Desmutagenic and Bio-antimutagenic Effects of the Extracts from Japanese Miso in Salmonella Assay

~

Mem. Fac. A gr. Kinki Iliv. 31 : 29 ... :H (199) 29

Desmutagenic and Bio-antimutagenic Effects of the Extracts from Japanese Miso in Salmonella Assay

Kentaro Yo HIK W

*

^{aoko 00}

*

Pramod ARYAL,

*

Takao TERA HIT ,* and Jiko HI HIY M * *

• D parlmenl 0/ Food alld ulrilion. FaCltlly 0/ Agricu!lur . Killki Iliver ily. akamachi. ara 631-8505. Japall .. Krolo Pre/eclurallll lilule 0/ Agricu!lural Biolechnology. Kilaill yalsuma. ika·cho. Kyolo 619-02. Japan

Synopsis

ing a a/monella/micro orne a ay the antimutagenic effects of specific components of the extracts from Japanese Mi 0 were investigated. Mi 0 exhibited an antimutagenic activity against ethyl methanesulfonate (E )·induc d mutag nicity. This activity wa r cogniz d in the fractions extracted with acetone and ether. The oybean-koji mi 0 how d mark dly antimutageni ff ct with both ether layer (fraction 1) and water layer (fraction 2). 0 mutagenicity or toxicity was ob erved for ahnonella lyphimuriu:l'n, TA100 using any of the ex- tracts. To study the mechanism of the activity, the extracts of oybean-koji miso were employed further for de mutagenic te t and bio-antimutagenic test. Fraction 1 extract was observed to exhibit both the desmutagenic effect and th bio-antimutagenic effect but fraction 2 wa ob erved to exhibit only the desmutagenic effect. It is

uggested that mi 0 i compo d of multiple compon nt with the antimutag nic activities.

Introduction activity against 3-amino-l-methyl imidazo [4,5-/]

quinoline (IQ) 21. The effect of miso diet on induc- Miso, a kind of fermented food having a history tion of -methyl- '-nitro- -nitrosoguanidine over 1300 years in Japan, has very much to do with (M G) mediated gastric tumors in rats have been Japanese daily life. The consumption of miso is in reported. It was reported that compared to the abundance for miso soup and others. The raw group taking the solution of aCI with M G the materials used for the preparation of miso are soy- incidence of gastric tumors in the group taking miso beans wheat barley and rice. iso is prepared by with M G was reduced 31.

microbial fermentation of the mixture of raw In this paper we report the desmutagenic and materials for a long period till it changes to th bio-antimutagenic effects of the ether and water peculiar components of ripe miso. layers of the acetone extracts of miso on ethyl There are some reports about the antimutagenic methanesulfonate (EM ) mediated induction of effects of miso. Yamamoto et al. have reported th His colonies of almonella typhimu1'l:um TAlOO.

suppression of the 3-amino-l-methyl-5H-pyrido

[4 3-b] indol (Trp-P-2) and 34-b nzpyrene (B [(r] P) Materials and Methods induced mutagenesis in almon lIa assay by free

unsaturated fatty acid uch as linoleic acid lino- Materials and reagents. The commercial products lenic acid and oleic acid in mi 0 1). A ahara tal. of rice-koji miso (white type, light yellow type and investigated the binding of mutagenic pyroly ates dark yellow type) barley-koji miso and soybean-koji and subsequent antimutagenic activities of microbi- mi 0, were purchased from a department store in al strains of miso. Bacteria and yeast show d the Osaka city, Japan and stored at 4 ~C until use. Ethyl large t antimutagenic effect toward Trp-P-2 but methanesulfonate (E ) was obtained from Al- most strains tested had no effective antimutagenic drich chemical Co. Bacto-agar was obtained from

30 K Yo HIKAWA et 01.

Difco Laboratories. Oxoid nutrient broth 0.2 was purchased from Unipath Ltd. All other chemicals and reagents used were of the commercially highest quality.

Preparation of the extmcts fmm Miso. Two hun- dred grams of the miso was extracted with 800 ml of acetone (4''C, 24h) and then filtered. After the filtrate was evaporated at 40 C under reduced pressure to yield 1/20 of the original volume, the concentrated solution was extracted three times with diethyl ether of twice the volume of the con- centrated solution. The solution was separated into the diethyl ether layer and the water layer and each layer was collected separately. The diethyl ether layer was evaporated at about 50l: under reduced pressure and the dry residue was dissolved in 10 ml of DMSO (fraction 1). The water layer was also evaporated at about 50 under reduced pressure until 2 ml. The solution was then dissolved in 8 ml of DMSO (fraction 2). These solutions were used after filtering using a membrance filter (0.45/1m) for sterilization.

Assay of antimutagenicity. The suppression of mutagenicity test was investigated using Salmonella typhimurium T AlOO, with slight modification of the mutagenic test as described by Maron and Ames 4). The mixture of 0.1 ml of the overnight culture of S. typhimurium T AlOO, 50 pi of 8%

EMS and the extracts of miso were thoroughly spread onto the minimal glucose plates. The plates were allowed for incubation at 3TC for 48h in an incubator and the number of His+ colonies were counted.

Assay of desmutagenicity. The desmutagenic effect of the extracts of miso was assayed using the Ames test. The mixture of 150 /11 of 8%EMS and 300 /11 of miso extracts in DMSO was incubated for 30 min at 37 ' with slight shaking, then heated at 100 for 3 min. The mixture was then filtered using a membrance filter (0.45 pm) and the filtrate was assayed. The mixture of 0.1 ml of the over- night culture of S. typhimurium T AlOO and 150 ttl of the filtrate was thoroughly spread onto the minimal glucose plates. The plates were allowed for incubation at 37 C for 48h in an incubator and the number of His+ colonies were counted. The procedure was as described by Mitsher et a1 51 ,

Shankel and Clarke ⁶¹ and Yoshikawa et al. ^71.

Assay of bio-antimutagenicity. After the mixture of 500 pi of the culture of S. tyhimurium T AlOO and 250plof 8%EMS was kept in water bath at 37 for 30 min with shaking, the mixture was washed three times with phosphate buffer (PB, 0.067M KHzP0^{4 /} 0.067M aZHP04 (3:4), pH 7.0) by centrifugation (1,100 x 9) to remove mutagen retained in suspen- sion. The bacterial cells were harvested and sus- pended in 0.5 ml of PB. The Ames assay was per- formed by pouring the mixture of 0.1 ml of the suspension and 0.1 ml of the extracts of miso in DMSO onto minimal glucose plates. There plates were incubated at 37 for 48h and the number of His+ colonies was counted.

Calculations of %of remaining mutagenicity.

Antimutagenic, desmutagenic and bio-antimuta- genic activities were expressed as % of remaining mutagenicity :

Remaining mutagenicity (%)= [(A-C)/(B-C)]

x 100. A and B are number of EMS-induced His+

revertants observed in the presence and absence of the extracts, respectively; and C is numbers of spontaneous His+ revertants observed in DMSO control.

Results and Discussion

The deleterious effect of ubiquitously occurring environmental mutagens and carcinogens have been investigated intensively. The suppression of formation of mutagens and carcinogens by fruits and vegetables lead to the understanding that the components of daily diet are capable of preventing the mutation leading to carcinogenesis 8-11).

In this paper, an investigation was carried out to understand the antimutagenicity of miso, a common diet in Japan, towards EM induced mutagenesis.

The acetone extract of 5 different miso was frac- tionated to ether layer (fraction 1) and water layer (fraction 2) by the addition of ether after the com- plete evaporation of acetone. Antimutagenicity of these fractions towards the EMS induced muta- genesis in Salmonella typhimu1'ium TAlOO is shown in Table 1. The decrease in the revertant colonies of S. typhimu1'ium T AlOO was observed to be de- pendent on the concentration of test samples. In addition, toxicity was not observed towards S.

typhimurium T AlOO in the test concentration. Out

Antimutagenic effects of Japanese Miso 31

Table 1. Antimutagenicity of extracts of miso against the mutagenicity of EMS in Salmonella typhimurium T AIOO test system

Extract of miso ^a)

(p Q/plate) white type Fraction 1

10 524±41.3 (55) 20 462±22.5 (46)

50 441 ±35A (44)

Fraction 2

10 778±47A (77)

20 727±57.3 (71) 50 658±69.1 (62)

Rice-koji miso light yellow type

476± 7.8 (48) 469±17.0 (47) 431 ± 18.0 (41)

736±25.0 (72) 787±29.0 (78) 647±27.9 (61)

EMS (33 nmol/plate)

dark yellow type

547±41.4 (58) 499±19.1 (53) 444±49.6 (43)

774±45.3 (76) 868±65.1 (88) 720±47.4 (70)

Barley-koji miso Soybean-koji miso

678±11.3 (78) 492±17.0 (50) 478± 4.9 (48)

447±42.7 (44) 464±41.0 (46) 329±22.6 (26)

826± 18.6 (83) 827 ± 11.0 (83) 793±23.3 (79)

649±42.9 (61) 486±66.6 (42) 425±31.6 (35) Values are mean colony counts±SD of three times. Remaining mutagenicity (%) of EMS-induced mutagenesis are in parentheses. Mean colony counts of EMS-induced His+ revertants in the absence of extracts were 829±46.0 (n=15) in experiments with fraction 1 and 927±50.4 (n=15) in experiments with fraction 2. Mean colony counts of spontaneous His+

revertants in the controls were 150±20A in experiments with fraction 1 and 136±15.0 in experiments with fraction 2.

a) ee the text for detail of fractionation.

of 5 different miso investigated, Soybean-koji miso exhibited higher degree of suppression of the remaining mutagenicity of EMS. The remaining mutagenicity in 50 tl~ /plate of the fraction 1 and 50 /I~ /plate of the fraction 2 was observed to be 26%

and 35%, respectively. Rice-koji miso and barley- koji miso are prepared by fermenting the mixture of boiled soybean, NaCI and koji prepared by in- oculating koji mold to rice or barley, respectively.

Soybean-koji miso is also prepared in similar way where soybean is used in preparation of koji 12).

The comparative high antimutagenicity exhibited by soybean-koji miso is proposed to be due to the constituents of soybean.

Mutation suppressibility of any compound has been interpreted as desmutagenic or bio-antimuta- genic depending upon the mode of its an tim uta- genicity. In order to elucidate the possible an- timutagenicity of Soybean-koji miso, the time of incorporation of test sample in the Salmonella / microsome assay was altered and investigated. On employing the sample prepared by allowing fraction 1 to react with EMS for 30 min at 37"C in Salm(Yn.ella / microsome assay, the revertant colonies of S.

typhimu1-ium T AIOO were fewer than those ob- served in control plates, indicating possible des- mutagenic effect. In addition to this, fraction 1 was

also observed to decrease the revertants of S.

typhimurium T AIOO preincubated with EMS for 30 min at 37"C (Fig. 1.). From these results, it is thought that fraction 1 exhibits both the desmuta- genic effect of inactivating EMS and bio-antimuta- genic effect of destabilizing DNA damage induced by EMS. Fatty acids and lipids are thought to be the major components in the diethyl ether layer of an acetone extract of miso. In one of the report by Yamamoto et al., it has been reported that free fatty acids exhibits the mutation suppressibility. From the results of fraction 1 of soybean-koji miso, it is thought that different components might be acting to exhibit desmutagenic and bio-antimutagenic ef- fect towards EMS induced mutagenesis.

The fraction 2 of soybean-koji miso was also in- vestigated in the similar pattern as that of fraction 1. In fraction 2 the desmutagenic effect was ob- served prominently while bio-antimutagenic effect was not so prominent (Fig. 2). In one of the reports, it is reported that the external and internal dialysate of water soluble fraction of miso exhibits the an- timutagenicity towards AF-2 induced mutagenesis

13) • Isoflavones such as daidzein and genistein,

which are found in considerable amount in the ex- tracts of miso, have been reported to inhibit the SOS response induced by M NG and Trp-P-l in

32 K Yo HIKAWA el al.

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of miso (fraction 1) against the mutagenicity of EMS in Salmonella typhimurium T A 100

Salmonella typhimurium TA1535/pSK1002 ¹⁴¹ .

At present further investigation is ongoing to understand the possible mechanism of suppression of mutagenicity by the fractions of miso with special attention on extraction and identification of an- timutagenic compound of miso.

References

I ) K. YAMAMOTO, T. KATO, M. ISHJJIMA and T. MIYAZAKI : Miso Sci. Teclmol., 42, 65-70 (1994) (in Japanese).

2) . ASAHARA, X. B. ZHA. G, and Y. OHTA : f. Sci. Food Agric., 58, 395-401 (1992).

3) H. WATA, ABE, M. TANIZAKI, Y. ANDO, K. F JIMOTO, T.

GoTOH, K. K RIS , Y. MA AOKA, . F jlMOTO and A. ITO : Miso Sci. Teclmol., 43, 214-218 (1995) (in Japanese).

4) D. M. MARO. and B. . AMES : Mulalioll Res., 113, 173-215 (1983).

- ) L. A. MITSHER, S. DRAKE, . R. GOLl..AP DI, J. A. HARRIS and D. M. SHA. KEL : In 'Antimutagenesis and anticar- cinogenesis mechanisms,' ed. by D. M. HANKEL, P. E.

HARTMA:;, T. KADA and A. HOLLAE DER. BASIC Life Sciences, 39, Plenum Press, ew York, pp. 153-165 (1986).

6) D. M. SHANKEL and C. H. CLARK: In "Antimutagenesis and anticarcinogenesis mechanisms ll,' ed. by Y.

KURODA, D. M. SflA, KEL and M. D. WATERS. Basic Life ciences, 52, Plenum Press, ell' York, pp. 457-460 (1990).

7) K. YOSHIKAWA, K. INAGAKI, R. MURAO, T. TERA HITA

125

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Extract of miso (/.11 )

• : Desmutagenicity of fraction 2 ----0---- : Bio - antimutagenicity of fraction 2

Fig. 2 Desmutagenic and bio-antimutagenic effects of extracts of miso (fraction 2) against the mutagenicity of EMS in Salmonella typhimurium T A 100

and J. SHISHIYAMA : Ann. Enviroll. Sci. Res. Insl.. Kinki Univ., 19, 1-5 (1991) (in Japanese).

8) T. KADA. K. MORITA and T. INOUE: Mulalion Res., 53, 351-353 (1978).

9) T.L 0 E, K. MORITA and T. KADA : Agri. BioI. Chem., 45, 345-353 (1981).

10) Y. SAMARU : Japan Food Sci., 3, 76-81 (1989) (in Japanese).

II) K. Yo HlKAWA, K. INAGAKI, T. TERA HITA, J. SHISHIYAMA, S. Kuo and D. M. Shankel: Mulalion Res., 371,65·71 (1996).

12) H. YOSHIJ : In "Handbook of fermentation' ed. by K.

oshiro, K. Yoshizawa, T. Mizunuma and M. Tadenuma.

Asakurasyoten, Tokyo, pp. 441-469 (1988).

13) M.IwAMA, M. IWASAKI, Y. MESHII and Y. SUGAYA : Presented at 64th meeting on Food Hyg. Soc. Japan,

ara, October 7-8, p.63 (1992) (in Japanese).

14) I. KIYOSAWA, J. MATSUYAMA, C. ARAI and T. ETOGUCHI:

ippon Shokuhin Kagaku Kogaku Kaishi, 42, 835-842 (1995).

(Received: September 30, 1997)

33

味噌抽 出液の消変異原効果 と生物的抗変異原効果

吉川賢太郎,*小 田奈央子,*

dA

' 近位大学段学部食品栄頚学科 目 京都肘飽潔資源研究所

要 約

Pr a mo R Y A L

,*寺下隆夫,*獅 山慈孝=

F 1r. 米味噌 ,安味噌,豆味噌 を用 い,それぞれアセ トン抽 出後,抽 出液 をエーテル可溶画分 ( (Fr.2)に分画 し,S.ly TAIO

)と水 可溶画分 Oを用 いて Amestest法でそれぞれの抗変異原効果 を検定 した｡ それぞ mu

' h p L rittm

r と は ともに E me

味噌で最 も大 きか った｡豆味噌の Fr.1は比較的鹿い消変異原効果 と比較的弱い生物的抗変 異原効 果を示 したが, には生物的抗変異原効果は認め られず,消変異原効果のみが確認 された｡

hly t F 2r.

1 . F F 2r.

れの味噌の thanesulf tonae(EMS )に対 して抗変 異原効果を示 し, この効果は豆