Comparison of Bioactive Components in Tempeh Produced by Three Different Rhizopus Starters and Immunomodulatory Effect of Tempeh on Atopic Dermatitis Mice

Abstract

Various food components, such as polyphenol and soluble dietary fiber, attenuate the decline of immunomodulatory function. We compared the bioactive components (isoflavone aglycone, total polyphenol, and low-molecular-weight soluble dietary fiber) in tempeh fermented with Rhizopus oligosporus (tempeh Rm), Rhizopus oryzae (tempeh Ro), or Rhizopus stolonifer (tempeh Rs). Tempeh Rs had the highest content for total isoflavone aglycone and low-molecular-weight soluble dietary fiber, which were 0.065 g and 2.5 g/100 g dry weight, respectively. Moreover, total polyphenol in tempeh Rs was 0.35 g/100 g dry weight, which was higher than that in unfermented soybean and tempeh Ro. We selected tempeh Rs and examined the therapeutic effects of dietary tempeh Rs on atopic dermatitis-like skin lesions induced by house dust mite in NC/Nga mice. The ingestion of tempeh Rs resulted in significant reductions in the skin severity scores for both ears and the immunoglobulin E concentration in experimental mice.

Introduction

Tempeh is a traditional non-salted soybean food from Indonesia that is fermented by Rhizopus species. Isoflavone aglycones, peptides, free amino acids, and gamma-aminobutyric acid are present in larger amounts in tempeh compared to in unfermented soybean (Aoki et al., 2003a; Baumann et al., 1991; Kameda et al., 2018a; Kameda et al., 2018b; Matsumoto and Imai, 1990). Tempeh has various physiological effects, which include modulating the colonic environment (Utama et al., 2013), improving lipid metabolism (Watanabe et al., 2006), promoting calcium absorption (Watanabe et al., 2008), reducing elevated systolic blood pressure in rats (Aoki et al., 2003b), and increasing the amounts of Bifidobacterium and Lactobacillus species in the rat cecum (Yang et al., 2018).

The regional standard for tempeh, as recently established in the Codex Alimentarius, defines the use of Rhizopus oligosporus, Rhizopus oryzae, and Rhizopus stolonifer as tempeh startersⁱ⁾. Rhizopus oligosporus has been used in several studies for to prepare tempeh (Aoki et al., 2003a; Aoki et al., 2003b; Baumann et al., 1991; Kameda et al., 2018a; Matsumoto and Imai, 1990; Utama et al., 2013; Watanabe et al., 2006; Watanabe et al., 2008) because it is a major tempeh starter (Dwidjoseputro and Wolf, 1970; Hesseltine., 1980). However, few studies have investigated the effects of tempeh prepared with R. oryzae or R. stolonifer.

Human life is maintained by activation and suppression of the immune response. Various diseases, such as infection, cancer, and atopic dermatitis (AD), develop because of an imbalance in the immune response. The immune system is also controlled by some nutrients and food components. Various food components, such as cocoa polyphenol, genistein (a type of isoflavone aglycone), and soluble dietary fiber, attenuate the decline of immunomodulatory function (Abril-Gil et al., 2012; Sakai et al., 2006; Yamada, 2001).

In this study, we compared the bioactive components (genistein, polyphenol, and low-molecular-weight soluble dietary fiber (LMWSDF)) in three types of tempeh fermented with R. oligosporus (tempeh Rm), R. oryzae (tempeh Ro), or R. stolonifer (tempeh Rs). Furthermore, to evaluate the immunomodulatory effects of tempeh Rs, we investigated the therapeutic effects of dietary tempeh Rs on AD-like skin lesions induced by the house dust mite in NC/Nga mice.

Materials and Methods

Microorganisms    Rhizopus microsporus var. oligosporus (hereafter referred to as R. oligosporus) NBRC 32002, R. oryzae NBRC 4716 and R. stolonifer var. stolonifer (hereinafter referred to as R. stolonifer) NBRC 30816 were obtained from the Biological Resource Center of the National Institute of Technology and Evaluation (Osaka, Japan) and used for tempeh production.

Tempeh preparation    Dehulled yellow soybeans were soaked in 0.2% acetic acid at 25 ± 5 °C overnight, boiled at 100 °C for 10 min, and cooled to below 40 °C after draining. Next, 100 g of boiled soybeans was inoculated with a spore suspension (about 3 × 10⁶ spores) of R. oligosporus, R. oryzae, or R. stolonifer. The inoculated soybeans were spread on polyethylene bags with small pinholes and incubated at 32 °C for 40 h. After cultivation, the tempeh obtained was sterilized by heat treatment at 90 °C for 30 min, lyophilized, and used to prepare tempeh extract. Tempeh powder (0.5 g) and methanol/acetone/water = 7/7/6 (5 mL) were mixed and incubated for 18 h at room temperature in the dark. The mixture was then centrifuged at 10 000 × g for 5 min to obtain the tempeh extract. The tempeh extract was used for isoflavone, total polyphenol analysis, and antioxidant activity.

Isoflavone analysis by HPLC    The levels of isoflavones in the tempeh extract were measured by high-performance liquid chromatography with ultraviolet detection at 254 nm (Kameda et al., 2018a). A reverse-phase InertSustain^® C18 column (4.6 × 250 mm, 5 µm; GL Sciences, Inc., Tokyo, Japan) was used for chromatographic separation. Solvents A (acetic acid/acetonitrile/water = 0.1:15:85) and B (acetic acid/acetonitrile/water = 0.1:35:65) were used as the mobile phase at a flow rate of 1.0 mL/min at 35 °C. The amount of solvent B was increased from 0 to 100% over 50 min and maintained at 100% for 10 min.

Total polyphenol analysis    The total polyphenol contents in each tempeh extract were measured by the Folin-Ciocalteu method (ISO 14502-1:2005).

Antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay    DPPH radical scavenging activity assay (Blios 1958; Sharma et al., 2009) was performed as follows: 2 700 µL of 75 µM DPPH/80% methanol solution was added to each 300 µL of tempeh extract. This mixture was incubated for 30 min at room temperature, after which the absorbance at 520 nm was measured. As a positive control, 0.2 mM Trolox/80% methanol solution was used.

Dietary fiber analysis    The dietary fibers (insoluble and high-molecular weight soluble dietary fiber) and LMWSDF contents in each tempeh extract were measured by enzyme-gravimetric and enzyme-HPLC methods (AOAC 2001.03), respectively.

Animals and diets    Ten-week-old male NC/Nga mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) and acclimated to the facility for one week prior to experimental use. Mice were housed under the following conditions: temperature, 23 ± 2 °C; humidity, 55 ± 10%; and reversed light schedule (white lights on: 7:30–19:30 h). The mice had free access to food and water. The experimental protocols used were approved by the Animal Care and Use Committee of Setsunan University (Osaka, Japan). After being fed a basal diet for 14 days, AD-like lesions were induced on mice skin using Biostir^® AD (Biostir, Inc., Kobe, Japan), which is an ointment containing the house dust mite Dermatophagoides farina, for 28 days. Mice were then divided into control and tempeh Rs groups (n = 10 per group) and fed control and tempeh Rs diets, respectively, for 14 days. The nutritional values of tempeh Rs powder were as follows: crude protein (45.4%), total nitrogen (7.3%), crude fat (24.8%), dietary fibers (20.9% containing 2.5% LMWSDF), ash (4.2%), total polyphenol (0.35% containing 0.024% daidzein and 0.041% genistein), and water (1.2%). The composition of the experimental diets is shown in Table 1. The protein levels in both diets were adjusted to 20%. The skin severity score was determined on days 0, 3, 7, 10, and 14 and was based on the scores of the dorsal skin and surfaces of both ears. The skin severity score was defined as the average of individual scores, which were 0 (none), 1 (mild), 2 (moderate), or 3 (severe) based on the evaluation of erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion. Blood samples were collected from the tail vein on days 0 and 14. Total plasma immunoglobulin (Ig) E levels were detected using a mouse IgE enzyme-linked immunosorbent assay kit (Yamasa Co., Chiba, Japan).

Table 1. Composition of experimental diets (%, w/w)

	Control	Tempeh Rs
Casein	20	0
Tempeh Rs	0	38.3
L-Cystine	0.3	0.3
Corn starch	36.65	36.15
α-Corn starch	12.2	12.0
Sucrose	10.0	10.0
Fat (soybean oil)	9.5	0
Cellulose (fiber)	8.0	0
Mineral mixture	3.5	3.5
Vitamin mixture	1.0	1.0
Choline bitartrate	0.25	0.25

Statistical analysis    Statistical analysis was performed using Student's t-test and paired t-test. P values < 0.05 were considered as statistically significant.

Results and Discussion

The types and amounts of isoflavones in tempeh Rm, tempeh Ro, and tempeh Rs were measured. The levels of daidzein and genistein in each tempeh were higher than those in the unfermented soybean (Fig. 1). Glycitein and its derivatives were not detected. Tempeh Rs showed higher yields of daidzein and genistein than tempeh Rm and Ro. After 40 h of fermentation, the amount of total isoflavone aglycones in tempeh prepared with R. stolonifer was higher than that in tempeh prepared with R. oligosporus and R. oryzae. Addtitionally, tempeh Ro and tempeh Rs have been reported to have significantly higher β-glucosidase activities associated with the production of isoflavone aglycones than tempeh Rm, suggesting higher production of isoflavone aglycones in tempeh Rs (Kameda et al., 2018a). In this study, the daidzein and genistein contents in tempeh Rs were 0.024 and 0.041 g/100 g dry weight, respectively, which were similar to previously reported values (Kameda et al., 2018a; Kameda et al., 2018b). Isoflavone aglycones are known to be absorbed faster and more rapidly than their glucosides (Izumi et al., 2000; Kano et al., 2006). Therefore, tempeh fermented with R. stolonifer (tempeh Rs) may be used to produce tempeh with excellent isoflavone absorption.

Fig. 1.

Isoflavone contents in unfermented soybean and three types of tempeh fermented with Rhizopus oligosporus, Rhizopus oryzae, or Rhizopus stolonifer.

Steamed soybeans were incubated with R. oligosporus, R. oryzae, or R. stolonifer at 32 °C for 40 h.

Next, the total polyphenol content in each tempeh was compared. The total polyphenol content was higher in all tempeh samples than in unfermented soybean (Fig. 2). The amounts of total polyphenol in tempeh Rm and tempeh Rs were 0.35 g/100 g dry weight, which were higher than those in unfermented soybean and tempeh Ro. The total isoflavone content was highest in unfermented soybean, followed by tempeh Rm, tempeh Rs, and tempeh Ro. However, the polyphenol content, except for total isoflavones, was highest in tempeh Rs, followed by tempeh Ro, tempeh Rm, and unfermented soybean. The antioxidant activities of tempeh Rm, tempeh Ro, and tempeh Rs were measured in a DPPH radical scavenging activity assay. The antioxidant activities of each tempeh were stronger than that of unfermented soybean (Fig. 3). Particularly, tempeh Rm and tempeh Rs showed stronger antioxidant activity than unfermented soybean and tempeh Ro. The strength of the antioxidant activity was similar to the amount of polyphenols. Previous studies reported the production of 6, 7, 4′-trihydroxyisoflavone and 3-hydroxyanthranilic acid, which are hydroxylated compounds of isoflavones, in tempeh prepared with R. oligosporus (Esaki et al., 1996; Ikehara et al., 1968). Therefore, the total isoflavone decrease in tempeh may be related to the conversion to isoflavone derivatives such as hydroxylated compounds of isoflavones during fermentation. It has been also reported that soybean-fermented foods such as miso and tempeh have higher antioxidative properties than unfermented soybeans because of the aglyconization of isoflavone (Ikeda et al., 1995). Soyasaponins in soybean are known to have strong antioxidant activities. These soyasaponins become soyasapogenol B, a type of aglycone, in tempeh (Kamo et al., 2014). The Folin-Ciocalteu method for total polyphenol analysis is used to detect hydroxyl groups. Therefore, the increased polyphenol and antioxidant activity in tempeh may be related to an increase in compounds containing hydroxyl groups such as isoflavone aglycone (daidzein and genistein), hydroxylated compounds of isoflavones, and soyasaponin aglycone (soyasapogenol B) produced during fermentation.

Fig. 2.

Polyphenol contents in unfermented soybean and three tempeh fermented with Rhizopus oligosporus, Rhizopus oryzae, or Rhizopus stolonifer.

Steamed soybeans were incubated with R. oligosporus, R. oryzae, or R. stolonifer at 32 °C for 40 h.

Fig. 3.

Antioxidant activites of unfermented soybean and three tempeh fermented with Rhizopus oligosporus, Rhizopus oryzae, or Rhizopus stolonifer.

Steamed soybeans were incubated with R. oligosporus, R. oryzae, or R. stolonifer at 32 °C for 40 h.

The dietary fiber content in each tempeh was examined. This value was highest in tempeh Rm, followed by in tempeh Ro and tempeh Rs, whereas each LMWSDF content was highest in tempeh Rs, followed by in tempeh Ro and tempeh Rm (Fig. 4). Dietary fiber-degrading enzymes, such as cellulases produced by R. oligosporus, R. oryzae, and R. stolonifer, have been reported previously (Kolarova et al., 2001; Kupski et al., 2014; Satoh et al., 2010). The increase in the LMWSDF content in each tempeh may be related to these enzymatic activities.

Fig. 4.

Dietary fiber contents in three tempeh fermented with Rhizopus oligosporus, Rhizopus oryzae, or Rhizopus stolonifer.

Steamed soybeans were incubated with R. oligosporus, R. oryzae, or R. stolonifer at 32 °C for 40 h.

Tempeh Rs had the highest contents of the components evaluated (0.024% daidzein, 0.041% genistein, 0.35% total polyphenol, and 2.5% LMWSDF). The skin severity score in spontaneous AD-developing NC/Nga mice after genistein administration (20 mg/kg body weight/day) for 56 days is significantly lower than that in control mice (Sakai et al., 2006). The IgE concentration in rat plasma is reduced by cocoa polyphenol (0.2%) consumption (Abril-Gil et al., 2012). Similarly, the IgE concentration in rat plasma is decreased by consumption of soluble dietary fibers (5%), such as pectin, chitosan, and glucomannan (Yamada, 2001). Thus, mouse AD may be improved by tempeh Rs consumption, and we investigated the effects of dietary tempeh Rs using AD-like skin lesions induced by house dust mite in NC/Nga mice.

Experimental diets were prepared for mice (Table 1). The tempeh Rs group was fed a diet of 38.3% tempeh Rs comprised of daidzein (0.009%), genistein (0.016%), total polyphenol (0.13%), and LMWSDF (0.96%). Food consumption was similar and not significantly different between the tempeh Rs and control groups. In addition, no difference in the dorsal skin severity score was observed between the control and tempeh Rs groups after 14 days of feeding (Fig. 5a). However, the skin severity score for both ears after 14 days of feeding was significantly lower in the tempeh Rs group than in the control group (Fig. 5b). As described previously, genistein has been reported to affect the skin severity score in spontaneous AD-developing NC/Nga mice (Sakai et al., 2006). Further, genistein and daidzein have been reported to modulate dendritic cell function and suppress allergic sensitization to peanuts (Masilamani et al., 2011). Therefore, one factor decreasing the skin severity score in the tempeh Rs group may be the effects of genistein and daidzein. Furthermore, the improvement in the score for the skin of both ears was better than that for the dorsal skin, which may be because ears can be scratched easily. The results also revealed no significant reduction in the plasma IgE concentration in the control group from days 0 to 14. However, the plasma IgE concentration was significantly lower after 14 days of feeding than on day 0 in the tempeh Rs group. After 14 days of feeding, the relative IgE concentration in the tempeh Rs group was significantly lower than that in the control group (Fig. 5c). The decrease in the plasma IgE concentration in the tempeh Rs group may have been caused by the effect of polyphenol and LMWSDF. The tempeh Rs containing a high polyphenol content (0.13%) in the diet decreased the IgE concentration in AD mice. In addition, the improvement in the cecal microflora suppresses IgE production (Cahenzli et al., 2013). Soluble dietary fibers, particularly LMWSDFs such as oligosaccharides, improve the cecal microflora (Morishita, 1999). The growth of Bifidobacterium and Lactobacillus and increased n-butyrate and propionate in the rat cecal have been reported after consumption of 20% tempeh Rs for 21 days (Yang et al., 2018). Cellulose nanofiber (CN) increased short-chain fatty acid production in vitro using human feces and increased bacterial diversity and Lactobacillaceae in mice fed a high-fat diet (Nagano et al., 2020a; Nsor-Atindana et al., 2020). Moreover, a synergistic action of CN intake and exercise was observed, increasing n-butyrate-producing bacteria in the intestinal microflora (Nagano et al., 2020b). Interestingly, the intestinal microflora also increased n-butyrate production following feeding of water-jet-treated okara (Nagano et al., 2020c). Furthermore, dietary soyasaponin increased regulatory T cells via the gut microbiota in mice (Nagano et al., 2019). Taken together, tempeh Rs may improve AD by increasing regulatory T cells through increased production of n-butyrate and propionate associated with improvements in the cecal microflora.

Fig. 5.

Effects of dietary tempeh Rs on atopic dermatitis-like skin lesions induced by house dust mite in NC/Nga mice.

a) b) Skin severity score (based on scores for the dorsal skin and surfaces of both ears) was defined as the average of individual scores, which were 0 (none), 1 (mild), 2 (moderate), or 3 (severe) based on the evaluation of erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion.

c) Decrease in plasma IgE level from days 0 to 14 showing the relative concentration of plasma IgE. This was measured using a mouse IgE ELISA kit.

* and ** indicate p < 0.05 and 0.01, respectively.

Furthermore, functional immunomodulatory peptides have been reported to decrease plasma IgE levels by silk and collagen-derived peptides and to stimulate the innate immune system by a soybean protein fraction digested with Rhizopus oryzae-producing peptidase R (Egusa et al., 2009; Ikegawa et al., 2012; Nishikimi et al., 2018). Thus, immunomodulatory peptides present in tempeh fermented with R. stolonifer (tempeh Rs) were composed of unique amino acid residues from soybean proteins and may improve AD in combination with the effects of isoflavone aglycone, total polyphenol, and LMWSDF.

In this study, the bioactive components with immunomodulatory functions were analyzed and compared in tempeh Rm, tempeh Ro, and tempeh Rs. The results revealed that tempeh Rs had the highest content among these components. Furthermore, tempeh Rs remarkably improved skin severity score and plasma IgE concentration. The beneficial effects of tempeh Rs may be related to its immunomodulatory function. Tempeh Rs may be a new functional food for treating AD; however, further studies are needed to identify the bioactive compounds and immunomodulatory function responsible for its properties and mechanisms underlying its effects. Compared to the human diet, the content of tempeh in the foods was in excess in this study. Our future studies will focus on the appropriate ratio of tempeh for human foods.

References

•  Abril-Gil,  M.,  Massot-Cladera,  M.,  Pérez-Cano,  F.J.,  Castellote,  C.,  Franch,  A., and  Castell,  M. (2012). A diet enriched with cocoa prevents IgE synthesis in a rat allergy model. Pharmacol. Res., 65, 603-608.
•  Aoki,  H.,  Uda,  I.,  Tagami,  K.,  Furuya,  Y.,  Endo,  Y., and  Fujimoto,  K. (2003a). The production of a new tempeh-like fermented soybean containing a high level of γ-aminobutyric acid by anaerobic incubation with Rhizopus. Biosci. Biotechnol. Biochem., 67, 1018-1023.
•  Aoki,  H.,  Furuya,  Y.,  Endo,  Y., and  Fujimoto,  K. (2003b). Effect of γ-aminobutyric acid-enriched tempeh-like fermented soybean (GABA-tempeh) on the blood pressure of spontaneously hypertensive rats. Biosci. Biotechnol. Biochem., 67, 1806-1808.
•  Baumann,  U.,  Bisping,  B., and  Rehm,  H.J. (1991). Content and release of amino acids during the fermentation of tempe by several strains of Rhizopus spec. In “Dechema Biotechnology Conference,” vol. 4, ed. By  D.  Behrens, Wiley Europe, Weinheim, pp. 205-208.
•  Blois  M.S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181, 1199-1200.
•  Cahenzli,  J.,  Köller,  Y.,  Wyss,  M.,  Geuking,  M.B., and  McCoy,  K.D. (2013). Intestinal microbial diversity during early-life colonization shapes long-term IgE levels. Cell Host Microbe, 14, 557-570.
•  Dwidjoseputro,  D. and  Wolf,  F.T. (1970). Microbiological studies of Indonesian fermented foodstuffs. Mycopathol., Mycol., Appl., 41, 211-222.
•  Egusa,  S. and  Otani,  H. (2009). Soybean protein fraction digested with neutral protease preparation, “Peptidase R”, produced by Rhizopus oryzae, stimulates innate cellular immune system in mouse. Int. Immunopharmacol., 9, 931-936.
•  Esaki,  H.,  Onozaki,  H.,  Kawakishi,  S., and  Osawa,  T. (1996) New antioxidant isolated from Tempeh. J. Agri. Food Chem., 44, 696-700.
•  Hesseltine,  C.W. (1989). Tempeh microorganisms — On molds of the genus Rhizopus. Food industry (Shokuhin Kogyo), 32, 34-40 (in Japanese).
•  Ikeda,  R.,  Ohta,  N., and  Watanabe,  T. (1995). Changes of isoflavones at various stages of fermentation in defatted soybeans. J. Jpn. Soc. Food Sci. Technol. (Nippon Shokuhin Kagaku Kogaku kaishi), 42, 322-327 (in Japanese).
•  Ikegawa,  Y.,  Sato,  S.,  Lim,  G.,  Hur,  W.,  Tanaka,  K.,  Komori,  M.,  Takenaka,  S., and  Taira,  T. (2012). Amelioration of the progression of an atopic dermatitis-like skin lesion by silk peptide and identification of functional peptides, Biosci. Biotechnol. Biochem., 76, 473-477.
•  Ikehara,  H.,  Wakaizumi,  M., and  Murata,  K. (1968). Antioxidant and antihemolytic activity of a new Isoflavone, “Factor 2” isolated from Tempeh. Agric. Biol. Chem., 32, 740-746.
•  Izumi,  T.,  Mariusz,  K.P.,  Osawa,  S.,  Obata,  A.,  Tobe,  K.,  Saito,  M.,  Kataoka,  S.,  Kubota,  Y., and  Kikuchi,  M. (2000). Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutr., 130, 1695-1699.
•  Kameda,  T.,  Aoki,  H.,  Yanaka,  N.,  Kumrungsee,  T., and  Kato,  N. (2018a). Production of isoflavone aglycone-enriched tempeh with Rhizopus stolonifer., Food sci. technol. Res., 24, 493-499.
•  Kameda,  T.,  Aoki,  H.,  Yang,  Y.,  Nirmagustina,  D. E.,  Iwamoto,  A.,  Kumrungsee,  T.,  Kato,  N., and  Yanaka,  N. (2018b). Benefical effects of dietary tempeh prepared with Rhizopus stolonifer on liver function in rats fed with a high-fat diet. J. Nutr. Sci. Vitaminol., 64, 379-383.
•  Kamo,  S.,  Suzuki,  S., and  Sato,  S. (2014). The content of soyasaponin and soyasapogenol in soy foods and their estimated intake in the Japanese, Food Sci. Nutr., 2, 289-297.
•  Kano,  N.,  Takayanagi,  T.,  Harada,  K.,  Sawada,  S., and  Ishikawa,  F. (2006). Bioavailability of isoflavones after ingestion of soy beverages in healthy adults. J. Nutr., 136, 2291-2296.
•  Kolarova,  N. and  Augustin,  J. (2001). Production of polysaccharide hydrolases in the genus Rhizopus. Folia Microbiol., 46, 223-226.
•  Kupski,  L.,  Pagnussatt,  F.A.,  Buffon,  J.G., and  Furlong,  E.B. (2014). Endoglucanase and total cellulase from newly isolated Rhizopus oryzae and Trichoderma reesei: production, characterization, and thermal stability. Appl. Biochem. Biotechnol., 172, 458-468.
•  Matsumoto,  I. and  Imai,  S. (1990). Changes of chemical composition of tempe during fermentation. J. Jpn. Soc. Food Sci. Technol. (Nippon Shokuhin Kogyo Gakkaishi), 37, 130-138 (in Japanese).
•  Masilamani,  M.,  Wei,  J.,  Bhatt,  S.,  Paul,  M.,  Yakir,  S., and  Sampson,  HA. (2011). Soybean isoflavones regulate dendritic cell function and suppress allergic sensitization to peanut. J. Allergy Clin. Immunol., 128, 1242-1250.
•  Morishita,  Y. (1999). Dietary components responsible for the stability and alteration of the intestinal microflora (2). J. Intestinal Microbiol. (Chonai Saikin-gaku Zasshi), 12, 57-72 (in Japanese).
•  Nagano,  T.,  Katase,  M., and  Tsumura,  K. (2019). Dietary soyasaponin attenuates 2, 4-dinitrofluorobenzene-induced contact hypersensitivity via gut microbiota in mice. Clin. Exp. Immunol., 195, 86-95.
•  Nagano,  T. and  Yano,  H. (2020a). Dietary cellulose nanofiber modulates obesity and gut microbiota in high-fat-fed mice. Bioact. Carbohydr. and Diet. Fibre, 22, 100214.
•  Nagano,  T. and  Yano,  H. (2020b). Effect of dietary cellulose nanofiber and exercise on obesity and gut microbiota in mice fed a high-fat-diet. Biosci. Biotechnol. Biochem., 84, 613-620.
•  Nagano,  T.,  Hirano,  R.,  Kurihara,  S., and  Nishinari,  K. (2020c). Improved effects of okara atomized by a water jet system on α-amylase inhibition and butyrate production by Roseburia intestinalis. Biosci. Biotechnol. Biochem., 84, 1467-1474.
•  Nishikimi,  A.,  Koyama,  YI.,  Ishihara,  S.,  Kobayashi,  S.,  Tometsuka,  C.,  Kusubata,  M.,  Kuwaba,  K.,  Hayashida,  O.,  Hattori,  S., and  Katagiri. (2018). Collagen-derived peptides modulate CD4⁺ T-cell differentiation and suppress allergic responses in mice. Immun. Inflamm. Dis., 6, 245-255.
•  Nsor-Atindana,  J.,  Zhou,  Y. X.,  Saqib,  M. N.,  Chen,  M.,  Douglas Goff,  H.,  Ma,  J., and  Zong,  F. (2020). Enhancing the prebiotic effect of cellulose biopolymer in the gut by physical structuring via particle size manipulation. Food Res. Int., 131, 108935.
•  Sakai,  T.,  Kogiso,  M.,  Mitsuya,  K.,  Komatsu,  T., and  Yamamoto,  S. (2006). Genistein suppresses development atopic-like dermatitis in NC/Nga mice. J. Nutr. Sci. Vitaminol., 52, 293-296.
•  Satoh,  Y.,  Okadome,  M.,  Te,  Li,  Fujii,  T., and  Ohno,  N. (2010). Production of carboxymethylcellulase by the tempeh manufacturing fungus, Rhizopus oligosporus IFO 32002, and its enzymatic properties. J. Home Econ. Jpn. (Nihon Kasei Gakkaishi), 61, 411-416 (in Japanese).
•  Sharma,  Om P and  Bhat,  Tej K. (2009). DPPH antioxidant assay revisited. Food Chem., 113, 1202-1205.
•  Utama,  Z.,  Okazaki,  Y.,  Tomotake,  H., and  Kato,  N. (2013). Tempe consumption modulates fecal secondary bile acids, mucins, immunoglobulin A, enzyme activities, and cecal microflora and organic acids in rats. Plant Foods Hum. Nutr., 68, 177-183.
•  Watanabe,  N.,  Endo,  Y.,  Fujimoto,  K., and  Aoki,  H. (2006). Tempeh-like fermented soybean (GABA-tempeh) has an effective influence on lipid metabolism in rats. J. Oleo Sci., 55, 391-396.
•  Watanabe,  N.,  Fujimoto,  K., and  Aoki,  H. (2008). Fermentation of soybean by Rhizopus promotes the calucium absorption ratio in rats. J. Sci. Food Agric., 88, 2749-2752.
•  Yamada,  K. (2001). Immunoglobulin production regulating activity of water-soluble dietary fibers. J. Jpn. Assoc. Dietary Fiber Res. (Nihon Shokumotsu Senni Gakkaishi), 5, 1-9 (in Japanese).
•  Yang,  Y.,  Kameda,  T.,  Aoki,  H.,  Nirmagustina,  D. E.,  Iwamoto,  A.,  Kato,  N.,  Yanaka,.  Okazaki,  Y., and  Kumrungsee,  T. (2018). The effect of tempe fermented with Rhizopus microsporus, Rhizopus oryzae, Rhizopus stolonifer on the clonic luminal environment in rats. J. Fuctional Foods, 49, 162-167.
• i) http://www.fao.org/fao-who-codexalimentarius/download/standards/13304/CXS_313Re_2015.pdf (Sep. 16, 2019)