Caption of Figures by nikeborome


									Anti-hypertensive effect of fermented soybean products

Jianping Wu1, Xiaolin Ding2, Rotimi E. Aluko3 and Alister D. Muir1

    Agriculture and Agric-Food Canada, Saskatoon Research Station, 107 Science Place,

Saskatoon SK S7N OX2, Canada.

Tel: 1 (306) 956-2840     Fax: 1 (306) 956-7247       Email:
    School of Food Science, Wuxi University of Light Industry, Wuxi, Jiangsu, PRC

    Department of Foods and Nutrition, University of Manitoba, Winnipeg, Manitoba,

Canada R3T 2N2.


Hypertensive patients are increasingly concerned about the side effects of synthetic drugs,

and are looking to alternatives to, or to ways to reduce drug use. Angiotensin converting

enzyme (ACE) inhibitors derived from food stuffs appear to offer hypertensive patients

an alternative to control high blood pressure without the side effects often associated with

ACE inhibitory drugs.

In vitro antihypertensive activities of seven major soy fermented products (Soy paste, soy

sauce, tempeh, natto, furu, douchi, soyogurt) were examined using a newly developed

HPLC method. It was found that the water extracts were more potent than aqueous

ethanol extracts; natto and douchi had the most potent activity among all these tested

samples. The IC50 values of most fermented soy products examined were in the range of

80-360 g powder/mL. ACE inhibitory activities of extracts of fermented soy foods

were comparable to the commercial products, which indicated the potential to develop

antihypertensive functional foods from fermented soy products. The most potent fraction

of the douchi extract was concentrated by gel filtration chromatography. The molecular

weight analysis of this fraction indicated the active compounds were less than 600


Key words: fermented soy products, douchi, antihypertensive activity, angiotensin I-

converting enzyme


Hypertension is the medical term for high blood pressure. It is defined in an adult as a

blood pressure greater than or equal to 140 mm Hg systolic pressure or greater than or

equal to 90 mm Hg diastolic pressure. Hypertension is worldwide and of epidemic

proportions, and affects 15% to 20% of all adults in the world. In North America, one in

four adults has high blood pressure. It adds to the workload of the heart and arteries, and

increases the risks of cardiovascular complications, coronary heart disease (which may

lead to a heart attack), stroke and even death. In 1998, hypertension killed 44 435

Americans and contributed to the deaths of about 210 000. Unfortunately, the cause of

90-95% of hypertensive cases is unknown. This is referred to as essential or primary

hypertension. In the remaining 5-10% of cases, the causes which are related to renal

parenchymal disorders, renal artery disease, endocrine and metabolic disorders, central

nervous system disorders, and narrowing of the aorta are known and blood pressure is

controlled by treating the cause. These cases are known as secondary hypertension.

One of the major factors responsible for elevated high blood pressure is the elevated

levels of angiotensin I converting enzyme (ACE). This enzyme plays a key physiological

role both in the renin-angiotensin system and kallikrein-kinin system in the regulation of

blood pressure by virtue of two different reactions that it catalyzes (Fig 1).       ACE

catalyzes the conversion of the inactive decapeptide angiotensin I (Asp-Arg-Val-Tyr-Ile-

His-Pro-Phe-His-Leu), to a powerful vasoconstractor and salt-retaining octapeptide

angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (Soffer 1976).             ACE is also

responsible for the inactivation of the vasodilator and natriuretic nonapeptide, bradykinin

(Yang et al 1970), a peptide which lowers blood pressure. Angiotensin II increases blood

pressure through contraction of smooth muscle by secretion of aldosterone (Laragh et al

1972).      Inhibitors of ACE with the potential to lower blood pressure were initially

isolated from snake venom (Ferreira, 1965). This discovery led to the development of

synthetic orally active drugs, such as captopril and enalapril etc (Ondetti et al 1977), that

inhibit the rennin-angiotensin system in humans. This class of drugs has emerged as one

of the most important to modern cardiovascular medicine (Opie, 1994).

Even though there are numerous high blood pressure medications, neither physicians nor

patients are fully satisfied with their treatment options. Because hypertension usually

needs life-long treatment, the side effects (cough, taste disturbance and skin rashes,

alternation in lipid metabolism etc) of synthetic drugs become a significant issue and is a

major factor driving the development of alternatives to drug therapy. In the late 1980’s

certain short peptides were found to inhibit ACE activity and this has led to growing

interest in the possibility of using food protein-derived peptides as part of a strategy to

control hypertension. The involvement of companies has made this possibility into a

reality.    The Nippon Synthetic Chemical Industry Co., Ltd (Japan) focused on the

development of “Katsuobushi” oligopeptides from dried bonito, while Davisco Foods

International (USA) launched a product containing whey protein derived ACE inhibitory

peptides.     Both Calpis Food Industry Co., Ltd. (Japan) and Valio (Finland) have

introduced fermented milk-based drinks with blood pressure lowering activity to the

market. There are different ways to produce ACE inhibitors, such as naturally produced

ACE inhibitors, enzymatic release of ACE inhibitory peptides from parent proteins, or by

synthesis, fermentation and protein engineering.

In a previous study we demonstrated that ACE inhibitory peptides released from soy

protein possessed potent in vivo activity against Spontaneously Hypertensive Rats (SHR)

(Wu and Ding, 2001). A dose of 100 mg/kg of body weight significantly lowered blood

pressure at a progressive rate.    Captopril, used as positive control, showed potent

antihypertensive effect, however we observed fluctuations in blood pressure during the

medication period. The relationship between the soy protein hydrolysate dose and blood

pressure reduction indicated that after reached a maximum reduction level, a further

increase in the dose did not result in a further lowering of the blood pressure. Excessive

reduction of blood pressure and the resulting fluctuation in blood pressure during

hypertension medication could result in dangerous consequences, even death. In spite of

mild activity, food protein-derived inhibitors are safe and cheap in price, and they appear

to act specifically to hypertensive people while having no blood pressure lowering in

non-hypertensive people.

Soybeans have been consumed for centuries by Asian populations, and more recently the

consumption of soy-products in the west has increased significantly in part due to

soybean’s associated health benefits such as cholesterol lowering effect etc. Fermented

soybean products prepared by traditional methods undergo a complicated microbial

mediated process, during which peptides and amino acids are generated. It is possible

that some of the peptides/amino acids, or other by-products formed during fermentation

might possess the ability to inhibit ACE, and thus have the potential to have the

antihypertensive activity in humans.      The occurrence of ACE inhibitors in some

fermented foods has already been investigated by Japanese and Korean scientists. For

example, ACE inhibitory activity was found in fermented soybean products such as soy

sauce (Kinoshita et al., 1993), natto (Okamoto et al., 1994) and soy paste (Shin et al 1995,

2001). Various kinds of fermented soy products possessed different activity levels, and

some of the components responsible for ACE inhibition were isolated and characterized.

The ACE inhibitor in soy sauce was identified as nicotianamine (non peptide substance)

by Kinoshita et al (1993), while in natto it was revealed that the large molecular weight

proteineous material was responsible for the activity (Okamoto 1993). The peptide His-

His-Leu was characterized by Shin et al (2001) as the major component in soy paste and

the peptide Ser-Trp was found in miso by Takahama et al (1993). However, most

previous works focused on single specific fermented soy products, and therefore

comparative studies among different kinds of fermented soy products is rare (Okamoto et

al 1995). The goal of this study was to examine the ACE inhibitory activity of a series of

soy fermented products, and purify the active fractions with the object of developing

functional foods from traditional fermented soy products.

Methods and Materials


Fermented soy products were purchased from local grocery stores or health foods stores

in Saskatoon, Canada.      Soyogurt was a product of Olympic Dairy products Ltd.

(Richmond, Canada); tempeh was from Green Cuisine Inc. (Victoria, Canada); natto was

from da shan tofu Co. Ltd (Japan); Jiang was produced by Yes’s company (Malaysia);

Miso was a product of ハナマル Co., Ltd (Japan); red furu was a product of Beijing Liu

Bi Ju Paste factory (Beijing, China); white furu was produced by GuangDong Mei Wei

Xian Spice Food Co., Ltd (Guangdong, China); soy sauce was a product of Kikkoman

Foods, Inc (USA); douchi was a product of Yangjiang salted black bean Co., Ltd (China).

Extraction of soluble peptides from fermented soy products

Solid fermented samples and soyogurt were mixed with 10 volumes of distilled water or

80% aqueous ethanol solution, and then homogenized. The slurries were stirred for 2

hours at 50C, centrifuged at 8000 rpm for 25 min to separate the supernatant, and freeze

dried. Liquid samples such as soy sauce were diluted with 5 times distilled water and

freeze dried directly. These freeze-dried samples were analyszed for ACE inhibitory


ACE inhibitory determination

ACE inhibitory activity was determined using the direct HPLC injection method

developed by Wu et al (2002). This method completely eliminates the interference from

hippuryl-histidyl-leucine (HHL) during quantitation of the hippuric acid (HA) peak area

by direct analysis of the enzyme reaction mixture, thus eliminating the time consuming

process of extracting HA with ethyl acetate as described by Cushman and Cheung (1971).

The HPLC method involves the use of a C18 reverse-phase column and a mobile phase

(acetonitrile/water/ trifluoroacetic acid (TFA)) which can be adapted readily for on-line

automatic assays. This method provides a simple, rapid and accurate method for the

assay of ACE-catalyzed reactions, especially in the presence of ACE inhibitors (Fig 2.).

The IC50 value was defined as the amount of inhibitory substance that resulted in 50%

inhibition of ACE activity in the reaction system. The percent inhibition versus peptide

concentrations (g/mL) curves, were constructed using at least five separate

determinations (duplicate determination).

Gel-filtration chromatography

Sephadex G-15TM (Pharmacia Biotech, Sweden) was packed manually into a 5  60 cm

column. This column was equilibrated and eluted with a 20% (v/v) aqueous ethanol

solution at a flow rate of 2.0 mL/min. 15 mL of 200 mg/mL douchi water extract was

applied and fractions were collected. The absorbance was monitored at 280 nm.

The molecular weight distribution of samples was also determined by gel filtration

chromatography using a Superdex Peptide 10/30 column (100-7000 daltons), eluted

isocratically with a acetonitrile : water (1:1, v/v) solution supplemented with TFA


Isolation of ACE inhibitory components from douchi water extract (DWE)

The most active fraction from Sephardic G-15 chromatography was further purified by

chromatography of a SephasilTM Peptide C18 ST 10/250 column. The column was eluted

with a two solvent system using a gradient of 5% of B (acetonitrile containing 0.05%

TFA) to 25% of B in 6 column volume at a flow rate of 5 mL/min, where the solvent A

was water containing 0.05% TFA. Fraction 4 was the most potent fraction (Fig 6).

Results and Analysis

Investigation of ACE inhibitory activity in different fermented soy products

The IC50 values of these fermented soy products were shown in figure 3. Generally,

aqueous ethanol extracts had higher IC50 values (less potent) than that of water extracts,

which indicated that most of ACE inhibitors were water soluble. In case of miso, the

water extract (IC50: 290 g powder/mL) was more than 10-times more potent than that of

the ethanol extract (IC50: 3160 g powder/mL). However, douchi and natto aqueous

ethanol extracts showed more potent ACE inhibitory activity than that of water extracts.

Water extracts of miso, jiang, douchi, natto, white furu and soy sauce had particularly

low IC50 values (170-360 g powder/mL). Red furu and tempeh showed relatively weak

ACE inhibitory activity. The highest activity was found in the natto ethanol extract (IC50:

80 g powder/mL).       No ACE inhibitory activity was detected in either extracts of


Okamoto et al (1994) reported an IC50 value of 400 g/mL for water extracts from natto,

this value was higher than ours. Subsequently, these authors (1995) reported an IC50

value of 190 g/mL, which was close to our result (Fig 3). Our result showed that

ethanol extract was 2 times more potent than the water extract. There was a report that

substances capable of reducing the blood pressure existed in ethanol extracts of dead

Bacillus natto (Hayashi et al 1977), however the contribution of soybean (or fermented

soybean proteins) to this activity was not identified.

Fermented soy pastes including Chinese Jiang and Japanese Miso are some of the most

important fermented oriental soyfoods. It was believed that jiang was the progenitor of

the many varieties of soy paste and soy sauce. Miso is made from soybeans mixed with

rice or barley, or from soybeans alone; whereas jiang is often made from soybeans and

wheat. Both water extracts of miso and jiang had moderate IC50 values (250 and 290 g

powder/mL respectively), whereas the values of the ethanol extracts were markedly high

(680 and 3160 g powder/mL respectively). The major components responsible for the

ACE inhibitory activity appear to be more water soluble than ethanol soluble. Okamoto et

al (1994) reported that soybean miso had an IC50 of 5350 g/mL, barley miso had an

IC50 of 2380 g/mL, while no activity was detected in rice miso; these values were

significantly higher than our values (Fig 3). The permeate obtained by passage of a cold

water soy paste (another name for jiang or miso) extract over PM-10 membrane was

reported to have an IC50 of 41.8 g/mL (Shin et al, 1995), however there was no

indication of the IC50 value of the crude cold water paste extract.

Soy sauce, whose origin could trace back to Chinese jiang, is a liquid extract pressed

from fermented soybeans mixed with wheat, and shows many similarities in its

preparation to jiang. The whole freeze-dried soy sauce had an IC50 of 360 g powder/mL

(Fig 3), which was relatively higher than the IC50 of water extracts of jiang and miso.

Due to differences in raw ingredients and methods of preparations of soy sauce, a wide

range of IC50 values were reported by Okamoto et al (1995). These values ranged from

710 to 17 800 g/mL. In our study, the IC50 of soy sauce was at least 2 times more

potent that previously reported (Okamoto et al 1995). Generally, the degree of hydrolysis

of soybean proteins is higher in soy sauce than in jiang, however Kinoshita et al (1993)

reported that the major component responsible for ACE inhibitory activity in soy sauce

was not a peptide.

The major difference in the production of red furu from white furu is the dressing mixture.

Angkak (red kojic rice) was included in red furu fermentation for the development of the

attractive red color and is absent in white furu. The results indicated (Fig 3) that the

aqueous ethanol extract and water extract were of white furu were more potent than red

furu extracts, which suggested that angkak was not contributing significantly to ACE

inhibitory activity.   Extracts from tempeh showed the second least ACE inhibitory

activity among all the tested traditional fermented soy foods. An IC50 value of 510 g

powder/mL was reported by Okamoto et al (1995), which was almost 3 times more

potent than our values. Unlike most fermented soy foods, which are usually used as

flavor agents, tempeh serves as a main dish or meat substitute and has been consumed for

centuries in Indonesia.

There were no reports on the ACE inhibitory activity from Douchi, which is another

fermented whole soybean food (produced using the strains of Aspergillus oryzae), known

as salted black beans in the West because of the black appearance after fermentation. It

was the first soyfood to be described in written records. The activity of aqueous ethanol

extract of douchi was weaker than aqueous ethanol extract of natto, while the activity of

their water extracts were similar (Fig 3). Since, there is a larger population consuming

douchi than natto, and there was no previous report of anti-hypertensive activity of

douchi, therefore, the douchi extract was investigated in more detail particularly.

Recently, douchi extract has been found to possess excellent alpha-glucosidase inhibitory

activity (Fujita et al 2000), which could lower the blood glucose level, and a douchi

extract has been marketed by Nippon Supplement, Inc. as an anti-diabetes supplement.

Alpha-glucosidase inhibitory activity was also detected in ACE inhibitory hydrolysate

from sardine muscle (Matsui, Oki and Osajima 1999).

Gel filtration chromatography of DWE

Five major fractions were collected by the gel filtration chromatography of douchi water

extract. The most potent fraction was indicated as G15F2 (Fig 4) with an IC 50 value of

78.6 g powder/mL (Table 1) and a protein content of 64.4%. Further purification of the

most active fraction resulted in a fraction (F4) with an IC50 value of 42 g powder/mL

(Fig 5).

Molecular weight distribution of douchi water extracts

In DWE, nearly 90% of the substances were less than 600 Daltons, while the ratio in

ethanol extract was 71.1%. After Sephadex G-15 chromatography, the active fraction

contained only components with molecular weight less than 600 daltons (Fig 6). These

results indicated that active components in douchi responsible for ACE activity were

small molecules of 600 daltons or less.

Discussion and Conclusion:

We have discovered that in the traditional spectrophotometric method developed by

Cushman and Cheung protocol, the ethyl acetate extraction could not separate the HHL

from the product of HA completely (Wu et al 2002). Since both HHL and HA have

spectrophotometric absorbance at 228 nm, the interference of HHL on the HA cannot be

ignored. The new HPLC method separates HA from HHL, and thus provided a simple,

rapid and accurate method for the assay of ACE inhibitory activity. Because different

ACE inhibitory assays have been used in the investigation of ACE inhibitory activity of

fermented soy products, care should be taken when comparing results between studies.

Whey protein hydrolysate (BioZate) developed by Davisco was reported to have an IC50

value of 450 g/mL, and “Katsuobushi” oligopeptides from dried bonito developed by

the Nippon Synthetic Chemicals had an IC50 of 80 g/mL (Fujita 2001). Soy protein

hydrolysate prepared by Wu and Ding (2001) was determined to have an IC50 of 130

g/mL (unpublished data) using the Wu et al (2002) method. All these products have

been shown to have in vivo antihypertensive activity in animal tests, or human trials. The

IC50 values of most of the fermented soy products examined were in the range of 80-360

g powder/mL. ACE inhibitory activities of extracts of fermented soy foods were very

comparable to these mentioned commercial products, which indicated the potential to

develop antihypertensive functional foods from fermented soy products. The potent

active components contained in fermented whole soybean products were mediated

through microorganisms. As these products have been consumed for centuries, the safety

is well established. Similarly, the extracts from douchi are derived from natural foods

using water or aqueous ethanol as medium under mild conditions, their safety should be

similar to douchi.

The identification and characterization of the active components are underway. Further

work will explore the source of these active components, investigate their in vivo activity

and develop functional foods from them.


Technical support from Krista Thompson, Kendra Reschny and Marina Choy are

especially acknowledged.


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Caption of Figures

Fig.1. Angiotensin converting enzyme and its regulation of blood pressure by virtue of
rennin-angiotensin-aldosterone system.

Fig.2. RP-HPLC chromatogram of determination of ACE inhibitory activity. Control: in
the absence of ACE inhibitors; Hydrolysate: in the presence of ACE inhibitors. HA;
hippuric acid, HHL; hippuryl-L-histidyl-L-leucine.

Fig.3. In vitro ACE inhibitory activity (IC50) of fermented soy products. All samples
were expressed as freeze-dried weight.

Fig.4. Sephadex G-15 (XK 50/60) chromatography of douchi water extract, injection
volume was 15 mL, detected absorbance was at 280 nm.

Fig.5. SephasilTM Peptide C18 ST 10/250 chromatography of G15F2 fraction at a flow
rate of 5 mL/min, detected absorbance was 214 nm.

Fig. 6. Molecular weight distribution of douchi water extracts determined by Superdex
Peptide 10/30 column.

Fig 1.

                     ACE Inhibitors
            Ang I                          Bradykinin

           Ang II
                                    Nitric oxide

         retention             BP

Fig 2.

              1.2                HHL







                    0   2   4    6       8      10    12      14   16

                                       Time (min)

Fig 3.

     IC50 (ug powder/mL)

                           3000.00        ethanol extract
                           2500.00        water extract
                                           Do g

                                         hi tto

                                         Te ru
                                        Re uru

                                            sa t
                                        So peh



                                       So gu






Fig 4.



1000               G15F2


         0   200   400          600   800   1000   mL

Fig 5.





               0.0   5.0    10.0   15.0    20.0   25.0   30.0   35.0

Fig 6.

              90   Douchi water extract
              80   Douchi ethanol extract
              70   DWE G15 F2


                   >3000                     600-3000        <600
                               Molecular weights (Daltons)

Table 1. Sephadex G-15 chromatography of douchi water extract

      Fractions        IC50 (g powder/mL)    Protein content (%)   Powder yield (%)
       G15 F1                 2477.1                  25.9                3.1
       G15 F2                  78.6                   64.4               47.1
       G15 F3                 2025.7                  43.2                31
       G15 F4                 364.3                   11.3               11.2
       G15 F5                 1554.3                  52.8                0.6


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