Evaluation of nutritional and physicochemical characteristics of soy yogurt by Lactobacillus plantarum KU985432 and Saccharomyces boulardii CNCMI-745

Nutritional yeast-produced soy yogurt has grown in demand, because of its unique nutritional and health benefits. It has low cholesterol, no lactose, and high levels of protein, probiotic yeast, vitamins, and minerals. In this work, Soymilk (12.5%) was prepared and fermented to produce soy yogurt. Growth curves, probiotic characteristics of Saccharomyces boulardii CNCMI-745 and Lactobacillus plantarum KU985432 were determined. The nutritional value of both yogurts was evaluated, including viable cell count, protein, vitamin B-complex, sugars, phenolic acids, and fatty acids, mineral content, stability, and storage. Analysis of the physicochemical composition of the yogurts included assessment of titratable acidity, antioxidant potential, viscosity, and moisture content. The probiotic viable count of the produced yogurts met the standards for commercial yogurts. S. boulardii CNCMI-745 displayed safety characteristics and high tolerance to heat, acid, and alkaline stress. The produced B vitamins increased in both yogurts. The total saturated fatty acids in Saccharomyces-yogurt decreased, while the unsaturated fatty acids increased. Saccharomyces-yogurt showed high antioxidant activity, phenolic acids, and crude protein content. Both yogurts demonstrated the same tendency for stability during 16 day-storage. In conclusion, using nutritional yeast in the production of soy yogurt increased its nutritional content more than probiotic lactic acid bacteria.

Probiotic properties.Stress tolerance tests were characterized for S. boulardii CNCMI-745 12 .The stress tolerance response of S. boulardii CNCMI-745 evaluated by subjecting live cells to various stress conditions.The culture of S. boulardii CNCMI-745, which grew on potato dextrose broth at 37 °C, was adjusted to about 0.6 at OD 600 .After 10-min centrifugation at 6000 rpm, yeast cells were re-suspended in various stress solutions after being washed twice with 20 mL of sterile, pH 7, 0.2 M sodium phosphate buffer.Yeast cells were then subcultured in potato dextrose broth and kept at 37 °C for 48 h.Controls grown on nutrient broth for 48 h at 37 °C were regarded as having 100% viability.Experiments were done in duplicate, expressed as mean ± SD and the results were compared to the control.
Safety aspects evaluation: Hematolytic activity.The hemolytic activity of yeast cells was used to assess their safety 13 .The technique involved plating actively growing cells onto Columbia-agar enriched with 5% (v/v) animal blood, which was used to measure the production of hemolysin.The plates were incubated aerobically at 37 °C for 24-48 h because anaerobic incubation might interfere with hemolytic activity.
Safety aspects evaluation: Histamine production.Decarboxylase agar medium was used to test the production of histamine 14 .Yeast cells were then streaked in duplicate on decarboxylase medium plates with and without histidine (as a control) and incubated for 4 days at 37 °C under aerobic conditions.
Production of soy yogurt.Soymilk was prepared according to the pervious method 15 .Soybeans were washed, soaked overnight in distilled water and then grinded with distilled water (1:8 w/v).The slurry was filtered through double-layered cheesecloth to separate insoluble residues.The soymilk was then autoclaved at 121 °C for 15 min.150 mL of the soymilk was inoculated with 10% of L. plantarum KU985432 or S. boulardii CNCMI-745 and left unshaken at 40 °C for 48 h.Before inoculation, the probiotic cultures were centrifuged at 4 °C and 5000 rpm for 10 min and washed twice with sterilized distilled water.
Acid production.The titratable acidity of both yogurts, which corresponds to the amount of sodium hydroxide (0.05 M) required to titrate a certain amount of the sample to a pH of 8.1, was also determined 16 .
Cell viability.The initial and final viable counts of L. plantarum KU985432 and S. boulardii CNCMI-745 were determined after 48 h of fermentation.Probiotic cultures were inoculated onto modified MRS agar for L. plantarum KU985432 and potato dextrose agar for S. boulardii CNCMI-745.All plates were incubated for 48 h at 37 °C.The count of viable cells was determined and expressed as CFU/mL 17 .
Stability and storage of yogurts.The viable cell count of L. plantarum KU985432 and S. boulardii CNCMI-745 used in preparing yogurt were monitored periodically for 16 days.Yogurt samples were preserved in the refrigerator.The viable cell count was determined after 48 h incubation by plate count agar 11 .
Extraction of soy yogurt.Extracts for total phenolic compounds and antioxidant activity were prepared using methanol.Ten grams from each yogurt sample was mixed with 100 mL methanol and homogenized using the Ultra-Turrax homogenizer.The homogenates were stored at 4 °C for 12 h before being centrifuged at 10,000 rpm for 20 min.The supernatants were recovered and stored at -20 °C until analysis.
Determination of total phenolic content.The total phenolic content was determined by Folin-Ciocalteu reagent according to Žilić et al. 19 .
Phenolic acids profile by HPLC.The sample (1 g) was hydrolysed with 20 mL of 2 M NaOH for 4 h at room temperature.The pH of the samples was adjusted to 2 with 6 M HCl.Then, phenolic compounds were extracted twice with 50 mL of a 1:1 mixture of ethyl ether and ethyl acetate.The organic phase was separated and evaporated at 45 °C and the samples were redissolved in 2 mL methanol.Chromatographic analysis of phenolic acid and isoflavones were performed by HPLC model 1100 series (Agilent Technologies, CA, USA) 20 .

Determination of water-soluble vitamins by HPLC.
The extraction solution was made by mixing 50 mL of acetonitrile with 10 mL of glacial acetic acid, and the volume was finally made up to 1000 mL with double distilled water.Each sample (10 g) was weighed and homogenized in a mortar with a pestle before being transferred to a conical flask with 25 mL of extraction solution and kept in a shaking water bath at 70 °C for 40 min.The sample was then chilled, filtered, and the volume was adjusted to 50 mL with extraction solution.After that, the sample was filtered through 0.45 m filter tips, and aliquots of 20 µL from this solution were injected into the HPLC by auto-sampler.The analysis and quantification of vitamins in samples were performed using an Agilent 1100 chromatographic system (Agilent Technologies, CA, USA) 20 .
Determination of sugars by HPLC-RID.The yogurt samples were extracted with 20 mL of deionized water and sonicated for 30 min.The final volume of solution was completed to 50 mL and filtrated through a 0.45 µm syringe filter.Sugars (glucose, fructose, and sucrose) were determined by Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler, a refractive index detector, and an SCR-101N analytical column.The mobile phase was water with a flow rate of 0.7 mL/min at 40 °C of the oven temperature.The injection loop was optimized for 5 µL.The concentrations of the products were determined from the peak area under the curve 21 .
Determination of fatty acid profile by GC-MS.The analysis of fatty acids was carried out using GC-MS system (Agilent Technologies) gas chromatograph (7890B) was equipped with mass spectrometer detector (5977A).The GC was equipped with HP-5MS column (30 m × 0.25 mm internal diameter and 0.25 μm film thickness).The derivatization of fatty acids was performed by 1% sodium methoxide in methanol 22 .Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.
Chemical composition.Moisture, protein (N × 6.25), fats (ether extract), ash, and crude fiber contents were determined according to 23 .Total carbohydrates were calculated by difference.Determination of minerals (Ca, K and Fe) was carried by atomic absorption, while phosphorus was determined by the spectrophotometer series 24 .

Results
Growth curve of L. plantarum KU985432 and S. boulardii CNCMI-745.L. plantarum KU985432 and S. boulardii CNCMI-745 growth curves are presented in Fig. 1a and b.The relation based on colony counting (Log CFU/mL) (Y-axis) against time (h) (X-axis) of these strains is given by the empirically derived equation Y = 0.0119X + 8.6458 and Y = 0.0068X + 6.9863, respectively.This equation described the growth pattern of the bacterial and yeast cells in MRS and PDA media at pH 6.5 and 37 °C, respectively.From measured growth curves (Fig. 1a,b), the exponential (Log) growth phase of L. plantarum KU985432 and S. boulardii CNCMI-745 was the same as observed between 8 and 48th h at 37 °C.

Probiotic properties of S. boulardii CNCMI-745. S. boulardii CNCMI-745 probiotic characteristics
were determined, just as they had been done for L. plantarum KU985432 by 10 .As depicted in Fig. 1c, S www.nature.com/scientificreports/9.0 for 3, and 6 h, cell survival ranged from 80 to 89%.It also managed to survive exposure to high temperatures up to 55 °C for 30 min and 70 °C for 15 min, respectively, with survival rates of 84 and 96%.However, the overall resistance to osmotic, surfactant and enzymatic stress was considerably lower, the exposure to 0.05% H 2 O 2 did not totally kill the cells, and it could survive and regrow again after sub-culturing the cells.Additionally, the safety characteristics were assessed to ensure the safe use of this probiotic yeast in food products.Fortunately, S. boulardii CNCMI-745 has no positive histamine production or blood hemolysis in the screening medium.It was a negative histamine producer and γ-hemolytic.
Production of soy yogurt.Viable cell count of Lactobacillus-yogurt increased from 1.2 to 1.7 × 10 8 CFU/ mL (Table 1).The final viable cell count of S. boulardii CNCMI-745 in the produced yogurt also increased from 1.475 to 1.795 × 10 8 CFU/mL.The highest titratable acidity was seen in soymilk fermented with L. plantarum KU985432, and the lowest was in Saccharomyces-yogurt compared to unfermented soymilk.www.nature.com/scientificreports/acid of soy milk increased.However, in Lactobacillus-yogurt and Saccharomyces-yogurt, the content of palmitic, trans-oleic, and cis-linoleic acids was reduced, although stearic acid was the same as in unfermented soymilk.In Saccharomyces-yogurt, the total saturated fatty acids decreased from 18.99 to 18.36%, whereas the unsaturated fatty acids increased from 81.01to 81.31%.
Vitamin B-complex.The changes in B vitamin (B1, B2, B6 and B9) in both soy yogurts are shown in Table 4.The thiamine (B1) content of unfermented soy milk was increased from 26.93 to 44.31 and 40.14 µg/g for both Lactobacillus-yogurt and Saccharomyces-yogurt at 48 h fermentation.Results showed that riboflavin content was increased to 3.13 µg/g in Lactobacillus-yogurt, followed by Saccharomyces-yogurt (2.28 µg/g), at 48 h fermentation, compared with unfermented soymilk, which contains 1.36 µg/g (Table 4).
The pyridoxine content (B6) and folate content (B9) of soymilk fermented by L. Plantarum KU985432 decreased more than S. boulardii (Table 4).Compared to unfermented soymilk, the folate level was about the same after fermentation by S. boulardii CNCMI-745.

Sugar profiles.
The change in reducing and non-reducing sugars showed an overall downward trend (Table 4).After 48 h of fermentation, the non-reducing sugar sucrose content of soymilk decreased in Lactobacillus-yogurt and Saccharomyces-yogurt, respectively, from 3.797 to 1.165 and 1.326 g/100g.Within two days of fermentation, the monosaccharides, fructose, and glucose were all nearly depleted.After, 48-h fermentation process, the glucose level of unfermented soy milk reduced from 1.140 to 0.447 and 0.28 g/100 g in Lactobacillusyogurt and Saccharomyces-yogurt, respectively.The fructose content of unfermented soymilk decreased as well (Table 4).After, S. boulardii CNCMI-745 fermentation, sugars were reduced more than L. plantarum KU985432 fermentation.

Physicochemical composition of soy yogurt.
The protein content in both soy yogurts indicated that fermenting soymilk with the two probiotic cultures increased its protein content (Table 5).Results also showed that unfermented soymilk samples had a lower fat content than Lactobacillus-yogurt and Saccharomyces-yogurt samples.Lactobacillus-yogurt and Saccharomyces-yogurt samples had almost the same fat contents.A sample of unfermented soy milk contained more ash (0.71%) than samples of lactobacillus-yogurt and Saccharomyces-yogurt. Lactobacillus-yogurt had less carbohydrate content than Saccharomyces-yogurt and unfermented soymilk (Table 5).Unfermented soymilk, and Lactobacillus-yogurt and Saccharomyces-yogurt had nearly the same moisture content of 95.75, 94.32 and 95.48%, respectively.For minerals content, both yogurts had higher minerals content than unfermented soymilk, except for Zn (Table 5).Lactobacillus-yogurt had the same content of Zn that was found in unfermented soymilk.
Stability and storage of soy yogurt.Figure 1d shows the changes in viable counts of L. plantarum KU985432 and S. boulardii CNCMI-745 in both yogurts.L. plantarum KU985432 and S. boulardii CNCMI-745 viable counts increased at the same rate until the end of the 16-day storage period at 6 °C.

Discussion
The field of fungal probiotics is one that is currently evolving.Yeast contains a massive and diversified population of microorganisms, which is drawing and extending the interest of researchers and companies 1 .In this work, S. boulardii CNCMI-745 as a probiotic yeast was used to produce soy yogurt, so its probiotic characteristics were evaluated.The same probiotic characteristics were found by Graff et al. 25 .They found that S. boulardii can survive at body temperature (37 °C), giving it the distinct benefit of being one of the few yeasts that thrive at human body temperatures.It can also tolerate gastric acid and bile salt.S. boulardii CNCMI-745 was also evaluated for its safety and it was negative histamine producer and γ-hemolytic.Similarly reported by Fernández-Pacheco et al. 26 Cell wall of S. boulardii is thicker than that of other yeasts.These cell wall features can explain some of its probiotic effects, such as tolerance to stress produced by fluctuations in regular and simulated gut pH values 8 .We had successfully produced two fermented soy-yogurts using L. plantarum KU985432 and S. boulardii CNCMI-745.viable cell count of L. plantarum KU985432 and S. boulardii CNCMI-745 in both yogurts increased.
Most LAB prefer to grow in a neutral pH, which is given by soymilk 27 .Similarly reported, the yeast S. boulardii growth in fermented soymilk reached a maximum after 48 h of incubation, ranging from 7.57 to 7.87 log CFU/ mL 28 .Across many countries, viable counts of yogurt products should be between 10 6 and 10 9 CFU/mL.As a result, both soy yogurts prepared in this work meet the commercial yogurt product criteria.Lactobacillus-yogurt showed a more acidic pH value than Saccharomyces-yogurt. Probiotic bacteria in soymilk produce galactosidase, which converts oligosaccharides (raffinose, stachyose, and sucrose) to lactic acid in varying amounts depending on the strain's galactosidase activity 27 , so pH of soy milk decreased during fermentation as the incubation period progressed.Soymilk fermented with S. boulardii CNCMI-745 expressed the highest radical scavenging activity compared to L. plantarum KU985432.It was also found that Saccharomyces-yogurt had the highest amount of total phenolic compounds followed by Lactobacillus-yogurt, compared to unfermented soymilk.So, the potent antioxidant activity of both yogurts may be due to that both yogurts had a higher content of total phenolics, and fatty acids than unfermented soymilk. in coincidence with that result, we found Saccharomyces-yogurt contained higher concentrations of phenolic acids like cinnamic acid, protocatechuic acid, gallic acid, p-hydroxybenzoic acid, and catechins than Lactobacillus-yogurt did.Cinnamic acid and its derivatives, particularly those with the phenolic hydroxyl group, are well-known antioxidants with various health benefits attributed to their high free radical scavenging abilities 29 .Protocatechuic acid, and protocatechuic aldehyde have been shown to have pharmacological effects both in vitro and in vivo, which include antioxidant activity 30 .Increased concentration of gallic acid in fermented yogurts could be du to that hydrolysable tannins of soy can be transformed to gallic acid 31 .Daidzein, and genistein increased also after 48h fermentation.That increment could be due to β-glucosidase which produced during probiotic soymilk fermentation.β-glucosidase hydrolyzes the glucosidic bond of glucosidic daidzin and genistin into their aglycone forms 32 .The extract of Cheonggukjang (traditional Korean fermented soybeans) containing genistein and daidzein had potent antioxidant activity in vitro 33 .The increased phenolic acid concentration during fermentation with L. plantarum KU985432 or S. boulardii CNCMI-745 was most likely due to microbial constitutive enzymes activating insoluble or bound phenolic acids, resulting in phenolic acids liberation 34 .However, the decline in coumaric acid content could be attributed to the formation of p-hydroxybenzoic acid due to the degradation processes 35 .In Saccharomyces-yogurt, the total saturated fatty acids decreased, whereas the unsaturated fatty acids increased.Twelve or more fatty acids were reportedly found in wine fermented by various species of Saccharomyces at various temperatures and sweet potato fermented by S. bouldardii with higher levels of myristic acid, stearic acid, linolenic acid, and Docosahexaenoic acid (DHA, omega-3) in comparison to the control 36 .Solid-state fermentation with L. casei increased the omega-3 fatty acids and decreased the saturated fatty acids content of soybean flour 37 .In Lactobacillus and Saccharomyces-yogurts, the thiamine (B1) content increased.This may be because the two probiotics can synthesize vitamin B1.Similarly reported, the thiamine level of fermented cashew apple juice by L. acidophilus was increased significantly throughout the fermentation period 38 .For riboflavin (B2) content, it also increased after fermentation by L. plantarum KU985432 and S. boulardii CNCMI-745.Similarly, L. acidophilus isolated from dairy samples could produce riboflavin 39 .Riboflavin could be synthesized by Lactobacillus spp isolated from a traditional sourdough 40 .Moreover, the increase in riboflavin concentration in fermented-probiotic products could be attributed to the riboflavin production pathway in probiotic bacteria 41 .The low level of pyridoxine (B6) and folate (B9) in Lactobacillus-yogurt could be ascribed to LAB requiring for their growth 38 .However, the amount of pyridoxine was increased in cash apple juice fermented by L. casei 38 .Furthermore, folate (B9) levels increased in fermented potato substrates produced by two L. sakei strains increased 42 .According to sugar analysis soy yogurts by HPLC-RID, S. boulardii CNCMI-745 fermentation resulted in a greater reduction in sugars than L. plantarum KU985432 fermentation.Yeast invertase can convert sucrose into fructose and glucose in samples associated with S. boulardii 44 .Reducing sugars are continuously used as fermentation substrates, which causes their content to decrease because probiotics require sugar metabolism for energy to maintain their propagation during fermentation 43,44 .

cFigure 1 .
Figure 1.(a) Time and log CFU/ml empirical relation of L. plantarum KU985432 in MRS medium; (b) Time and log CFU/ml empirical relation of S. boulardii CNCM I-745 in PDA medium; (c) Probiotic properties of S. boulardii CNCM I-745; (d) The change of L. plantarum KU985432 and S. boulardii CNCM I-745 viable counts in yogurts samples produced with soy milk during storage.

Table 2 .
Changes of phenolic acids in soy yogurt.*ND not detected.

Table 3 .
Changes of fatty acids in soy yogurt.*NDnot detected.Fatty acid (

Table 4 .
Changes of B vitamins and sugars in fermented soy yogurt.

Table 5 .
Physicochemical composition of soy yogurt.