AU609631B2 - Prevention of aflatoxin contamination in cereals and nuts by applying iturin a thereto - Google Patents

Description

gO0 1 A B COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 NAME ADDRESS OF APPLICANT: Morinaga Co., Ltd.

33-I, Shiba 5-chome, Minato-ku Tokyo 108 Japan a a a a a 4 0 a a a a NAME(S) OF INVENTOR(S): Norio KIMURA Makoto ONO This documnent contains the amnendments made under t Sectioni 49 and is corrct for printingo.

a a t.

.40 0 a a ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys I Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Prevention of afiatoxin contamination in cereals and nuts by applying iturin A thereto The following statement is a full description of this invention, including the best method of perfonning it known to me/us:- .4 0 0 4.

a a" Insert place and date of signature.

Signature of Declarant(s) (no attestation required).

Note: Initial all alterations.

4. The basic application.......... referred to in paragrapn J oi tia uecLaratiun XeXX the first application.......... made in a Convention country in respect of the invention the subject of the application.

Declared at Tokyo, Japan this 24th day of July, 1989 MORINAGA CO., LTD.

Akio Matsuzaki, President a DAVIES COLLISON- MELBOURNE and CANBERRA, Representative Director I LI' c; This invention relates to a method for preventing an aflatoxin contamination in cereals and nuts by applying thereto a cyclic peptide mixture, named iturin

A.

o o on o0 oO 0 0 0 a s a a 0 o 0 0 00O 00 a an 0 0 0 0 0 0 o 0 00 0 Aflatoxin is one of the most potent carcinogens among those hitherto known (Nihon Rinsyo, 39 135 (1981)).

Aflatoxin is a metabolite secreted by the aflatoxin-producing fungi such as Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, and several homologues thereof including aflatoxin B 1 and aflatoxin G 1 are also known.

Among cereals such as wheat, barley, rice, maize (corn), etc. and nuts such as hazelnut, almond, Brazil nut, peanut, etc., those contaminated with the aflatoxin have been found (cf. Am. Assoc. Cereal Chemists Inc., 595 (1974); Am. Assoc. Cereal Chemists Inc., 603 (1975); JAOCS, 980A, December (1981); J. Agric. Food Chem., 26 249 (1978); Dtsch Lebensm. Rudsch., 76, 47 (1980); Lebensm.

-Wiss. u. -Technol., 14, 252 (1981)). The aflatoxin- R 1 RA LU S C; 12, ~r

I

producing fungous microbes are supposed to possibly spread around the areas where these crops are cultivated and yielded. Particularly, in tropical and subtropical zones quite many fields have been found to be infected and contaminated with those aflatoxin-producing fungi.

When the cereals and nuts are grown in a field infected with the aflatoxin-producing fungi, the possibility that the yielded cereals and nuts are as well contaminated with the aflatoxin is likely very high.

Since such cereals and nuts contaminated with the aflatoxin provide a serious health problem, a lot of countries strictly regulate the importation of the contaminated cereals and nuts so as not to enter their territories. It is an urgent measure to prevent the aflatoxin contamination in the cereals and nuts and to provide the cereals and nuts free from those carcinogenic aflatoxins.

Sa It is known that the aflatoxins may be removed in 0 soil (cf. Soil Sci. Soc. Am. 44 1237 (1980)), and the adsorption of the aflatoxins to the soil and the a participation of some microbes in the removal of the aflatoxins are supposed to be caused therefor. Further, it has been reported that many microbes may experimentally reduce the quantity of aflatoxin in an aqueous test solution into which the aflatoxin is dissolved (cf. J.

Bact., 93, 464 (1967); J. gen. Microbiol., 54, 185 (1968); I i I Naturwissenschaften, 62, 537 (1975); Proc. Japan. Assoc.

Mycotoxicol., 12, 33 (1980)). Among those microbes, Flavobacterium aurantiacum, Bacillus megaterium, Corynebacterium rubrum, Penicillium islandicum, Stachybotrys lobulata, Cunninghamella echinulata, Streptococcus lactis and so on have hitherto been informed as the microbes moderately eliminating the aflatoxins in the test solution.

However, many of the reductions of the aflatoxins by the microbes hereinabove mentioned are caused due to the adsorption of the aflatoxin to cell walls of the microbes (cf. J. gen. Microbiol., 54. 185 (1968); J. Bacteriol., 93, 464 (1967)) and besides the removal of the aflatoxin took place in an experimental test solution containing it.

Referring to those publications, the removal of the aflatoxin from the cereals and nuts growing in the field and the prevention of the aflatoxin contamination in those cereals and nuts have not yet been actually accomplished by making use of the microbes hereinbefore mentioned.

Moreover, the removal of the aflatoxin from the yielded cereals and nuts may require a large amount of microbes, which is unsuitable for foodstuffs. In addition, it is difficult to completely eliminate the aflatoxins which are present within cores of the cereals and nuts. Besides, it is unknown whether or not these microbes are harmless to human being when applied to the foodstuffs. They possibly include microbes associated with hygienic problems such as pathogenicity. Moreover, there may be a species such as Streptococcus lactis which demands a specific nutritional requirement, and accordingly is unsuitable for treating the food cereals and nuts.

The following substances have also been tested to prevent the aflatoxin contamination: Ammonia (Transactions of the ASAE, 1160 (1977)); Propionic acid (Poult. Sci., 56, 1630 (1977)); Sodium bisulfite Food Prot., 43, 571 (1980)); Benzoic acid derivatives (Microbios, 31, 93 (1981)); Cinnamon Food Sci., 42, 1107 (1977)); Spices Lebensm. Unters. Forsch., 171, 344 (1980)); and Pesticides.

Among these substances, ammonia and propionic acid are practically employed to prevent the contamination.

However, ammonia is strictly limited to the treatment of feedstuff owing to the insufficient preventive activity S and severe coloration upon the treated crops. The prevention with propionic acid is only effective at storage of the crops and is not applicable at pre- and post-harvest.

A substance named iturin A represented by the following formula R- (CH 2

-CHCH

2 CO-Asn-Tyr-Asn NH (I) L Ser'-Asn-Pro-G In wherein R is an alkyl group, is known as a peptide mixture having antifungal activity against phytophathogenic fungi Antibiotics, XXIX, 1043 (1976); Tetrahedron Lett., 23, 3065 (1982); Japanese Patent Application Laid-Open (KOKAI) No. 59-212416).

Iturin A is identified as a mixture of eight peptides wherein each R of the formula is ethyl, propyl, 1-methylpropyl, 2-methylpropyl, butyl, 3-methylbutyl, pentyl, or 3-methylpentyl.

It is known that Bacillus subtilis ATCC 10774 produces bacillomycin-B which is identical to iturin A (Proc. Soc. Exp. Biol. Med., 67, 539 (1948)).

o However, it is unknown that iturin A inhibits both the growth of the aflatoxin-producing fungi and their production of the aflatoxins. Moreover, -he prevention of the aflatoxin contamination with iturin A has hitherto not been reported.

The present inventors have investigated microorganisms present in the soil at various regions in order to develop a method for preventing the contamination in cereals and nuts with the aflatoxin while using a microbe which is safe and harmless to the human being even when applied to the foodstuffs, and as a consequence found strains belonging to Bacillus subtilis which has so far closely been connected to the field of foodstuff industry, which strains can reduce the quantity of the aflatoxin in the aqueous solution as well as inhibit both the growth of the aflatoxin-producing fungi and their production of the aflatoxins.

Now, the inventors find that some strains of Bacillus subtilis produce iturin A and the substance strongly inhibits both the growth of the aflatoxin-producing fungi and their production of the aflatoxins. Furthermore, iturin A is found to be potent for preventing the aflatoxin contamination in the cereals and nuts.

on the other hand, R. Mann et al. Lebensm.

Unters. -Forsch., 163, 39 (1977)) describe that there are strains capable of eliminating the aflatoxins among B.

subtilis (ATCC 6633 and ATCC 9372). R. Mann et al.

further describe that 40 to 50% of the aflatoxin was reduced in an aflatoxin solution for 20 days. But, at such a degree of reduction, the substantial removal of the aflatoxins from the cereals and nuts growing in the field can not be expected. Suh also discloses one of the above strain (ATCC 6633) suppress the aflatoxin production by Aspergillus flavus. However, the extent of the suppression is still insufficient. The strain ATCC 6633 is known to produce another antibiotic mycosubtilin which differs from iturin A (Eur. J. Biochem., 77, 61 (1977)).

D. T. Wicklow et al. (Phytophathology, 70, 761 (1980)) report that Aspergillus flavus may be inhibited in its proliferation and aflatoxin-producing activity under So" the co-existence of Aspergillus niger or Trichoderma or o viride which is known not to produce iturin A.

0 So According to one aspect of the invention, provided is So 0 a method for preventing the aflatoxin contamination in the cereals and nuts by applying to them iturin A mentioned S above, which permits to supply the cereals and nuts -o o without contamination with the aflatoxins and to cultivate 0 them in a field infected with the aflatoxin-producing fungi.

Further, according to other aspect of the invention, g o a composition for preventing an aflatoxin contamination in 0 0 cereals and nuts, comprising an agriculturally effective amount of iturin A defined above, is also provided.

Detailed Description of the Preferred Embodiments: Any iturin A prepared according to either a chemical or a biological method can be applied in the present invention. The substance may be suitably produced via microbiological process using a strain of Bacillus subtilis capable of producing iturin A.

The example of the strain of Bacillus subtilis include a strain NK-330, a strain NK-C-3, a strain ATCC 10774, and a strain IAM 1145. The strains NK-330 and NK-C-3 were deposited at Fermentation Research Institute, Tsukuba, Ibaraki in Japan, with accession numbers FERM P-9162 (date of deposit; January 30, 1987) and FERM P-9404 (date of deposit; June 6, 1987), respectively, which were then converted on November 27, 1987 to accession numbers FERM BP-1580 and FERM BP-1581, respectively, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent SProcedure.

The culturing for production of iturin A using above microbes may be usually carried out in normal liquid medium such as Potato dextrose culture medium, nutrient broth culture medium, optionally with adding mineral salts such as dipotassium hydrogen phosphate and magnesium sulfate, at 20 to 30 0 C for 2 to 7 days under aerobic condition with aeration. After the incubation, the bacterial cells were removed by centrifugation or filtration to obtain a supernatant. After concentrating the supernatant, iturin A can be isolated by a suitable combination of a precipitation with an acid, an extraction with an organic solvent such as ethanol, and a partition or adsorption chromatography over silica gel.

The prevention of aflatoxin contamination in cereals and nuts can be achieved by applying thereto iturin A thus obtained according to the present invention. The method of the present invention also comprizes the application of a solution or composition containing iturin A, a liquid culture thereof without isolating iturin A, and a strain of B. subtilis capable of producing iturin A per se.

Among the target crops, examples of the cereals include maize (corn), rice, wheat, barley, foxtail millet.

The nuts may be peanut, pistatio nut, almond, hazelnut, and Brazil nut. Particularly important crops are maize (corn), peanut, pistatio nut and almond in view of the high risk of the aflatoxin contamination.

According to the present invention, the control of the contamination may be achieved by treating the cereals and nuts with iturin A not only during their storage but also before and after harvest. In addition, by spraying over and penetrating into the field in which the cereals and nuts are growing, their protection from the aflatoxin contamination is feasible. The contamination may also be Ii i presented by spraying over and penetrating into the field into which the cereals and nuts are to be seeded, or treating the seeds thereof with iturin A before sowing.

The words "before harvest" is equal to so called "pre-harvest" which means the term of from fruition to harvest. "After harvest", "post-harvest" means the term of from harvest to storage.

The necessary amount of iturin A depends on the procedure of applying iturin A. In case of treating the field before sowing the cereals and nuts, it is preferable to apply iturin A in an amount of 1 g to 100 g per 1 ha, 11 13 or a strain of 10 to 1013 cells per 1 ha. At spraying over the cereals and nuts under growing or before harvest, preferable amount of iturin A is in a range of 1 g to 50 g 11 12 per 1 ha, or a strain of 10 to 1012 cells per 1 ha is suitably applied. The seeds of the cereals and nuts may be coated by dipping them into a solution of 1 to 50 mg/l of iturin A or 10 to 108 cells/l of the strain. After harvest or during storage, the contamination of the J cereals and nuts may be effectively prevented by an addition of iturin A in an amount of at least 0.005% by .J weight, preferably, 0.01 to 0.05% by weight.

Iturin A may be applied to the crops as a composition containing an agriculturally effective amount of the substance, which constitutes other aspect of the present invention. The composition may be in a form of a solution or suspension diluted with an agriculturally acceptable diluent such as water or other solvents, and also, after formulation, a wettable powder, emulsion, powder, or granule. The applying form may be selected according to the application timing and procedure.

The wettable powder may contain 5 to 20% by weight of iturin A, supported on an agriculturally acceptable diluent (so called support) such as clay, bentonite, silica, white carbon or attapulgite together with an auxiliary substance such as a sulfuric acid ester salt of higher alcohol, a sulfuric acid ester salt of higher alkyl ether, an alkylbenzenesulfonic acid salts, a phosphoric acid ester salt of higher alcohol, an addition product of higher alcohol with ethylene oxide, fatty acid ester of sorbitane and a higher fatty acid salt, and a spreader such as sodium dinaphthylmethanesulfonate, condensate of formaline, a lignin sulfonic acid salt, a gum and methyl cellulose.

SFor preparing the emulsion, iturin A may be emulsified together with an agriculturally acceptable diluent (so called liquid support) such as ethylene glycol and glycerin as well as an auxiliary substance such as an alkyl dimethylbenzylammonium chloride in a mixture of a solvent such as methanol, ethanol, isopropanol, acetone and methyl ethyl ketone with a polar solvent such as dimethylformamide and dimethyl sulfoxide.

j NH (I) L Ser-Asn-Pro-G In /2 The wettable powder and emulsion thus obtained may be applied to the crops after diluting them with water.

At the direct treatment with a strain of Bacillus subtilis, the strain mixed with an agriculturally acceptable diluent such as branched dextrin and dried may be suspended together with an above spreader in water so as to adjust the concentration within a range of 10 to 7 cells/ml.

In the case of the liquid culture, the culture may be first formulated by drying it with an above diluent and adding a spreader thereto. Then, the formulated culture may be suspended and/or dissolved into water upon its application to the crops.

The present invention Will-be further described in detail with referring to Examples and Comparative Examples as below.

In the following tables, N.D. means that the aflatoxin was not detected.

12 Examples: Preparation of iturin A Into 100 ml of flask was added 50 ml of culture medium shown in Table 1 and then sterilized at 120°C for minutes. One loopful of a Bacillus subtilis strain NK-330 was inoculated to the medium and incubated at over-night. The resulting culture (50 ml) was inoculated to 7 liters of sterilized above culture medium placed in a liter jar fermentor and incubated at 30°C for 3 days under stirring (300 rpm) and aeration (1/2 vvm). The culture (7 liters) was centrifuged to afford 9.5 g of k, bacterial cells and 6 liters of bacterial cell-free liquid. The liquid was concentrated to 300 ml by means of evaporator. After adding 1.2 liters of 99.5% ethanol to the concentrate, the whole was stirred to extract active substances and centrifuged to separate into an extract and a precipitate. The precipitate was further treated with liters of 80% ethanol and centrifuged to separate an extract. The extracts were combined and dried under reduced pressure to afford 35 g of a brown substance. The brown substance was dissolved into 500 ml of water and pH of the solution was adjusted to 4.0 whereby precipitation occurred. The precipitate was freeze-dried to afford 4.18 g of a brown powder.

r r 1 rii i- Table 1 Potato 200 g Distilled water 1000 g After boiling them in a boiling water for ca.

minutes, the resulting mixture was filtered through gauze to remove solid mass of potato remained.

Glucose 20 g pH 6.7-6.8 The brown powder was dissolved into 200 ml of water of which pH was adjusted to alkaline by adding dilute ammonia. After adding 4.2 g of active carbon, the mixture was heated to 60°C under shaking to adsorb active substances on the active carbon. After filtering, the active carbon was washed with 200 ml of water and then the active substances were eluted with 500 ml of 80% propanol.

The eluate was concentrated to dryness under reduced pressure to afford 770 mg of a slightly brown powder.

After dissolving ca. 300 mg of the powder into 5 ml of ethanol, the ethanolic solution was subjected to column chromatography (silica gel 60 column; 47 mm (inner diameter) x 350 mm) using 1.2 liters of a mixed solvent of ethanol/acetic acid/water 17/1/2 as an eluent. The i_ c -i r I I I, I_ I I I !i ll eluate was fractionated into a volume of 15 ml and the active substances were eluted between 30th and fractions. These fractions were collected and concentrated to dryness by means of an evaporator under reduced pressure. The above operation was repeated three times to give 115 mg of a white powder (roughly purified iturin After dissolving 5 mg of the powder containing active substances into methanol, the solution was subjected to reverse phase liquid chromatography (column: iBondasphare ODS, eluent: acetonitrile/water 2/3, flow rate: 8 ml/min, detecting wavelength: 210 nm) and the separated active fractions were collected. This chromatographic purification was repeated 20 times to give 79.1 mg of purified iturin A in total as a white powder.

The purified iturin A was separated into six fractions as main components at the above reverse phase liquid chromatographic separations. The six fractions were referred to as A, B, C, D, E and F in an order of the retention times and weighed 22.2 mg, 15.7 mg, 23.1 mg, 10.7 mg, 4.9 mg, and 2.5 mg, respectively.

Each substance was hydrolyzed in 6 N hydrochloric acid at 110°C for 20 hours to afford an amino acid mixture which shows, upon the detection by TLC, spots of aspartic acid, glutamic acid, proline, tyrosine, serine, and an spot positive to ninhydrin test which is suspected to be due to an abnormal amino acid having longer retention time than that of any normal amino acid. At the analysis by means of automatic amino acid analizer, three molecules of aspartic acid, one molecule of glutamic acid, one molecule of tyrosine, one molecule of proline, one molecule of serine and four molecules of ammonia were detected per one molecule of each substance. This result means each substance of A to F consists of three molecules of asparagine, one molecule of glutamine, one molecule of tyrosine, one molecule of proline, one molecule of serine.

Table 2 shows FAB-mass spectra (the values of parent peak, 1H-NMR spectra (only the peak of methyl protons at around 6 0.9 is cited), and UV spectra max in methanol) of these substances. In the 1H-NMR spectra, each substance, A to F, shows nearly identical spectrum pattern each other except for the pattern of methyl group in the IH-NMR spectra. The spectrum pattern of substance A is coincident with that of an authentic sample.

Based on these results, it is confirmed that the substances of A to F are identified as the compounds having the following side chains.

A) R -CH2-CH2-CH 3 B) R -CH(CH 3

)-CH

2

-CH

3 C) R -CH 2

-CH(CH

3 2 D) R -CH2-CH2-CH2-CH 3 E) R -CH2-CH2-CH(CH3) 2 F) R -CR 2-CH 2-CR 2-CR 2-CR Table 2 Compd. MW (FAB- Constructing 1 H-NMR UV (MeOH) MASS) amino acids 6 (CHR 3 X mx A 1043 AspNH 2 1 Glu(NH 2 0.9(3H,t) 278 Tyr, Pro, Ser B 1057 i C 1057 (6H,d) D 1057 (3H,t) E 1071 "(6H,d) F 1071 (3H,t) k: INHIBITING ACTIVITY OF ITURIN A TO GROWTH OF ASPERGILLUS PARASITICUS NRRL 2999 Example 1: Into a round laboratory dish (diameter: 90 mm) was placed a Potato Dextrose agar medium containing 0.01% roughly purified iturin A obtained above. One loopful of a concentrated spore suspension (ca. 10 spores/ml) of Aspergillus parasiticus NRRL 2999 was inoculated on the central part of the plate culture in the dish and incubated at 25 0 C. The diameter of colony was determined with the lapse of time and tabulated in Table 3.

For comparison, the same spore suspension was inoculated on a plate culture of the same Potato Dextrose agar medium in which iturin A was not added (Control).

Table 3 Diameter of Colony (mm) After 3 days After 8 days Control 28 90 (dish full) 0.01% iturin A 3 12 n C ii INHIBITING ACTIVITY OF ITURIN A TO AFLATOXIN PRODUCTION BY ASPERGILLUS PARASITICUS NRRL 2999 IN CULTURE MEDIUM Example 2: To a Potato Dextrose agar medium containing 0.01% roughly purified iturin A obtained above was inoculated about 50 spores of Aspergillus parasiticus NRRL 2999 per one round laboratory dish and incubated at 25 0 C. The concentrations of aflatoxins B and G 1 in the medium were determined with the lapse of time and tabulated in Table 4, together with the results adding no iturin A (control).

Table 4 Concentration of aflatoxins

B

1 (ppb) G 1 (ppb) 4 days 9 days 4 days 9 days Control 9720 8000 0.01% iturin A N.D. N.D. N.D. N.D.

C CI not determined IC LI1U INHIBITING ACTIVITY OF ITURIN A TO AFLATOXIN PRODUCTION BY ASPERGILLUS PARASITICUS NRRL 2999 IN PEANUT Example 3: Small grains of peanut, variety of US Florunner, harvested in 1986 in USA were soaked in water for ca. 2 hours (rate of water absorption: about 50%) and thereafter g of the soaked peanut were introduced into an Erlenmeyer flask. Iturin A was added to the peanut grains in an amount of 0.005, 0.01 and 0.1% oy weight per flask and then Aspergillus parasiticus NRRL 2999 was inoculated thereto about 20 spores per flask. Incubating at the amounts of aflatoxins B 1 (Table 5-1) and G 1 (Table 5-2) in the peanut grains were determined with the lapse of time. It was found that in the peanut to which iturin A was added the production of aflatoxins B 1 and G 1 was remarkably reduced as compared to those of Comparative Example 1. That is, iturin A effectively inhibited the production of aflatoxins B 1 and G 1 In this connection, Comparative Example 1 determined the amounts of the aflatoxins provided that iturin A was not added, but only A. parasiticus NRRL 2999 was a inoculated.

i, i

K

Table 5-1 Amount of Aflatoxin B 1 (ppm) Days 5 7 Ex. 3 (0.005%) 0.01 0.05 0.91 trace trace trace N.D. N.D. N.D.

Comp. Ex. 1 11.30 17.80 24.70 Table 5-2 Amount of Aflatoxin G 1 (ppm) Days 5 7 Ex. 3 (0.005%) 0.12 0.87 1.56 trace trace trace N.D. N.D. N.D.

Comp. Ex. 1 12.90 23.50 37.50 n n o c INHIBITING ACTIVITY OF ITURIN A TO AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS NRRL 3357 IN PEANUT Example 4: Small grains of peanut, variety of US Florunner, harvested in 1986 in USA was treated in a similar manner as described in Example 3. Thereafter 15 g of the soaked 21 II III L~j I I !w-"iiii w nl iniiniu i i~ 11111111 1 CI I .r i- peanut were introduced into an Erlenmeyer flask. Iturin A was added to the peanut grains in an amount of 0.005, 0.01 and 0.1% by weight per flask and then Aspergillus flavus NRRL 3357 was inoculated thereto about 20 spores per flask. Incubating at 25 0 C, the amounts of aflatoxin B 1 (Table 6) in the peanut grains were determined with the lapse of time. It was found that in the peanut to which iturin A was added the production of aflatoxin B 1 was remarkably reduced as compared to those of Comparative Example 2. That is, iturin A effectively inhibited the production of aflatoxin

B

1 In this connection, Comparative Example 2 determined the amounts of the aflatoxins provided that iturin A was not added, but only A. flavus NRRL 3357 was inoculated.

Table 6 Amount of Aflatoxin B 1 (ppm) Days 5 7 Ex. 4 (0.005%) 0.21 0.55 1.75 S trace trace trace S N.D. N.D. N.D.

Comp. Ex. 2 15.20 30.60 48.7 cZa o Soo 0 0O

I

INHIBITING ACTIVITY OF ITURIN A TO AFLATOXIN PRODUCTION BY ASPERGILLUS PARASITICUS NRRL 2999 IN CORN <ample About 15 g of commercially available corn grains for raw eating was introduced into an Erlenmeyer flask.

Iturin A was added to the peanut grains in an amount of 0.005, 0.01 and 0.1% by weight per flask and then Aspergillus parasiticus NRRL 2999 was inoculated thereto about 20 spores per flask. Incubating at 25°C, the amounts of aflatoxins B 1 (Table 7-1) and G 1 (Table 7-2) in the corn grains were determined with the lapse of time.

It was found that in the corn, to which iturin A was added Sthe production of aflatoxins B and G .was remarkably reduced as compared to those of Comparative Example 3.

That is, iturin A effectively inhibited the production of S, aflatoxins B and G In this connection, Comparative Example 3 determined the amounts of the aflatoxins provided that iturin A was not added, but only A. parasiticus NRRL 2999 was inoculated.

i Table 7-1 U Un a o Amount of Aflatoxin B 1 (ppm) Days 5 7 Ex. 5 (0.005%) 0.01 0.07 0.14 trace trace trace S N.D. N.D. N.D.

Comp. Ex. 3 3.20 4.50 6.70 Table 7-2 Amount of Aflatoxin G 1 (ppm) Days 5 7 Ex. 5 (0.005%) trace trace 0.10 trace trace trace N.D. N.D. N.D.

Comp. Ex. 3 4.20 6.70 9.10 a a a a 4 INHIBITING ACTIVITY OF ITURIN A TO AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS NRRL 3357 IN CORN Example 6: About 15 g of commercially available corn grains was introduced into an Erlenmeyer flask. Iturin A was added i to the corn grains in an amount of 0.005, 0.01 and 0.1% by weight per flask and then Aspergillus flavus NRRL 3357 was inoculated thereto about 20 spores per flask. Incubating at 25 0 C, the amounts of aflatoxin B 1 (Table 8) in the corn grains were determined with the lapse of time. It was found that in the corn to which iturin A was added the production of aflatoxin B 1 was remarkably reduced as compared to those of Comparative Example 4. That is, iturin A effectively inhibited the production of aflatoxin

B

1 In this connection, Comparative Example 4 determined the amounts of the aflatoxins provided that iturin A was not added, but only A. flavus NRRL 3357 was inoculated.

Table 8 Amount of Aflatoxin B 1 (ppm) Days 5 7 Ex. 6 (0.005%) trace 0.11 0.54 trace trace trace N.D. N.D. N.D.

Comp. Ex. 4 5.80 7.10 12.10 af :4 C INHIBITING ACTIVITY TO AFLATOXIN PRODUCTJON BY ASPERGILLUS PARASITICUS NRRL 2999 IN PEANUT Example 7: Small grains of peanut, variety of Natalcommon, harvested in 1986 in South Africa were soaked in water for ca. 2 hours (rate of water absorption: about 50%) and thereafter 15 g of the soaked peanut were introduced into an Erlenmeyer flask followed by sterilization at 121 0 C for minutes in an autoclave. The strain NK-330 (Example or the strain ATCC 10774 (Example 7-2) of Bacillus subtilis about 200 cells per flask, was inoculated to the sterilized peanut together with Aspergillus parasiticus NRRL 3357 about 20 spores per flask. Incubating at 25 0

C,

the amounts of aflatoxins B 1 (Table 9-1) and G 1 (Table 9-2) in the peanut grains were determined with the lapse of time. It was found that in the peanut to which B.

subtilis NK-330 or ATCC 10774 was added the production of aflatoxins 3 and G was remarkably reduced as compared to those of Comparative Example 5-1, 5-2 and 5-3. That is, the strains effectively inhibited the production of aflatoxins

B

1 and G

I

In this connection, Comparative Example 5-1 determined the amounts of the aflatoxins provided that the B. subtilis strains were not added, but only A.

parasiticus NRRL 2999 was inoculated. In Comparative Examples 5-2 and 5-3, the B. subtilis strain was replaced with B. subtilis (strain) IAM 1026 and Pseudomonas aeruginosa (strain) IAM 1514, respectively. These strains showed only low inhibition of the production of aflatoxins

B

1 and

G

1 Table 9-1 Amount of Aflatoxin B 1 (ppm) Days 3 5 7 Ex. 7-1 trace 1.70 2.80 Ex. 7-2 3.00 4.30 3.29 Comp. Ex. 5-1 4.70 59.3 43.3 Comp. Ex. 5-2 1.80 38.2. 34.9 Comp. Ex. 5-3 4.15 7.60 11.8 Table 9-2 Amount of Aflatoxin G 1 (ppm) Days 3 5 7 Ex. 7-1 Ex. 7-2 Comp. Ex. 5-1 Comp. Ex. 5-2 Comp. Ex. 5-3 trace trace 6.84 4.72 trace 3.54 5.87 61.9 51.8 10.1 5.81 7.31 52.7 44.1 13.4 i- INHIBITING ACTIVITY TO AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS NRRL 3357 IN PEANUT Example 8: Small grains of peanut, variety of Natalcommon, harvested in 1986 in South Africa were treated in a similar manner as described in Example 7 and thereafter g of the soaked peanut were introduced into an Erlenmeyer flask followed by sterilization at 121°C for 15 minutes in an autoclave. The strain NK-330 (Example or the strain ATCC 10774 (Example 8-2) of Bacillus subtilis about 200 cells per flask, was inoculated to the sterilized peanut together with Aspergillus flavus NRRL 3357 about spores per flask. Incubating at 25 0 C, the amounts of aflatoxin B 1 (Table.10) in the peanut grains were determined with the lapse of time. It was found that in the peanut to which B. subtilis NK-330 or ATCC 10774 was added the production of aflatoxin B 1 was remarkably reduced as compared to those of Comparative Examples 6-1, 6-2 and 6-3. That is, the starins effectively inhibited the production of aflatoxin B 1 In this connection, Comparative Example 6-1 determined the amounts of the aflatoxin provided that the B. subtilis strains were not added, but only A. flavus NRRL 3357 was inoculated. In Comparative Examples 6-2 and 6-3, the B. subtilis strain was replaced with B. subtilis (strain) IAM 1026 and Pseudomonas aeruginosa (strain) IAM 1514, respectively. These strains showed only low inhibiing activity to the production of aflatoxin B 1 Table Amount of Aflatoxin

B

1 (ppm) Days 3 5 7 Ex. 8-1 trace 1.47 2.75 Ex. 8-2 2.85 5.76 6.33 Comp. Ex. 6-1 7.98 72.8 51.6 Comp. Ex. 6-2 3.09 48.2 31.1 Comp. Ex. 6-3 4.57 7.69 17.8 INHIBITING ACTIVITY TO AFLATOXIN PRODUCTION BY ASPERGILLUS PARASITICUS NRRL 2999 IN CORN Example 9: About 15 g of commercially available corn grains for raw eating were introduced into an Erlenmeyer flask. The strain NK-330 (Example 9-1) or the strain ATCC 10774 (Example 9-2) of Bacillus subtilis about 200 cells per flask, was inoculated to the corn together with Aspergillus parasiticus NRRL 2999 about 20 spores per flask. Incubating at 25 0 C, the amounts of aflatoxins B 1 (Table 11-1) and G 1 (Table 11-2) in the corn grains were 29 =--~7;31111110*1111 IL determined with the lapse of time. It was found that in the peanut to which B. subtilis NK-330 or ATCC 10774 was added the production of aflatoxins B 1 and G 1 was remarkably reduced as compared to those of Comparative Examples 7-1, 7-2 and 7-3. That is, the starins effectively inhibited the production of aflatoxins B1 and

GI'

In this connection, Comparative Example 7-1 determined the amounts of the aflatoxins provided that the B. subtilis strains were not added, but only A.

parasiticus NRRL 2999 was inoculated. In Comparative Examples 7-2 and 7-3, the B. subtilis strain was replaced with subtilis strain IAM 102'6 and Pseudomonas aeruginosa strain IAM 1514, respectively. These strains showed only low inhibition of the production of aflatoxins

B

1 and G 1 STable 11-1 0 Amount of Aflatoxin B 1 (ppm) Days 3 5 7 Ex. 9-1 trace 0.98 3.27 Ex. 9-2 1.16 3.85 4.88 Comp. Ex. 7-1 4.20 45.1 39.4 Comp. Ex. 7-2 2.00 30.4 29.1 Comp. Ex. 7-3 3.01 9.31 14.3 Table 11-2 Amount of Aflatoxin G 1 (ppm) Days 3 5 7 Ex. 9-1 trace 4.02 5.20 Ex. 9-2 trace 6.02 8.09 Comp. Ex. 6-1 7.83 70.11 58.2 Comp. Ex. 6-2 4.55 53.8 40.7 Comp. Ex. 6-3 1.19 14.5 20.7 INHIBITING ACTIVITY TO AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS NRRL 3357 IN CORN Example About 15 g of commercially available corn grains for raw eating was introduced into an Erlenmeyer flask. The strain NK-330 (Example 10-1), or the strain ATCC 10774 (Example 10-2) of Bacillus subtilis about 200 cells per flask, was inoculated to the corn together with o Aspergillus flavus NRRL 3357 about 20 spores per flask.

Incubating at 25 0 C, the amounts of aflatoxin B (Table 12) 1 in the corn grains were determined with the lapse of time.

It was found that in the corn to which B. subtilis NK-330 or ATCC 10774 was added the production of aflatoxin B 1 was remarkably reduced as compared to those of Comparative Examples 8-1, 8-2 and 8-3. That is, the starins effectively inhibited the production of aflatoxin B 1 In this connection, Comparative Example 8-1 determined the amounts of the aflatoxin provided that the B. subtilis strains were not added, but only A. flavus NRRL 3357 was inoculated. In Comparative Examples 8-2 and 8-3, the B. subtilis strain was replaced with B. subtilis (strain) IAM 1026 and Pseudomonas aeruginosa (strain) IAM 1514, respectively. These strains showed only low inhibiing activity to the production of aflatoxin B 1 Table 12 Amount of Aflatoxin E 1 (ppm) Days 3 5 7 Ex. 10-1 trace 0.82 2.42 Ex. 10-2 3.26 7.87 8.99 Comp. Ex. 8-1 8.96 78.35 62.7 Comp. Ex. 8-2 2.98 47.3 40.3 Comp. Ex. 8-3 3.88 12.2 21.9 INHIBITING ACTIVITY TO AFLATOXIN PRODUCTION BY ASPERGILLUS PARASITICUS NRRL 2999 IN PEANUT EMBEDDED IN SOIL Example 11: About 25 g of commercially available culture soil was introduced into a 500 ml Erlenmeyer flask and sterilized in the autoclave at 121 0 C for one hour. Then, Aspergillus parasiticus (strain) NRRL 2999, about 20 spores per flask, as well as Bacillus subtilis (strain) NK-330 (Example 11-1) or ATCC 10774 (Example 11-2), about 200 cells per flask, was inoculated thereto. Thereafter, 15 g of the sterilized peanut of Example 7, which was harvested in South Africa was further added thereto and embedded in the culture soil. The incubation took place at 25 0 C. The amounts of aflatoxins B 1 and G 1 accumulated in the peanut were determined and the results were tabulated in Tables 13-1 and 13-2.

As shown in the tables, in Examples 11-1 and 11-2 in which B. subtilis NK-330 or ATCC 10774 was added, the production of the aflatoxins was remarkably inhibited as compared to Comparative Example 9 in which the strains were not inoculated.

Table 13-1 Amount of Aflatoxin B (ppm) Days 3 5 7 Ex. 11-1 trace 1.45 3.42 Ex. 11-2 2.27 6.97 7.99 Comp. Ex. 9 9.96 88.75 52.7 Table 13-2 Amount of Aflatoxin G 1 (ppm) Days 3 5 7 Ex. 11-1 trace 1.42 3.42 Ex. 11 4.16 5.37 9.19 Comp. Ex. 9 7.96 88.5 72.7 INHIBITING ACTIVITY TO AFLATOXIN PRODUCTION BY ASPERGILLUS FULAVUS NRRL 3357 IN PEANUT EMBEDDED IN SOIL Example 12: About 25 g of commercially available culture soil was introduced 'into a 500 ml Erlenmeyer flask and sterilized in the autoclave at 121 0 C for one hour. Then, Aspergillus flavus (strain) NRRL 3357, about 20 spores per flask, as well as Bacillus subtilis (strain) NK-330 (Example 12-1) or ATCC 10774 (Example 12-2), about 200 cells per flask, was inoculated thereto. Thereafter, 15 g of the a, sterilized peanut of Example 7, which was harvested in South Africa was further added thereto and embedded in the culture soil. The incubation took place at 25°C. The amounts of aflatoxin B 1 accumulated in the peanut were determined and the results were tabulated in Table 14.

As shown in the table, in Examples 12-1 and 12-2 in which B. subtilis NK-330 or ATCC 10774 was added, the

I

production of the aflatoxin was remarkably inhibited as compared to Comparative Example 10 in which the B.

subtilis strains were not inoculated.

Table 14 Amount of Aflatoxin B 1 (ppm) Days 3 5 7 Ex. 12-1 trace 2.43 3.69 Ex. 12-2 5.02 6.47 10.9 Comp. Ex. 10 8.48 83.5 66.7

Claims (14)

1. A method for preventing an aflatoxin contamination in cereals and nuts, comprising applying an effective amount of iturin A represented by the following formula to the cereals and nuts: R- (CH 2 8 -CHCH

2 CO-Asn-Tyr-Asn NH (I) 0 0 L Ser-Asn-Pro-G In o o 0 0 "o"P in which R is an alkyl group. 0 2. A method as claimed in claim 1, wherein said iturin A is sprayed over and penetrated into the field, followed by sowing seeds of the cereals and nuts in the S field treated. 0 00 0o

3. A method as claimed in claim 1, wherein said oo iturin A is applied before or after harvest of the cereals and nuts.

4. A method as claimed in claim 1, wherein said 0*4040 0 iturin A is sprayed over and penetrated into the field on which the cereals and nuts are growing.

A method as claimed in claim 1, wherein said iturin A is applied to the cereals and nuts by sowing their seeds which are coated with iturin A in advance.

6. A method as claimed in claim 1, wherein said o^F~ iturin A is applied to the cereals and nuts during their storage.

7. A method as claimed in claim 1, wherein the cereals and nuts are selected from maize (corn), peanut, pistatio nut or almond,

8. A method as claimed in any one of the preceding claims 3 to 6, wherein said iturin A is applied in an o amount of 0.005 to 0.1 by weight based on the cereals Q 0 0. and nuts to be treated.

S9. A method as claimed in claim 1, wherein said S iturin A is produced by a strain of Bacillus subtilis capable of producing iturin A.

10. A method as claimed in claim 9, wherein the strain of Bacillus subtilis capable of producing iturinA.A is applied directly to the cereals and nuts.

11. A method as claimed in claim 9, wherein a liquid culture of the strain of Bacillus subtilis containing iturin A is applied directly.

12. A method as claimed in claim 9, wherein the strain of Bacillus subtilis is a strain NK-330 (FERM o BP-1580) or a variant thereof capable of producing iturin A.

13. A method as claimed in claim 9, wherein the strain of Bacillus subtilis is a strain NK-C-3 (FERM BP-1581) or a variant thereof capable of producing iturin A. 37

14. A composition for preventing an aflatoxin contamination in cereals and nuts, comprising an agriculturally effective amount of iturin A represented by the formula R- (CH2 )8 -CHCH 2 CO-Asn-Tyr-Asn NH (I) Se r-As n--Pro--G I n wherein R is an alkyl group, and agriculturally acceptable diluent, auxiliary substance or a mixture thereof. 39 A method as claimed in claim 1, or a composition as claimed in claim 14, substantially as hereinbefore described with reference to the Examples. DATED this 29th day of January, 1991 MORINAGA CO., LTD. By Its Patent Attorneys DAVIES COLLISON 0 0~ o 0 (4, 004 S 00 44 00 4 (4 o 05 I 0 5 0 45 0 4 0414 (4 5 (4 4 (440 4 0 0 0 4 00 04 a- :1 4, 910129,ejhspe.015,39235.spe,39