Antioxidative, ACE inhibitory and antibacterial activities of soy milk fermented by indigenous strains of lactobacilli

Soy milk, a derivative of soybean, is an alternative to dairy beverage, but its acceptability is limited worldwide due to unpleasant beany flavour. Fermentation may, however, improve the nutritional and sensory values of soy milk. Thus, post‐fermentation improvement in functional attributes of soy milk were investigated via antioxidant, ACE inhibitory, and antimicrobial activities in this study using five test and one control strains of lactobacilli. Results indicated that soy milk fermented by Lactobacillus rhamnosus strain C25 (LR C25) effectively scavenged more than 60% of the ABTS, DPPH and Hydroxyl radicals generated in in vitro models. Moreover, all the strains showed significantly higher (p < 0.05) antioxidant activity as compared to unfermented soy milk in all three assays performed. Further, Lactobacillus plantarum strain C6 (LP C6) fermented soy milk displayed significantly higher (p < 0.05) percent ACE inhibitory activity (68.40 ± 0.93%) as compared to other tested Lactobacillus isolates, reference strain and unfermented soy milk. It was also observed that LP C6 strongly inhibited the growth of indicator strain of E. coli in the agar well diffusion assay. These strains can therefore be further explored in the preparation of beneficial soy foods and bioactive food supplements for wellbeing.


| INTRODUCTION
Economically, soybean (Glycine max L. Merrill) is the world's most important leguminous crop, supplying millions of people with vegetable protein, and hundreds of chemical ingredients. Traditionally, soybean is used to make various fermented and unfermented products such as miso, soy cheese, soy sauce, soy yogurt, tamari, tempeh, and soymilk, especially in the Asian subcontinent (Jayachandran & Xu, 2019;Tamang, 2015). Among them, soy milk is an aqueous extract of soybeans produced by soaking, grinding, and filtering. Soy milk is a perfect emulsion of oil, water, and protein. It includes sufficient proteins, iron, unsaturated fatty acids and other nutrients and low fat and carbohydrates (Singh, Vij, & Hati, 2014). Soy milk is also one of the richest sources of isoflavones, generally referred to as phytoestrogens because of their resemblance to estrogens (Cao, Green-Johnson, Buckley, & Lin, 2019).
Although, soy milk is a perfect nutrient supplement, but its acceptability limited worldwide due to beany flavor and flatulence.
Fermentation is now recognized as the best way to reduce soy milk's beany taste and flatulence along with enhancing soy milk's functional properties by increasing bioactive components and decreasing antinutritional components. In addition, fermentation is used to improve the bioavailability of soy vitamins, minerals, isoflavones and proteins (Cao et al., 2019;Singh & Vij, 2018). Refer to their reported beneficial effects on human nutrition and health, fermented soybean products are getting more attention now days.
Last few decades witnessed the use of lactic acid bacteria as a probiotic and a starter organism for the development of health beneficial foods. Although, Bacillus subtilis and Aspergillus are the microorganisms that are widely used to ferment various soy products (Jayachandran & Xu, 2019), now specific strains of lactobacilli have been recognized as a starter for the fermentation of soy milk (Dobreva, Dragnev, Mladenova, & Danova, 2019). Lactobacilli can degrade soy proteins into simpler forms like oligopeptides, di-peptides, and tri-peptides during fermentation resulting in improving the functionality of the protein. Enzymatic machinery of lactobacilli can hydrolyze soy complex oligosaccharides which are principally responsible for its beany flavor (Hati et al., 2013). Fermentation also enriches soy milk with functional attributes. It has reported that fermented soybean possess potent antioxidant (Singh & Vij, 2018) and angiotensin-converting enzyme (ACE) inhibitory activities (Wu & Ding, 2002), which are the major cause of most of the new generation disorders such cancer, cardiovascular and other related diseases. In this way, soy fermented milk and their bioactive components can be a possible alternative for the prevention of several cardiovascular and life-style diseases. This study was therefore designed to determine the functional attributes of soy milks fermented by selected strains of Lactobacillus. Three important activities viz. antioxidative, antihypertensive and antimicrobial were selected to appraise the possibility to develop a soy based functional fermented product.

| Antioxidative activity
Reaction mixture was prepared by mixing 1 ml brilliant green (0.435 mm) with 2 ml FeSO 4 (0.5 mm) and 1.5 ml of 3% H 2 O 2 . A 1 ml aliquot of hydrolyzate was added to reaction mixture and incubated at room temperature for 20 min. The change in absorbance was measured at 624 nm by UV-1800 spectrophotometer. The results were expressed as; Hydroxyl radical scavenging activity (Hydroxyl RSC) (%) = (A 624 nm blank -A 624 nm sample/A 624 nm blank) × 100.

| ACE inhibitory activity
ACE inhibitory activity was analyzed according to the method given by Cushman and Cheung (1971) with minor modifications. A 50 μl hydrolyzate was mixed with 50 μl of ACE (50 mU/ml) and preincubated for 10 min at 37 C. Then 150 μl of 4.15 mm HHL (Hippuryl-L-Histidyl-L-Leucine) solution was mixed with above mixture and incubated for 30 min at 37 C. The reaction was terminated by the addition of 500 μl of 1 M HCl. The hippuric acid liberated by the ACE was then extracted with 1.5 ml ethyl acetate by centrifugation at 3000 x g for 10 min followed by heat evaporation at 95 C for 10 min. The residue containing hippuric acid was dissolved in 1 ml of deionized water and the absorbance of the solution was measured at 228 nm (UV-1800 spectrophotometer). For blank all components except ACE was added and for control all components except sample was added. The extent of inhibition was calculated as follows; ACE

| Antibacterial activity
Antibacterial activity was measured using an agar well diffusion assay described by Schillinger (1989) with slight modifications. Nutrient agar (NA) plates were prepared and allowed to solidify. A 100 μl aliquot of the active test pathogen was mixed in soft agar (0.7% agar) and overlaid on pre-solidified NA plates. The inoculated plates were allowed to dry, and equidistant wells (6 mm) were punched out with sterile glass borer. The wells were filled with 100 μl of hydrolyzate, and uninoculated soy milk served as a control. The plates were pre-incubated at 4 C/2 h to facilitate the diffusion of hydrolyzate, followed by overnight incubation at 37 C. The antibacterial activity was measured as clear zone of inhibition extending around the well. Likewise, the percent DPPH RSC of LR C25 (64.0544 ± 1.10) and LR C28 (63.88 ± 0.71) was almost similar, both showed significantly higher (p < 0.05) activity among the tested bacterial strains and unfermented control (Figure 1b). Conversely, LP C6 and LR C34 were observed with lower DPPH RSC in comparision to reference bacterial strain NCDC 288. Moreover, no significant difference in percent DPPH RSC was found between LR C8 and NCDC 288. In DPPH RSC assay fermented hydolyzates were allowed to react with a stable DPPH radical in a methanol solution. The reduction of DPPH radical was followed by monitored in decrease of absorbance at 550 nm (Brand-Williams et al., 1995). Thus, two of our strains viz. LR C25 and LR C28 potentially inhibit the free radical produced by DPPH.
control ( Figure 1c). Overall, all the Lactobacillus strains in our study were efficiently able to inhibit more than 50% of hydroxyl radicals.
Differences in radical scavenging activity of soy milks may be due to generation of diverse free radicals in all the three assays performed.
Free radical scavenging capacities of soy milk may be principally due to bioactive components such as bioactive peptides, isoflavones, saponines and phenolic compounds, generated during fermentation by specific strains of lactobacilli. In this context, L. plantarum and L. rhamnosus strains were reported to actively grow in soy milk and enhances antioxidant activity by increasing phenolics and isoflavone aglycones (Subrota, Shilpa, Brij, Vandna, & Surajit, 2013;Xiao et al., 2015). Similar to our work, Xu and co-workers also studied antioxidant capacities of the twenty-seven fermented soybean products, who found highest activity in black bean product "Douchi". In addition, eleven L. plantarum strains isolated from traditional Chinese fermented products were analyzed and found that L. plantarum C88 showed the highest hydroxyl radical and DPPH scavenging activities, with inhibition rates of 44.31% and 53.05%, respectively. Further, when L. plantarum C88 was administered to mice suffering from oxidative stress, the serum superoxide dismutase activity, glutathione peroxidase activity and the total antioxidant capacity in liver increased significantly (Xu, Du, & Xu, 2015). Similarly, Lactococcus acidophilus fermented soymilk also reported to improve antioxidant capacities in hyperlipidemia rats (Chen, Wu, Yang, Xu, & Meng, 2017). A cocktail of probiotic (Bifidobacterium bifidum, Lactobacillus casei, and Lactobacillus plantarum) fermented soymilk also reduced the production of reactive oxygen species in high-fat diet mice . Bhatnagar, Attri, Sharma, and Goel (2018) reported ABTS radical scavenging activity ranging from 62 to 67% in soy milk fermented by Lactobacillus rhamnosus GG, Brevibacilllus aydinogluensis and Brevibacillus thermoruber strains. Likewise, Wang, Yu, and Chou (2006) documented strain specificity in antioxidant activity of fermented soy milk.
They found that reducing activity and scavenging effect of superoxide anion radicals varied with the starters used. Moreover, Zhao and Shah (2014), reported highest antioxidant activity in soy milk fermented by a Lactobacillus rhamnosus among all the Lactobacillus sp. studied. In addition, Marazza, LeBlanc, de Giori, and Garro (2013) reported that soy milk fermented by Lactobacillus rhamnosus exhibited higher antioxidant activity than the unfermented soymilk. Also, the fermented soymilk extracts were able to inhibit the oxidation of DNA induced by Fenton's reagent.

| ACE inhibitory activities of fermented soy milks
ACE inhibitory activity of fermented soy milk was investigated using HHL as a substrate and shown in Figure 2. The activity of LP C6, 68.40 ± 0.93%, was found significant higher (p < 0.05) from other tested Lactobacillus isolates, reference strain and unfermented soy milk. Additionally, the percent ACE inhibitory activity of LR C8, LR C25 and LR 28 was 56.52 ± 1.22, 65.52 ± 1.40 and 62.92 ± 0.52, respectively. The inhibition recorded by LR C8, LR C25 and LR C28 was significantly higher (p < 0.05) from reference strain NCDC 288.
The ACE inhibitory activity of all the fermented soy milks was significantly higher (p < 0.05) in comparison to unfermented soy milk. It has been known that; several ACE inhibitory peptides are released from F I G U R E 1 Antioxidative activity of fermented soy milks. Panel "a" ABTS radical scavenging capacity (ABTS RSC); panel "b" DPPH radical scavenging capacity (DPPH RSC); panel "c" Hydroxyl radical scavenging activity (Hydroxyl RSC). Graphs represents the mean ± SEM of each experiment performed in triplicate, values with different letters differ significantly (p < 0.05) in all panels the inactive soybean precursor proteins by the action of microbial proteases during the fermentation (Singh & Vij, 2017). In this context, the higher ACE inhibitory activity of most of the strains as compared to unfermented soy milk indicated that some ACE inhibitory peptides were released during fermentation. Several previous studies support this statement that soy milk fermented by specific strains of lactic acid bacteria is a good source of ACE inhibitory peptides (Singh & Vij, 2017;Tsai, Lin, Pan, & Chen, 2006). Likewise, Tsai and coworkers (2006), documented ACE-inhibitory effect of tri-peptide (Val-Pro-Pro and Ile-Pro-Pro) in animal model obtained from fermented soy products. Similar to our study, more than 55% ACE inhibition was reported in fermented soybean extracts and soy whey medium (Lye, Kuan, Ewe, Fung, & Liong, 2009;Pyo & Lee, 2007). Moreover, Ma, Cheng, Yin, Wang, and Li (2013) reported more than 60% increment in ACE inhibitory activity followed by fermentation. A recent study also reported around 80% ACE inhibitory activity of lactobacilli fermented soy milk after 5 days of storage under refrigeration conditions (Mishra, Hati, Das, & Prajapati, 2019). Bhatnagar et al. (2018) also found ACE inhibitory activity (41.66%) in fermented soy milk hydrolysate of Lactobacillus paracasei CD4 strain. In another study, the ACE inhibitory activity of L. fermentum fermented soy milk was observed to reached 60% after 20 h of fermentation (Myagmardorj, Purev, & Batdorj, 2018).

| Antibacterial activities of fermented soy milks
Antibacterial activity of fermented soy milks showed weak to strong inhibition against tested pathogenic microorganisms (  (Singh et al., 2015). Some previous reports support this statement that specific strain of Lactobacillus species such as L. plantarum and L. rhamnosus are able to produces some antimicrobial compounds (Lin & Pan, 2017;Zhang, Wu, et al., 2017).
Also, unfermented soy milk used as control in our study did not show inhibition against any of the pathogenic microorganism (Table 1)

| CONCLUSION
Based on the results from this study we may infer that selected strains of Lactobacillus generated some biofunctional components during soy milk fermentation. The functional activities (antioxidative, ACE inhibitory and antibacterial) of fermented soy milk may be due to a specific bioactive component or cumulative effect of these bioactive compounds. The enhanced bioactivities of fermented soy milk in our study as compared to unfermented soy milk possibly support this statement.
Therefore, these Lactobacillus isolates may be used for the development of health beneficial soy foods or bio-therapeutics. The present study did not, however, describe the bioactive components and specific mechanisms that regulate these effects. Further research may therefore be conducted to classify the biofunctional compounds in fermented soy milk and their impact in the in vivo system.

ACKNOWLEDGEMENTS
BPS wish to thank "Ministry of Social Justice and Empowerment, Government of India" and "University Grants Commission (UGC), India" for providing research fellowship during Ph.D. Also, the necessary support provided by Director, ICAR-National Dairy Research Institute, Karnal, India for conduction of this work is highly acknowledged.

CONFLICT OF INTEREST
No conflict of interest declared.

AUTHOR CONTRIBUTIONS
BPS design the study, performed the experiments, analyzed the data, and wrote the manuscript. BB reviewed and edited the manuscript.
SV supervised the concept and reviewed manuscript.
F I G U R E 2 ACE-inhibitory activity of fermented soy milks. Graphs represents the mean ± SEM of each experiment performed in triplicate, values with different letters differ significantly (p < 0.05)

ETHICS STATEMENT
This article does not contain any human and animal subjects for experiment.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.