Impact of Storage on Probiotic Viability, Nutritional and Sensory Quality of Fermented Soymilk Produced from Different Soybean Varieties

Background: In recent years, there has been an increase in consumer demand for beverages made from fermented soybeans. Unfortunately, the nutritional imbalance and flavour profiles of plant-based milk substitutes frequently prevent people from consuming them. Fermentation can aid plant-based milk substitutes’ taste profiles, nutritional characteristics, texture and microbiological safety in order to produce more valued and delicious products without adding additional, frequently viewed as fake, ingredients. Methods: The implications of probiotic fermentation to improve the nutritive and sensory quality of fermented soymilk are assessed. Three soybean varieties as food grade soybean (Krune), low KTI soybean (V-23) and black soybean (VL-Bhatt 65) are used for the formation of soymilk which further fermented by three probiotic strains as Lactobacillus rhamnosus , Lactobacillus acidophilus and Weissella confusa . The fermented soymilk samples were stored for 28 th days at 4  C to analyse the changes in chemical composition and sensory quality. Result: High protein content was found in BA (6.85±0.13 g/100g), while the consumer preferred the LR with best overall ranking (6.25±0.05) in hedonic scale. The viability count of probiotic was highest (9.44±0.17 log cfu/ml) in BA after storage. The study suggested that soymilk with enormous number of biomolecules or pigments showed better viability of probiotic after storage and L. rhamnosus fermented soymilk preferred by the consumer over others probiotic fermented soymilk.


INTRODUCTION
Soybean is the prominent crop in India which majorly grow in Madhya Pradesh. Soybean contain profound amount of protein, which is a good source for vegetarian peoples. Hence, different soy-based food products as soymilk and tofu are consumed all around the world Ahmad et al., 2021). Apart from the high-quality protein soybean also possess isoflavones which play key role in therapeutic property of soymilk. For people with lactose intolerance, vegetarians, or people who have a milk allergy, it offers a plentiful and affordable source of nutrients (Maheshwari et al., 2021;Sasi et al., 2022). Additionally, it offers customers health advantages like hypolipidemic, anticholesterolemic and antiatherogenic qualities, as well as the ability to minimise allergenicity (Sasi et al., 2022). On the other hand, because the human intestinal tract lacks the enzyme α-galactosidase, soymilk has unfavourable qualities due to its indigestible oligosaccharides such stachyose and raffinose (Donkor and Shah, 2008). These indigestible carbohydrates can be converted into gas and other unpleasant symptoms by GI-residual bacterial groups (Rodrigues et al., 2020). In addition, some anti-nutritional factors also found in soybean as phytic acid and trypsin inhibitor . Phytic acid impedes the bioavailability of bivalent cations as Ca, Mg, Fe and Zn while trypsin inhibitor affects the proteolytic enzyme trypsin to digest protein (Tangyu et al., 2019) (Vadodariya et al., 2022).
Probiotics are defined as living microorganisms that, when given to a host in sufficient amounts, confer health benefits on that host. For example, probiotics can change the balance of the intestinal microflora, stop the growth of pathogenic bacteria, promote healthy digestion and immune function, lessen the impact of allergens, improve lactose intolerance and boost resistance to infection (Zhu et al., 2020). Probiotics have been used for decades in dairy products including yoghurt, kefir and cultured buttermilk, which causes the development of acids and the release of  (Xia et al., 2019). Probiotics contained in plant-based products are becoming more and more prevalent recently.
The fermentation of soy milk with lactic acid bacteria (LAB) was discovered to improve the nutritional and bioactivity values of ingested foods, as well as the quality, nutritive value and sensory aspects of soy milk. The aim of this study is to understand the changes in chemical composition, bacterial viability and sensory profiling of fermented soymilk during 28 th days of storage at 4C.

Soybean seeds
The experiment was design and executed during March 2020 to July 2022 at the Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi and Division of Food Science and Post-harvest Technology, ICAR-Indian Agricultural Research Institute, New Delhi. Three types of soybean variety were used for the preparation of soymilk. Food grade soybean (krune), Low KTI soybean (V-23) and black soybean (VL-Bhat 65) were procured from Division of Genetics, ICAR-IARI, New Delhi, ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora, Uttarakhand respectively.

Chemicals and bacterial strains
All of the chemicals used for the viability investigation and proximate analysis were purchased from Sigma Aldrich in the USA.
For the fermentation of soymilk, three probiotic bacterial strains-Lactobacillus rhamnosus 1136 (LR), Lactobacillus acidophilus 1132 (LA) and Weissella confusa 30082b (WC)were obtained from the National Centre for Microbial Resource in Pune, India, the Division of Microbiology at the ICAR-Indian Agricultural Research Institute in New Delhi, India and the RIKEN Bioresource Research Centre, In MRS broth, the acquired bacterial cultures were kept alive.

Preparation of soymilk
Using the hot-water extraction method, soymilk was made from food-grade, low KTI and black soybeans. After being cleaned, soybean seeds were steeped in distilled water all night. The swollen soybean seeds were manually dehulled, crushed in hot water (80C) at a ratio of 1:10 and then filtered through two layers of cheesecloth to get rid of any insoluble residues (Donkor and Shah, 2008). The extracted soymilk was put in a glass bottle and autoclaved for 15 minutes at 121C to sanitise it. To be used later, the autoclaved soymilk is bottled, cooled to room temperature and kept at 4C.

Fermentation of soymilk
All three probiotic strains-LR, LA and WC were multiplied twice in MRS broth at 2% (v/v) each and incubated for 24 hours at 37C for LR and LA and 30C for WC. Temperatures of 37C and 30C, respectively, are ideal for the unhindered growth of the Lactobacillus and W eissella species.
Autoclaved and stored soymilk was independently inoculated (1% v/v means 1 mL bacterial culture in 100 ml soymilk) with three different bacterial strains for soymilk fermentation. The inoculation samples were fermented at 30C (WC) and 37C (LR, LA), respectively, for 24 hours. To ascertain the impact of fermentation on storing fermented soymilk at 4C for 28 days, unfermented samples incubated under the identical experimental conditions were utilised as a reference. Overall methodology adopted for sample preparation represented in Fig 1.

Determination of viability of probiotic after 28 days of storage at 4C
The plate dilution method with MRS agar was used to determine the viability of all three probiotic strains (Man et al., 1960). Each sample of fermented soymilk at 48 hr of fermentation and at 28 days after storage was serially diluted and plated in two separate dishes. The plates were then incubated for 48 hours at 37C (LR and LA) and 33C (WC) respectively. To calculate the results, colony forming units per millilitre (cfu/ml) were utilised.

Determination of pH and titratable acidity of fermented soymilk after storage
At 48 hours and 28 days of fermentation, the samples pH was assessed using a pH meter. By utilizing 0.1 N NaOH, titratable acidity was measured as a percentage of lactic acid (%TA). 0.1 N NaOH was used to titrate soymilk that had been diluted 1:1 with distilled water using 3-4 drops of phenolphthalein as an indicator. %TA, or titratable acidity, was calculated using the formula below:

Determination of proximate analysis of fermented soymilk after storage
The proximate analysis executed by using AOAC methods (AOAC 2000). All the analysis proceeds in triplicate. 1 g of samples was used for the nitrogen content estimation by using the Kjedahl method and the protein amount was approximated by multiplying the results by a conversion factor (5.71 for soy). The following equations were used for the calculation of % nitrogen and % of protein content in the soymilk sample.
Here: (V S -V B ) = consumed volume of 0.1N HCl Total carbohydrate content determined by difference to obtained total content of 100 g. To determine the moisture content, 5 g of the samples were dried in an oven at 70C % Titratable acidity (%TA) = Volume of 0.1 N NaoH consumed for neutralization 0.09 100 Volume of soyamilk  Specific gravity Weight of sample (mg) 100 % Protien = 5.71 % Nitrogen Impact of Storage on Probiotic Viability, Nutritional and Sensory Quality of Fermented Soymilk Produced from Different Soybean...
till weight of the sample became constant. Ash content in soymilk samples was determine by AOAC method. 2 g of sample heated or burn out at 550C in muffle furnace, until completely ashes. The calorie value of soymilk samples was determined by using Atwater conversion factor (protein4, fat9, carbohydrate4) (Santos et al., 2019).

Sensory analysis of fermented soymilk after storage
For sensory acceptance tests, a standardised 9-point hedonic scale (1 for extremely dislike; 9 for extremely like) was used (Albuquerque et al., 2017). 50 consumers (untrained panellists, consisting of men and women with ages ranging from 25 to 50) from the faculty, including teachers, students, staff and visitors from other places, were recruited based on interest and consumption patterns for soy product on each sampling day (at 48 hr and at 28 th days) of soymilk and fermented soymilk.

Statistical analysis
All experiments were performed in triplicate and results were expressed as mean±standard deviation (SD). Statistical analyses were carried out using SPSS 10.0 Statistics software and MV app (Julkowska et al., 2019). Tukey's test was used at a significance level of p<0.05.

Viability of bacterial strain of stored fermented soymilk
Lactobacillus rhamnosus (LR), Lactobacillus acidophilus (LA) and Wessilla confusa (WC) were significantly remained constant in fermented soymilk after 28 th days of storage at 4C. The viability of probiotic strains after 28 th days of storage of fermented soymilk at 4C was shown in Fig 2. The colony forming unit (CFU) of L. rhamnosus fermented food grade soymilk (FR), L. acidophilus fermented food grade soymilk (FA) and W. confusa fermented food grade soymilk (FW) was 6.1, 6.5 and 5.6 log cfu/ml respectively at 28 th days of storage. In the case of low KTI soybean milk fermented with same bacterial strains, the CFU was reported 5.8 log cfu/ml for LR, 6.1 log cfu/ml for LA and 5.2 log cfu/ml for LW. The cfu count in L. rhamnosus, L. acidophilus and W. confusa fermented black soymilk as BR, BA and BW were reported 9.23 log cfu/ml, 9.44 log cfu/ml and 8.56 log cfu/ml respectively. Among all three probiotic strains L. acidophilus showed better cell viability after storage period at 4C in comparison to L. rhamnosus and Weissella confusa. Similarly, among the three soybean varieties, black soybean milk showed better viability of bacterial strains than food grade soybean and low KTI soybean variety. The higher  viability of bacterial strains even after 28 th days storage at 4C indicates that black soymilk harbour plant bioactive compounds as anthocyanin or other phenolic compounds which act as source of hydrocarbon and energy for the growth of bacterial strain during storage (Krishnan et al., 2018;Janpaeng et al., 2018;Kumar et al., 2019;Krishnan et al., 2020;Bhartiya et al., 2020).

Changes in pH and titratable acidity (%TA) of stored fermented soymilk
During the storage pH of the fermented soymilk decreased while titratable acidity (%TA) increased ( In reverse to pH, the %TA of the samples enhanced after storage. %TA of unfermented food grade soybean milk (FC) observed 1.41, which increased during storage in fermented soymilk 1.59 (FR), 1.75 (FA) and 1.65 (FW). In low KTI soybean milk % TA observed 0.97 (LC), which increased in fermented sample during storage 1.32 (LR), 1.5 (LA) and 1.41 (LW). %TA of fermented samples of black soymilk raised from 1.94 (BS) to 2.34 (BR), 3.98 (BA) and 3.37 (BW ).
The reduction of pH in fermented samples indicates that bacterial strains producing some short chain fatty acids by metabolizing the large hydrocarbon molecules as RFOs (raffinose family oligosaccharides) which led to pH reduction (Nazhand et al., 2020) (Sasi et al., 2022). Among the three mentioned bacterial strains L. acidophilus (LA), fermented soymilk samples showed lower value of pH and higher value of %TA in comparisons to remaining probiotic strains.

Changes in chemical composition of stored fermented soymilk
Chemical composition as protein, total fat, carbohydrate, moisture, ash and energy content of non-fermented and fermented soymilk determined by the AOAC methods (AOAC 2000) and the obtained results are shown in Table 1 and Fig 3 (A-D). The protein content in non-fermented soymilk were reported as 1.94 g/100g (FC), 2.26 g/100g (LC) and 2.05 g/100g (BS) which are low in comparison to fermented soymilk (Fig 3A). The higher amount of protein content 6.85 g/100g was found in L. acidophilus fermented black soymilk (BA). High bacterial fermentation may cause the enhancement of protein content (Hong et al., 2012). The total fat content in non-fermented soymilk were reported as 1.21 g/100 g (FC), 1.87 g/100 g (LC) and 2.53 g/100 g (BS) which are high in comparison to fermented soymilk ( Fig 3B). The low amount of total fat 0.7 g/100g was found in L. acidophilus fermented low KTI soymilk (LA). The L. acidophilus fermented soymilk may be responsible for the substantial decrease in total fat (Lee et al., 2015). The higher amount of total carbohydrate 5.41 g/100g was found in L. acidophilus fermented food grade soymilk (FA) (Fig 3C) while the high energy value 49.75 Kcal was reported in W. confusa fermented black soymilk (BW) (Fig 3D). During the fermentation moisture content in the sample declined while the ash content raised. Hence high amount of moisture content was observed in non-fermented low KTI soymilk sample (LC) was 93.67 g/100 g and low amount of moisture content 88.87 g/100 g was reported in L. acidophilus fermented black soymilk sample (BA). Similarly, the high ash content 0.79 g/100 g was reported in L. acidophilus fermented black soymilk (BA), while low ash content 0.21 g/100g was observed in non-fermented low KTI soymilk (LC).

Sensory profiling of stored fermented soymilk
Along with the nutritive and health-promoting qualities, fermented products must also have sensory qualities that appeal to consumers. The sensory profiles of stored soymilk samples are shown in Fig 4. The lowest colour value (Fig 4A) was found in non-fermented black soymilk (BS-3 unit), while the highest colour value was found in non-fermented food grade soymilk (FC-5.8 unit), W. confusa fermented food grade soymilk (FW-6.0 unit), non-fermented low KTI soymilk  (LC-5.9 unit), L. rhamnosus fermented low KTI soymilk (LR-6.1 unit) and L. acidophilus fermented low KTI soymilk (LA-6.23 unit). The high colour value of low KTI soybean milk over food grade soymilk and black soymilk might be due to the presence of lower amount of fat in these samples which responsible for the off-white colour of the product. L. rhamnosus fermented food grade soymilk (FR) showed the lowest value (1.1 unit) of mouthfeel (Fig 4B), while the highest value of mouthfeel observed in L. acidophilus fermented black soymilk (BA-5.2 unit). The higher value of mouthfeel might be due to the presence of excess protein content in the BA sample which precipitate in acidic pH during storage. L. acidophilus fermented low KTI soymilk (LA) showed the higher value (5.3 unit) of consistency (Fig 4C), while for the taste point of view W. confusa fermented low KTI soymilk (LW) and L. acidophilus fermented black soymilk (BA) showed higher value of taste 5.1-unit, 4.9 unit, respectively ( Fig 4D). L. rhamnosus fermented low KTI soymilk (LR) showed the high flavour value (6.3 unit) (Fig 4E). Higher value of overall acceptance observed in L. rhamnosus fermented low KTI soymilk (LR-6.25 unit) followed by L. acidophilus fermented food grade soymilk (FA-6.19 unit) (Fig 4F). L. rhamnosus secretes exopolysaccharides (EPS), which enhance the texture of fermented products, hence L. rhamnosus fermented low KTI soymilk (LR) demonstrated the best consumer approval (Li et al., 2014). Additionally, the fragrance of the L. rhamnosusfermented soymilk was pleasant, which encouraged more people to choose the L. rhamnosus fermented low KTI soymilk (LR) sample.

CONCLUSION
The research described in this article has demonstrated that during storage of fermented soymilk, probiotic strain, in addition to soybean variety, also affects the pH, titratable acidity, chemical composition and particularly sensory parameters. Black soybean among the three soybean varieties demonstrated higher probiotic activity and improved chemical composition during storage of fermented soymilk. However, consumers appreciated fermented soymilk made from black soybeans less. Therefore, this study may be able to entice researchers to look into blending various pigmented soybean varieties or to separate bioactive ingredients and add them to soymilk for improved overall fermented soymilk performance.