A review on the functional properties of fermented soymilk

Soy products have been used for ages in various forms, including soymilk, soy cheese, tempeh, tamari, soy sauce, soy yogurt, and soy mayonnaise. They are rich in isoflavones, and their consumption is associated with several health benefits, especially protection from gastrointestinal disorders, cardiovascular diseases, and cancers (Asif & Acharya, 2013; Zhang et al., 2003; Messina, 2016; Pisani et al., 1999).

the antioxidant capacity of the fermented soymilk via β-carotene bleaching and DPPH methods showed that CRL981 mediated fermentation process improved the bioactivity of the soymilk. Moreover, the capability of CRL981 to produce β-glucosidase increased the isoflavone aglycones content in the fermented soymilk (Marazza et al., 2012).
Soymilk fermented using Streptococcus thermophilus grx02 showed higher antioxidant activity in DPPH assay and inhibited the formation of malondialdehyde (MDA). The supplementation of fermented soymilk significantly reduced alanine transaminase (ALT), aspartate transaminase (AST) activities in alcohol-fed mice. The content of glutathione and alcohol dehydrogenase, superoxide dismutase activities were increased in the liver of the mice supplemented with fermented soymilk compared to the control (Xu et al., 2012).
Soymilk fermented with L. paracasei KUMBB005 showed high antioxidant activity in DPPH and ferric reducing antioxidant power (FRAP) assays (Usha Rani & Pradeep, 2015). Similarly, soymilk fermented with L. fermentum LAB 9 displayed more significant antioxidant activity in DPPH assay compared to control (Fakri et al., 2016). Yamamoto et al. reported that soymilk fermented with Leuconostoc mesenteroides MYU 60 or Pediococcus pentosaceus MYU 759 or Lactobacillus plantarum MYU 74 or Lactobacillus reuteri MYU 220 improved greatly in reducing the production of reactive oxygen species. Notably, soy milk fermented with P. pentosaceus MYU 759 had more antioxidant capacity than the other fermented soymilks and control (Yamamoto et al., 2019).

Anti-proliferative activity
The anti-mutagenic activity of soymilk fermented with S. thermophilus and B. infantis (Hsieh & Chou, 2006;Hsieh et al., 2007), L. acidophilus, and B. longum (Hsieh & Chou, 2006) has been studied. The anti-mutagenic property of the soymilk increased after its fermentation. The degree of its bioactivity depended on the starter culture used for the fermentation process and the type of mutagen tested. The fermented soymilk showed more significant anti-mutagenic activity against 3,2'-dimethyl-4amino-biphenyl, a known potent mutagen. S. thermophilus and B. infantis mediated fermentation process effectively improved the anti-mutagenic property of soymilk against 4-nitroquinoline-Noxide and 3,2'-dimethyl-4-amino-biphenyl mediated mutagenicity (Hsieh & Chou, 2006;Hsieh et al., 2007).
The fermented soymilk was prepared using Weissella cibaria FB069 or L. rhamnosus GG as a starter culture. A synbiotic formulation was made with FB069 or GG mediated fermented soymilk and xylooligosaccharides (XOS). The addition of XOS improved the dextran, folate, γ-aminobutyric acid, and aglycone content in soymilk fermented with FB069. The synbiotic fermented soymilk effectively suppressed the proliferation of HCT116 and Caco-2 cell lines. The anti-proliferative property of the synbiotic soymilk was associated with TLR4/NF-κB signaling pathway. The expression of MyD88, TLR4, NF-κB, and MD2 was reduced in synbiotic soymilk treated in HCT116 cells and Caco-2 (Le et al., 2020). Similarly, Qian et al. reported the anti-proliferative activity of ethanolic extract and water extract of soymilk fermented with L. casei 16 in HT-29 cells and Caco-2 cells. The inhibitory activity was in a dose-depended manner. Both extract treatment suppressed the proliferation of cell nuclear antigen (it plays a role in cell cycle regulation and DNA replication) expression in HT-29 cells (Qian et al., 2020).
Japan Public Health Center-based Prospective study did not report that there is any link between consuming soy products (both fermented and unfermented soymilk) and liver cancer risk (Abe et al., 2020). Kwon et al. reported that fermented soymilk prepared with Kefir culture and Rhodiola extracts could improve type II diabetic condition. The in vitro study showed that the fermented soy milk reduced α-amylase inhibitory activity. The fermentation process enriched the product with bioactive phytochemicals like polyphenolic compounds (Kwon et al., 2006).

Anti-diabetic activity
Soymilk fermented with Bacillus subtilis improved the diabetic condition in streptozotocin-induced diabetic mice via α-glucosidase and α-amylase inhibition. The protective activity was higher in fermented soymilk compared to unfermented soymilk (Yi et al., 2009). Similarly, Ju and Han demonstrated that the soymilk fermented together with bokbunja (Rubus coreanus Miquel) extract using B. subtilis exhibited more inhibitory activity against α-glucosidase than the soymilk fermented alone. 5% treatment of fermented soymilk with bokbunja extract significantly reduced the high postprandial blood glucose levels in diabetic mice model. The results suggested that the addition of other phytochemical-rich fruit preparation could increase the protective nature of the fermented soymilk (Ju & Han, 2010).
L. rhamnosus CRL981 mediated fermented soymilk significantly alleviates the consequences of diabetes and improved the lipid profile and total antioxidant capacity in streptozotocin-induced diabetic mice. The bioactivity was possibly attributed to the high isoflavones content and high β-glucosidase activity. The study claimed that the selection of appropriate starter culture for the production of fermented soy products is an important key factor for developing functional food products (Marazza et al., 2013).
The supplementation of soymilk (SM), fermented soymilk (FSM), and fermented soymilk with omega-3 (FSMO3) significantly reduced the blood glucose level, total cholesterol, triglyceride, and malondialdehyde in streptozotocin-induced diabetic mice. Notably, FSMO3 intervention improved the body weight, SOD activity, hemoglobin concentration, and red blood cells count in diabetic mice. The study results suggested that FSMO3 have protective action against diabetic condition (Mohammadi Sartang et al., 2015).
Soymilk fermented together with Cuminum Cyminum essential oil (FSMC) using L. plantarum A7 improved the fasting blood glucose level and total cholesterol content in diabetic rat model. Also, the levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were decreased and increased, respectively, in diabetic rats supplemented with FSMC (Babashahi et al., 2020). The supplementation of soy yogurt reduced the blood glucose level and improved the expression of Glut4 in rats. The study supports that the regular consumption of fermented soy products could provide health benefit (Setiawan et al., 2020).

Immunomodulatory Properties
Soymilk fermented with L. helveticus R0052, B. longum R0175, and S. thermophilus ST5 have been reported to have immune enhancing property. The soy milk treated with mixed and single strains reduced the production of IL-8 in tumor necrosis factor-α (TNFα)-treated intestinal epithelial cells (Wagar et al., 2009).
Sirilun et al. reported that the soymilk fermented using lactic acid bacteria increased the expression of IL-10 in vitro.
The study claimed that probiotic-lactic acid bacteria mediated fermented soymilk could be used as food supplement to improve immune health (Sirilun et al., 2017).

Anti-inflammatory Activity
The methanol extract of Streptococcus salivarius subsp. thermophilus CCRC 14805 and/or B. infantis CCRC 14603 mediated fermented soymilk showed anti-inflammatory activity in vitro. The fermented soymilk extract inhibited iNOS expression in lipopolysaccharide (LPS)-induced macrophages. The fermented soymilk extract suppressed the expression of TNF-α, IL-6, IL-11β, and PGE2 (prostaglandin E2) in LPS-induced RAW 264.7 macrophages (Liao et al., 2010). Wu et al. also reported that L. plantarum mediated fermented soymilk has anti-inflammatory activity in LPS-induced macrophages. The protective action was possible through inhibition of NF-κB activation. In detail, fermented soymilk treatment reduced the expression of TNF-α, IL-6, and IL-1β. The fermented soymilk attenuated the production of NO, PGE2, and reactive oxygen species (ROS) in LPS-induced macrophages (Wu et al., 2013).

Antilipidemic activity
Leuconostoc kimchii, Leuconostoc citreum, and L. plantarum mediated fermented soymilk (Soypro TM ) intervention reduced the total cholesterol (TC), LDL-cholesterol (LDL-C) levels in high-fat diet-induced obese rats. The triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) values were not changed in Soypro TM supplemented group compared to the control. The in vitro experiments showed that the cellular TG level was reduced in adipocyte 3T3-L1 cells upon Soypro TM treatment (Kim et al., 2008).
Soymilk fermented with or without Momordica charantia using L. plantarum NTU 102 or L. paracasei subsp. paracasei NTU 101 was prepared. The preparation contained 25% of milk to increase the acceptability of the fermented product. The anti-atherosclerosis property of the preparation was tested using hyperlipidemic hamsters. The test animals were supplemented with fermented soymilk product for eight weeks. The results showed that the NTU 102 mediated fermented soymilk supplementation significantly reduced the ratio of LDL-C/HDL-C; whereas, NTU 101 mediated fermented soymilk with M. charantia increased the SOD and TAC of the animals. The study concluded that NTU 101 mediated fermented soymilk with or without M. charantia can prevent hyperlipidemia-induced atherosclerosis and oxidative stress (Tsai et al., 2009).
The consumption of soy product (soy yogurt) fermented by Lactobacillus delbrueckii subsp. delbrueckii SNC33 reduced the liver weight and hepatic TG levels in the rat model. Further, the soy yogurt intervention reduced the plasma cholesterol level and increased the expression of β-oxidation-related genes in rat liver. Notable down-regulation of the SREBP-1 gene and lipogenesis-associated enzymes was observed in the group treated with soy yogurt compared to the control. The results suggested that soy yogurt consumption could prevent hepatic lipid accumulation (Kitawaki et al., 2009). The SNC33 mediated fermented soy product intervention upregulated the CYP7al expression and suppressed the fatty acid synthesis related genes and SREBP-2 in rats. The protective action was noticed after five weeks of supplementation and was observed only in the fermented soymilk-treated group (Kobayashi et al., 2012a;Kobayashi et al., 2012b).
An eight-week supplementation of Bacillus subtilis KCCM42923 mediated fermented soymilk reduced the plasma TC, LDL-C levels in apolipoprotein E knockout mice. In contrast, body weight, organ weight, TG, and HDL-C levels of the mice were not affected. The results showed that the KCCM42923 mediated fermented soymilk helps to control the plasma cholesterol level (Seo et al., 2010).
L. plantarum P-8 mediated fermented soymilk (LP8FS) intervention significantly increased the HDL-C level and reduced the TC and LDL-C levels in hyperlipidemic experimental rats. The fecal excretion of bile acids, TG, and TC was also observed in the LP8FS treatment group. The LP8FS treated group had improved liver function and reduced oxidative stress. The LP8FS intervention improved lipid metabolism and positively altered the gut microbiota in hyperlipidemic rats . Likely, L. plantarum A7 mediated fermented soymilk (LA7FS) intervention also improved the HDL-C level in streptozotocinnicotinamide-induced diabetic Wistar rats. In comparison, there were no significant changes in LDL-C and other biomarkers among the LA7FS group and control (Babashahi et al., 2015).
Lactococcus acidophilus MF204 mediated fermented soymilk (MFFSM) treatment regulated the lipid levels and normalized the TG, HDL-C, and LDL-C levels in hyperlipidemic rats. Arteriosclerosis and coronary risk indexes were reduced in MFFSM treated group compared to the other groups. The total antioxidant capacity of rats given MFFSM supplement was improved (Chen et al., 2017). Probiotics (L. plantarum, L. casei, and B. bifidum) mediated fermented soymilk (PFSM) was prepared and its hypolipidemic property was evaluated. PFSM intervention significantly reduced the high-fat diet-induced weight gain, liver fat accumulation, ROS production, and hyperlipidemia in the mice model. Additionally, the intervention of PFSM reduced the ALT, LPS, and TNF-α levels and increased the serum leptin level. The results showed that the supplementation of PFSM could protect the high-fat diet-induced consequences in mice (Zhang et al., 2017).
The fermented soymilk treatment significantly reduced the lipid accumulation in palmitic acid-induced lipogenic HepG2 cells. The suppression of SREBP-1 expression, induction of NRF-2 expression, and increased phosphorylation of ERK were observed in fermented soymilk treated cells. The fermented soymilk reduced the palmitic acid-induced ROS generation in HepG2 cells. The results indicated that FSM could prevent lipid accumulation and reduce ROS generation (Ahn et al., 2018).
L. acidophilus and L. casei mediated fermented soymilk supplementation significantly reduced the plasma glucose level and improved the insulin level in hypercholesterolemic rats. Fermented soymilk intervention increased the HDL level and decreased the LDL level. The safety study suggested that fermented soymilk consumption was safe and helped to prevent cardiovascular diseases and manage metabolic disorders (Ahsan et al., 2019).

Skin health
The hairless ovariectomized mice were fed with fermented soymilk. The fermented soymilk supplementation effectively reduced the epidermal thickness and erythema intensity of dorsal skin and also reduced the cyclobutane pyrimidine dimers levels in dorsal skin. The isoflavone levels were increased in dorsal skin and serum in fermented soymilk supplemented mice compared to control. The study claimed that the supplementation of fermented soymilk protects the skin from UV irradiation and photo-damage in experimental hairless mice (Kano et al., 2016).
B. breve Yakult and L. mali mediated fermented soymilk consumption improve facial wrinkles in healthy women. An eight-week intervention study showed that fermented soymilk consumption significantly improved the average depth and maximum depth of skin wrinkles in study subjects. The urine isoflavone level was also increased in the treatment group compared with the control, which indicates that the fermented soymilk consumption aid in delaying skin aging .
The supplementation of fermented soymilk containing L. casei Shirota improved the skin conditions in terms of elasticity, dryness, pigmentation, coarseness, moisture, and stratum corneum morphology. The urine isoflavone level was increased, and the abundance of Lactobacillaceae and Bifidobacteriaceae was also increased in the treatment group. The level of Enterobacteriaceae and Porphyromonadaceae were reduced significantly after the consumption of fermented soymilk. The study suggested that fermented soymilk's regular consumption confers skin health to premenopausal healthy women (Nagino et al., 2018).

Other bioactivities
The whey obtained from LAB (L. casei, L. acidophilus, L. bulgaricus, S. thermophilus, and B. longum) mediated fermented soymilk (rich in peptides and γ-aminobutyric acid) was administrated to the spontaneously hypertensive rat for eight weeks. The intervention significantly reduced the systolic blood pressure in the experimental rats (Tsai et al., 2006). L. plantarum CCFM8610 or L. bulgaricus CCFM8004 mediated fermented soymilk was given as supplement to the experimental mice with or without cadmium for eight weeks. CCFM8610 fermented soymilk facilitates the fecal cadmium excretion, increased the antioxidant capacity, and reversed hepatic and renal damage effectively than non-fermented soymilk and CCFM8004-fermented soymilk. The results suggested that CCFM8610-fermented soymilk could protect from chronic cadmium toxicity (Zhai et al., 2015).
L. fermentum LAB 9-fermented soymilk significantly improved the LPS-induced memory defects in experimental mice (Fakri et al., 2016). L. plantarum TWK10-fermented soymilk could enhance learning and memory in deoxycorticosterone acetate-salt hypertension-induced vascular dementia rats (Liu et al., 2016). Miyake et al. reported that the consumption of fermented soy products effectively lowers depressive symptoms during pregnancy (Miyake et al., 2018).
The ethanol extract of TWK10-fermented soymilk (ETFSM) effectively reduced oxidative stress in vitro. ETFSM intervention significantly exhibited the wound healing activity in streptozotocin-induced diabetic rats and decreased inflammation-associated proteins. The results indicated that the topical application of ETFSM helps to accelerate wound healing (Chuang et al., 2019). The administration of ETFSM and water extract of TWK10-fermented soymilk enhances the antioxidant capacity and improves cognitive function in type 2 diabetic rats (Liu et al., 2020).
The protective nature of riboflavin-producing L. plantarum CRL 2130 mediated fermented soymilk (RLFSM) was studied using trinitrobenzene sulfonic induced colitis mouse model. RLFSM treatment significantly reduced the weight loss, intestinal damages, microbial translocation, and cytokine levels in the treated mice compared to controls. The study suggested that RLFSM might have anti-colitis property (Levit et al., 2017).
L. rhamnosus mediated fermented soymilk supplementation increased the isoflavone metabolites (O-desmethylangolensin and equol.) and short-chain fatty acids (propionic acid) content in mice. The fecal enzyme activity and amendment in gut microbiota were improved upon fermented soymilk administration. The study proved that the consumption of L. rhamnosus mediated fermented soymilk could improve the antibiotic-mediated gut microbial damages (Dai et al., 2019).

Conclusion
As per the literature survey, fermented soymilk has been reported for its antioxidant, antiproliferative, antidiabetic, hypolipidemic, immune-modulatory, memory improvement, and wound healing activities, etc. (Figure 1). However, most of the reports are based on in vivo and in vitro studies. A minimal number of studies have been carried out on human subjects. This research gap has to be filled to conclude that fermented soymilk products provide health benefits to human beings. Thus, further detailed studies are required in human subjects and there is a need to develop functionally superior fermented soymilk using potent starter cultures. Babashahi