Biological functions of compounds from Bacillus subtilis and its subspecies, Bacillus subtilis natto

1 INTRODUCTION

Post-pandemic (COVID-19) era, there is a rise in demand of sources for food, processing by-products are being valorized to identify useful compounds to improve future food security, of which cereal processing are a well-known example (Boyacι-Gündüz et al., 2021; Galanakis, 2022). Bran, germ, and husk of cereals are rich in functional macromolecules such as arabinoxylan, β-glucan, pectin, protein and small molecules such as ferulic acid and vitamins. Therefore, by-products from processing are widely used for biofuels, bioplastics, biopolymers, nutraceuticals, pharmaceuticals and fermentation applications. However, Galanakis noted that these by-products are currently not being used efficiently enough (Galanakis, 2022). In this global situation, increasing needs are arising for the development of new sources of useful materials not only limited to grains.

Bacillus subtilis (B. subtilis) is a species that forms rod-shaped, aerobic, gram-positive bacteria, 2–6 μm long, and less than 1 μm in diameter (Errington & Aart, 2020; Turnbull, 1996). B. subtilis has been closely associated with the development of human scientific technology since 1872, explained in detail by Cohn (1872) and used as a model bacterium for differentiation experiments in 1876, disproved the theory of spontaneous generation (Cohn, 1876). Compared to cultured cells, and so forth, B. subtilis has a significantly faster growth rate, under culture conditions with a doubling rate in about 20 min at 30–35°C (Errington & Aart, 2020). In addition to these advantages, B. subtilis has the advantage of easy genetic manipulation and availability of abundant experimental data (proteome, transcriptome, metabolome, and genome sequence data) from previous reports (Zeigler et al., 2008). Due to these advantages, B. subtilis has been used in metabolic engineering, genetic engineering, and other fields until today (Brantl & Müller, 2021; Jameson & Wilkinson, 2017; Y. Liu, Li, et al., 2017; Xiang et al., 2020).

From past to present, various compounds have been chemically synthesized and their beneficial functions have been discovered. However, the process of chemical synthesis is high cost, requires environmental considerations, and has safety concerns (Kharissova et al., 2019). Microorganisms have the potential to solve these problems. Du et al. stated that there are three main advantages of using microorganisms instead of chemical synthesis regarding obtaining the target compounds. First, “ability to conduct the synthesis process through environmentally friendly routes, avoiding the use of heavy metals, organic solvents, and strong acids and bases.” Second, “ability of enzymes, the most dominant factor in microbial reactions, contribute to the suppression of by-product formation because of their relatively high substrate specificity.” And third, “ability of microorganisms to produce compounds with complex structures that are difficult to obtain through chemical synthesis” (Du et al., 2011). With these advantages, methods for obtaining beneficial compounds using microorganisms (“Microbial cell factories”) are being developed worldwide (Y. Wang et al., 2021). Many of the compounds produced by microorganisms already have a significant role in daily lives of the humans (Xiang et al., 2019). Among the technologies related to “Microbial cell factories,” B. subtilis has been highly studied for producing beneficial compounds because of its attractive advantages (rapid growth rate, simple genetic manipulation, abundant accumulated past experimental data), to use them in agriculture, cosmetics, dyes, foods, pharmaceuticals, and industrial chemicals, leading to one of the recent research fields of great interest (Liu, Ding, et al., 2017; Y. Wang et al., 2021; Xiang et al., 2019). During the developmental process of B. subtilis (intercellular communication, biofilm formation, spore formation, etc.), a variety of specialized low molecular weight compounds, proteins, carbohydrates, and nucleic acids are produced (Branda et al., 2005). However, it has been noted that there are several specialized metabolites produced by B. subtilis that have not yet been fully identified (Schoenborn et al., 2021; Straight & Kolter, 2009).

B. subtilis has been used in humans not only as an experimental model and “Microbial cell factories” but also as a fermented soybean food. Despite their limited availability, fermented soybean food utilizing B. subtilis are consumed (Bin et al., 2013; Gopikrishna et al., 2021). Among these, natto, produced by fermentation of soybeans by “Bacillus subtilis natto (a subspecies of Bacillus subtilis),” has been consumed in Japan as a traditional fermented food for at least a thousand years (Akbulut et al., 2020). Natto is attracting interest as a potential functional food for health, and has been reported to exhibit antithrombotic, antihypertensive, antiosteoporotic, antidiabetic, antioxidant, antiobesity, LDL-lowering, and cholesterol-lowering activities (Chan et al., 2021; Kozioł-Kozakowska & Maresz, 2022; P. Wang, Gao, et al., 2020; Y. Yang et al., 2021; L. Yuan et al., 2022). Recently, development of new products of natto for the human health function has been under consideration (Chakrabarti et al., 2018; Shimakage et al., 2012).

The above paragraph suggests that the use of B. subtilis for bioactive compound production and as a functional food has been gaining attention. However, while there have been reports focusing on individual compounds produced by B. subtilis, to the best of the authors' knowledge, there are few compiled information on the bioactive compounds produced by B. subtilis, in particular, Bacillus subtilis natto (B. subtilis natto). Therefore, this review summarizes compounds and its functions of (1) B. subtilis in the first half and (2) B. subtilis natto in the second half, thereby describing the status of compounds produced by B. subtilis and its subspecies, B. subtilis natto. Hopefully, this review will help those interested in the unique compounds produced by B. subtilis and its subspecies B. subtilis natto, and functions of their fermented foods.

2 FUNCTIONAL COMPOUNDS PRODUCED BY B. SUBTILIS

This chapter describes some of the most popular compounds produced by B. subtilis. Table 1 shows typical reports to date on beneficial compounds produced by B. subtilis.

Bacteriocins such as bacillomycin, bacilysocin, fengycin, mejucin, rhizocticin, subtilosin, and surfactin produced by B. subtilis have antibiotic and antimicrobial functions (Hamley, 2015). Among them, the lipopeptide surfactin has been heavily investigated and is commercially available as a potential alternative to antibiotics and antimicrobial agents (Horng et al., 2019; Lilge et al., 2022). Furthermore, through genetic manipulation, it is easy to obtain the lipopeptides with a variety of chemical structures from B. subtilis (Peypoux et al., 1999). Details on the various antibiotics and antimicrobials produced by B. subtilis have been summarized in other reviews (Hamley, 2015; Ngalimat et al., 2021).

In this section, compounds available from B. subtilis other than bacteriocins are summarized. l-ornithine is a compound reported to exhibit potential biological activities, such as release of growth hormone, suppression of obesity, promotion of wound healing, hepatoprotection, and reduction of bitter taste in beverages. By expressing a gene encoding arginase, which hydrolyzes l-arginine to l-ornithine and urea, into B. subtilis 168, produced l-ornithine at high efficiency (M. Wang et al., 2015). Low molecular weight hyaluronan is known to play important physiological roles such as anti-cancer, stimulation of fibroblast proliferation, and collagen synthesis, P. Jin et al. (2016) efficiently produced low molecular weight hyaluronic acid with a specific structure from sucrose using B. subtilis 168. They also confirmed that the production of this low molecular weight hyaluronic acid can be three-fold increased by coexpression of specific commitment genes and manipulation of downregulation of glycolytic pathway. Low molecular weight mannan stimulates cells of the immune system. However, efficient methods for producing low molecular weight mannans remain difficult. As one approach to solving this problem, P. Jin et al. (2021) synthesized low molecular weight mannan from a low-cost source of glucose by genetic recombination technology of B. subtilis. Menaquinone-4, also called vitamin K2, has an important role in cardiovascular and bone health, but is known to be difficult to obtain because of the difficulty in achieving organic synthesis. P. Yuan et al. (2020) genetically modified the metabolism of B. subtilis to improve the production of this menaquinone-4. H. Yang et al. (2020) achieved efficient biosynthesis in B. subtilis by enhanced gene expression that boosted the synthetic pathway of 5-methyltetrahydrofolate (major form of folate in human plasma) with dihydrofolate as a precursor. They reported that the production efficiency of 5-methyltetrahydrofolate was 178.2-fold higher than that of the original B. subtilis 168. β-glucanase is a compound that has been shown to be effective in the prevention and treatment of plant diseases and is expected to be an important biotechnological aid in the brewing industry. Niu et al. (2018) reported that to optimize the production of β-glucanase in B. subtilis, they increased its production by 1.94-fold by genetic recombination technology. Maltogenic amylases are widely used in the food industries, because of their effectiveness in improving the retention of softness of puffed food and extending shelf life. Ruan et al. cloned the coding gene for maltogenic amylases-producing bacteria and overexpressed it into B. subtilis WB600. Maltogenic amylases produced by overexpressed B. subtilis exhibited beneficial results such as improved bread quality, increased bread volume during storage, reduced hardness, and extended shelf-life (Ruan et al., 2021). Lactic acid is used as a preservative, seasoning, acidifier, and pH adjuster in the food industry. It is known that 90% of the total lactic acid produced worldwide is derived from microbial fermentation (Kadam et al., 2006). While lactic acid is in demand in a variety of industries, in the food industry its use is as a acidifier, preservative, seasoning, and pH adjuster. Gao et al. (2012) produced UV mutants of B. subtilis and found that they could produce 99.3 g/L and 183.2 g/L lactic acid in 12 and 52 h, respectively, with a substrate conversion yield of 98.5% when using fed-batch culture with initial 30 g/L glucose. 1-deoxynojirimycin, a compound also found in mulberry leaves and other plants, is known to have α-glucosidase inhibition, prevent diabetes mellitus, and is expected to be used in health foods. Onose et al. have screened strains producing 1-deoxynojirimycin among 750 Bacillus species and related genera and reported that the highest production was found in Bacillus amyloliquefaciens AS385 and B. subtilis B4, which could be applied to produce food and pharmaceutical grade 1-deoxynojirimycin (Onose et al., 2013). Recently, environmental pollution caused by microplastics has become a problem, and an efficient method of degrading plastics is needed, and the potential use of B. subtilis is under investigation as a solution to this problem. N. Wang, Guan, et al. (2020) reported that polyethylene terephthalate can be degraded by B. subtilis with knockout of a gene that maximizes expression of polyethylene terephthalate hydrolase (PETase), an enzyme that degrades polyethylene terephthalate.

This chapter summarizes research reports up to the present on individual bioactive compounds produced by B. subtilis and their applications. Individual beneficial compounds produced by B. subtilis probably provide important biological activities, and it is becoming clear that B. subtilis has the potential as a useful production source to produce such compounds. However, it is estimated that it will take more time to technically establish a “Microbial cell factory” of B. subtilis to operate on an industrial scale challenging on how to reduce the production costs of microbial production in factories (Du et al., 2011). Further development of such industrial technologies would greatly enhance the usefulness of B. subtilis.

3 FUNCTIONAL COMPOUNDS PRODUCED BY B. SUBTILIS NATTO

Nattokinase, which has fibrinolytic activity, has been reported to decrease blood coagulation in vivo and helps prevent atherosclerosis (Chen et al., 2018; Dabbagh et al., 2014). Ku et al. (2009) used B. subtilis natto strains isolated from commercial natto products to develop a method for producion of nattokinase in a low-cost culture by optimizing the conditions of the culture with a statistical evaluation method using the response surface methodology (RSM). Levan is a polymer of fructose linked by fructofuranoside linkages, found in a variety of plants and microorganisms. Several reports have investigated the optimization of various culture conditions (sucrose concentration, medium pH, metal ions, and agitation speed) to produce the highest amount of levan using B. subtilis natto (Shih et al., 2010; Vieira et al., 2021). Shih et al. compared the strains that could produce the highest amount of levan among B. subtilis natto Takahashi, B. subtilis natto ATCC 7058, B. subtilis natto ATCC 7059, B. subtilis natto IFO 13169, and B. subtilis natto IFO 3335 (Shih et al., 2005). As a result, they reported that B. subtilis natto Takahashi produced the highest amount of levan. Poly-γ-glutamic acid is a polypeptide in which the d-glutamic acid or l-glutamic acid monomer is γ-amide-linked (Bajaj & Singhal, 2011). Because of its biodegradability, low toxicity, and nonimmunogenic properties, poly-γ-glutamic acid is used for industrial purpose, such as agriculture, food, pharmaceuticals, and cosmetics (Restaino et al., 2022). B. subtilis has been reported to produce poly-γ-glutamic in a medium environment with a sodium chloride concentration of 3%–6%, but too high a salt concentration inhibits growth of B. subtilis natto (Nguyen et al., 2018; Ogawa et al., 1997). Therefore, it is considered essential to grow at the appropriate concentration. Milk-clotting enzyme is used to coagulate milk to produce cheese. The selection of specific coagulation enzymes in cheese production is an important step in the process of milk coagulation and is known to affect cheese yield, texture, and flavor (Nicosia et al., 2022). Shieh et al. used B. subtilis natto Takahashi to determine the optimal culture conditions affecting the production of milk coagulase (Shieh et al., 2009). Among vitamin K, long-chain menaquinones, such as menaquinone-7, have relatively long circulating times in the blood and have been reported to correlate inversely with the risk of arterial calcification with the intake of longer-chain vitamin K (Bus & Szterk, 2021; Schurgers & Vermeer, 2002). The potential to reduce osteoporosis and cardiovascular disease has led to the promise of a stable supply technology for menaquinone-7 (Mandatori et al., 2021). To achieve this, H. Wang et al. (2019) optimized the culture conditions of B. subtilis natto to increase menaquinone-7 production by 37%. Additionally, Fang et al. (2019) optimized the purification conditions of menaquinone-7 for the large-scale production of menaquinone-7 from fermentation of B. subtilis natto and established a technique to obtain over 96% purity menaquinone-7 with a 99.3% recovery rate by two-step extraction with ethanol. Details of beneficial compounds produced by B. subtilis natto are shown in Table 2.

This chapter focused on the beneficial compounds produced by B. subtilis natto, a subgroup of B. subtilis. 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. Yuan et al., 2022). On the other hand, several reports have focused on compounds contained not in B. subtilis natto but in natto as a whole. Dipicolinic acid (also called as 2,6-pyridinedicarboxylic acid) is an antibiotic produced by B. subtilis and is known as a compound with antiblood coagulation properties. Ohtsugi et al. (2005) found that this dipicolinic acid was present in 100 g of commercial natto in an amount of 20.55 ± 13.67 mg. It is reported that the water-soluble fraction of natto promotes apoptosis of melanoma cells (Chou et al., 2021). Japan is one of the countries with longest life expectancy among the world, and “Washoku (traditional Japanese cuisine)” was registered as UNESCO's Intangible Cultural Heritage in 2013, which has raised attention to the relationship between daily consumption of Japanese food and health. Natto, fermented food produced by B. subtilis natto, is sometimes included in Japanese food. However, the definition of Japanese food has yet to be defined, and the dietary patterns of Japanese are changing. Therefore, it is still unclear what mechanism can be associated with the contribution of natto to health (Miyazawa, Abe, et al., 2022). A pack of natto contains about 19 g of protein (Afzaal et al., 2022). Since the general recommended daily allowance (RDA) of protein for adults is 0.8 g/kg/day, so eating natto at three meals a day can be considered to provide the required human protein, suggesting that natto may be a useful food source of protein (Trumbo et al., 2002). Several cohort studies have investigated the effects of daily natto consumption on health, reported that it was associated with reduced risk of osteoporotic fractures, decreased tooth loss, reduced cardiovascular disease-related mortality (Iwasaki et al., 2021; Katagiri et al., 2020; Kojima et al., 2020; Nagata et al., 2017). On the other hand, nevertheless, since natto is not a single molecule but an aggregate of numerous molecules, it seems that the evaluation of the overall biological activities of natto, might has an aspect that is difficult to explain with the current science technology. However, with the recent development of AI technology, novel approaches have begun to be established to explain the physiological effects of such “foods” that are composed of multiple molecular species (Lemay et al., 2021; Miyazawa, Hiratsuka, et al., 2021, 2022). Novel technologies through such interdisciplinary interventions may in the future make it possible to attempt to bring two separate interpretations of “food” as a whole and as “individual nutrients” closer together. Such considerations are beyond the scope of this review, but they will need to be considered and evaluated.

4 FUTURE PERSPECTIVES

The Food and Agriculture Organization of the United Nations (FAO) defines food security as “Food security exists when all people, at all times, have physical and economic access to sufficient, safe and nutritious food that meets their dietary needs and food preferences for an active and healthy life.” (Boyacι-Gündüz et al., 2021; Food and Agriculture Organization, 2006). However, in terms of food security, high-value products such as fruits, vegetables, and seafood require a great effort in their production (Cullen, 2020). Furthermore, severe pandemics, such as that caused by coronavirus (COVID-19), have been reported to be a serious threat to food security, especially in low- and -mid-income countries (Boyacι-Gündüz et al., 2021). It is also concerned that pandemic reduced availability of labor by 25% or more would cause severe food shortages worldwide (Galanakis, 2020; Huff et al., 2015). The impact of severe pandemic caused by COVID-19 has intensified the need for preparedness to avoid or reduce future frequency of food and health crises (Galanakis, 2020). Food demand for functional foods with bioactive components is attracting more attention in postpandemic era than before (Galanakis, 2021). Galanakis proposed four key issues that should be addressed for the food system during such a pandemic, one of which is “as consumers are looking to protect themselves and their immune system by adopting healthier diets, the availability of bioactive ingredients of food and functional foods may become critical, as the demand for these products may increase” (Galanakis, 2020). This suggests, developing new agri-food innovations in food security that are related to Industry 4.0 (Galanakis et al., 2021). For example, a type of polyphenol, such as flavonoids, is gaining attention as a potential compound that may contribute to this issue (Galanakis et al., 2020). However, there is still no evidence that the compounds produced by B. subtilis natto, such as those presented in this review, are directly involved in the immune system and inhibit infection or reduce symptoms of viruses such as COVID-19 (L. Wang, Wang, et al., 2020). Therefore, further studies are needed to discover whether B. subtilis natto has the potential to contribute to this issue.

5 CONCLUSION

B. subtilis has attracted interest from various research fields for its use as “Microbial cell factories” due to the bioactive compounds it produces. And the bioactive substances produced by B. subtilis is itself an attractive research target and has the potential to effectively solve future health, environmental, and energy problems. In addition, the unique bioactive compounds produced by B. subtilis natto is considered to affect the health function of natto, which is consumed by humans daily. Overall, natto is a natural source of bioactive compounds and is expected to be applied to functional foods. Many related studies reported so far have focused on the physiological effects of individual compounds in natto, but with the use of AI and other technologies, technology will be developed in the future to explain the health functions of natto, which is composed of various food ingredients.

AUTHOR CONTRIBUTIONS

Taiki Miyazawa: Writing – original draft. Chizumi Abe: Writing – original draft. Maharshi Bhaswant: Writing – review and editing. Ryoichi Ikeda: Writing – review and editing. Ohki Higuchi: Writing – review and editing. Teruo Miyazawa: Supervision; Writing – review and editing.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

ETHICS STATEMENT

None declared.