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Natto and miso: an overview on their preparation, bioactive components and health-promoting effects
Abstract
Natto and miso are two major traditional fermented soy foods in Japan. Natto is prepared by fermenting cooked soybeans with Bacillus subtilis natto. The beans of natto have a sticky outlook, slippery texture, sour aroma and nutty flavour. Bioactive components of natto are nattokinase, bacillopeptidase F, vitamin K2, dipicolinic acid and γ-polyglutamic acid. Miso is a fermented soybean paste widely used to make miso soup. The paste is produced by fermenting cooked soybeans with koji (steamed rice inoculated with Aspergillus oryzae). Bioactive compounds of miso include isoflavones and phenolic acids. In this review, the preparation, bioactive components, and health-promoting properties of natto and miso are highlighted. Sources of information referred were from Google Scholar, J-Stage, Science Direct, PubMed, PubMed Central and PubChem
... To make natto, soybeans are submerged in water, steamed/boiled, and fermented at 40 • C for 16 h, followed by 24 h of maturation in the refrigerator. Bacillus subtilis Natto, derived from rice straw, activates fermentation (Chan, Wong, Kezuka, Oshiro, & Chan, 2021). The final product is viscous and has a characteristic flavour profile. ...
... Some examples of these components are bacillopeptidase, nattokinase, dipicolinic acid, vitamin K2 (menaquinone-7), and gamma-polyglutamic acid (γ-PGA). γ-PGA holds the potential to promote lipid metabolism, calcium absorption and lowering blood sugar levels (Chan et al., 2021;Diez-Ozaeta & Astiazaran, 2022). Animal studies and human trials demonstrate that natto possesses anti-thrombotic and anti-coagulant properties. ...
... Animal studies and human trials demonstrate that natto possesses anti-thrombotic and anti-coagulant properties. Furthermore, natto exhibits anticarcinogenic activity, antibacterial spectrum, antiinflammatory and hypocholesterolemic effects, b-galactosidase activity, anti-diabetic effect, antiallergic properties, antioxidative properties, apoptosis protection, and many more (Afzaal et al., 2022;Chan et al., 2021). ...
... γ-GP are typically present in foods rich in protein and are formed through natural enzymatic processes during fermentation, aging, or enzymatic reactions (Table 5). Foods, such as miso and natto, contain γ-GP formed during fermentation (Chan et al. 2021;Yang et al. 2024a). The aging process in cheese promotes the formation of γ-GP, which adds depth and intensity to the umami and kokumi tastes of these products (Araya-Morice et al. 2021; Hillmann and Hofmann 2016). ...
Taste‐active peptides (TAPs) impart significant organoleptic attributes of food products. Their diverse taste profiles, such as umami, bitter, sweet, salty, sour, and kokumi, hold great potential for various food and nutraceutical applications. The perception of taste is influenced by TAPs' interactions with taste receptors, which are governed by their structural features, including amino acid composition, sequence, charge, molecular weight, and bond configurations. Beyond taste, many TAPs exhibit bioactive properties with potential health benefits that may find applications for mitigating chronic diseases, such as diabetes, cardiovascular disease, inflammation, obesity, and cancer. Understanding these characteristics enables the development of peptides with tailored tastes and functional benefits. This review aimed to provide a comprehensive understanding of the structural determinants of TAPs, their receptor interactions, and their dual role in taste and health. Furthermore, it highlights advancements in computational modeling and machine learning for TAP identification and characterization. Future advancements on TAPs are expected to significantly influence how the food industry addresses both flavor and health aspects.
... It has a distinctive smell, slippery texture, and strong flavor. Regarding its possible health benefits, natto presents many bioactive components generated during the fermentation process, such as nattokinase (NK), bacillopeptidase F (BPF), vitamin K2 (menaquinone-7), dipicolinic acid (DPA), and γ-polyglutamic acid (γ-PGA), which have the potential to increase immunity to infections [60]. ...
Urinary tract infections (UTIs) are a prevalent global health issue, often requiring antibiotic treatment, which contributes to antimicrobial resistance. This narrative review explores the potential of probiotics and plant-based foods as alternative or complementary preventive strategies against UTIs. Fermented foods, such as yogurt, kefir, and kombucha, contain probiotic strains that can modulate the gut and urogenital microbiota, enhancing resistance to uropathogens. Likewise, plant-based foods, including cranberry, garlic, bearberry, juniper, and nettle, possess bioactive compounds with antimicrobial, anti-inflammatory, and diuretic properties. Laboratory and clinical studies suggest that these natural interventions may reduce the incidence of UTIs by inhibiting pathogen adhesion, modulating immune responses, and promoting urinary tract health. However, despite promising findings, inconsistencies in study methodologies, dosage standardization, and long-term efficacy warrant further investigation. Future research should focus on optimizing probiotic formulations, standardizing plant-based supplement dosages, and assessing potential food–drug interactions to establish evidence-based guidelines for UTI prevention.
... In addition to these widely studied examples, global fermented foods such as miso and natto from Japanese cuisine, kefir from Eastern Europe, and dosa from India provide unique probiotic strains and bioactive compounds. Miso and natto, for instance, contain Bacillus subtilis, which has been shown to promote immune regulation and reduce markers of systemic inflammation (Oshiro et al., 2021). Kefir is particularly rich in lactic acid bacteria and yeast, offering a broad spectrum of probiotics with antimicrobial and gut-stabilizing properties (Tingirikari et al., 2024). ...
The gut microbiome plays a pivotal role in human health, influencing digestion, immunity, and disease prevention. Beneficial gut bacteria such as Akkermansia muciniphila, Adlercreutzia equolifaciens, and Christensenella minuta contribute to metabolic regulation and immune support through bioactive metabolites like short-chain fatty acids (SCFAs). Dietary patterns rich in prebiotics, fermented foods, and plant-based bioactive compounds, including polyphenols and flavonoids, promote microbiome diversity and stability. However, challenges such as individual variability, bioavailability, dietary adherence, and the dynamic nature of the gut microbiota remain significant. This review synthesizes current insights into gut bacteria’s role in health, emphasizing the mechanisms by which dietary interventions modulate microbiota. Additionally, it highlights advancements in microbiome-targeted therapies and the transformative potential of personalized nutrition, leveraging microbiota profiling and artificial intelligence (AI) to develop tailored dietary strategies for optimizing gut health and mitigating chronic inflammatory disorders. Addressing these challenges requires a multidisciplinary approach that integrates scientific innovation, ethical frameworks, and practical implementation strategies.
... Japan, fermented soybean products are referred to as natto and miso (Chan et al., 2021). In Korea, they are known as cheonggukjang (Piao and Eun, 2020), while in traditional Chinese fermented food, the term is douchi (Zhou et al., 2024). ...
Background. There is a growing interest in harnessing bioactive peptides derived from food-grade raw materials , especially for the development of functional foods. Tempeh milk protein hydrolysates (TMPHs) are interesting sources of bioactive peptides. In the current study, TMPHs were characterized by their bioactive peptides, dietary polyphenols and antioxidant activity. Material and methods. TMPHs were made from Rhizopus oligosporus fermented tempeh with fermentation times of 0, 1, 2, 3 and 4 days, designated as TMPH0, TMPH1, TMPH2, TMPH3 and TMPH4, respectively. The morphological appearance of tempeh was examined by scanning electron microscopy. The analyzed characteristics of TMPHs were physicochemical properties, total soluble proteins (TSP), trichloroacetic acid (TCA)-soluble peptides, bioactive peptides, antioxidant activity, total phenolic compounds (TPCs) and total flavonoid compounds (TFCs). Results. The morphological appearance and microstructure of tempeh with varied fermentation times were different under SEM due to the growth of R. oligosporus. The pH of TMPHs remained constant at 7 for samples with fermentation times of 0, 1 and 2 days and continuously decreased for those fermented for 3 and 4 days. However, total acidity continuously increased from the first day of fermentation. TMPH3 had the highest level of total soluble proteins and trichloroacetic acid (TCA)-soluble peptides. SDS-PAGE analysis demonstrated that TMPHs consist of various protein fractions with large molecular weights from 25 kDa and with lower molecular weights. Their SDS-PAGE patterns were different, indicating protein alteration during fermentation. In DPPH and ABTS assays, TMPH3 exhibited the strongest antioxidant activity. These results agreed with those from the analysis of total phenolic compounds (TPCs) and total flavonoid compounds (TFCs) showing that TMPH3 had the highest TPC and TFC.
... Miso has several bioactive compounds that are responsible for its health-promoting properties. These include vitamins, minerals, phenolic acids (syringic acid and vanillic acid), and isoflavones (genistein and daidzein) (Chan et al., 2021). Isoflavones isolated from miso have shown antiproliferative effects on certain cancer cell lines. ...
One of the greatest threats to global health is cancer. Probiotic foods have been shown to have therapeutic promise in the management of cancer, even though traditional treatments such as radiation therapy, chemotherapy, and surgery are still essential. The generation of anticarcinogenic compounds, immune system stimulation, and gut microbiota regulation are a few ways that probiotics when taken in sufficient quantities, might help health. The purpose of this review is to examine the therapeutic potential of probiotic foods in the management of cancer. Research suggests that certain strains of probiotics have anticancer effects by preventing the growth of cancer cells, triggering apoptosis, and reducing angiogenesis in new tumors. Probiotics have shown promise in mitigating treatment-related adverse effects, such as diarrhea, mucositis, and immunosuppression caused by chemotherapy, improving the general quality of life for cancer patients. However, there are several factors, such as patient-specific features, cancer subtype, and probiotic strain type and dosage, which affect how effective probiotic therapies are in managing cancer. More research is necessary to find the long-term safety and efficacy characteristics of probiotics as well as to clarify the best ways to incorporate them into current cancer treatment methods.
Graphical representation showing the role of probiotic foods in cancer management.
... The ability to identify multiple probiotic strains that can be used in a given fermented food can facilitate the production of multi-strain probiotic fermented products that also fulfill the health goal of maintaining and increasing gut microbial diversity [4,[56][57][58]. For example, the cardiovascular health benefit associated with Bacillus subtilis in natto [49,59,60] can be combined with the cholesterol-lowering effect of Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus plantarum or Lactobacillus reuteri [61,62] by screening for their ability to survive and grow together in the specific conditions dictated by natto ingredients and fermentation conditions. Using the approach demonstrated here, our overall goal is to stimulate the application of this model in the large-scale production of probiotic fermented products characterized by different thermal treatments, sources of raw material, and aging conditions typical of many commercial fermented foods already largely known and widely consumed (Kefir, cheeses, labne, feta, sauerkraut, pickles, olives, kimchi, gochujang, ssamjang, doenjang, natto, miso, etc.). ...
The past few decades have demonstrated how important the human gut microbiota is for human health. Because of this, the use of microbiota-modulating dietary interventions such as probiotics and prebiotics is growing in popularity amongst consumers, food manufacturers, healthcare professionals, and regulators. In particular, there is interest in making a wider variety of foods with probiotic properties. However, as a solution for food manufacturers to produce fermented foods compatible with the “probiotic foods” label definition, we used an impedometric analysis to identify the survival and growth capacity of microbial strains in specific environmental contexts. Using this approach, manufacturers can more effectively select the strains with the highest growth rate for use in probiotic fermented food production trials. To provide a proof of concept, we tested three Lacticaseibacillus rhamnosus probiotic strains growing in milk at different temperatures. We quantified the probiotic’s growth using species-specific primers and quantitative real-time PCR. Overall, our results demonstrate the feasibility of this type of model in facilitating the production of probiotic fermented foods by allowing manufacturers to select strains able to grow under specific conditions. Our model can be used to develop, increase, and target the beneficial health properties of a multitude of fermented foods produced worldwide.
... The molecular weight of γ-PGA produced by Bacillus sp. varies from 10 5 to 10 6 Da and different molecular weights can be used depending on the required applications [90,91]. Reducing the molecular weight to about 10 4 Da is an important step in γ-PGA production for drug delivery applications, since the lower molecular weight polymers demonstrate more homogeneity in the size properties [92]. ...
Vaccination is an innovative strategy for cancer treatment by leveraging various components of the patients' immunity to boost an anti-tumor immune response. Rationally designed nanoparticles are well suited to maximize cancer vaccination by the inclusion of immune stimulatory adjuvants. Also, nanoparticles might control the pharmacokinetics and destination of the immune potentiating compounds. Poly-γ-glutamic acid (γ-PGA) based nanoparticles (NPs), which have a natural origin, can be easily taken up by dendritic cells (DCs), which leads to the secretion of cytokines which ameliorates the stimulation capacity of T cells. The intrinsic adjuvant properties and antigen carrier properties of γ-PGA NPs have been the focus of recent investigations as they can modulate the tumor microenvironment, can contribute to systemic anti-tumor immunity and subsequently inhibit tumor growth. This review provides a comprehensive overview on the potential of γ-PGA NPs as antigen carriers and/or adjuvants for anti-cancer vaccination.
... The process of making natto goes through the stages of cleaning and sorting dry soybeans, soaking, sorting wet soybeans, steaming, inoculating the Bacillus subtilis natto, transferring into containers, then continuing with the fermentation process and sorting the products. Usually, natto is eaten for breakfast along with rice (Sahirman, 2019;Chan et al., 2021). Both products have a slightly slippery (sticky) texture and have a distinctive and strong flavor (Liu et al., 2021). ...
Sere kedele is a traditional food from fermented soybeans produced by people on the southeast coast of Bali. The purpose of this study was to determine the nutritional content and sensory properties of sere kedele from various producers in Gianyar Regency. The survey results showed that in Gianyar Regency there are 4 producers of sere kedele, namely 2 producers in Blabatuh Village (Banjar Pokas and Banjar Teruna), 1 producer in Sukawati Village and 1 producer in Buruan Village. Sere kedele products were sampled from all producers and then analyzed for its nutritional content and sensory properties. The design used was Completely Randomized Design (CRD) with 4 replications. The results of the analysis showed that the water content of sere kedele ranged from 57.68% to 60.84%, ash content from 2.24% to 3.06%, fat content from 7.84% to 12.40%, dissolved protein content from 2.18% to 4.30%, carbohydrate content from 22.77% to 26.51%, glutamic acid content from 0.52 % to 6.97 % , total microbes from 10.78 log cfu/ml to 11.50 log cfu/ml, total lactic acid bacteria from 10.54 log cfu/ml to 10.77 log cfu/ml.
... The compounds produced by B. subtilis natto are contained in the finally produced natto. Therefore, it seems that the beneficial health functions of natto that have been reported in the past are largely due to the components produced by B. subtilis natto (Chan et al., 2021;Kozioł-Kozakowska & Maresz, 2022;Wang, Gao, et al., 2020;Y. Yang et al., 2021;L. ...
Natto, fermented food produced by Bacillus subtilis natto (B. subtilis natto), is widely consumed around the world, and its potential health functions are attracting attention. Natto has been reported to have a variety of bioactive compounds such as levan, menaquinone‐7, nattokinase, and others. Such bioactive compounds produced by B. subtilis natto are deeply involved in the function of natto, because they are consequently contained in natto. This review summarizes bioactive compounds and its functions of (1) B. subtilis and its subspecies, (2) B. subtilis natto. Overall, natto is a natural source of bioactive compounds and is expected to be applied to functional foods. Natto, produced by fermentation of soybeans by B. subtilis natto (a subspecies of B. subtilis), has been consumed as a traditional fermented food for at least a thousand years. Natto is attracting interest as a potential functional food for health. This review summarizes compounds and its functions of B. subtilis natto.
... Recent studies on PGA-rich fermented soybean foods have shown an ability of PGA to suppress increased postprandial blood glucose levels [16]. PGA is capable of regulating blood pressure due to its ability to inhibit angiotensin-converting enzyme (ACE) activity [17] and is associated with intestinal calcium absorption as well [18]. It is also a biological glue that has major applications in wound healing and treatment of dental caries [12]. ...
This present work is aimed to screen the PGA-producing Bacillus spp. from naturally fermented soybean foods of Arunachal Pradesh in India and to characterize the poly-glutamic acid (PGA) extracted from Bacillus spp. A total of 50 isolates were screened for high stickiness from 165 bacterial isolates isolated from 34 samples viz., grep chhurpi, peha/paeha, peron namsing, and peruñyaan. Based on 16S rRNA gene sequence analysis, 50 isolates were identified as Bacillus spp. Viscosity of 50 species of Bacillus were measured, out of which 7 species of Bacillus showing ≥0.03 Pa.s (30 cP) of viscosity were selected viz., Bacillus velezensis GC1-42, B. siamensis GC4-36, B. subtilis PH3-21, B. subtilis PN1-14, B. subtilis PH6-29, B. tequilensis PN9-22, and B. safensis subsp. safensis PY1-19 for PGA production. Amino acid analysis of PGA extracted from seven species of Bacillus showed high molecular weight (˃600 kDa). B. safensis subsp. safensis PY1-19, isolated from peruñyaan, showed 78.9% of glutamic acid, antagonistic properties against pathogenic bacteria and had the ability to produce phytase with no hemolytic activity. Hence, this strain was selected as a potential starter to prepare peruñyaan at laboratory, and the final product showed high stickiness and viscosity with production of PGA of around 11 g/L.
... After that, the beans are mashed and kneaded along with Koji, salt, and water. The paste is then packed into a sealed glass jar and kept in a cool, dark place to ferment at a temperature of 25-30°C (Chan et al., 2020). Soybeans, rice, salt, and Koji are some of the main components in the production of miso. ...
Consumer demand for fermented foods with a well‐balanced nutrient profile has been increasing owing to their ability to prevent chronic diseases as well as their functional, nutritional, and nutraceutical benefits. Among those functional foods, miso is a well‐known traditional fermented food with a distinctive savory flavor and aroma that is most commonly used as a seasoning in miso soup. Among different fermented products, miso is derived from soybeans and grains as a result of the activities of Koji enzymes and beneficial microbes. Additionally, the microbial community of miso is thought to be crucial in enhancing its distinct flavor and texture as well as its nutritional properties. Despite the importance of microorganisms in the production of miso, there has been relatively little research done to characterize and describe the nutritional and medicinal potential of miso. In this review, the potential therapeutic properties, i.e., anticancer, antimicrobial, and antiobesity, of miso have been discussed comprehensively. This review envisions the production technology, its history, microbial population, nutritional properties, and the potential health benefits of miso associated with its consumption. Miso is a well‐known traditional fermented food. In addition, this review envisions the production technology, its history, microbial population, nutritional properties and the potential health benefits of miso associated with its consumptions. In this review, the potential therapeutic properties i.e., anti‐cancer, anti‐microbial, anti‐obesity of miso have been discussed comprehensively. Furthermore, the positive effects of miso consumption, eliminating the radioactive effects, and regulating the cholesterol levels in the human body, are the limelight of this article.
... Nattokinase is shown to be responsible for anti-thrombotic and anti-coagulative activities. The anti-thrombotic effect of NK can be used for the treatment of cardiovascular diseases, and such a compound also acts on amyloid degradation related to Alzheimer's disease and on the suppression of atherosclerosis, heart attack, and stroke in sick patients [179,180]. The health benefits of miso are associated with the presence of isoflavones, such as 8-OH-daidzein, 8-OH-genistein, 6-OH-daidzein, which have strong antioxidant activity, this is related to a series of beneficial effects on human health [181]. ...
In the growing search for therapeutic strategies, there is an interest in foods containing natural antioxidants and other bioactive compounds capable of preventing or reversing pathogenic processes associated with metabolic disease. Fermentation has been used as a potent way of improving the properties of soybean and their components. Microbial metabolism is responsible for producing the β-glucosidase enzyme that converts glycosidic isoflavones into aglycones with higher biological activity in fermented soy products, in addition to several end-metabolites associated with human health development, including peptides, phenolic acids, fatty acids, vitamins, flavonoids, minerals, and organic acids. Thus, several products have emerged from soybean fermentation by fungi, bacteria, or a combination of both. This review covers the key biological characteristics of soy and fermented soy products, including natto, miso, tofu, douchi, sufu, cheonggukjang, doenjang, kanjang, meju, tempeh, thua-nao, kinema, hawaijar, and tungrymbai. The inclusion of these foods in the diet has been associated with the reduction of chronic diseases, with potential anticancer, anti-obesity, antidiabetic, anticholesterol, anti-inflammatory, and neuroprotective effects. These biological activities and the recently studied potential of fermented soybean molecules against SARS-CoV-2 are discussed. Finally, a patent landscape is presented to provide the state-of-the-art of the transfer of knowledge from the scientific sphere to the industrial application.
... Indeed, B. subtilis G8 and B. subtilis BEST195 did not possess any mutation in their swrA CDS and degQ promoter sequences that might disrupt γ-PGA production, as observed in B. subtilis 168 (Fig. 4). The lack of such a mutation shows that B. subtilis G8 is capable of producing γ-PGA, which contributes to the bulk of natto's mucilage and consequently its sticky consistency (Hsueh et al. 2017;Chan et al. 2021). Collectively, these results strongly suggest that B. subtilis G8 is a natto-derived B. subtilis strain that is able to produce nattokinase and γ-PGA (Kada et al. 2013). ...
Dikson, Victor H, Jong D, Sanjaya A, Samantha A, Jo J, Pinontoan R. 2022. Whole-genome analysis of Bacillus subtilis G8 isolated from natto. Biodiversitas 23: 1293-1300. Bacillus subtilis-fermented soy-based food is associated with multiple health benefits. Various bacterial strains have been isolated from it, includingB. subtilisG8, recent isolation from Japanese natto commercially available in Indonesia. Both 16S rRNA gene sequencing and fibrinolytic activity characterization have been performed and published in prior studies. After comparison to the genome of a natto-isolated reference strain (i.e., B. subtilisBEST195), the B. subtilis G8 genome showed a similar guanine-cytosine (GC) content, predicted number of coding sequences (CDS) and predicted number of tRNA genes, but had a shorter sequence length and fewer predicted rRNA genes. Further analysis using multiple genome alignment with Mauve, average nucleotide identity (ANI) matrix calculation, and phylogenetic inference indicated that B. subtilis G8 was more related to natto-derived B. subtilis than to cheonggukjang-derived B. subtilis and B. subtilis 168. Finally, sequence analyses of a gene encoding nattokinase as well as two genes regulating poly-gamma-glutamic acid (?-PGA) production in B. subtilis G8, B. subtilis BEST195 and B. subtilis 168 clearly indicated that B. subtilis G8 is able to produce nattokinase and ?-PGA, which both contribute to natto’s fermentation process. Therefore, it is proposed that B. subtilis G8 should be reclassified as B. subtilis subsp. natto G8 to reflect that it is a natto-derived B. subtilis strain.
... Miso has received considerable attention as a potential approach to reduce the risk of chronic illness, such as cardiovascular disease, and its long-term consumption has also been associated with a possible lower risk of mortality [19]. Recent studies have reported the possible utilization of food by-products, such as grain brans, and their solid-state fermentation by fungus, in order to develop functional foods, enhanced in bioactive compounds [20]. ...
Several fruit by-products may exert a beneficial role on oxidative stress and inflammation modulation, providing essential bioactive components, such as polyphenols and carotenoids. Recently, the potential bioactivity of miso has been reported. The aim of this dietary intervention–clinical study was to evaluate the acute effect of a novel, functional miso-type sauce based on legumes, on postprandial biomarkers of oxidative stress and inflammation. In this randomized, cross-over design, intervention–clinical trial, 14 healthy volunteers, aged 20–30 years old, consumed a rice meal rich in fat and carbohydrates (258 g), containing a legume-based sauce. After a 1-week washout period, the same subjects consumed the same meal, containing the novel fermented miso-type sauce, enhanced with 50% carotenoid-rich, fruit peel extract. Differences in postprandial total plasma antioxidant capacity according to the FRAP method, serum lipids, glucose, uric acid levels, and antithrombotic activity in platelet-rich plasma were evaluated before, 30 min, 1.5 h, and 3 h after consumption. The results showed that, in comparison to the control group, consumption of the novel sauce resulted in a significantly increased total plasma antioxidant capacity 3 h after consumption (p < 0.05). In addition, we observed a significant attenuation of triglycerides concentration increase in the last 1.5 h in the functional group (p < 0.05). A significant decrease in serum aggregation was found at 30 min and 3 h after functional sauce intake in comparison with the baseline (p < 0.05). Finally, LDL-cholesterol concentrations were significantly reduced 3 h after the functional meal consumption, in comparison with baseline values (p < 0.05). The remaining biomarkers did not show statistically significant differences (p > 0.05). Further investigation is needed in order to validate the current results.
Fermentation has been used for centuries to preserve food and to obtain products with new, attractive sensory characteristics. Fermented products are a source of dietary fiber, vitamins, bioactive compounds, and probiotic bacteria with health-promoting properties. This review provides a comprehensive overview of the effects of fermented fruits, vegetables, and legumes on metabolic disturbances characterizing metabolic syndrome (MetS). Furthermore, the chemical composition, microbial communities, and molecular mechanisms of action of fermented plant foods are discussed. Fermented fruits and vegetables, including table olives, caper fruits, and kimchi, contain polyphenols and probiotic bacteria, which are beneficial in terms of obesity and impaired glucose and lipid metabolism. Fermented legumes are a valuable source of bioactive peptides and isoflavone aglycones. Among fermented soybean products, natto stands out due to the presence of γ-polyglutamic acid, which improves glucose tolerance and the lipid profile, and nattokinase, an enzyme that acts as an angiotensin-converting enzyme inhibitor. Potential future studies focused on developing functional fermented foods and easy-to-use supplements based on fermented plant products are suggested.
There has been a major growth in the development of plant-based meat alternatives (PBMA) in recent years. However, current PBMA often contain ultra processed ingredients and numerous additives to be able to mimic animal-based meat (ABM) including the meaty (umami) flavour, characteristic firm/chewy structure and juicy mouthfeel. In this review, the potential of ancient fermentation techniques as a minimally processed alternative to ABM and current PBMA are explored. Fermented foods including tempeh, natto, dawadawa and ugba are naturally high in protein and umami flavours. The nutritional, aroma, flavour and techno-functional properties are provided and discussed in the context of ABM and PBMA. The fermented foods have potential to be used as whole foods ingredients, or their constituents can be used as ingredients in plant-based foods. Particularly the umami flavours and high protein content combined with the naturally occurring high water holding capacity (WHC), solubility and other material properties make fermented legume foods suitable candidates for use in high-protein plant-based foods. Understanding the sensory characteristics and material properties generated during legume fermentation and their similarities to ABM can aid in stimulating innovations in food technology to obtain a new generation of less-processed PBMA with limited additives.
Natto is a traditional fermented soybean-based food that has been an integral part of Japanese cuisine for several centuries. Although there have been extensive studies on the cognitive benefits of soybeans, only limited studies have examined the effects of natto on cognitive function. This study investigated the potential cognitive benefits of natto in senescence-accelerated mouse-prone 8 (SAMP8) mice. After 12 weeks of oral administering natto fermented for 18 h, the spatial learning and memory performance were improved compared with those in SAMP8 control mice. Furthermore, activation of the brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element-binding protein (CREB) signaling and N-methyl-D-aspartate receptor (NMDAR)-calcium/calmodulin-dependent protein kinase II (CaMKII) cascade was observed in the hippocampus of SAMP8 mice that were fed natto. Additionally, natto administration upregulated trace amine-associated receptor 1 (TAAR1) as a modulator of NMDAR. These findings suggest that natto ameliorates cognitive decline by activating the TAAR1-mediated CaMKII/CREB/BDNF signaling pathway in the hippocampus of SAMP8 mice.
A rapidly growing population, resource scarcity, and the future sustainability of our food supply are among the major concerns of today’s food industry. The importance of resilient food crops that will sustain in the future is imperative, and legumes are ideal future food crops owing to their rich nutrient profile, cost-effective production and resource usage efficiency. Furthermore, they have the potential to meet the protein needs of the future. There are however several limitations associated with legumes in terms of their sensory, nutritional, and functional properties, which make them challenging for the food industry to use. In this review, these challenges are discussed in detail with particular reference to fermentation as a strategy for overcoming them. A major focus is on examining the potential application of fermentation for modifying techno-functional properties, such as foaming and emulsifying properties, solubility, and water and oil binding capacities of legume substrates. In many studies, fermentation has been demonstrated to enhance the techno-functional, sensory and nutritional attributes of various legume substrates. Future studies must focus on developing scalable fermentation processes to utilize the technology for improving the techno-functional and sensory properties of legume-based ingredients at industrial scale.
High salt intake increases blood pressure, and dietary salt intake has been clearly demonstrated to be associated with hypertension incidence. Japanese people consume higher amounts of salt than Westerners. It has been reported that miso soup was one of the major sources of daily salt intake in Japanese people. Adding salt is indispensable to make miso, and therefore, in some cases, refraining from miso soup is recommended to reduce dietary salt intake. However, recent studies using salt-sensitive hypertensive models have revealed that miso lessens the effects of salt on blood pressure. In other word, the intake of miso dose not increase the blood pressure compared to the equivalent intake of salt. In addition, many clinical observational studies have demonstrated the absence of a relationship between the frequency of miso soup intake and blood pressure levels or hypertension incidence. The mechanism of this phenomenon seen in the subjects with miso soup intake has not been fully elucidated yet. However, in basic studies, it was found that the ingredients of miso attenuate sympathetic nerve activity, resulting in lowered blood pressure and heart rate. Therefore, this review focused on the differences between the effects of miso intake and those of the equivalent salt intake on sympathetic nerve activity, blood pressure, and heart rate.
PurposeThe aim of this study was to compare the impact of fermented and unfermented soy intake, based on the following soy-derived products: tofu, soymilk, natto, and miso, on the risk of liver cancer among Japanese adults.Methods75,089 Participants of the Japan Public Health Center-based Prospective Study (JPHC Study) were followed from the time of the 5-year follow-up questionnaire until the end of 2012–2013. Subjects with available data on hepatitis B virus (HBV) and hepatitis C virus (HCV) infection status from blood samples (n = 14,016) and those who were anti-HCV antibody (anti-HCV) or hepatitis B virus antigen (HBsAg) positive (n = 1033) were also analyzed separately. Cox proportional hazard models were employed to calculate hazard ratios (HRs) and 95% confidence intervals (95% CIs).ResultsDuring 1,145,453 person-years, 534 newly diagnosed cases of liver cancer were identified in the JPHC Study. For miso intake among men, the multivariate-adjusted highest versus lowest quartile HR was 0.65 (95% CI, 0.48–0.89); p for trend = 0.006. Results were similar in those who were anti-HCV or HBsAg positive, 0.24 (0.08–0.70); p for trend = 0.004 highest versus lowest tertile. For the sub-analysis among only participants with known hepatitis infection status and HCV and HBsAg adjustment, a similar association was observed. In the multivariate complete cohort analysis, among women, the highest intake of fried tofu was inversely associated with the risk of liver cancer, HR = 0.45 (0.26–0.80); p for trend = 0.014.Conclusions
We observed no association between total soy intake, fermented and unfermented, and risk of liver cancer, and only an inverse association between miso intake and liver cancer among men.
The natto containing high levels of gamma-polyglutamic acid (γ-PGA) was recently developed. We investigated the effect of γ-PGA-rich natto consumption on postprandial glycemic excursion in humans. A randomized crossover meal test study was performed on healthy volunteers aged 20–64 years using the following test meals: (1) white rice (WR), (2) low-γ-PGA natto meal (WR + low-γ-PGA natto), and (3) high-γ-PGA natto meal (WR + high-γ-PGA natto). Blood samples were obtained at each visit before and for 120 min after loading. The incremental area under the curve (IAUC) of blood glucose and insulin levels was calculated and compared among the test meals. The blood glucose’s IAUC at 0–120 min, the primary endpoint, was 20.1% and 15.4% lower for the high- and low-γ-PGA natto meal than for the WR, with a significant difference only between the high-γ-PGA natto meal and WR (p < 0.05). The blood glucose’s IAUC at 0–15, 0–30, and 0–45 min was lower for the high-γ-PGA natto meal than for the low-γ-PGA natto meal (all p < 0.05). The possibility that high-γ-PGA natto might suppress blood glucose elevations in the early phase after eating is indicated.
Objective
To investigate the association between several types of soy products and all cause and cause specific mortality.
Design
Population based cohort study.
Setting
Japan Public Health Centre-based Prospective Study, which includes 11 public health centre areas in Japan.
Participants
92 915 participants (42 750 men and 50 165 women) aged 45 to 74 years.
Exposures
Intake of total soy products, fermented soy products (natto and miso), non-fermented soy products, and tofu from a five year survey questionnaire.
Main outcome measures
All cause and cause specific mortality (cancer, total cardiovascular disease, heart disease, cerebrovascular disease, respiratory disease, and injury) obtained from residential registries and death certificates.
Results
During 14.8 years of follow-up, 13 303 deaths were identified. In the multivariable adjusted models, intake of total soy products was not significantly associated with total mortality. Compared with the lowest fifth of total soy product intake, the hazard ratios in the highest fifth were 0.98 (95% confidence interval 0.91 to 1.06, P trend =0.43) in men and 0.98 (0.89 to 1.08, P trend =0.46) in women. Intake of fermented soy products was inversely associated with all cause mortality in both sexes (highest versus lowest fifth: 0.90 (0.83 to 0.97), P trend =0.05 in men, and 0.89 (0.80 to 0.98), P trend =0.01 in women). Natto showed significant and inverse associations with total cardiovascular disease related mortality in both sexes.
Conclusions
In this study a higher intake of fermented soy was associated with a lower risk of mortality. A significant association between intake of total soy products and all cause mortality was not, however, observed. The findings should be interpreted with caution because the significant association of fermented soy products might be attenuated by unadjusted residual confounding.
We newly manufactured miso rich in angiotensin-converting enzyme (ACE) inhibitory activity (Marukome MK-34-1, shinki miso) and investigated its antihypertensive properties in rat models of genetic hypertension. ACE inhibitory activity was tenfold higher in shinki miso than in commercially available Marukome Nenrin miso (nenrin miso). The inhibitory activity of shinki miso was confined to <3 kDa fractions and was detected in several fractions with high polarity by C18 high-performance liquid chromatography. Systolic blood pressure (SBP) increased age-dependently in stroke-prone spontaneously hypertensive rats (SHRSP/Izm) given a 0.6% (w/v) NaCl solution (salt solution group) that matched the salt content of the miso solutions. This SBP increase was attenuated in both the 5% nenrin and 5% shinki miso solution groups compared to the salt solution group. The reduction in SBP was greater in rats fed shinki than in rats fed nenrin miso. Similarly, in a salt-induced hypertension model with Dahl rats, the 5% nenrin miso solution attenuated the rising SBP observed in the salt solution group. Moreover, combining 5% nenrin miso with 5% shinki miso (2:1, v/v) (awase miso group) significantly decreased the SBP per gram salt intake by 8% compared with the nenrin miso treatment. However, there were no differences in urinary Na excretion between the nenrin and awase miso groups. In conclusion, we produced a new miso with potent ACE inhibitory activity that reduced spontaneous and salt-induced hypertension. These results suggest that salt sensitivity is decreased by the addition of shinki miso to nenrin miso.
Objective: To evaluate the efficacy of oral nattokinase (NK) in the reduction of common carotid artery intimal medial thickness (CCA-IMT) and carotid artery plaque size and in lowering blood lipids, and to explore the underlying mechanism of actions of NK and its potential clinical use. Methods: All enrolled patients were from the Out-Patient Clinic of the Department of TCM at the 3rd Affiliated Hospital of Sun Yat-sen University. Using randomised picking method, all patients were randomly assigned to one of two groups, NK and Statin (ST) group. NK Group-patients were given NK at a daily dose of 6 000 FU and ST Group-patients were treated with statin (simvastatin 20 mg) daily. The treatment course was 26 weeks. CCA-IMT, carotid plaque size and blood lipid profile of the patients were measured before and after treatment. Results: A total of 82 patients were enrolled in the study and 76 patients (NK 39, ST 37) completed the study. Following the treatments for 26 weeks, there was a significant reduction in CCA-IMT and carotid plaque size in both groups compared with the baseline before treatment. The carotid plaque size and CCA-IMT reduced from(0.25±0.12)cm² to (0.16±0.10)cm² and from (1.13±0.12)mm to (1.01±0.11)mm, repectively. The reduction in the NK group was significantly profound (P<0.01, 36.6% reduction in plaque size in NK group versus 11.5% change in ST group). Both treatments reduced total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG). While the reduction in both groups was shown to be statistically significant (P<0.01), the reduction of TC, LDL-C and TG in ST group was significantly greater (P<0.05). In addition, NK significantly increased the level of high-density lipoprotein cholesterol (HDL-C) (P<0.05), in contrast, HDL-C in the ST group did not change. The lipid lowering effect observed in the NK group was not correlated to the reduction of CCA-IMT and carotid artery plaque size (r=0.35, P=0.09). Conclusions: Our findings from this pioneer clinical study suggests that daily NK supplementation is an effective way to manage the progression of atherosclerosis and potentially may be a better alternative to statins which are commonly used to reduce atherosclerosis and further to prevent cardiovascular attack and stroke in patients. The mechanism underlying the reduction of carotid atherosclerosis by NK may be independent from its lipid-lowering effect, which is different from that of statins.
Natto, a fermented soybean product, has been consumed as a traditional food in Japan for thousands of years. Nattokinase (NK), a potent blood-clot dissolving protein used for the treatment of cardiovascular diseases, is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. NK has been extensively studied in Japan, Korea, and China. Recently, the fibrinolytic (anti-clotting) capacity of NK has been recognized by Western medicine. The National Science Foundation in the United States has investigated and evaluated the safety of NK. NK is currently undergoing a clinical trial study (Phase II) in the USA for atherothrombotic prevention. Multiple NK genes have been cloned, characterized, and produced in various expression system studies. Recombinant technology represents a promising approach for the production of NK with high purity for its use in antithrombotic applications. This review covers the history, benefit, safety, and production of NK. Opportunities for utilizing plant systems for the large-scale production of NK, or for the production of edible plants that can be used to provide oral delivery of NK without extraction and purification are also discussed.
Our aim was to determine the quantitative effects of a single-dose of Nattokinase (NK) administration on coagulation/fibrinolysis parameters comprehensively in healthy male subjects. A double-blind, placebo-controlled cross-over NK intervention study was carried out in 12 healthy young males. Following the baseline blood draw, each subject was randomized to receive either a single-dose of 2,000 FU NK (NSK-SD, Japan Bio Science Laboratory Co., Ltd) or placebo with subsequent cross-over of the groups. Subjects donated blood samples at 2, 4, 6 and 8 hours following administration for analysis of coagulation/fibrinolysis parameters. As a result, D-dimer concentrations at 6, and 8 hours, and blood fibrin/fibrinogen degradation products at 4 hours after NK administration elevated significantly (p < 0.05, respectively). Factor VIII activity declined at 4 and 6 hours (p < 0.05, respectively), blood antithrombin concentration was higher at 2 and 4 hours (p < 0.05, respectively), and the activated partial thromboplastin time prolonged significantly at 2 and 4 hours following NK administration (p < 0.05 and p < 0.01, respectively). All the changes, however, were within the normal range. In conclusion, thus, a single-dose of NK administration appears enhancing fibrinolysis and anti-coagulation via several different pathways simultaneously.
The fermented soybean product natto is a popular traditional food in Japan and is considered a health supplement. NKCP®, a natto-derived dietary food supplement whose main component is bacillopeptidase F, has antithrombotic, fibrinolytic, and blood pressure-lowering effects. We examined whether daily intake of NKCP® effectively improves subjective symptoms in patients with lifestyle diseases in this cross-over, double-blind study. Fermented soya extract with subtilisin NAT (nattokinase) as the main component was used as an active placebo. A 4-week course of NKCP® significantly decreased the visual analog scale (VAS) score for shoulder stiffness from 42.3 to 32.4 (P=0.009), the VAS score for low back pain from 25.5 to 18.8 (P=0.02), and the VAS score for coldness of the extremities from 33.1 to 25.7 (P=0.002). However, no significant difference was found in the VAS score for headache. After a 4-week course of active placebo, no significant changes in the VAS score were found for any symptoms. The significant improvement in the symptoms secondary to blood flow disturbance was caused by the improvement in blood flow by NKCP®. The use of dietary supplements based on the Japanese traditional food natto helps to relieve subjective symptoms for patients with lifestyle diseases receiving medical care.
This review describes effects of miso with reference to prevention of radiation injury, cancer and hypertension with a twin focus on epidemiological and experimental evidence. Miso with a longer fermentation time increased crypt survival against radiation injury in mice. When evaluating different types of miso provided by different areas in Japan, miso fermented for a longer period increased the number of surviving crypts, and 180 days of fermentation was the most significant. Dietary administration of 180-day fermented miso inhibits the development of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and rat colon cancers in F344 rats. Miso was also effective in suppression of lung tumors, breast tumors in rats and liver tumors in mice. The incidence of gastric tumors of groups of rats given NaCl was higher than those of the groups given miso fermented for longer periods. Moreover, the systolic blood pressure of the Dahl male rat on 2.3% NaCl was significantly increased but that of the SD rat was not. However, the blood pressures of the rats on a diet of miso or commercial control diet (MF) did not increase. Even though miso contains 2.3% NaCl, their blood pressures were as stable as those of rats fed commercial diet containing 0.3% salt. So we considered that sodium in miso might behave differently compared with NaCl alone. These biological effects might be caused by longer fermentation periods.
The large scale fermentation of γ-polyglutamic acid (γ-PGA) by Bacillus subtilis (natto) was done using a 30-liter jar fermenter. A stable cultivation without foaming could be done with addition of 3% NaCl to the medium. The γ-PGA productivity became higher with increasing speed of agitation and amounts of glutamic acid added to the broth. Finally, we were able to obtain about 35 mg/ml of γ-PGA under the optimum conditions. The glutamic acid added to the medium was efficiently converted into γ-PGA in the stationary phase. To discover the role of L-glutamic acid added to the medium for γ-PGA biosynthesis by Bacillus subtilis (natto), the radioactivity incorporated into γ-PGA from 14C-L-glutamic acid was measured. As a result, radioactive γ-PGA was detected in the medium. Then, the glutamic acid in the medium was transported into the cells and actually polymerized as the glutamic acid unit of γ-PGA.
Long-term ripened miso has a characteristic mouthfulness and continuity of flavour, which is heightened from 11 months of ripening. Focussing on its main components, changes of protein and sugar were investigated from 10 days up to 20 months of ripening. Protein and sugar changes during ripening did not correlate with sensory evaluation results. From visual observation of miso colour and colorimetric analysis of the water-soluble fraction, it was apparent that the Maillard reaction was occurring during 5–11 months of ripening. By fractionation and evaluation of umami, mouthfulness and continuity, we were able to identify a water soluble fraction of 20 month ripened miso with a molecular weight of 1000–5000 which was coloured and appeared to be a peptide that has undergone the Maillard reaction. From these results, the Maillard-reacted peptide was considered to be a key substance which gives the characteristic flavour (mouthfulness and continuity) of long-ripened miso.
We examined the effects of soybeans, a soy product (miso) and biochanin A, an isoflavone derivative, on N-nitroso-N-methylurea (MNU)-induced rat mammary carcinogenesis. Seven-week-old female CD/Crj rats received a single i.v. dose (40 mg/kg body weight) of MNU. After administration of MNU, rats were fed diet containing 0% (control), 2% or 10% soybeans, or 10% miso as a soy-supplemented diet, or 10 or 50 mg/kg biochanin A. All rats were observed for 18 weeks after MNU administration. At 18 weeks, the multiplicity (mean tumors/rat) of palpable mammary tumors was significantly decreased in the 10% soybean (1.1) and 10% miso (1.2) diet groups compared to the control (2.2) (P<0.05, respectively). In the biochanin A-supplemented diet groups, the incidence (percentage of rats with tumors) was significantly decreased in the 50 mg/kg (32%) diet group compared to the control (80%) (P<0.01), and the multiplicity was significantly decreased in both the 10 mg/kg (0.7) and 50 mg/kg (0.5) diet groups compared to the control (2.2) (P<0.01 and P<0.001, respectively). The proliferative cell nuclear antigen labeling index of mammary tumors was significantly decreased in both biochanin A-supplemented diet groups compared to the control. The present results indicate that soybeans, miso, and biochanin A are useful for the prevention of mammary cancer.
More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.
Although isoflavones, such as those found in soy, have been shown to inhibit breast cancer in laboratory studies, associations between consumption of isoflavone-containing foods and breast cancer risk have been inconsistent in epidemiologic studies. We evaluated the relationship between isoflavone consumption and breast cancer risk among women in the Japan Public Health Center-Based Prospective Study on Cancer and Cardiovascular Diseases (JPHC Study).
In January 1990, 21 852 Japanese female residents (aged 40-59 years) from four public health center areas completed a self-administered questionnaire, which included items about the frequency of soy consumption. Through December 1999 and 209 354 person-years of follow-up, 179 women were diagnosed with breast cancer. Cox proportional hazards regression was used to estimate the relative risks (RRs) and 95% confidence intervals (CIs) for breast cancer in relation to consumption of miso soup, soyfoods, and estimated isoflavones. All statistical tests were two-sided.
Consumption of miso soup and isoflavones, but not of soyfoods, was inversely associated with the risk of breast cancer. The associations did not change substantially after adjustment for potential confounders, including reproductive history, family history, smoking, and other dietary factors. Compared with those in the lowest quartile of isoflavone intake, the adjusted RRs for breast cancer for women in the second, third, and highest quartiles were 0.76 (95% CI = 0.47 to 1.2), 0.90 (95% CI = 0.56 to 1.5), and 0.46 (95% CI = 0.25 to 0.84), respectively (P(trend) =.043). The inverse association was stronger in postmenopausal women (P(trend) =.006).
In a population-based, prospective cohort study in Japan, frequent miso soup and isoflavone consumption was associated with a reduced risk of breast cancer.
We isolated from soybean miso 8-hydroxyglycitein and 6-hydroxydaidzein as DPPH-radical scavengers, and elucidated their chemical structures by mass spectrometric, and (1)H- and (13)C-NMR spectrosopic analyses. These compounds showed DPPH-radical scavenging activity as high as that of alpha-tocopherol, 8-hydroxygenistein and 8-hydroxydaidzein. This is the first report of the isolation of 8-hydroxyglycitein from a natural source.
Natto, steamed soybeans fermented by Bacillus subtilis natto, is a traditional Japanese food. We derived a purified protein layer, called NKCP as a trade mark, from B. subtilis natto fermentation. In the present study, we examined the fibrinolytic and antithrombotic effects of NKCP and identified its active component to clarify the fibrinolytic effect of NKCP observed in preliminary clinical trials previously. The active component of NKCP was identified as a 34-kilodalton protein designated bacillopeptidase F. NKCP showed direct degradation of artificial blood clot in saline. The protease activity, accounting for the fibrinolytic effect of NKCP, was examined with a chromogenic substrate for plasmin. Dose-dependent prolongations of both prothrombin time and active partial thromboplastin time were observed in rats with intra-duodenum administration of NKCP. Our in vitro and in vivo studies suggest that NKCP has both a fibrinolytic effect and an antithrombotic effect similar to heparin. Because NKCP is derived from food and has safety data demonstrated by previous animal experiments and preliminary clinical trials, NKCP is considered as safe for clinical use.
The purpose of this study was to compare the effects of miso and sodium chloride (NaCl) on blood pressure in both sexes of Dahl and SD rats. The systolic and diastolic blood pressures (SBP/DBP) were measured at 2, 4, 8 and 12 weeks of treatment with a miso diet including 2.3% NaCl, a high-sodium diet including 2.3% or 1.9% NaCl, or a normal diet including 0.3% NaCl (MF diet; Oriental Yeast Co., Tokyo, Japan). The rats were autopsied after 12 weeks on a diet. DBP in male Dahl rats was significantly increased by the 2.3% NaCl diet as compared with that in the MF group (p < 0.01) or miso group (p < 0.05) from 4 weeks of treatment. Thereafter, SBP and DBP in both the high NaCl groups were significantly increased when compared with the MF or miso group. SBP in female Dahl rats on 2.3% NaCI was significantly increased from 8 weeks after treatment. Nephropathy was observed in both sexes of Dahl rats but not SD rats. These results show that blood pressure was not increased by the miso diet.
A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan.
Background:
The direct association between intake of Japanese fermented soybeans, namely natto, and bone mineral density (BMD) is known. However, the association with osteoporotic fractures has not been studied.
Objective:
This study aimed to investigate whether habitual natto intake is associated with a risk of osteoporotic fractures.
Methods:
This prospective cohort study included 1417 postmenopausal Japanese women who were enrolled in the Japanese Population-Based Osteoporosis cohort study in 1996, 1999, 2002, and 2006 and were aged ≥45 y at baseline. The intake of natto, tofu, and other soybean products was surveyed with use of a FFQ at baseline. Fractures were ascertained in follow-up surveys conducted in 1999, 2002, 2006, and 2011/2012. Osteoporotic fracture was the primary outcome and was defined as a clinical fracture occurring without strong external force, diagnosed with radiographs by a medical doctor. HRs with 95% CIs were estimated with Cox proportional hazard models.
Results:
During the 17,699 person-years of follow-up (median, 15.2 y), 172 women experienced osteoporotic fractures. After adjustment for age and BMD at the total hip, the HRs compared with those of < 1 pack (approximately 40 g)/wk natto intake were 0.72 (95% CI: 0.52, 0.98) and 0.51 (95% CI: 0.30, 0.87) for 1-6 and ≥7 packs/wk, respectively. After further adjustment for BMI, history of osteoporotic fractures, history of myocardial infarction or stroke, diabetes mellitus, current smoking, alcohol intake, frequency of tofu and other soybean product intakes, and dietary calcium intake, the HRs were 0.79 (95% CI: 0.56, 1.10) and 0.56 (95% CI: 0.32, 0.99) for 1-6 and ≥7 packs/wk, respectively. Frequency of tofu or other soybean product intakes had no association with the risk of osteoporotic fractures.
Conclusions:
Habitual natto intake may be associated with a reduced risk of osteoporotic fractures independent of confounding factors, including BMD, in Japanese postmenopausal women. This trial was registered at umin.ac.jp as UMIN 000032869.
The history of fermented foods and drinks dates back more than 4000 yr. Wine already existed around 5000 B.C., and the original forms of soy sauce and fermented milk existed around 3000 to 2000 B.C. Microorganisms obtained from the environment were put to use for the fermentation and maturation of fermented foods. Regional differences in starting materials, climate, culture, and other environmental factors have developed unique fermented products in various parts of the world. At the same time, regional and racial differences have had a big effect on whether some fermented products are considered good tasting or bad. This chapter describes the traditional Japanese fermented soybean product miso, and its effects on human health and metabolism.
Background and hyposesis:
Soybean isoflavones have been shown to reduce the risk of cerebral infarction in humans according to epidemiological studies. However, whether intake of miso can reduce the incidence of stroke in animal models remains unknown. In this study, we investigated the effects of soybean paste (miso) in an animal model of stroke.
Methods:
Stroke-prone spontaneously hypertensive rats (SHRSP) were fed a miso diet (normal diet 90%, miso 10%; final NaCl content 2.8%), a high salt diet (normal diet and NaCl 2.5%; final NaCl content 2.8%), or a low salt diet (normal diet; final NaCl content 0.3%).
Results:
Kaplan-Meier survival curves revealed a significantly lower survival rate in the high salt group compared to the miso group (P = 0.002) and the low salt group (P ≤ 0.001). Large hemorrhagic macules were found in the cerebrum in the high salt group, whereas none were found in the other 2 groups. There were also fewer histological and immunohistochemical changes in the brain and kidneys in the miso group compared to the high salt group.
Conclusion:
Our results suggest that miso may have protective effects against stroke despite its high salt content.
Background:
Whether soy intake is associated with a decreased risk of cardiovascular disease (CVD) remains unclear. A traditional Japanese soy food, natto, contains a potent fibrinolytic enzyme. However, its relation to CVD has not been studied.
Objective:
We aimed to examine the association of CVD mortality with the intake of natto, soy protein, and soy isoflavones in a population-based cohort study in Japan.
Design:
The study included 13,355 male and 15,724 female Takayama Study participants aged ≥35 y. At recruitment in 1992, each subject was administered a validated semiquantitative food-frequency questionnaire. Deaths from CVD were ascertained over 16 y.
Results:
A total of 1678 deaths from CVD including 677 stroke and 308 ischemic heart disease occurred during follow-up. The highest quartile of natto intake compared with the lowest intake was significantly associated with a decreased risk of mortality from total CVD after control for covariates: the HR was 0.75 (95% CI: 0.64, 0.88, P-trend = 0.0004). There were no significant associations between the risk of mortality from total CVD and intakes of total soy protein, total soy isoflavone, and soy protein or soy isoflavone from soy foods other than natto. The highest quartiles of total soy protein and natto intakes were significantly associated with a decreased risk of mortality from total stroke (HR = 0.75, 95% CI: 0.57, 0.99, P-trend = 0.03 and HR = 0.68, 95% CI: 0.52, 0.88, P-trend = 0.0004, respectively). The highest quartile of natto intake was also significantly associated with a decreased risk of mortality from ischemic stroke (HR = 0.67, 95% CI:0.47, 0.95, P-trend = 0.03).
Conclusion:
Data suggest that natto intake may contribute to the reduction of CVD mortality.
Objectives:
We investigated the antihypertensive mechanism of long-term Miso soup consumption in Dahl salt-sensitive (Dahl S) rats with salt-induced hypertension.
Material and methods:
Female Dahl S rats fed a low-salt (0.3% NaCl) diet were divided into three groups: (1) six rats given water, (2) six rats given 0.65% (w/v) saline solution or (3) eight rats given 5% (w/v) Miso soup containing 0.65% (w/v) saline solution. They were followed for 12 weeks. Variables in the plasma or 24-h urine were determined. Systolic blood pressure (SBP) was measured by the tail-cuff method.
Results:
The SBP increased in an age-dependent manner in Dahl S rats drinking saline solutions. The elevation of SBP was significantly attenuated in Dahl S rats given Miso soup although the ultimate cumulative salt loading was much greater in the Miso group than those given the saline solutions. This SBP reduction in the Miso group was associated with an increase in fractional excretion of Na (FENa) and free water clearance in the kidney. Urinary dopamine excretions were increased in the Miso group compared with that in the saline group. The increase in urinary dopamine excretions was associated with a decrease in brain oxidative stress. Urinary dopamine excretions were an independent predictor of SBP in the Miso group.
Conclusions:
Long-term consumption of Miso soup attenuated blood pressure elevation in Dahl salt-sensitive rats with salt-induced hypertension. The blood pressure reduction was due to, at least in part, constituent(s) of the Miso that increase natriuresis and diuresis and enhance dopaminergic nervous activity in the kidney.
The effects on blood coagulation of dipicolinic acid (DPA, 2,6-pyridinedicarboxylic acid), an anti-bacterial substance known to be produced by Bacillus subtilis natto and contained in natto, a traditional Japanese fermented soybean food, were studied. It was found that addition of DPA with a final concentration of 5 × 10 -3 M results in substantial inhibition of platelet aggregation. DPA inhibition was found to be far stronger than that resulting from addition of aspirin. Furthermore, the clotting reaction of thrombin-fibrinogen was also found to be inhibited by DPA. It was confirmed by examination of thromboelastogram patterns that the coagulation of whole rat blood was completely inhibited by addition of 5 × 10 -3 M DPA. From the point of view of the blood coagulation system, these results show that DPA contained in natto may be effective in the prevention of thrombosis.
Foods containing bioactive components are receiving increasing attention due to their functionality in disease prevention and treatment. Soybean products contain a plethora of bioactive phytochemicals such as isoflavones, saponins, phytic acids, phytosterols, trypsin inhibitors, and peptides. Research has implicated soybean phytochemicals as functioning in cholesterol reduction, cardiovascular disease prevention, diabetic symptoms prevention, bone loss prevention, and cancer prevention. However, some bioactive compounds in soybean are reported to have some adverse effects to health also. Nevertheless, the benefits of soybean bioactive components exceed by far their potential adverse effects. The main objective of this article is to discuss the different soybean bioactive components and their implications to human health.
Guided by their DPPH radical-scavenging activity, nine compounds were isolated from soybean mise. Of these, 8-hydroxydaidzein, 8-hydroxygenistein and syringic acid had as high DPPH radical-scavenging activity as that of alpha-tocopherol. The antiproliferative activity of four of the isolated isoflavones toward three cancer cell lines was examined. 8-Hydroxygenistein showed the highest activity (IC50 = 5.2 mu M) toward human promyelocytic leukemia cells (HL-60).
Natto is a popular traditional Japanese food made by fermenting steamed soybeans with Bacillus subtilis natto. It has long been thought that natto is effective in preventing hypertension. But there have not been any reports demonstrating in vivo antihypertensive effects of natto. In this study, we demonstrate for the first time the antihypertensive activity of natto in vivo. We determined optimal conditions for producing natto with the highest angiotensin I converting enzyme (ACE) inhibitory activity. An ACE inhibitor partially purified from natto was orally administered as a single dose (1 mg, 10 mg, and 100 mg/kg body weight) in spontaneously hypertensive rats, and then their blood pressure was measured five times every hour after administration. The administration of the inhibitor, even at the lowest dose, resulted in a significant decrease in blood pressure 4 h after administration. Thus, the ACE inhibitor from natto appears to moderately reduce blood pressure relative to increases in dosage.
We investigated the effects of a water extract of natto on the aging of the nematode Caenorhabditis elegans. The water extract significantly prolonged the adult lifespan of the wild-type worms and rendered them resistant to oxidative and thermal stress. In addition, treatment with natto extract significantly delayed the accumulation of lipofuscin, a characteristic of aging cells. Our findings suggest that components of natto have a beneficial anti-aging effect in vivo.
Bacillus natto (Bacillus subtilis natto) was cultivated, and an analysis was conducted after performing lysozyme treatment and water extraction of the culture supernatant and the B. subtilis natto cells. The intracellular existence of a large amount of water-soluble vitamin K (Menaquinone-7: MK-7) was established. The existence of small amounts of other types of vitamin K2 including MK-4 and PK was also confirmed in the culture solution (water-soluble fractions). The amount of water-soluble menaquinone-7 in Bacillus was 85 µg/g wet weight of the bacteria, and the amount was equivalent to more than 100 times as much as that contained in the culture solution (0.02 µg).Gel filtration using Sephacryl S-200HR revealed that the molecular weight of the water-soluble menaquinone-7 is approximately MW 200,000, and isoelectric focusing revealed a behavior similar to that of protein, with a pI of about 4.2. A rabbit antibody was prepared with this water-soluble vitamin K as the antigen. By using the Ouchterlony method, the antibody showed a reaction (precipitation line) with the water-soluble menaquinone-7 prepared from both the intracellular fraction and the extracellular culture solution, and it was found through the antigen–antibody reaction that the menaquinone-7 disappears from the supernatant of the reactant (the water-soluble menaquinone-7 is thus neutralized). Based on these results, it was inferred that the vitamin K produced by B. subtilis natto becomes water-soluble by forming an intracellular complex with protein and is released in the extracellular fraction during the culture process.
We investigated the effects of long-term miso soup drinking on salt-induced hypertension in Dahl salt-sensitive (Dahl S) rats.
Dahl S rats were divided into four groups that consumed 1) water, 2) a 0.9% NaCl solution, 3) a 1.3% sodium NaCl solution, or 4) miso soup containing 1.3% NaCl. They were followed for 8 wk. Systolic blood pressure and hypertensive organ damage were determined.
Systolic blood pressure increased in an age- and dose-dependent manner in Dahl S rats drinking salt solutions. The systolic blood pressure increase was significantly less in the Dahl S rats that drank miso soup, although the ultimate cumulative salt loading was greater than that in the Dahl S rats given the 1.3% NaCl solution. This blood pressure decrease was associated with a morphologic attenuation of glomerular sclerosis in the kidney and collagen infiltration in the heart. Urinary protein excretions were less in the miso group than in the rats given the 1.3% NaCl solution. The fractional excretion of sodium was increased and that of potassium was decreased in Dahl S rats given the 1.3% NaCl solution, and these effects were reversed in rats given miso soup toward the values of the control.
We found that long-term miso soup drinking attenuates the blood pressure increase in salt-induced hypertension with organ damage. This may be caused by a possible retardation of sodium absorption in the gastrointestinal tract or by the direct effects of nutrients in the miso soup from soybeans. The decrease was associated with decreases in cardiovascular and renal damage.
To determine whether the antihypertensive effect of nattokinase is associated with the protease activity of this enzyme, we compared nattokinase with the fragments derived from nattokinase, which possessed no protease activity, in terms of the effect on hypertension in spontaneously hypertensive rats (SHR). In the continuous oral administration test, the groups were given a basic diet alone (control), the basic diet containing nattokinase (0.2, 2.6 mg/g diet) or the basic diet containing the fragments derived from nattokinase (0.2, 0.6 mg/g diet). The group fed the basic diet containing high-dosage nattokinase (2.6 mg/g diet) showed significant reductions in systolic blood pressure (SBP), diastolic blood pressure (DBP) and plasma fibrinogen level, compared with control group and no influence on activities of renin and angiotensin-converting enzyme (ACE, EC 3.4.15.1), and plasma angiotensin II level in the renin-angiotensin system. The treatment of the basic diet containing high-dosage fragments (0.6 mg/g diet) significantly decreased SBP, DBP and plasma angiotensin II level in plasma but the treatment did not influence on plasma fibrinogen level. These results suggest that nattokinase and its fragments are different from each other in the mechanism to reduce hypertension. Nattokinase, retained its protease activity after absorbance across the intestines, may decrease blood pressure through cleavage of fibrinogen in plasma. The fragments, which absorbed as nattokinase-degradation products, prevents the elevation of plasma angiotensin II level to suppress hypertension.
2-Furanmethanethiol, a compound contained in many kinds of food, was identified for the first time in five types of miso fermented soybean paste (red salty rice miso, thin-colored salty rice miso, weak salty rice miso, barley miso, and soy miso) by specific extraction of volatile thiols using p-hydroxymercuribenzoate. In the triangle test with red salty rice miso, which included a higher concentration of 2-furanmethanethiol, and thin-colored salty rice miso, which included a lower concentration of 2-furanmethanethiol, it was shown that the aroma of thin-colored salty rice miso was similar to that of red salty rice miso by adding 2-furanmethanethiol into thin-colored salty rice miso. In addition, a quantitative descriptive analysis (QDA) clearly shows that 2-furanmethanethiol contributed to the intensity of three odor qualities "thick, complex", "sweet", and "pleasant aroma like coffee beans" in six odor qualities and was a very important component of miso aroma.
Daily intake of soybean paste soup was found to significantly reduce standardized mortality rates for gastric cancer in an ongoing large-scale prospective study of 122,261 males and 142,857 females aged 40 and above in 29 Japanese health center districts, 1966-1978. The gastric cancer standardized mortality rates were 171.9, 210.2, 240.0, and 255.9 per 100,000 males, and 77.8, 85.3, 97.5, and 113.6 per 100,000 females in daily, occasional, rare, and noningesters, respectively. These relationships remained significant when observed by age group, district, socioeconomic status, dietary pattern, and smoking habit. The risk-reducing effect has also been observed in case-control studies in the past, for both males and females and in urban and rural areas. This beneficial effect could arise from selected compounds such as protease inhibitors and/or other nutritious substances included in the soybean, but it is also possible that it merely reflects the effect of some frequent accompaniment to soybean paste soup, such as green-yellow vegetables.
Natto is a traditional Japanese fermented food made by fermenting boiled soy beans with Bacillus natto. Its contents of inhibitors against the angiotensin converting enzyme (ACE, EC3.4.15.1) were investigated. Relatively strong inhibitory activity (IC50: 0.4 mg/ml, 11.8 inhibition units/g natto) was detected in natto extracts and the inhibitory activity observed in the viscous fraction was more potent than in the bean extract. Two groups of inhibitors in the viscous material, high and low molecular weight inhibitors, were resolved by dialysis test. The inhibitor of high molecular weight was a protein with low IC50 value (0.12 mg/ml). The two types of low molecular weight inhibitors were detected in ethanol extracts (IC50: 0.53 mg/ml and 0.95 mg/ml) and they were found to be stable over a wide range of pH and temperature up to 100 degrees C. They were different in the mode of ACE inhibition. One is competitive, and the other noncompetitive against the hydrolysis of Bz-Gly-His-Leu by ACE.
Nattokinase is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro. In this study, we investigated the thrombolytic effect of nattokinase on a thrombus in the common carotid artery of rat in which the endothelial cells of the vessel wall were injured by acetic acid. When a section of occluded vessel was stained for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, the antigen was localized around the surface of the occluded blood vessels. This result suggests that the occlusive thrombosis was caused by platelet aggregation. In addition, thrombolysis with urokinase (UK; 50000 IU/kg, i.v.) or tissue plasminogen activator (tPA; 13300 IU/kg, i.v.) in our model was observed to restore the blood flow over a 60 min monitoring period. The results indicate that our chemically induced model is useful for screening and evaluating a thrombolytic agent. We evaluated the thrombolytic activity of nattokinase using this model and compared it with fibrino(geno)lytic enzyme, plasmin or elastase. On a molar basis, the recovery of the arterial blood flow with nattokinase, plasmin and elastase were 62.0 +/- 5.3%, 15.8 +/- 0.7% and 0%, respectively. The results indicate that the thrombolytic activity of nattokinase is stronger than that of plasmin or elastase in vivo.
The effect of dietary vitamin K2 (menaquinone-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12mg/100g diet) or menaquinone-7 (MK-7; 18.1mg/100g diet) for 24 days; MK-4 and MK-7 were equal in molar concentrations. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. In separate experiments, OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 microg MK-7/100g diet) without or with added MK-7 (37.6 microg/100g diet) for 77 days. Feeding produced a significant elevation of MK-4 and MK-7 concentrations in the serum of OVX rats. In this case, a significant increase in the femoral MK-4 content was observed but MK-7 was not detected in the femoral tissues. OVX-induced decreases in the femoral dry weight and femoral calcium content were significantly prevented by the feeding of diets containing natto with MK-7 added (37.6 microg/100g diets). This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4, which is formed by degradation of MK-7.
We prepared natto (fermented soybeans) mucilage containing poly-gamma-glutamic acid (gamma-PGA) from commercial natto. The effect of natto mucilage on calcium (Ca) solubility in vitro and in vivo was investigated. Ca solubility in vitro increased with an increase in the amount of natto mucilage, due to inhibition of the formation of an insoluble complex of Ca with phosphate by natto mucilage. Rats were fed with 5 g of soybean protein isolate, natto, mucilage-free natto, or natto mucilage diet for 1.5 h. Small intestinal contents were collected 2.5 h after ingestion. In the lower half of the small intestine, both the amount and the percentage of soluble Ca of intestinal contents were significantly higher (P < 0.001) in rats fed with natto mucilage diet than in those fed with the other diets. Natto mucilage also increased Ca solubility in vivo. These results suggested that gamma-PGA is responsible for the increasing effect of natto mucilage on Ca solubility.
The oxygen radical scavenging activity of natto (fermented soybeans) and its inhibitory effect on the oxidation of rat plasma low-density lipoprotein (LDL) in vitro were investigated to evaluate the usefulness of the antioxidant properties of natto, which has been shown to have antioxidant activity. Natto was separated into three water-soluble fractions: high-molecular-weight viscous substance (HMWVS; Mw > 100 000), low-molecular-weight viscous substance (LMWVS; Mw < 100 000), and soybean water extract (SWE). LMWVS had the strongest radical scavenging activity for hydroxyl and superoxide anion radicals, as assessed by electron spin resonance. The increase of conjugated dienes in LDL oxidized by copper and an azo pigment was depressed by the addition of LMWVS and SWE. These results demonstrate that natto fractions have inhibitory effects on LDL oxidation as a result of their radical scavenging activity.
To determine if the risk of hepatocellular carcinoma (HCC) is reduced by consumption of soya foods, we conducted a case-control study within a cohort of Japanese A-bomb survivors. We compared the prediagnosis consumption of isoflavone-rich miso soup and tofu to HCC risk, adjusting for hepatitis B (HBV) and C (HCV) viral infections, the major HCC risk factors in this population. The study included 176 pathologist-confirmed cases of HCC diagnosed in 1964-1988 and 560 controls who died of diseases other than liver cancer. We examined dietary information collected at least 2 years before diagnosis or death and tissue-based measures of viral hepatitis. Using logistic regression, crude ORs were 0.5 (95% CI 0.29-0.95) and 0.5 (95% CI 0.20-0.99) for high vs. low miso soup and tofu intake, respectively. Adjusting for year of birth, sex, HBV, HCV and other factors, the OR for miso soup was unchanged at 0.5 (95% CI 0.14-1.55), and miso results were similar when ORs were recalculated separately for earlier and later birth cohorts to assess consistency of results. The adjusted OR for tofu was 0.9 (95% CI 0.20-3.51). We also found a statistically significant (p < 0.0001) interaction between sex and HCV, with risk of HCC being substantially higher for women. We conclude that consumption of miso soup and other soya foods may reduce HCC risk.
The present study was designed to investigate the effects of fermented miso (fermented soybean paste) on the induction of colon tumors by azoxymethane (AOM) in male F344 rats. A total of 91 rats, 6 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. The animals were placed on diets one week before the first AOM dose: commercial normal control MF diet or a diet containing 10% 2-year, 180-day fermented, or 3-4-day fermented miso. There were no differences in body and organ weights, and no aberrant crypt foci (ACF) among carcinogen-treated groups at week 25. The rates of tumor incidence were 45%, 85%, 75% and 60% with the 2-year, 180-day, and 3-4-day fermented miso and MF, respectively, and those for colon tumors were 34%, 55%, 60% and 55%, respectively. The size of well-differentiated adenocarcinomas and total (well differentiated and signet ring cell) adenocarcinomas in the 180-day fermented miso group was significantly smaller than that in the 2-year fermented miso and MF+AOM groups. Nuclear staining of beta-catenin in colon tumors was increased for the 3-4-day fermented miso compared to the 180-day fermented miso. Cdx2 staining tendency was decreased in colon tumors and adenocarcinomas compared to normal mucosa and ACF, which stained in 100% of cases. In addition, the PCNA index was significantly reduced in the 180-day group compared with those groups receiving the 3-4-day fermented miso and MF diet. The germinal region was also decreased. The present results indicate that dietary supplementation with 180-day fermented dietary miso could act as a chemopreventive agent for colon carcinogenesis.
Japanese fermented soybeans (natto in Japanese), which contain a large amount of menaquinone-7, may help prevent the development of osteoporosis. We assessed the possibility of an association between habitual natto intake and bone mineral density (BMD) and BMD change over time in healthy Japanese women who participated in a large representative cohort study (Japanese Population-based Osteoporosis Study: JPOS study). The BMD was measured at the spine, hip, and forearm in 944 women (20-79 y old) at baseline and at a follow-up conducted 3 y later. Dietary natto intake was assessed by a FFQ on both occasions. Additional covariates including age, height, weight, lifestyle factors, dietary calcium intake, and the intake of other soybean products, were also measured. The total hip BMD at baseline increased (P for trend = 0.0034) with increasing habitual natto intake in the postmenopausal women, although this was not the case at other skeletal sites. There were significant positive associations between natto intake and the rates of changes in BMD at the femoral neck (P < 0.0001) and at the distal third of the radius (P = 0.0002) in the postmenopausal women. The association in the femoral neck persisted even after adjusting for covariates. No significant association was observed between the intake of tofu or other soybean products and the rate of BMD change in the postmenopausal women. Natto intake may help prevent postmenopausal bone loss through the effects of menaquinone 7 or bioavailable isoflavones, which are more abundant in natto than in other soybean products.
Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.
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