Functional Effects of Japanese Style Fermented

J OURNAL OF B IOSCIENCE AND B IOENGINEERING?2005, The Society for Biotechnology, Japan V ol. 100, No. 3, 227–234. 2005

DOI: 10.1263/jbb.100.227

REVIEW

Soy Sauce (Shoyu) and Its Components

Shigehiro Kataoka1

Quality Assurance Department, Kikkoman Corporation, 399 Noda, Noda-shi, Chiba 278-0037, Japan1

Received 24 March 2005/Accepted 18 May 2005

The functional effects of Japanese style fermented soy sauce (shoyu) have been studied. Soy

sauce promotes digestion, because the consumption of a cup of clear soup containing soy sauce en-

hances gastric juice secretion in humans. Soy sauce possesses antimicrobial activity against bac-

teria such as Staphylococcus aureus, Shigella flexneri, Vibrio cholera, Salmonella enteritidis, non-

pathogenic Escherichia coli and pathogenic E. coli O157:H7. Soy sauce also contains an anti-

hypertensive component. An angiotensin I-converting enzyme inhibitor having antihypertensive

effects was found in soy sauce. The active compound was identified as nicotianamine, which

comes from soybeans. Soy sauce exhibits anticarcinogenic effects. Giving diets containing soy

sauce to mice inhibit benzo[a]pyrene (BP)-induced forestomach neoplasia. The anticarcinogenic

compounds in soy sauce were identified. The flavor components of Japanese style fermented soy

sauce, such as 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), which is a char-

acteristic flavor component of Japanese style fermented soy sauce and 4-hydroxy-2,5-dimethyl-

3(2H)-furanone (HDMF) and 4-hydroxy-5-methyl-3(2H)-furanone (HMF) exhibit antioxidant ac-

tivities and anticarcinogenic effects on BP-induced mice forestomach neoplasia when fed follow-

ing carcinogen exposure. The feeding of a diet containing 10% soy sauce to male C3H mice for

13months also reduces the frequency and multiplicity of spontaneous liver tumors. HDMF and

HEMF also exhibit anticataract effects in the spontaneous cataract rat (ICR/f rat). Fermented soy

sauce contains three tartaric isoflavone derivatives called shoyuflavones. These shoyuflavones

were shown to have inhibitory activities against histidine decarboxylase, which produces hista-

mine, a mediator of inflammation, allergy and gastric acid secretion. Soy sauce also exhibits anti-

platelet activity. β-Carbolines were isolated from soy sauce as the active compounds. Soybeans

and wheat, which are the main raw materials of soy sauce, are allergenic foods. However, recent

studies by enzyme-linked immunosorbent assay showed the absence of soybean and wheat aller-

gens in soy sauce.

[Key words:fermented food, shoyu (soy sauce), 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF),

antioxidant, anticarcinogen, antimicrobial activity, allergy]

Japanese style fermented soy sauce (shoyu) is a typical traditional Japanese fermented food. Shoyu is the Japanese name for soy sauce and the most popular liquid condiment used in Japanese cuisine as well as in cuisine of other orien-tal countries. Soy sauce increases the appetite, imparts a de-licious flavor and promotes digestion.

Generally, soy sauce is divided into two categories: fer-mented soy sauce and chemical soy sauce, which is pro-duced by the acid hydrolysis of raw materials. Chemical soy sauce is not recognized as soy sauce by the Japanese gov-ernment. Chemical soy sauce is used only as an extender of fermented soy sauce in Japan. Fermented soy sauce, semi-chemical soy sauce, in which the acid hydrolysis-derived product is subjected to fermentation, and blended soy sauce, which is produced by mixing fermented soy sauce with chemical soy sauce and/or enzymatically hydrolyzed vege-table proteins, are produced in Japan. Fermented soy sauce is of high quality. About 83% of all soy sauce products con-sumed in Japan in 2002 was of this type.

Japanese style fermented soy sauce is prepared by digest-ing mold-cultured soybeans and wheat, called koji in Japan, at different ratios, usually about 50:50, in the presence of about 17% sodium chloride and concurrently fermenting the mash with lactobacilli and yeasts. After 6–8 months the well-aged mash is pressed and the liquid part obtained is pasteurized to make the final product.

Five varieties of soy sauce are available in Japan: (i) koikuchi (a dark-colored common soy sauce produced using soybeans and wheat in almost the same quantities as the raw

e-mail: skataoka@mail.kikkoman.co.jp

phone: +81-(0)4-7123-5592fax: +81-(0)4-7123-5952

227

KATAOKA J. B IOSCI . B IOENG .,

228materials of shoyu koji), (ii) usukuchi (a light-colored soy sauce produced using soybeans and wheat in almost the same quantities as the raw materials of shoyu koji, and us-ing moromi prepared with or without steamed rice or rice saccharified by koji mold, which is subjected to control in the manufacturing process so that the color is not deep-ened), (iii) tamari (produced using soybeans alone or soy-beans with a small quantity of wheat as the raw materials of shoyu koji), (iv) saishikomi (produced using soybeans and wheat in almost the same quantities as the raw materials of shoyu koji, and using moromi prepared with kiage instead of salt water; kiage is raw soy sauce that is not pasteurized)and (v) shiro (an extralight-colored soy sauce produced us-ing wheat with a small quantity of soybeans as the raw ma-terials of shoyu koji).

The annual production of soy sauce in Japan has been about 1 million kiloliters in recent years. Soy sauce is ex-ported from Japan to more than 110 countries.

Many substances are formed during the fermentation and production processes, such as the heat treatment of raw ma-terials and the pasteurization of the liquid part of the mash.The functional effects of fermented foods have been focused on in recent years (1–4). Some 83% of all soy sauce prod-ucts consumed in Japan is koikuchi shoyu, which has a dark reddish-brown color and a strong flavor. In this paper, I re-view the functional effects of Japanese style fermented soy sauce (the common soy sauce koikuchi shoyu) and its com-ponents.

I.PROMOTION OF GASTRIC JUICE SECRETION It was reported that soy sauce promotes gastric juice se-cretion in humans (5). A clear soup containing 25 ml of soy sauce in 300ml of hot water was given to each of 15 people and the amount of gastric juice secreted was measured. The average amount of secreted gastric juice was 46.8ml, which was almost the same as that (44.0ml) in the positive control given Katsch –Kalk ’s caffeine solution (0.07% caffeine solu-tion), which is a commonly used in inducing gastric juice secretion. I n chemical soy sauce, the average secretion amount was 33.4ml in the same people. Therefore, it seems

that Japanese style fermented soy sauce promotes digestion to a greater extent than chemical soy sauce.

II.HYPOTENSIVE EFFECT

I reviewed two reports on the effects of soy sauce on blood pressure. Kajimoto (6) reported that soy sauce de-creases blood pressure. When 25 to 30ml of soy sauce was administered to dogs, whose body weights were 6–10kg,their blood pressures decreased 1min after the administra-tion and returned to the baseline level within 1h. Kajimoto suggested that soy sauce contains a substance that promotes histamine absorption as the causative agent of the blood pressure decrease effect.

Kinoshita et al . (7) found an inhibitory compound of the angiotensin I-converting enzyme (ACE) in soy sauce. ACE catalyzes the hydrolysis of angiotensin I resulting in the gen-eration of the potent vasoconstrictor angiotensin I I , which regulates arterial blood pressure (8, 9). ACE inhibitory ac-tivity in soy sauce was fractionated into two major fractions of high molecular weight (Hw) and low molecular weight (Lw) by gel filtration chromatography after treating with ethanol. The Hw fraction decreased blood pressure in hy-pertensive rats after oral administration, while the Lw frac-tion did not (Table 1). Blood pressure decreased 1–8 h after the administration of the Hw fraction and returned to the baseline level within 24h in spontaneously hypertensive rats (SHR) and two-kidney Goldblantt hypertensive rats (2KGH rats). The Lw fraction had no effect on blood pressure in either type of rat. The main ACE inhibitor in the Hw frac-tion was purified by high-performance liquid chromatogra-phy and was identified as nicotianamine (N -[N -(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidine-2-car-boxylic acid) (Fig. 1). The IC 50 of nicotianamine for ACE was 0.26μM. I t was one-tenth of that of the widely used antihypertensive drug Captopril.

Blood pressure decreased to 24, 20 and 19mmHg at 1h,4h and 8h respectively, after a single administration of 20mg/kg body weight of the purified inhibitor (nicotianamine)to SHR. Kinoshita et al . (7) found that soybeans contain a large quantity of nicotianamine. They suggested that the

TABLE 1. Relationship between oral administration of Hw or Lw fraction and blood pressure decrease in SHR a

and 2KGH rats b

Group No. of rats

Blood pressure decrease after administration c

(mmHg)

1h 4h 8h 24h

SHR

Captopril, 10mg/kg 439.3±16.731.0±28.822.0±9.710.3±18.6Hw, 3.6g/kg 522.3±6.624.7±6.423.6±5.80.9±0.9Lw, 6.0g/kg 5 1.1±2.40.2±0.50.1±0.3 1.1±1.5Distilled water 4 1.8±1.90.8±0.9 1.3±2.1?4.6±1.82KGH rats

Captopril, 10mg/kg 454.8±15.848.0±20.429.8±8.812.8±18.4Hw, 3.6g/kg 322.1±16.824.8±11.338.8±5.8 4.4±4.5Lw, 6.0g/kg 4 1.1±2.40.2±0.50.1±0.30.3±0.5Distilled water 40.8±0.90.7±0.8 1.5±2.6 2.2±2.9

A single oral administration of the test substances dissolved in distilled water was given, and the control rats were given the same amount of dis-tilled water. Tail systolic blood pressure was measured by the tail cuff method (10).a

Spontaneously hypertensive rats (SHR).b

Two-kidney Goldblantt hypertensive rats (2KGH rats).c

Blood pressure decrease in each group is given by (blood pressure before administration) ? (blood pressure after administration). Values are shown as mean ±SD. Adopted from Ref. 7.

FUNCTIONAL EFFECTS OF SOY SAUCE V OL . 100, 2005229

origin of the nicotianamine in soy sauce is soybeans.

III.ANTIMICROBIAL ACTIVITY

Soy sauce has been expected to possess the ability to in-hibit the deterioration of food and has been used to preserve and season raw fish and vegetables. The effects of soy sauce on food-borne and other common pathogens were studied.Akiba et al . (11) reported that soy sauce with p -hydroxy-butyl benzoate as a preservative killed pathogens such as Escherichia coli , Shigella flexneri , Salmonella typhi , Sal-monella paratyphi A, Salmonella enteritidis and Vibrio cholera within 6 h (Table 2). When the soy sauce did not contain preservatives, it took 48h to kill them. Shimizu et al .(12) also reported that soy sauce containing a preservative killed pathogens and food-borne pathogens like Staphylococ-cus 24, 32, Salmonella 87, E. coli O-26, Shigella flexneri 2,Shigella sonnei and Proteus OX19 within 24h. Yamamoto et al . (13) characterized the fate of nonpathogenic E. coli and Staphylococcus aureus in soy sauce not containing pre-servatives. The cell numbers of 1×103, 1×105, and 1×107 of bacteria in 1ml of soy sauce decreased to undetectable level within 4–6h, 24–48h and 5–7d, respectively. They showed that the antimicrobial activity of soy sauce is mainly based on the combined effects of NaCl, ethanol, pH, preservatives and temperature (14).

Masuda et al . (15) reported on the effect of soy sauce on the survival of enterohemorrhagic E. coli O157:H7. Four soy sauce samples, namely, (i) a koikuchi, (ii) a koikuchi that was a semichemical soy sauce and contained preserv-atives (sodium benzoate and p -hydroxy-butyl benzoate),(iii) an usukuchi (light-colored), and (iv) a low-salt soy sauce, were examined (Fig. 2). The salt concentrations in koikuchi, usukuchi, and low-salt soy sauce are generally

17.5%, 19% and 9%, respectively. When the incubation tem-perature was 30°C, the cell number of E. coli O157:H7 de-creased to an undetectable level (<20CFU/ml) within 9d in all the soy sauce samples. The soy sauce with the highest antimicrobial activity was the koikuchi type containing pre-servatives (sample ii), which decreased the cell number of the bacterium to an undetectable level within 24h. This rapid decrease was due to the preservatives sodium ben-zoate and p -hydroxy-butyl benzoate. Usukuchi (sample iii)and koikuchi (sample i) without preservatives decreased the bacterial population to an undetectable level within 3 and 5d, respectively. The low-salt soy sauce without preserva-tives (sample iv) required 9d to do so. The cell numbers of E. coli O157:H7 decreased in all of the type of soy sauce at 18°C, but more slowly than at 30°C. No decrease in E. coli O157:H7 cell numbers was observed at 4°C. These results also indicate that salt content and temperature are important factors that affect determining the antimicrobial activity of soy sauce against bacteria.

The above results indicate that bacteria, such as food-borne and other common pathogens, are killed in soy sauce at room temperature even without preservatives.

IV .ANTICARCINOGENIC EFFECT

Mortality from gastric cancer is much higher in Japan than in Europe or the United States (16). Wakabayashi et al .(17) reported that Japanese style fermented soy sauce con-tains precursors of bacterial mutagens that become direct-acting mutagens following their reaction with nitrite. They postulated that these precursors may be related to the rela-tively high incidence of gastric cancer in Japan, which is about eightfold higher than that observed in the United States (18). However, there are reports that soy sauce has anticar-cinogenic effects. Benjamin et al . (19) showed that feeding Japanese style fermented soy sauce with or without nitrite to mice in the tumor initiation stage inhibits benzo[a]pyrene (BP)-induced forestomach neoplasia. BP is a strong carcin-ogen and is present in cigarette tar and the burnt parts of foods. Giving a diet containing soy sauce to mice inhibits BP-induced forestomach neoplasia (Fig. 3). Both the num-ber of neoplasms per animal and the incidence of neoplasia

FIG. 1. Structure of nicotianamine. Adopted from Ref. 7

TABLE 2. Antimicrobial activity of soy sauce with or without preservative against pathogens

Pathogen Cell number/ml 0 time Preservative

Incubation time (h)136

2448

Escherichia coli

2.6×108

?2000–0

Shigella flexneri 2.7×10

+61002500?650000–4400010000

Salmonella typhi 3.4×10

+450?11000–12000

Salmonella paratyphi A 1.6×10

+700?45–100Salmonella enteritidis 2.3×108

+200830?9000–320800Vibrio cholera 1.7×108

+0?0

The pathogen was mixed with a soy sauce with or without p -hydroxy-butyl benzoate as a preservative. The mixture was incubated at room temperature (18–20°C).b

+, With preservative; ?, without preservative. Adopted from Ref. 11.

KATAOKA J. B IOSCI . B IOENG .,

230are reduced by increasing the intake of soy sauce. The max-imal level of inhibition was observed at 20% soy sauce in the diet. Exposure to nitrite neither enhances nor diminishes the anticarcinogenic effects of soy sauce in the diet (data not shown). They also found that soy sauce contains a substan-tial antioxidative activity.

Ito et al . (20) examined the suppressive effects of diets containing soy sauce or miso (fermented soybean paste) on spontaneous liver tumors in male C3H mice. Diets contain-ing 10% soy sauce or 10% miso were fed to the mice for 13months. I n this long-term feeding experiment, both miso and soy sauce significantly reduce the frequency and multi-plicity of liver tumors in mice (Table 3). They presumed that the estrogenic effect of isoflavone may be a causative factor for tumor prevention in the case of the mouse liver.However, soy sauce does not contain high amounts of water insoluble isoflavones, because isoflavones, such as genistin

and daidzin, transform during fermentation into their agly-cone forms, genistein and daidzein, which are less soluble in water than genistin and daidzin. The isoflavone concen-tration is approximately 5ppm in soy sauce. Therefore, iso-flavone may not be the main causative factor for tumor pre-vention, but 4-hydroxy-3(2H )-furanone derivatives, which will be described in this section, and shoyuflavones, which are present in Japanese style fermented soy sauce at about 60ppm and will be described in section VI, may affect spon-taneous liver tumorigenesis in male C3H mice.

Nagahara et al . (21) found that a flavor component of Japanese style fermented soy sauce, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H )-furanone (HEMF) (Fig. 4), is a potent antioxidant (Table 4) and one of the active anticarcinogens.HEMF was first isolated from a natural product and it has an intense, sweet aroma and a shoyulike flavor (22). There-fore, HEMF is believed to be the principal and characteristic flavor compound in Japanese style fermented soy sauce.HEMF is biosynthesized by some soy sauce yeasts during soy sauce fermentation (23). It occurs at high concentra-tions (>100ppm) in soy sauce. Diets containing 0–75ppm HEMF were fed to mice for 17wk following exposure to

FIG.2.Survival of E. coli O157:H7 in soy sauce; incubated at 30°C, 18°C and 4°C. Symbols: closed squares, sample i (koikuchi); closed circles, sample ii (koikuchi, semichemical soy sauce and contained preservatives); closed triangles, sample iii (usukuchi, light-colored); closed rhombuses, sample iv (low-salt soy sauce); open circles, control. Control: 0.1 M phosphate-buffered saline (pH 7.0). Adopted from Ref. 15

FIG.3.Effect of dietary soy sauce on the BP-induced forestomach neoplasia. Symbols: closed circles, neoplasms/animal; triangles, % in-cidence. Values are means ±SEM (n =25 mice per group). Data for treatment without nitrite are shown. Adopted from Ref. 19

FIG.4.Flavor component 4-hydroxy-3(2H )-furanones of Japa-

nese style fermented soy sauce. Adopted from Refs. 21 and 24.

TABLE 3.Inhibitory effects of dietary miso or soy sauce on liver tumors in male C3H mice

Treatment

No. of mice

Weight (g)

Liver tumor

Body Liver Incidence (%)Multiplicity

Control 2839.3±5.20 2.5±1.2889 2.86

Miso 30 40.2 ±3.20 2.1 ±0.80 32

a 1.06 b

Soy sauce 30 35.0 ±5.58 a 1.7 ±0.35 a 38a 0.63 a

a,b

Significantly different from control by p <0.05 and p <0.01, respectively. Adopted from Ref. 20.

FUNCTIONAL EFFECTS OF SOY SAUCE V OL . 100, 2005231

BP. HEMF is an effective inhibitor of BP-induced mouse forestomach neoplasia (Fig. 5). A diet containing 25 ppm HEMF was sufficient to substantially reduce the incidence of neoplasia and the number of neoplasms per mouse. HEMF was effective when fed at 4mg/kg body weight/d.

Kataoka et al . (24) reported on the anticarcinogenic activ-ity of other flavor compounds in soy sauce. Soy sauce con-tains other structurally similar flavor components, namely,4-hydroxy-5-methyl-3(2H )-furanone (HMF) and 4-hydroxy-2,5-dimethyl-3(2H )-furanone (HDMF) (Fig. 4). The con-centrations of HDMF and HMF are approximately below 10ppm and 100–300ppm in soy sauce, respectively. These compounds are found in many foods, that is, HDMF in pineapples (25), strawberries (26) and beef broth (27), and HMF in beef broth (27) and wild raspberries (28). HDMF and HMF in soy sauce are formed chemically by the Maillard reaction between sugars and amino acids during heating processes, such as the heat treatment of raw mate-rials and the pasteurization of liquid part of mash (29, 30).These furanones were investigated for their antioxidative activities. HMF and HDMF, as well as HEMF, were found to be antioxidants (Table 4). The order of potency is HEMF > HDMF > HMF. HEMF and HDMF are more potent than ascorbic acid. These furanones inhibit lipid peroxidation by oxygen radicals. HEMF scavenges singlet oxygen and its scavenging potency is greater than that of methionine, a singlet oxygen scavenger (data not shown). It was reported that the formation of reactive oxygen species (ROS), such as hydrogen peroxide (H 2O 2) and superoxide anion radicals,by human polymorphonuclear leucocytes (PMNs) stimu-lated by 12-O -tetradecanoylphorbol-13-acetate (TPA) in-duces oxidative DNA damage in vitro and in vivo (31, 32).TPA is a potent tumor promoter. The inhibition of H 2O 2 for-mation is a characteristic of many chemopreventive agents including tamoxifen, sarcophytols and epigallocatechin gal-late (33–35). Some of these compounds are antioxidants and inhibitors of tumor promotion (33, 34).

The effects of 4-hydroxy-3(2H )-furanones on H 2O 2 gen-erated by human PMNs stimulated by TPA were studied.HDMF is the most potent, showing about 90% inhibition at 90μM. The IC 50 values for HDMF, HEMF and HMF are 39, 44 and 55 μM, respectively (Fig. 6). This order corre-lated with their anticarcinogenic activity against BP-induced mouse forestomach neoplasms.

The anticarcinogenic activities of HDMF and HMF against

BP-induced mouse forestomach neoplasia were examined.Diets containing HDMF or HMF were given to mice in the tumor promotion stage, as was HEMF. These furanones showed anticarcinogenic effects (Fig. 7). The treatment groups (except for the group fed 25ppm HMF) showed sig-nificant reductions in the number of tumors per mouse.HDMF at 50 and 75ppm in the diet significantly reduced tumor incidence. HDMF is more potent than HMF. HMF and HDMF, similar to HEMF, are effective as anticarcino-gens when fed to mice following carcinogen exposure, and hence act at the postinitiation stage. This is also supported by the inhibitory activity of 4-hydroxy-3(2H )-furanones against H 2O 2 generation by human PMNs stimulated by the potent tumor promoter TPA. As part of their anticarcino-genic action, these 4-hydroxy-3(2H )-furanones may affect ROS concentration, and prevent oxidative DNA damage caused by ROS at the postinitiation stage.

TABLE 4.Antioxidant activity of flavor components

of Japanese style fermented soy sauce Sample Antioxidant activity (neq./ml)a Weight

(mg solids/ml)Specific

activity

(neq./mg)

HDMF 3.20.2512.8HMF 2.30.259.3

HEMF

3.8 0.25 15.2Ascorbic acid

c ,d

101.0 10.0 10.0Values are means of triplicate assays.a

Nanoequivalents/ml sample.b

Nanoequivalents/mg solid.c

See Ref. 21.d

10,000ppm in water.Adopted from Ref. 24.

FIG.5.Inhibition of BP-induced frestomach neoplasia by HEMF.Symbols: closed circles, neoplasms/animal; open circles, % incidence.For neoplasms per animal, all treatment groups were significantly dif-ferent from the control group (P ≤0.05). Values are means ±SEM (n =25–27 mice per group). Adopted from Ref. 21.

FIG.6.Reduction of H 2O 2 in TPA-stimulated human neutrophils by 4-hydroxy-3(2H )-furanones. Symbols: triangles, HDMF; open cir-cles, H EMF; closed circles, H MF. Samples were incubated 15min prior to addition of 25nM TPA followed by incubation for 30min at 37°C. Values are means ±SEM (n =3–5). Adopted from Ref. 24

KATAOKA J. B IOSCI . B IOENG .,

232V .ANTICATARACT EFFECT

Cataracts are the most common age-related changes in the eye crystalline lens tissue. One of the most plausible causes of cataracts is oxidative stress due to aging (36). Epi-demiological studies suggest the usefulness of natural anti-oxidants such as vitamins C and E against human cataracts (37, 38). The antioxidative compounds 4-hydroxy-3(2H )-furanones were examined for their anticataract effect on two cataract models in vitro and in vivo (39). HMF, HDMF and HEMF were examined for their anticataract effects on the galactose-induced cataract model in vitro . Treatment with HDMF and HEMF (1.0mg/ml) significantly reduced the in-cidence of opaque lenses from Wister rats, which were in-cubated for 7d with a high concentration of galactose. The effect of HDMF and HEMF was apparent, even at a low concentration (0.1mg/ml), but there was no effect on the lenses treated with HMF, which has a weaker antioxidative activity than HDMF and HEMF. An other experiment was performed using spontaneous cataract rats (ICR/f rats), the hereditary cataract model. A saline solution of 4-hydroxy-3(2H )-furanone (1.0%) was dropped three times a day on both the eyes of 7-week-old ICR/f rats for 3 weeks. The lens opacification of the ICR/f rats was observed with a slit lamp microscope. On the 21st day of this experiment, 56% of the lenses in the control group had developed opacities, which were judged to be in the cataract condition. On the other hand, the groups treated with HDMF and HEMF showed delayed cataract formation; the incidence of cataracts being 21% and 44%, respectively (Fig. 8). HMF did not have any ability to prevent cataracts in the lenses of ICR/f rats.

Mizuno et al . (40) reported that the increase in the levels of serum lipid peroxides may result in the increase in the levels of superoxide radicals in the aqueous humor and then in the lens, and that superoxide radicals may affect the pro-tein SH group that are converted to form S-S bonds. They concluded that the formation of S-S bonds in the lens pro-teins and the acceleration of protein aggregation may be a cause of cataract formation in the ICR/f rat lens. Yagi et al .

(41) proposed that increased levels of lipid peroxides in blood may be a direct cause of cataractogenesis. Koga et al .(42) showed that HEMF and HDMF exhibit a high suppres-sive effect on lipid peroxidation in human plasma. The in-hibition of spontaneous cataract formation by HDMF and HEMF is suggested to have been caused by their antioxida-tive activity against lipid peroxidation or superoxide radi-cals in the lens tissue.

VI.OTHER FUNCTIONAL EFFECTS

From the chemometric pattern recognition analysis of HPLC profiles of soy sauce, three tartaric isoflavone deriva-tives, named shoyuflavones, were found (Fig. 9) (43). Shoyu-flavones A, B, and C are derivatives of daidzein, genistein,and 8-hydroxygenistein, respectively. These shoyuflavones are specific to fermented soy sauce, because they have not been found in soybeans or any other soy products. There-fore, it was estimated that shoyuflavones are produced dur-ing fermentation. These shoyuflavones showed inhibitory activities against histidine decarboxylase (HDC), resulting in the production of histamine from L -histidine (44). Hista-mine is a representative mediator of inflammation, allergy,gastric acid secretion, and neurotransmission. In the studies

FIG.7.Inhibition of BP-induced frestomach neoplasia by HMF and HDMF. Symbols: open squares, neoplasms/neoplasm-bearing animal;closed squares, neoplasms/animal; closed circles, % incidence. Values are means ±SEM (n =28–30 mice per group). *P ≤0.05; **P ≤0.1.Adopted from Ref. 24

FIG.8.Anticataract effect of 4-hydroxy-3(2H )-furanones on spon-taneous cataract rats (ICR/f rats). Each 4-hydroxy-3(2H )-furanone was dissolved in saline at a concentration of 1.0%. Topical eye drops of this solution were applied three times a day to both eyes of the rats for 3weeks. Lens opacity was scored from stages 0 to 5, with stage 4 being judged to be the cataract condition. Adopted from Ref. 39.

FIG.9.Structures of shoyuflavones A, B, and C isolated from soy sauce. Adopted from Ref. 43

FUNCTIONAL EFFECTS OF SOY SAUCE V OL . 100, 2005233

of the inhibitory activities of shoyuflavones against HDC obtained from mouse mastocytoma P-815 cells, shoyu-flavones B and C were more potent inhibitors than (+)-cat-echin and their own aglycones, that is, daidzein and genistein. The inhibition of HDC activity by shoyuflavones may attenuate excessive gastric acid secretion and inflam-mation, which results in the prevention of allergic reactions.Shoyuflavones are more water soluble than their own agly-cones, and fermented soy sauce contains approximately 60ppm of shoyuflavones. On the other hand, the isoflavones in soy sauce are present at approximately 5ppm.

Soy sauce also exhibits antiplatelet activity (45). Aque-ous twofold diluents of soy sauce completely inhibited both the collagen- and epinephrine-induced aggregation of hu-man platelets. The active components are 1-methyl-1,2,3,4,-tetrahydro-β-carboline (MTBC) and 1-methyl-β-carboline (MBC) (Fig. 10). The concentrations in commercially avail-able soy sauces are 28–85ppm for MTBC and 0.3–4.2ppm for MBC. MTBC shows mean concentrations of 4.6, 4.2,28.6, 11.6 and 65.8μg/ml to produce a 50% inhibition of the maximal aggregation response induced by epinephrine,platelet-activating factor, collagen, adenosine 5′-diphosphate and thrombin, respectively. β-Carboline analogs were found to inhibit platelet aggregation (46, 47). Among them, MTBC is the most active inhibitor of platelet aggregation by all the tested inducers. Soy sauce is a functional seasoning with a potent preventive effect on thrombus formation.

VII.ALLERGENICITY OF SOY SAUCE Food allergy has become a serious problem globally. The major allergenic foods are eggs, milk, wheat, soybeans and rice in Japan (48). Soybeans and wheat are the two major raw materials of soy sauce, but soy sauce is a hypoaller-genic food. Recently, some studies of the allergenicity of soy sauce have been performed. A major soybean allergen,Gly mBD 30k, present in soybean products was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) (49). The allergen occurred at a high concentration in soy milk, tofu, kori-dofu and yuba. No allergens were found in fermented foods such as miso, soy sauce and natto.Another major soybean allergen, Gly mBD 28k, was mea-sured by sandwich ELISA (50). This study showed the same result as that mentioned above. In fermented foods such as miso, natto and soy sauce, the allergen was not detected.Tanabe et al . reported (51) on the degradation of wheat allergens in Japanese soy sauce. They used immunoblotting,inhibition ELI SA and direct inhibition ELI SA using sera from five children with wheat allergy to examine the degra-dation of wheat allergens in soy sauce. They confirmed that no wheat allergens were detected in 10 items of commercial

soy sauce in Japan. These results indicate that the soybean and wheat allergens are completely degraded during the fer-mentation by microbial proteolytic enzymes. Therefore, it was concluded that no soybean and wheat allergens are con-tained in soy sauce.

I have reviewed the physiological and functional effects of Japanese style fermented soy sauce. Fermented foods are improved not only in their nutritional properties compared with the original foods but also in their functional compo-nents, which are beneficial to human health. Some harmful ingredients, such as allergens, are eliminated from fermented foods such as soy sauce and miso. I anticipate further devel-opments in studies on the functional effects of fermented foods.

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