mg/100 g of Cholesterol; 1.7 g/100 g of Dietary Fibre; 36.7 mg/100 g of sodium; 156.7 mg/100 g of Calcium; 4.3 mg/100 g of Iron; and 2043.3 kJ/100 g (613 cal/100 g). As prebiotic agents were used fructooligosaccharides (FOS) (Raftilose 95Ò- Orafti Inc., São Paulo, SP, Brazil) and polydextrose (LitesseÒ, Danisco-DupontÒ, Cotia, SP, Brazil). As anti-foaming was added in the fermentation medium silicon dioxide (ProceedingsÒ, Jundiaí, SP, Brazil). Some assays were added with commercial sucrose

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(União, Sertãozinho, SP, Brazil) and calcium lactate (calcium; 2-hydroxypropanoate – Purac, São Paulo, SP, Brazil) as buffer agent.

the experimental design and initial counts of each inoculum strain. Samples of all treatments were collected at the end of fermentation (0 day). Other samples were stored under refrigeration (4–6 °C) and removed after 10, 20 and 30 days, respectively, in duplicate.

2.2. Probiotic cultures and activation 2.4. Experimental design The micro-organisms in freeze dried DVS (Direct Vat Set) form were L. rhamnosus LR32 LAB strain (LR) and the standard probiotic inoculum: L. acidophilus LAC4 (LAC), L. paracasei subsp paracasei LBC81 (LBC) and Bifidobacterium longum BL04 (BL) (DaniscoDupontÒ, Cotia, SP, Brazil). DVS (Direct Vat Set) cultures were reactivated in modified medium of sterile (116 °C/4 min) propagation containing MRS broth (Sigma-Aldrich, São Paulo-SP, Brazil, De Man, Rogosa, & Sharpe, 1960), replacing glucose with sucrose 120 g/L and yeast extract per 60 g/L of soybean extract for adaptation of probiotic strains (according to Mondragón-Bernal et al., 2012). After cooling the culture medium, 0.5% v/v L-cysteine (10% m/v cysteine hydrochloride -Sigma-Aldrich, São Paulo-SP, Brazil, adapted from LourensHattingh & Viljoen, 2001) was added and to decrease oxyreduction potential 0.3% v/v ascorbic acid solution (17% w/v) aseptically by filtration on 2 lm pore discs (MilliporeÒ, Merck S/A, São Paulo, SP, Brazil, adapted from Lourens-Hattingh & Viljoen, 2001). The envelopes with lyophilized cultures were opened under aseptic conditions and 1 g of each bacterium was suspended in 10 ml of the propagation medium and reactivated for 3 h at 37 °C. The 10 ml with the culture activated were diluted in poured into 90 ml of the medium obtaining an amount of 3 log cycles less than the initial population. Each individually diluted culture was divided into fixed volumes in sterile eppendorfs and frozen at 18 °C. The cell viable counts were determined by poor plate technique after defrosting for direct application as inoculums in the media to be fermented. 2.3. Preparation of fermented products from soy extract with probiotic cultures 2.3.1. Preparation of fermented products for kinetic formation of EPS study With the goal to know the molecular size and classified EPS producing as homopolysaccharide (HoPSs) or heteropolysaccharide (HePSs) was made e kinetic study in three fermentation times (0, 8 and 16 h) for the control synbiotic soy food formulated with soy extract (10%), sucrose (12%), prebiotics (2% FOS and 2% Polydextrose) and silica as anti-foaming agent (5 ppm) in triplicates. Suspensions were sterilized (116 °C/4 min) and cooled to 40 °C. Then the medium was aseptically inoculated with mixtures of lactic acid cultures and fermented, without stirring, at 37 °C. The probiotic inoculum was 6.7 log CFU/mL for total count of soy medium being the proportion was 10:10:20:60 (LAC:LBC:LR:BL) parts of each strain. EPS producing by probiotic mixture were quantified by the Anthrone method and identified qualitatively by molecular size by gel permeation in HPLC. 2.3.2. Preparation of fermented products for experimental design According to Plackett & Burman experimental design (Rodrigues & Iemma, 2014), suspensions of soy extract were prepared, added of silica as an anti-foaming agent (5 ppm), FOS and/ or polydextrose, with and without sucrose, were sterilized (116 °C/4 min) and cooled to 40 °C. Then the medium was aseptically and slowly added drop by drop of Calcium lactate (sterilized solution 1% m/v) and inoculated with mixtures of lactic acid cultures and fermented, without stirring, at 37 °C, until pH between 4.8 and 4.3 was reached (approximately 16 h). The probiotic’s inoculum was 6.7 log CFU/mL of soy medium and the quantities and proportion of each probiotic bacteria was made according with

The Plackett & Burman experimental design was applied for 12 trials and 4 central points in a total of 16 assays (Rodrigues & Iemma, 2014), with 6 independent variables: X1 = rate of LR and standard probiotic mix (30:10/60; 20:20/60; 10:30/60 being (LAC + LBC):LR/BL); X2 = sucrose (0; 6; 12% m/v); X3 = calcium lactate as a buffering agent (0; 0.25; 0.5 g/100 mL); X4 = soy extract (6; 7.5; 10% m/v); X5 = fructooligosaccharides (FOS) (0; 2; 4% m/ v) and X6 = polydextrose (0; 2; 4% m/v), where LAC: L. acidophilus LAC4; LBC: L. paracasei subsp. paracasei LBC81; BL: Bifidobacterium longum BL04; LR: L. rhamnosus LR32; FOS: fructooligosaccharides. Trials were done in duplicate and results were calculated through the means of values obtained. The dependent variables or responses were: Total Counts (log CFU/mL); Bifid Counts (log CFU/mL); EPS concentration (g/L); syneresis (% v/v); rheological parameters: initial shear stress - so (Pa), consistency coefficient k, flow behaviour index – n, and apparent viscosity - ga (Pa s); pH (was also controlled during shelf-life). Fermentations were finalized when the pH of the medium reached around 4.5 and samples were then analyzed. Statistical analyses and regressions adjusted to rheological models (Eqs. (A.1) to (A.4)) were done using STATISTICA 8.0 software (Statsoft, 2008). The matrix applied for the Plackett & Burman experimental design in this study can be observed in Results and Discussion in Table 2. 2.5. Analytical methodology pH Measurement: pH of samples was measured through a calibrated Mettler Toledo 320 pH Meter potentiometer. Replicates were prepared simultaneously only with the objective to measure pH. The acquisition of pH values was made in aseptic conditions into the laminar flux chamber with calibrated and sanitized potentiometer with alcohol 70% and sterilized-deionized water. The pH variation (DpH) was calculated between the first and last days of storage by the difference between the values measured at these times (DpH = pH (0 day.) - pH (30th day). Syneresis Measurement: The volume of serum separated from the fermented medium was measured in volumetric tubes and the percentage of syneresis was calculated. Counts culture medium: The methodology to count mixed probiotics in complex media was development in the Bio-Process Laboratory in University of Campinas/Brazil (Mondragón-Bernal, Costa, Rodriguez, & Maugeri, 2005). The pour plate method was used to count cultures with Agar MRS medium (Sigma-Aldrich, São Paulo-SP, Brazil; De Man et al., 1960) added of 0.01% (m/v) aniline blue (BL not pigment with aniline blue), incubation during 72 h at 37 °C in aerobic and anaerobic conditions using jars (Merck S/A, São Paulo, SP, Brazil) and anaerobic generators (Anaerobac – Probac, São Paulo, SP, Brazil). This method consist in the viable counts by differences on colonies morphology observed by stereoscopy (stereoscopy Citoval 2 – Carl Zeiss), more easily perceived when aniline blue is added in MRS media and colonies of lactobacilli and bifid bacteria can be differentiated. The morphologies of colonies are: BL (colony’s diameter 0.1–3.0 mm, lentil shape with regular edges and white/cream colour); LAC (colony’s diameter 0.1– 3.0, irregular edges light blue with central protrusion and like an egg yolk dark blue); LBC (Robust and thick, pigmented discs of homogeneous intense blue, homogeneous central contours and central protuberance, disc with diameter of 1.0–5.0 mm and the colour of medium are blue intense too); and LR has similar charac-

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teristics as LBC. This similitude caused difficult for difference viable counts when both of them (LBC and LR) are mixture. Total Count (TC) is the result of anaerobic growth (lactobacilli + bifid bacteria) because lactobacilli are anaerobic facultative genus while bifid bacteria are strict anaerobic. The count of Total lactobacilli (LC) is the count in aerobic condition and the count of bifid bacteria (BC) is the difference into anaerobic and aerobic conditions. Determination of rheological properties:11276965737870112769 65737870 Rheological properties of samples were analysed through a Carri-Med CSL2 500 controlled stress Rheometer (TA Instruments, New Castle, DE, USA), with a 6-cm conical cup at 1.59°, with stress control. Stress Factor: 0.0177; deformation factor: 28,7; truncating: 67 mm. They were performed in triplicate and rheological models were adjusted and selected according to Eqs. (A.1) to (A.4) through the correlation coefficient estimated (R2) and chi-squared test (v2) with data from the second runs. Kinematic (g) and apparent viscosity (gap) (Pa s) were calculated for 100 s 1, being the average of the deformation force (c) for chewing food, through Eqs. (A.1.1) to (A.5.1) (Bourne, 2002). Determination of Exopolysaccharides (EPS): - Extraction of Raw EPS: The separation of the EPS from the nonEPS components, particularly enzymes, proteins, and cells in complex samples were made by thermic denaturation and deactivated in a bath at 100 °C for 10 min. For each mL of sample, 0.01 ml of 1 M citric acid was added, centrifuged to 9640g for 10 min/5o C (Sorvall RC-28S Superspeed Centrifuge) for the removal of cells and proteins and filtrated. The surface matter was divided for analysis of EPS and reducing sugars. After these treatments the methodology to extract the EPS was done. EPS were precipitated by adding 5 volumes of cooled ethanol during 12 h at 4 °C. The sample was centrifuged at 907g (3000 rpm) for 20 min; the precipitate was re-dissolved by adding 2 more volumes of cooled alcohol and it was left for one night at 4 °C. It was again centrifuged at 907g for 20 min, the supernatant being removed afterwards. Following, the residual alcohol was evaporated during 3 h at 60 °C in oven and the raw EPS was re-dissolved in deionized water for later analysis (methodology adapted from Grattepanche et al., 2007; RuasMadiedo, Gueimonde, Reyes-Gavilán, & Salminen, 2006; RuasMadiedo, Hugenholtz, & Zoon, 2002; Savadogo et al., 2004). - EPS quantification by the Anthrone method (Trevelyan, Forrest, & Harrison, 1952): In a test tube, 4 mL of Anthrone (Merck Millipore) reactive were added (0.2 g/100 mL of H2SO4) and 1 mL of a previously diluted sample (0.01–0.1 g/L). Tubes were incubated in double boiler during 10 min and cooled in ice bath. Absorbance was measured with a Beckman Coulter DU640 spectrophotometer at 600 nm (Silva, Monteiro, Alcanfor, Assis, & Asquieri, 2003). The standard curve was plotted using Dextran of 9300 Da molecular weight (Sigma–Aldrich Ò). With the object to eliminate interference compounds in the final result, EPS quantification was made on each component of the complex matrix used to formulate the synbiotic samples with the same methodology. The results were subtracted to calculate the real quantity of EPS formed through mass balance. All dilution factors were considered in the calculation of final EPS (Goh et al., 2005). - Identification of EPS, oligosaccharides and sugars by molecular size by HPLC: For each mL of sample (soy extract or symbiotic sample), 0.01 ml of 1M citric acid was added, centrifuged to 9640g for 10 min/5 °C (Sorvall RC-28S Superspeed Centrifuge) for the removal of cells and proteins and filtrated. The surface matter was conveniently diluted with Milli Q water (Milli-QÒ- Water Purification System, Millipore, Bedford, MA, USA) in the concentration range of 10–100 mg/L with the aid of the automatic diluent (Dilutor Dispenser 402, Gilson, São Paulo, SP, Brazil) e

filtered through 2 mm (Millipore) before injection into the chromatograph. EPS producing by probiotic mix, oligosaccharides and sugars was identified qualitatively by molecular size in HPLC, model Varian 9010 (Varian Inc. Scientific Instruments, Palo Alto, CA, USA), through columns of permeation in gel in series GPC6000, GPC4000 and GPC3000 (TSKgel - SigmaAldrichÒ), using ultrapure water (Milli-QÒ- Water Purification System, Millipore, Bedford, MA, USA) as eluent, a flow of 1.0 mL/min and a detector by refraction index. The standards injected were: dextrans of 165KDa, 79KDa and 43KDa; polydextrose (Litesse IIÒ); fructooligosaccharides (Raftilose-95Ò); stachyose (ICN Biomedicals Inc.); raffinose (Sigma-Aldrich), sucrose (Merck), glucose (Merck), fructose (Merck) and galactose (Sigma-Aldrich) (adapted from Hernalsteens, 1999). Reducing Sugars (AR): The analysis of AR in kinetic study was done by the acid 3.5-dinitrosalicylic (DNS) method (Miller, 1959). After proper dilution, the absorbance of the samples was measured on a Beckman Coulter DU640 spectrophotometer to 540 nm. AR was calculated using linear regression obtained from standard curve made with glucose (Merck). 3. Results and discussion 3.1. Kinetic and identification of EPS producing in synbiotic soy food Heteropolysaccharides (HePSs) are compounds with two to eight monosaccharides (the most frequent are glucose, galactose, rhamnose and fructose) and these are produced in quantities bigger than 2 g/L (De Vuyst & Degeest, 1992). Homopolysaccharides (HoPSs) are compounds with only one kind of monosaccharide being fructose or galactose the most common. A kinetic study to investigate the EPS-producing by probiotic mixture was done with the goal to recognize the molecular mass and general characteristics of them. This experiment used a control sample contained soy extract (10%), sucrose (12%), prebiotics (2% FOS and 2% Polydextrose) and fermented with probiotic mixture in the proportion 10:10:20:60 (LAC:LBC:LR:BL) parts of each strain. Table 1 shows the results of the kinetic study for EPS-producing in the control synbiotic soy food during 0, 8 and 16 h of fermentation. It can be observed that the Total Counts (TC) obtained was above than 9 log CFU/mL and above than 8 log CFU/mL for Bifidobacterium longum BL04 in the final time. These viable counts obey the Brazilian regulation for probiotic food (Brasil, 2002). The apparent viscosity (gap) increased with time (from 0.056 to 0.077 Pa s with 100 s 1of deformation for chewing food) and it is Table 1 Kinetic study in the control sample of synbiotic and fermented soy food. Time (hours)

Total counts (log UFC/mL) Bifid counts (log CFU/mL) EPS (g/L) AR (g/L) pH Syneresis (% v/v) so (Pa) k (Pa sn) n gap (Pa s) (100 s 1) Model adjusted with R2 > 09999

0

8

16

6.92 ± 0.26 6.45 ± 0.50 3.68 ± 0.18a 14.98 ± 0.18 6.8 ± 0.2 nd

7.19 ± 0.32 6.95 ± 0.23 3.03 ± 0.16b 15.49 ± 0.17b 6.6 ± 0.1 nd 0.447 ± 0.063 0.062 ± 0.009 0.868 ± 0.016 0.038 ± 0.002 HerschelBulkley

9.30 ± 0.09 8.31 ± 0.18 3.54 ± 0.13a 15.17 ± 0.19a 5.0 ± 0.1 19.5 ± 0.5 0.347 ± 0.059 0.089 ± 0.008 0.957 ± 0.005 0.077 ± 0.008 HerschelBulkley

a

0.124 ± 0.013 0.826 ± 0.018 0.056 ± 0.010 Pseudoplastic

Results of EPS with equal letters (superscript) doesn’t have significant differences (P<0.05). nd: non determinate. EPS: Exopolysaccharides. AR: Reducing Sugars. so: yield shear stress; k: consistency coefficient; n: flow behaviour index; gap: apparent viscosity. Pa s: Pascal second. R2: Coefficient of determination.

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proportional to bacterial growth and to pH decrease (from 6.8 to 5.0). The medium showed changes from its initial rheological behaviour as pseudo-plastic fluid to final Herschel-Bulkley fluid. The consistency index (k) diminished however the fluidity index (n) increased along the fermentation time. Between 8 and 16 h fermentation the synbiotic prototype needed initial shear stress to begin to fluid and so decreased from 0.447 to 0.347 Pa. On the other hand, the EPS producing didn’t show significance changes throughout fermentation and it was observed that the biopolymer formation was not relationed to probiotic growth. During the first 8 h of fermentation it was observed a little decrease of EPS producing, indicating some bacterial activity on the structure of biopolymer. This situation can be explained when the RS kinetic is compared with EPS kinetic because a small increase of the RS occurred in 8 h and after 16 h RS decreased with a respective increase of EPS. It can be assumed that there was a release of reducing sugars in the first 8 h and a consumption of them to form EPS by probiotic activity. The mean value of EPS-producing was 3.4 g/L in the synbiotic control. Through the chromatograms (Fig. 1), it is possible to observe that there is microbial activity in the formation of compounds of high molecular mass, during 16 h of fermentation, through the interactions of sugars, oligosaccharides and proteins present in the soy extract and in the formulated medium. The EPS producing by LR + LBC + LAC + BL in mixed culture was greater than 165 KDa and showed yet a pick with molecular mass proximal to raffinose (504.4 g/mol). The retention times for standards can be visualised in Fig. 3(a). Note that in the chromatogram of pure soybean extract 10% w/v (Fig. 3(b)), the presence of a large area (peak 1) having a molecular mass (m.m.) larger than 165 KDa, a second area (peak

2) close to 43 kDa and a peak 3 around 9 kDa. In Fig. 1(c) the EPS producing kinetic by LR+ mixed probiotic culture added of sucrose and soluble fibres is showed, and the changes and peaks formation with different molecular mass can be observed. It becomes visible that in the fermented medium formulated with soy extract, an area was formed of molecular mass between 11 and 39 KDa and yet several peaks with molecular mass near to oligosaccharides (667–504 g/mol). Therefore high molecular mass biopolymers with characteristic as HePS and EPS (2.5 and 19 min of retention time) were produced by mixture of probiotic inoculum in complex soy medium. 3.2. Experimental design Placket & Burman to study the significant variables on stability of synbiotic soy food during shelf-life With the goal to know the effects of six independent variables on the diverse important stability responses of the synbiotic soy foods during refrigerated storage was done a saturated Plackett & Burman experimental design for a total of 16 assays. This experimental design allowed select some prototypes that had the satisfactory stability responses during 30 days of shelf-life as, for example, products with or without sucrose, acceptable levels of probiotics and fibres or with fewer expenses. Table 2 shows the matrix and responses for Total Counts (TC), Bifid Counts (BC) and final pH values for Plackett & Burman experimental design. 3.2.1. Probiotic counts A functional product must contain between 106 and 108 colony-forming units per gram (CFU/g) of end product of viable

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

(a)

(b)

Dextran 165KDa (26 min) Dextran 79KDa (26,5 min) Dextran 43KDa (27 min) Dextran 39KDa (28 min) Dextran 11KDa (30 min) Dextran 9,300 Da (30.25 min) Polydextrose (33.75 min) Fructooligosaccharides (34.75 min) Stachyose (34.75 min) Raffinose (35.25 min) Sucrose (35.5 min) Glucose (36.75 min) Galactose (37.25 min) Fructose (38.1 min)

(c) 0h 8h 16 h

1

2

3 4

5 2

3 4

5

67

Fig. 1. (a) Retention times for molecular mass of standards (b) Chromatogram of macromolecules in soy extract medium (10 g/100 mL): Area A (peaks 1,2, 3 and 4) with higher molecular mass (m.m.) showed >165 KDa, peak 4 close to 79 KDa and peak 5 adjacent to 1 KDa. (c) Kinetic EPS producing by LAC:LBC:LR:BL (10:10:20:60) culture + sucrose12% + soy extract 10% + Prebiotic 4%. The molecular mass for oligosaccharides are: Polydextrose average molar mass 2000 g/mol (Kibbe, 2000); Fructooligosaccharides (nystose) 666.58 g/mol; Stachyose 666.58 g/mol; Raffinose 504.44 g/mol (PubChem, 2016).

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40.0% 35.0%

% Syneresis (v/v)

30.0% 25.0% 20.0% 15.0% 10.0% 5.0% 0.0% 0

5

10

15

20

25

30

Time (days) Assay 1

Assay 2

Assay 3

Assay 4

Assay 5

Assay 6

Assay 7

Assay 8

Assay 9

Assay 10

Assay 11

Assay 12

Assay 13

Assay 14

Assay 15

Assay 16

Fig. 2. Syneresis percentage during shelf-life in assays for fermented soy and synbiotic foods.

16.000 14.000

EPS (g/L)

12.000 10.000 8.000 6.000 4.000 2.000 0.000 0

10

20

30

Time (days) Assay 1

Assay 2

Assay 3

Assay 4

Assay 5

Assay 6

Assay 7

Assay 8

Assay 9

Assay 10

Assay 11

Assay 12

Assay 13

Assay 14

Assay 15

Assay 16

Fig. 3. Responses for EPS quantification (g/L) during 30 days of shelf-life for fermented synbiotic soy food prototypes from Plackett & Burman experimental design.

cells to get most advantageous therapeutic effects; however, various international legislations recommend a minimum probiotic population ranging from 108 up to 109 CFU/daily serving portion of the food product (Brasil, 2002; Martinez et al., 2015). From Table 2, it can be noticed that the populations of probiotics, after 30 days of shelf-life remained at viable counts bigger than 8 log CFU/mL. In the assay 2, with high levels of LR, sucrose, soy extract and without prebiotic agents, the highest score on the 30th day with 11.18 log CFU/mL of total counts was obtained. It is observed that the total counts of all trials are in accordance with legislation. However, in the treatments with higher viable counts the medium pH was less than 4.0, leading to sensorial problems. For bifid bacteria counts, it was found that practically constant values (7.7 log CFU/mL) were obtained in most trials, and in some assays viable

counts reached more than 10 log CFU/mL. In other assays, as number 1 and 5, the greatest oscillations of bifid strain over the storage condition were evidenced; perhaps due to the greater proportion of LR and this strain compete with BL and limits the availability of the carbon sources in the absence of sucrose and prebiotics as in the assay 1. Assay 7 with a smaller proportion of LR, 12% sucrose and 4% polydextrose oscillated positively as there was a considerable increase in bifid bacteria at the beginning of storage (8.43 log CFU/mL) until 10 days thereafter (10.57 log CFU/mL). This behaviour can be explained by the a-galactosidase, b-galactosidase and b-2,1-D-fructano-fructanohidrolyse activity of bifid bacteria on oligosaccharides, bio-providing carbon through simple sugars for LR, and, the lactobacilli, in a symbiotic relationship, provide peptides for bifid species due to their proteolytic activity causing the growth of both (Hou et al., 2000).

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Table 2 P&B experimental design’s results for the fermented and synbiotic soy extract food prototypes during 0, 10, 20 and 30 days on the shelf-life. Assays

Independent variables xi x1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 1

x2

1 1

1 1 1

1 1 1

1 1 1

1 1 1 1 1 1

1 1 1 0 0 0 0

x3

1 1

1

1 1

1 1 1 1 1 1 1

1 0 0 0 0

x5

1 1

1 1

1 0 0 0 0

x4

1

1

1 1

1 1 1 0 0 0 0

x6

1 1

1

1 1 1

0 Days

1

1

1

1 1

1

1 1 1

1

1 1 0 0 0 0

1 1 1

1 1 1 1 0 0 0 0

10 Days

20 Days

30 Days

Total count log CFU/ mL

Bifid count log CFU/ mL

Final pH

Total count log CFU/ mL

Bifid count log CFU/ mL

Final pH

Total count log CFU/ mL

Bifid count log CFU/ mL

Final pH

Total count log CFU/ mL

Bifid count log CFU/ mL

Final pH

8.77 9.66 9.88 9.28 10.47 10.19 10.05 9.50 10.55* 10.26 10.32 8.98 10.20 10.07 10.35 10.32

8.29 9.17 9.35 8.62 9.67 8.81 8.34 8.73 10.52* 10.03 9.34 8.84 9.58 9.34 8.96 9.89

4.98 4.66 4.72 4.84 4.55 4.57 4.66 4.66 4.54 4.36 4.52 4.95 4.82 4.79 4.61 4.63

9.60 10.48 10.06 10.05 11.06* 10.75 10.73 9.87 9.43 10.15 10.28 8.68 10.25 10.60 9.38 10.34

8.00 9.54 9.13 8.85 10.52 9.54 10.57* 9.47 9.24 9.10 10.13 8.63 9.65 9.94 8.69 9.89

4.54 4.17 4.25 4.41 4.10 4.26 4.35 4.30 4.03 4.04 4.04 4.48 4.20 4.10 4.25 4.29

10.41 10.37 10.66 10.56 10.49 10.76 11.15* 10.40 10.52 10.40 10.54 9.64 10.78 10.67 10.59 10.74

9.88 9.36 10.26 10.17 7.70 8.90 10.82* 9.32 9.44 9.61 9.75 8.40 9.72 9.80 10.16 9.66

4.40 4.04 4.02 4.33 3.95 4.12 4.20 4.22 3.94 3.88 3.91 4.35 4.01 3.96 4.15 4.19

11.01 11.18* 10.06 9.91 10.17 9.92 10.35 10.05 9.60 8.59 9.79 9.26 9.06 9.33 9.30 9.15

9.18 9.92* 9.49 8.85 8.48 8.60 9.86 9.39 9.44 7.85 9.00 8.71 7.70 8.40 8.95 8.46

4.38 3.95 3.97 4.29 3.89 4.06 4.12 4.18 3.96 3.88 3.83 4.28 3.99 3.95 4.10 4.14

P&B: Plackett & Burman. Independent variables xi = coded levels ( 1; 0; +1), where real values are: x1 = rate LAC+LBC:LR/BL (30:10/60;20:20/60; 10:30/60); x2 = Sucrose (0; 6; 12% m/v); x3 = Calcium lactate (0; 5; 10 g/L); x4 = Soy extract (6; 8; 10% m/v); x5 = FOS (0; 2; 4% m/v); x6 = Polydextrose (0; 2; 4% m/v). (*) Best responses.

Crittenden et al. (2001) developed yogurt inoculated with S. thermophilus DS 224 and bifid bacteria strains and composed of solids of defatted milk 14%, fat 2.1%, sucrose 6% and the prebiotic resistant starch (Hi-maizeTM) 1% and inulin 1%. They found that Bifidobacterium lactis B94 and B. lactis SD 920 maintained higher viable counts (7 log CFU/mL at 4 °C for 5 weeks) in symbiotic yogurt whereas B. adolescentis B97 were less than 2 log CFU/mL in 4 weeks. Heenan, Adams, Hosken, and Fleet (2004) developed a soy nonfermented dessert with Lactobacillus acidophilus MJLA1, L. rhamnosus 100-C, L. paracasei ssp. Paracasei 01, Bifidobacterium lactis BBDB2, B. lactis BB-12, and, the survival of them during 6 months of storage showed 107 CFU/g or bigger final counts, but Saccharomyces boulardii 74012 decreased to below 106 CFU/g. In the current study, the trials with some of the carbon sources at the top level and without calcium lactate, except assay 7, caused favourable results for total and bifid counts, and might have kept more than 11 and 10 log CFU/mL, respectively, over storage time. Similar observations were found by Zielin´ska, Danuta, Antoni, and Motyl (2014) who has storage fermented soy beverage at 5 °C during 28 days and did not observe significantly change in the number of Lactobacillus casei KN291 in the beverage. But, at temperatures of 10, 15 and 20 °C there was a variation in the counts of viable cells. 3.2.2. pH The pH had a slight variation in the various tests, remaining within an interval of 4.0 to 5.0 between 10 and 30 days of shelflife (Table 2). The most significant change in pH occurred in the first 10 days of storage for all samples. Tests 1 and 12 (4.38 and 4.28) show the highest pH and tests 10 and 11 the lowest ones (3.88 and 3.83) at the 30th day. The pH-lowering during storage was an expected behaviour by high activity of lactic acid probiotic cultures that are also found in high numbers. With the addition of calcium lactate as a buffering agent, it was expected that changes in pH were lower. In general, it was observed that the change in pH (DpH) between times zero and 30 days of storage varied from 0.48 (for the assays 8 and 10) to 0.75 (for assay 3). It can be seen that smaller variations (<0.6 pH units) occurred in most cases in the tests containing 10 g/L lactate (assays 1, 3, 4, 6, 7 and 8). However, exception was the assay 3 that despite containing lactate showed the greatest variation in pH, probably associated with lower soy and higher levels of sucrose and FOS that increased lactic

acid activity by probiotic culture. The other assays without the addition of lactate had variations in pH > 0.6. Bianchi, Rossi, Gomes, and Sivieri (2014) also observed a reduction in pH during 28 days of storage in fermented beverage formulated with aqueous extracts of quinoa and soy. However, even with the decrease in pH of the beverage during the shelf-life, the viability of probiotic bacteria was maintained. Li et al. (2014) reported that soymilk fermented with Lactobacillus spp, the pH at 4 °C storage during 21 days were significantly (p < 0.01) 26.10% lower than that recorded at 0 day of storage (near to 4.8 pH). 3.2.3. Syneresis The control of syneresis is regarded as an extremely important for the process due to the visual appearance of the final product, which is often rejected by customers. This defect is caused when the gel formed cannot retain the liquids of the product in the three-dimensional arrangement and are expelled, comparable to tears. It is commonly said that ‘‘the product is crying”. Syneresis responses for the experimental design can be observed in Fig. 2. Assays 2, 5 and 9 had the lowest percentage of syneresis and were considered the best ones. In these assays were kept levels below than 5% syneresis. This result is very good when compared with the syneresis of symbiotic control (19.5%). The main reason for this phenomenon is attributed to the absence of calcium lactate in all assays. The trials 10 and 11 without calcium lactate, also had low syneresis, below than 10%, but showed more fluctuations during storage, the trial 11, reached 10% on the 10th day and then decreased and held at 6.33%. Laneuville and Turgeon (2014), in experiments with acid milk gels containing 0.01% of anionic exopolysaccharide (EPS) produced by Lactobacillus rhamnosus RW-9595M in combination with different commercial polysaccharides, they found lower syneresis (2.5%) than control without EPS (3.7%), however, 0.05% EPS increased syneresis to over 17%, although no significant changes in gel strength. When anionic polysaccharides as xanthan or j-carrageenan, with stiff molecular structures were added, the protein self-aggregation and syneresis were induced in one week at cool storage. 3.2.4. EPS quantification The EPS quantification responses for the twelve experiments from the screening design (Fig. 3) were practically constant (between 0.5 and 6.0 g/L), being the mean of 3.8 g/L (Table 4) for

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all assays during 30 days of storage. The exception for the 10th day was to assays 2, 3, 4 and 7, that increased until 11.6 g/L. Assay number 7, formulated whit sucrose 12%, calcium lactate 1%, soy extract 10% and polydextrose 4% was quite different from the others, increasing during the storage and reaching the biggest concentration of EPS (16 g/L) in the final shelf-life. The assay 2 showed a lower percentage of syneresis also at day 10 (1.63%), then parallel to the decrease of EPS until 30th day (3.36 g/L), syneresis was increased (4.85%) too and it was formulated with higher levels of LR, sucrose, and soy. These results showed a relationship between the responses and the independent variables. In the meantime, the assay 5, formulated without lactate and polydextrose, over the 30 days of storage showing less syneresis, (0.90% at 10th day) and an average production of EPS (4.65 g/L). Still the majority of values for EPS producing in assays were major than the mean value of 3.4 g/L obtained with the control symbiotic soy product. Macedo et al. (2002) obtained up to 2.78 mg/L of EPS from L. rhamnosus RW-9595M in basal minimal media composed by serum and yeast extract enriched with vitamins, salts and amino acids. These authors explained that EPS production by lactic bacteria is influenced by the composition of the growth media, particularly amino acids, minerals, vitamins and nitrogenous bases, which is related to the production of biomass, without exceptions, in addition the methodology used can be else interfered in the results. In the current study, EPS concentrations were obtained up to 16.20 g/L, which is high when compared to the other results. Bianchi et al. (2014) showed that the presence of EPS and the higher total solid content in the product helped in the product firmness, hence the higher the consistency index, thus explain the high viscosity. Results reported by Grattepanche et al. (2007), showed a concentration of EPS in fermented milk 3 times smaller in mixed cultures of lactobacilli than with pure culture of L. rhamnosus RW-9595M. Conversely, in the current study, the concentration of EPS was bigger in the fermented food by LR in the presence of the mixed culture of probiotics (LAC + LBC + BL). Li et al. (2014) reported 0.64 g/L of EPS when soymilk was fermented with pure strain of L. rhamnosus 6005 after 21 days of storage at 4 °C and they observed that EPS produced by lactobacilli at 25 °C of storage decreased 50% in average. The EPS quantified (0.38% m/v in average) together with 4% (m/ v) added of prebiotics additives in the synbiotic soy food, confirm the functionality of these products, since legislation explain that foods are ‘‘high fibre source”, when containing 3,0 g/100 mL (Brasil, 1998).

3.2.5. Rheological properties and apparent viscosity Apparent viscosity (gap) is also an extremely important factor on final product quality. A too liquid fermented product is not well accepted by most persons, who associate yogurt or similar to a more consistent and creamy texture. Table 3 shows rheological models (according to Appendix A – Eqs. (A.1) to (A.5)) with a better adjustment (a greater explained variation coefficient – R2 – and a lower chi square – v2) for each of the experimental design tests, and the so, k and n rheological parameters, plus the calculation of gap for a 150 s 1 deformation (for food chewing) (Bourne, 2002). It can be observed that the majority of tests have behaviour of a Herschel-Bulkley fluid, associated to tests that have in their formulation the 10% level of soy extract. A few tests showed a pseudo-plastic behaviour, since they had negligible so; there was one case of Bingham Plastic behaviour in assay 9, on day zero, and the Dilatant case with assay 12. Bingham plastics are fluids that require initial shear stress (so) so that it can flow and have a linear relationship between shear stress and shear rate. Dilatant fluid means that the flow index is greater than unity, and the apparent viscosity increases with the shear rate. The

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trial 12 was formulated with all variables in the lower level; i.e. without sucrose, prebiotics and lactate. From Table 3 it is possible to note that the assay 1 (atypical), 5 and 7 showed the largest apparent viscosities (gap) and that the viscosity in assay 1 decreases from time 0 to 10 days (57.4– 2.7 mPa s), reaching values significantly lower than the other tests. The viscosity of assay 7 increases during the whole shelf-life (from 18.6 to 88.5 mPa s), and the gap of the assay 5 increases a little bit until the 10th day (34.7 mPa s), but remains practically constant afterwards. Another rheological property that was analysed was the initial shear stress (so), i.e., the stress required for the product flow to occur. One can note from Table 3 that the largest initial shear stresses belong to tests 2 and 5, but test 2 has an increase on the stress value in the first 10 days and, thereafter, there is a decrease until the 30th day. Test 5 presents a small increase until the 10th day, but the value remains constant until completing 30 days of shelflife. According Bianchi et al. (2014), the apparent viscosity is reduced after the application of high shear rates and the viscous behaviour becomes near-Newtonian, which implies an almost permanent breakdown of the structure. This is not completely permanent since the viscosity increases slightly when the fluid is stored for a long period, because it is a liquid containing gel fragments. And the apparent viscosity decrease at temperatures between 10 and 25 C. Li et al. (2014) observed the same behaviour in soy milk fermented with L. plantaruam or L. rhamnosus and gap approximately 100 mPa s with shear rate >10 s 1 in 21 days of storage at 4 C. This behaviour was observed in the product developed in the current study. The fluidity (n) and consistency (k) indexes were also analyzed, the results of which are in Table 3. One can note that k increases from the 10th day for assays 2, 5, 7 and 11, and in other tests its values remain constant. On the other hand, n decreases in assays 2, 5, 10 and 11 during the whole shelf-life, and in assay 12 there is a decrease until the 10th day but this test has the lowest values of k and the highest of n, which is explained because the treatment had all variables at the 1 level of the experimental design, becoming the worst of tests in relation to stability. In other tests n remains practically constant. 3.2.6. Effects estimation Table 4 summarizes the main effects of variables on responses, shows the coefficients of determination (R2), and the effects of significant variables (p  0.1) in boldface, for each of the eight responses of the Plackett & Burman experimental design related to the stability of the synbiotic product for 0, 10, 20 and 30 days of shelf-life at 4–6 °C. For Total Count (TC), through the calculation of means, the main effects and the standard error (p  0.10), one can analyse that for the finished product with 0 days of shelf-life, with a average of 9.93 log CFU/mL, calcium lactate had a negative effect, i.e., increasing the lactate concentration, the TC is decreased. FOS, polydextrose and sucrose had a positive effect, indicating that, increasing the addition of these prebiotics and sucrose (within the study limits), the higher TC is. However, for 30 days of shelf-life, with a mean of 9.80, none of the variables had a significant effect, indicating that in this period of time microbial stability had been reached. However, for the maintenance of the bifid bacteria (B. longum BL04), calcium lactate had a significant negative effect and only FOS had a positive effect. After 30 days of shelf-life, none of the variables had a significant effect on the maintenance of the bifid bacteria, showing that counts of BL until the 30th day are stable. In relation to EPS (Table 4) it can be seen that sucrose, followed by polydextrose, are the unique variables that affected EPS with positive effect (p  0.10) in the end of fermentation (day zero on

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Table 3 Rheological models adjusted to P&B experimental design tests. Assays

Models 0 days

R2

so (Pa)

k (Pa
sn)

n

gap (Pa s) (c = 150 s 1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Pseudoplastic Herschel-Bulkley Pseudoplastic Pseudoplastic Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Pseudoplastic Bingham plastic Newtonian-dilatant Herschel-Bulkley Dilatant Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Pseudoplastic

0.99982 0.99984 0.99996 0.99992 0.99841 0.99997 0.99990 0.99885 0.99968 0.99991 0.99995 0.99977 0.99997 0.99989 0.99900 0.99990

– 0.421 – – 0.742 0.037 0.034 – 0.231 – 0.244

0.068 0.025 0.018 0.011 0.038 0.025 0.043 0.023 0.007 0.003 0.018 0.001 0.021 0.031 0.030 0.027

0.967 0.876 0.870 0.924 0.824 0.846 0.828 0.832 1.007 1.063 0.860 1.299 0.877 0.824 0.840 0.830

0.0574 0.0159 0.0099 0.0082 0.0207 0.0116 0.0186 0.0098 0.0088 0.0050 0.0106 0.0032 0.0117 0.0134 0.0139 0.0114

Assays 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Models 10 days Newtonian-dilatant Herschel-Bulkley Pseudoplastic Pseudoplastic Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Pseudoplastic Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Dilatant Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley

R2 0.99940 0.99992 0.99984 0.99975 0.99994 0.99999 0.99998 0.99910 0.99990 0.99994 0.99995 0.99965 0.99999 0.99994 0.99996 0.99997

so (Pa)

k (Pa sn) 0.003 0.126 0.017 0.013 0.123 0.020 0.099 0.026 0.019 0.005 0.047 0.002 0.028 0.035 0.026 0.020

n 1.015 0.721 0.884 0.917 0.684 0.901 0.838 0.839 0.887 0.982 0.766 1.127 0.857 0.838 0.865 0.889

gap (Pa s) (c = 150 s 1)

Assays 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Models 30 days Pseudoplastic Herschel-Bulkley Pseudoplastic Pseudoplastic Herschel-Bulkley Herschel-Bulkley Pseudoplastic Pseudoplastic Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Dilatant Herschel-Bulkley Herschel-Bulkley Herschel-Bulkley Pseudoplastic

R2 0.99969 0.99774 0.99991 0.99980 0.99943 0.99997 0.99963 0.99944 0.99975 0.99988 0.99873 0.99970 0.99987 0.99986 0.99999 0.99998

so (Pa)

k (Pa sn) 0.003 0.852 0.025 0.008 0.379 0.023 0.334 0.029 0.057 0.038 0.287 0.001 0.032 0.060 0.022 0.030

n 0.999 0.513 0.848 0.980 0.538 0.879 0.735 0.806 0.732 0.746 0.512 1.179 0.851 0.780 0.887 0.849

gap (Pa s) (c = 150 s 1)

0.089 0.084 0.088 – – 1.779 – – 1.443 0.052 0.187 – 0.555 0.064 0.849 0.071 0.120 0.073 0.052

1.034 – 1.372 0.041 – – 0.618 0.248 0.336 0.104 0.112 0.052 –

so: yield shear stress (Pa), k = consistency coefficient, n = flow behaviour index, gap: Apparent viscosity calculated at deformation c = 150 s

the shelf); though, after 30 days, only sucrose had a positive effect and FOS had a small negative effect on response. However, one observes that EPS content is not associated to the product stability or to the quantity of the L. rhamnosus inoculum (without effect on response), but it is possible to observe that the EPS quality had an influence on the final product stability. For apparent viscosity (gap) on day zero of the shelf life (Table 4), there were no variables with a significant effect; but after 30 days in storage,% sucrose and soy extract variables had a significant positive effect on gap, the mean increasing from 0.02 to 0.03 Pa s, indicating that during storage metabolic activity by lactic bacteria continues to occur and it can cause changes on pH, on the structures of soy protein, on EPS, and, as a consequence, on the consistency and viscosity of fermented products. Analysing syneresis responses, calcium lactate had a significant positive effect on the syneresis response, while sucrose, FOS and polydextrose had significant negative effects. The lactate was cho-

0.0027 0.0429 0.0093 0.0088 0.0347 0.0127 0.0450 0.0111 0.0139 0.0052 0.0202 0.0032 0.0141 0.0158 0.0135 0.0117 0.0030 0.0678 0.0111 0.0077 0.0456 0.0125 0.0885 0.0110 0.0200 0.0122 0.0260 0.0028 0.0157 0.0206 0.0127 0.0139 1

(for chewing food).

sen as process variable in order to improve the stability of pH and decrease the syneresis problems. However the results showed the opposite, with an increasing of syneresis in average from 13% to 18% along the 30 days of shelf-life. The calcium lactate also showed highly effects on stability related to viable counts and rheological properties, but samples had a slight improvement in pH stability during shelf-life. It should be considered that the activity of the microorganisms during storage produce changes in acid concentration and consequently in pH. According with Chang (2003), a buffer is a solution that can resist pH change upon the addition of acidic and alkaline components. It is able to neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable. This is important for processes and/or reactions which require specific and stable pH ranges, i.e. shelf-life of foods. Buffer solutions have a working pH range and capacity which dictate how much acid/base can be neutralized before pH changes, and the amount by which it

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Table 4 Summary of effects for the independent variables and explained variation coefficients on responses through Plackett & Burman on stability of soy synbiotic product during shelflife. Independent variables

Estimated effects on responses Y1 = Syn

Y2 = EPS

Y3 = TC

Y4 = Bif

Y5 = gap

Y6 = so

Y7 = k

Y8 = n

0 days R2 Mean x1 = Prop LR x2 = %Suc x3 = %CaLact x4 = %SE x5 = %FOS x6 = %Polyd

0.8795 0.13 0.01 0.08 0.13 0.03 0.06 0.04

0.6972 3.66 0.32 2.47 0.03 0.89 0.24 1.82

0.8482 9.93 0.10 0.54 0.42 0.18 0.63 0.56

0.7621 9.22 0.09 0.06 0.91 0.07 0.76 0.27

0.4098 0.016 0.010 0.000 0.008 0.002 0.008 0.010

0.8182 0.136 0.101 0.195 0.256 0.177 0.039 0.117

0.5139 0.063 0.112 0.093 0.117 0.100 0.109 0.112

0.6295 0.902 0.057 0.141 0.086 0.078 0.076 0.047

10 days R2 Mean x1 = Prop LR x2 = %Suc x3 = %CaLact x4 = %SE x5 = %FOS x6 = %Polyd

0.8606 0.15 0.03 0.08 0.13 0.06 0.08 0.03

0.5520 4.73 0.98 6.19 2.98 2.40 0.75 0.35

0.7632 10.11 0.51 0.93 0.16 0.35 0.25 0.27

0.7050 9.44 0.27 1.02 0.27 0.61 0.21 0.35

0.9054 0.019 0.002 0.024 0.008 0.020 0.007 0.001

0.8709 0.329 0.288 0.612 0.745 0.496 0.121 0.256

0.9129 0.038 0.014 0.061 0.024 0.052 0.013 0.015

0.8046 0.875 0.020 0.162 0.038 0.132 0.034 0.003

20 days R2 Mean X1 = Prop x2 = %Suc x3 = %CaLact x4 = %SE x5 = %FOS x6 = %Polyd

0.8401 0.18 0.03 0.09 0.12 0.07 0.05 0.08

0.7465 3.16 0.92 3.79 2.70 1.78 0.48 1.88

0.7696 10.54 0.01 0.34 0.33 0.18 0.09 0.33

0.5519 9.56 0.39 0.01 0.85 0.00 0.52 0.63

n/d n/d n/d n/d n/d n/d n/d n/d

n/d n/d n/d n/d n/d n/d n/d n/d

n/d n/d n/d n/d n/d n/d n/d n/d

n/d n/d n/d n/d n/d n/d n/d n/d

30 days R2 Mean x1 = Prop LR x2 = %Suc x3 = %CaLact x4 = %SE x5 = %FOS x6 = %Polyd

0.8100 0.18 0.01 0.07 0.16 0.05 0.06 0.02

0.7093 3.72 1.61 5.11 2.33 2.53 1.32 2.74

0.5648 9.80 0.28 0.51 0.45 0.43 0.52 0.60

0.4552 8.89 0.50 0.32 0.33 0.52 0.38 0.26

0.7500 0.026 0.003 0.037 0.009 0.032 0.014 0.006

0.9003 0.271 0.347 0.376 0.664 0.457 0.082 0.264

0.8172 0.107 0.016 0.214 0.118 0.135 0.077 0.010

0.6823 0.802 0.027 0.211 0.146 0.144 0.061 0.075

*Shown in boldface, variables with a significant effect (p < 0.1) on responses for stability, calculated through the residual SS. Syn = syneresis (% v/v); EPS = exopolysaccharide (g/L); TC = total count (logCFU/mL); Bif = bifid bacteria count (logCFU/mL); gap = apparent viscosity (Pa
s); so = yield shear stress (Pa); k = consistency coefficient (Pa
sn); n = flow behaviour index (dimensionless); Prop LR = proportion of L. rhamnosus;%Suc = Sucrose;%CaLact = Calcium lactate;%SE = Soy extract;%FOS = Fructooligosaccharides;% Polyd = Polydextrose; n/d = non determined.

will change. Beynon and Easterby (1996) explain that the acidulants with higher molarity like lactic acid have a greater buffering capacity. The pKa of the acidulant is other factor involved; the closer the buffered pH is to the acid pKa, the higher is the buffering capacity. Lactic acid has a pKa equal to 3.86, it is a monoprotic acid, and therefore its capability to control pH is smaller when compared to poliprotic acids. The equilibrium reaction of acid lactic and lactate is represented as CH3CH(OH)CO2H H++ CH3CH(OH) CO2- and the equilibrium constant is Ka = 1.38  10 4 (Manning & Gramatges, 2013). Silva, Abreu, and Assumpção (2012) studied the effects on apparent viscosity, water retention capacity and syneresis of soy extract (14.8 g/L) and 2% of Bifidobacterium lactis in yogurts from goat milk during 29 days of storage. Soy extract caused viscosity and water retention increase (from approximately 500 to 1700 cP) and syneresis reducing (from 30% to 25%) in yogurts. The addition of B. lactis did not cause significant changes in the rheological characteristics of the products. In the current study the reduction of syneresis was from 30% to 5% approximately. In average the prototypes got 18% of syneresis during 30 days of storage at 4–6 °C. According to Table 4, variables with significant effects on yield shear stress (so) were, at time zero, sucrose and soy extract both

with positive effect, calcium lactate and polydextrose with negative effects at 10% significance. For this reason, some assays such as 6, 7 and 10, formulated with polydextrose and lactate, had the lowest values of so; however, assays such as 2 and 5 prepared with a +1 level of sucrose (12%), soy extract (10%) and without lactate yielded the highest values of so. Assay 9, containing polydextrose (4%) and soy extract (10%) at the +1 level, without the addition of sucrose at and calcium lactate showed an intermediate value of so. After 30 days, all variables, with the exception of FOS, had an effect on so, and the rate of L. rhamnosus, sucrose and soy extract affected positively so (Table 4). Thus, tests like 2 and 5, inoculated with the condition (10:30/60 LAC + LBC:LR/BL) of the LR, showed the highest values of so, a parameter that defines HerschelBulkley fluids, characteristic of most milk yogurts. One can observe that the mean of so increases from 0.16 to 0.27 Pa from 0 to 30 days on the shelf, according to the gap. As it can be observed from Table 4 for rheological indexes k and n, there was the same phenomenon as for gap on day zero on the shelf-life; there were no variables with a significant effect on them; after 30 days in storage, sucrose had a positive effect on k and negative on n; k is also positively affected by soy extract and negatively by the lactate. Means of k increased from 0.06 to 0.11 during the 30 days on the shelf-life, while means of n decreased

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from 0.90 to 0.80. One can also observed that the general trend is an increase in viscosity during shelf-life.

Model type

4. Conclusions Newtonian The EPS producing was a HePS with molecular mass of 39 KDa. Sucrose and soy extract had a significant positive effect in most stability responses during shelf-life. These variables favour the maintenance of probiotics and rheological characteristics. Calcium lactate highly affects stability of the symbiotic soy food in relation to syneresis, viable counts and rheological properties. The prototypes had a small improvement in pH stability and bifid counts during 30 days of shelf-life. The addition of calcium lactate buffer increased the syneresis of the fermented product, while sucrose and polydextrose decreased it. The best tests for the stability study during 30 days of shelf-life for the soy extract based synbiotic product were characterized by not containing calcium lactate and containing quantities of sucrose (12%) and soy extract (10%). The prototype containing 10 portions of LR, 10%SE, 4% FOS, 4% Polydextrose, without sucrose and lactate, and a prototype containing 10 portions of LR, 12%Sucrose, 6% SE, 4% Polydextrose, without FOS and lactate generated good results in general stability. These tests yielded the smallest syneresis, Herschel-Bulkley characterization, best pH and viable counts stabilization during shelf-life. Counts of added probiotics had little changes, keeping excellent concentrations and assuring the product’s functionality. Variables with the best effect on stability were soy extract, sucrose and the FOS and polydextrose prebiotics, being possible to add them at the quantities of the central points, or greater, for the preparation of diet or zero sucrose products.

Replicates Equation Viscosity (⁄) adjustment to model

Linear adjustment s = k
c, s0 = 0 Pseudoplastic Power law adjustment s = k
cn, n<1 Dilatant Power law adjustment s = k
cn, n>1 Bingham Linear Plastic adjustment s = k
c+ s0, s0 > 0 HerschelAdjustment s = s0 + k
cn, Bulkley n<1

Equation

A.1

g = s/c = k

A.2

gap = k
c(n

1)

A.2.1

A.3

gap = k
c(n

1)

A.3.1

A.4

gap = (s/c) + k A.4.1

A.5

gap = (s0/c) + k
c(n 1)

A.1.1

A.5.1

k = consistency coefficient (Pa sn); n = flow behaviour index (dimensionless); s0 = yield shear stress (Pa); s = shear stress (Pa); c = shear rate (s 1); g = kinematic viscosity (Pa s); gap = apparent viscosity (Pa s). (⁄). The g and gap was calculated for 150 s 1, being the average of the deformation force for chewing food (Bourne, 2002).

Conflict of interest The authors have declared no conflict of interest. Data in brief Studies were conducted to verify the kinetics of growth, carbon fonts consumption, EPS formation and rheological behaviour in different mediums with soy extract by Lactobacillus. rhamnosus sp (LR) added or not with sucrose and prebiotic, with and without probiotic standard mixed inoculum during fermentation. It was observed that the LR showed in front of several Lactic Acid Bacteria (LAB) strains the best growth in soy extract with high sucrose concentrations and the best capacity to produce polysaccharides (EPS). Acknowledgements This study was supported by grants from the Higher Education Personnel Improvement Coordination (CAPES), the National Council for Scientific, Technological Development (CNPq), the Student Program Post Graduate Agreement (PEC-PG) of Brazil, by contributions from the Engineering Biochemicals Processes Laboratory (LEB) – University of Campinas and by grants from the ProceedingsÒ company, as well as by donations to Ó Olvebra Industrial S/A, DuPontTM DaniscoÒ, and the Beneo Orafti. We would like to thank Dra. Maria Isabel Rodrigues for her support in the Plackett & Burman experimental design and Dra. Fátima de Almeida Costa by the contribution with chromatography analyses. Appendix A. Equations Equations A. Rheological models adjusted to symbiotic food soy samples and equations to calculate kinematic and apparent viscosity in Plackett & Burman assays.

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