ベトナム産コーヒー豆から分離されたAspergillus sectionsNigri及びCircumdatiのカビ毒産生性とコーヒー豆のカビ毒

汚染

誌名誌名 JSM Mycotoxins

ISSNISSN 02851466

巻/号巻/号 651

掲載ページ掲載ページ p. 1-6

発行年月発行年月 2015年1月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

JSM M ycotoxins 65 ( l) , 1 - 6 (20 15) http:/ /dx.doi.org/ 1 0.2520/myco.65.1

Research Pa~er

JSM Mycotoxins

www.jstage.jst.go.jp/browse/myco

Mycotoxin contamination of Vietnamese coffee beans caused by Aspergillus sections Nigri and Circumdati

Ruiko Hashimoto 1, Hiroyuki Nakagawa2, Yoshiki Onji3, Katsuyoshi Asano3,

Koji Yokoyama4, Haruo Takahashi4·5

1Chiba Prefectural Institute of Public Health, 666-2 Nitona·cho, Chuou·ku, Chiba 260-8715, Japan 2National Food Research Institute, National Agriculture and Food Research Organization, 2·1-12 Kannondai , Tsukuba, lbaraki 305-8642, Japan 3Nara Prefectural Institute of Public Health, 1 000 Ohdono, Sakurai, Nara 633-0062, Japan 4Medical Mycology Research Center (MMRC), Chiba University, 1-8-1 lnohana, Chuou-ku, Chiba 260-8673, Japan 5Nationallnstitute of Health Science, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 , Japan

Keywords

Aspergillus section Circumdati; Aspergillus section Nigri; fumonisin; mitochondorial cytochrome b gene; ochratoxin; ~-tubulin gene; Vietnamese coffee

Correspondence Ruiko Hashimoto, Chiba Prefectural Institute of Public Health, 666-2 Nitona-cho, Chuou-ku, Chiba 260-8715, Japan E-mail: r.hshmt2@pref.chiba.lgjp

(Received April 19, 2014, accepted March 12, 2015)

Introduction

Abstract Sixteen samples of Vietnamese coffee beans were examined for the

presence of mycotoxigenic fungi that produce ochratoxins and fumonisins. Species of the strains isolated from the beans were tentatively identified by morphology as Aspergillus niger species complex (isolation frequencies of the beans: 56.6%), Aspergillus carbonarius (3.3%), and Aspergillus species in section Circumdati (2.9%). The strains randomly selected from the species were correctly identified by sequencing of the ~-tubulin and/ or mitochon­drial cytochrome b gene. All of the strains of A. carbonarius and Aspergillus

westerdijkiae, identified in section Circumdati, produced ochratoxin A (OTA). On the other hand, only one out of the 41 strains of A. niger produced a detectable level of OTA. Therefore, A. carbonarius and A. westerdijkiae, rather than A. niger, are likely to be the main sources of OTA contamination in the beans. With regard to A. niger, 37 out of the 41 strains produced fumonsin B2 (FB2). LC-MS/ MS analysis of the 16 bean samples showed that 3 samples were contaminated with OTA and/ or FB2; one Arabica sample with OTA (2.3 1-Jg/ kg), another with FB2 (55 1-Jg/ kg), and one Robusta sample with both OTA (6.3 t-Jg/ kg) and FB2 (49 1-Jg/ kg) . These results demonstrate that Vietnamese coffee beans are commonly infected with OTA- and FBrproducing fungi and occasionally co-contaminated with these mycotoxins.

The coffee bean is one of the most important agri­cultural products in tropical and subtropical countries such as Brazil and Indonesia. In recent decades, Viet­nam has become a major coffee-producing country, especially for the Robusta (Coffea canephora var. robusta) 1l_ The product is exported worldwide to coun­tries including Japan, USA and EU. Ochratoxin A (OTA) is a natural contaminant in green coffee beans and causes a negative economic impact to the coffee indus­try 2l because it exhibits nephrotoxic, carcinogenic, ter­atogenic, genotoxic, and immunosuppressive effects on humans and animals1l,3l.4l_

section Circumdati (Aspergillus ochraceus group, yellow aspergilli)3l,4l. A. niger and Aspergillus carbonarius are ochratoxigenic species in section Nigri. A. carbonarius

has been considered as one of the main sources of OTA contamination because it produces relatively larger amounts of OTA2'·3L4l. Ilic et al. reported that A. niger

was the sole ochratoxigenic species isolated from Viet­namese coffee beans and that 8.7% of these isolates produced OTA 1l. However, their identification of the species is based on only morphology, and A. niger may be considered as a species complex5

' ·6

' . Molecular techniques are necessary for correct identification of the species.

A. ochraceus was previously considered as another main source of OTA contamination. Section Circumdati

was recently revised by Frisvad et al_?l, and some new ochratoxigenic species, such as Aspergillus westerdi-

Previous reports demonstrated that OTA contami­nation in coffee beans was caused by Aspergillus sec­tion Nigri (Aspergillus niger group, black aspergilli) and

2 H ASHIMOTO eta!.

jkiae and Aspergillus steynii, have been described within

the section. Noonim et al. reported that A. ochraceus

was not detected in Thai coffee beans with reference to

the recent classification of section Circumdati4l. There­

fore, correct identification of the fungi is of great

importance to clarify the species responsible for OTA

contamination in coffee beans. In addition to OTA, A. niger has been reported to

produce fumonisins8l, which were firstly discovered in

corn as carcinogenic mycotoxins produced by Fusarium

proliferatum. Previous study showed that approxi­

mately 67% of A. niger isolates from Thai coffee beans

produced fumonisin B2 (FB2) and fumonisin B4 (FB4)9l.

Since A. niger often infects coffee beans, fumonisin

contamination is also of great concern in Vietnamese

coffee beans.

The objective of this study is to clarify the causative

fungi of the mycotoxin contamination after correct

identification by molecular techniques and to know the

level of contamination in Vietnamese coffee beans for

reducing human health risk.

Materials and Methods

Sample of coffee bean

We collected the green coffee bean samples with

the cooperation of the Western Highland Ag ricultural

and Forestry Science Institute (WASI) in Buon Ma Thout

at Dac Lac plateau in the central highlands of Vietnam

in October 2010. Sixteen coffee samples, consisting of

13 Robusta samples and 3 Arabica samples, were col­

lected in total.

Detection of black and yellow aspergilla from

the sample

One-hundred beans from each coffee sample were

taken, disinfected with alcohol, and distributed to 10

petri dishes (5 beans on each dish) of dichloran rose

bengal chloramphenicol (DRBC) agar and dichloran

18% glycerol (DG18) agar. These plates were incubated

at 25 ·c for 7 days. Black (section Nigri) and yellow

(section Circumdati) aspergilla were isolated on Czapek

agar plates. The isolates were tentatively identified as

A. niger or A. carbonarius by morphological criteria (Pitt

and Hocking10l and Samson et al. 11 l, especially in orna­

mentation of conidial wall24l). Yellow aspergilli was

inoculated to the plates of malt extract agar (MEA) and

Czapek yeast autolysate agar (CYA), and incubated at

25 •c and 37 •c for 7 days for the tentative identifica­

tion 7L10l·11 l. Representative isolates of both aspergilla

were transferred to potato dextrose agar (PDA) slant for

the further studies.

JSM Mycotoxins

Species identification of the strains by DNA analyses

Yellow aspergilli and A. carbonarius were examined

for the species identification by sequencing of ~-tubu­lin 13)·25) and cytb genes12l·23l. The putative A. niger

strains were identified by the cytb gene analysis. After

culturing in potato dextrose broth, DNA was extracted from the isolates using the Gen Toru Kun DNA extrac­

tion kit for yeast (TaKaRa Bio Inc., Japan) according to the manufacturer's instructions. PCR amplification of

the ~-tubulin gene was performed with primers Bt2a and Bt2b 13)·25) and the cytb gene with primers E1m4

and rE2m412l. The amplified DNA fragments were

sequenced with the Dye Terminator Cycle Sequencing

Ready Reaction Kit (Applied Biosystems Division of Per­kin-Elmer, Japan Co., Ltd.) and the ABI Prism 3700 DNA

Sequencer. Sequences were aligned using CLUSTALW in Molecular Evolutionary Genetics Analysis (MEGA) v. 4,

and phylogenetic trees were prepared by the neighbor­joining method and un-weighted pair group method. For the gene analyses, the following strains were used as reference; A. westerdijkiae (IFM 60979, CBS 112803),

Aspergillus melleus (IFM 60743, CBS 546.6), A. ochra­ceus (IFM 60744, CBS 547.65), A. carbonarius (IFM 47662)

and Aspergillus foetidus (IFM 57758, CBS 101708).

Mycotoxin production of fungal strains

For ochratoxigenic species, conidial suspension of

the strain (1 x 108 CFU/mL) was prepared in sterile

water containing 0.01% (v/v) Tween 80 after incubation on PDA slant at 25 ·c for 7 days, and the suspension

was inoculated on 15 g of autoclaved barley grains in a

200-mL Erlenmeyer flask . Then the flask was incubated at 20 •c for 7 days 14)·15). After the incubation, 60 mL of

acetonitrile/water mixture (3:2, v/ v) was added to the culture, and the mycotoxin was extracted as described previouslyl 5l. The mycotoxin was analyzed by LC-MS/

MS according to a previous report8l. Aspergillus foeti­dus (IFM 57758) was examined as a reference strain.

For examination of fumonisin production, conidial

suspension of the A. niger strain was prepared as men­tioned above and inoculated on 20 g of autoclaved rice

grains in a 200-mL Erlenmeyer flask, and the flask was incubated at 2s•c for 7 days8l·15l·16l. Then, 50 mL of

methanol/water mixture (3:1, v/v) was added to the cul­

ture, and the mycotoxin was extracted and analyzed by

LC-MS/MS as described below. Fusarium proliferatum (IFM 58832) was used as a reference strain.

LC-MS/MS analysis of mycotoxins in culture

extract

Chromatographic separation was performed using

a Waters alliance liquid chromatograph coupled with a SCIEX API 3000 triple mass spectrometer with a turbo

spray ion source (ESI) . For ochratoxins, a 150 x 2.1 mm

Vol. 65, No. l, l - 6 (2015)

i.d. (31-1m) Inertsil ODS-3 V column (GL Sciences, Tokyo) was used for separation. The mobile phase was pro­grammed with a 30-90% acetonitrile in 5 mM ammo­nium acetate linear ramp for 10 min at a flow rate of 0.20 mL/min. MS/MS measurement was carried out with ESI at negative polarity in multiple reaction moni­toring (MRM) mode with the following transitions; OTA quantifier m/z 402.1 -> 357.9 and qualifier m/z 402.1-> 166.9; OTB quantifier m/z 368.1 -> 324.0 and quali­fier m/z 368.1 - > 280.1, respectively. For fumonisins, a 150 x 2 mm i.d. (3 !Jm) TSK gel ODS 100 V column (Tosoh, Tokyo) was used. The mobile phase was pro­grammed with a 30-90% acetonitrile in 0.1% formic acid linear ramp for 10 min at a flow rate of 0.20 mL/ min. MS/MS was with ESI positive and in MRM mode; FB1 quantifier m/z 722.5 - > 334.5 and qualifier 722.5 ->

352.2; FB2 quantifier m/z 706.3 -> 336.4 and qualifier m/z 706.3 -> 318.4; and FB4 quantifier m/z 690.5 ->

320.5 and qualifier 690.5 -> 338.5, respectively26l.

Analysis of mycotoxins in coffee beans

Coffee bean samples were milled with a Waring Laboratory Blender (Model 7012 S, Waring Commer­cial, Connecticut, USA) to yield finely ground powder. For ochratoxins, 4 g of the powder was extracted in a 50-mL plastic centrifugation tube with 20 mL of MetOH/3% NaHC03 (1:1, v/v) by homogenization. After centrifugation, a portion of the supernatant (5 mL) was diluted up to 50 mL in a measuring flask with PBS (phosphate buffer saline) containing 0.01% (v/v) Tween 20 (PBST20), and filtered with a glass filter (Whatman GF/A 110 mm). For OTA determination, 10 mL of the resulting solution was loaded on the immu­noaffnity column Ochra-king (Horiba Co., Ltd., Kyoto Japan) that was constituted in advance with the same PBST20. After loading, the column was washed with the PBST20 (3 mL x 2 times) and 10 mM ammonium acetate aqueous solution (3 mL x 2 times), successively. There­after, the remaining solution was pushed out of the col­umn by air stream. OTA was eluted from the immunoaffnity column with 3 mL of MeOH containing 2% acetic acid (v/v). The eluent was collected in a glass tube vial, and dried up under the nitrogen gas stream at 40 oc. The residue was re-dissolved in 0.2 mL of ace­tonitrile/water/acetic acid (5:94:1, v/v/v) and subjected to LC-MS/MS analysis as described above. For fumoni­sins, 10 mL of the filtered solution were loaded on another immunoaffnity column (Fumoni-Prep, R-Bio­pharm. Rhone Co., Ltd) constituted in advance with the same PBST20. After loading, the column was washed with the PBST20 and 10 mM ammonium acetate aque­ous solution in the same way, and the remaining solu­tion was pushed out by air stream. Fumonisins were eluted from the immunoaffnity column with 3 mL of MeOH. The eluent was collected, dried up, and the res­idue was re-dissolved in 0.2 mL of acetonitrile/water/ acetic acid (5:94:1, v/v/v) for the analysis by LC-MS/MS

3

under the conditions as described above.

Results and Discussion

Detection of black and yellow aspergilla from

the sample

Black aspergilli (section Nigri) were detected at high frequency in almost all of the Vietnamese coffee bean samples examined (Table 1). The predominant fungus was A. niger that was presumptively identified by morphology. The incidence of this fungus varied considerably among the coffee samples (6-100%, 56.6% on average), which was in agreement with the previous reports from Vietnam3l and other countries2l, 4

'. In addition to A. niger, putative A. carbonarius was occasionally found in 3.3% (averagely) of the sample beans. From the mycotoxigenic section Nigri, 49 strains of putative A. niger and 11 strains of putative A. carbon­

arius were randomly chosen for further characterization (see the next section).

Yellow aspergilli, A. ochraceus and its related spe­cies in section Circumdati, were detected in 2.9% (aver­agely) of the sample beans (Table 1) although previous report showed that no ochratoxigenic species other than A. niger was present in the Vietnamese samples 1l. Ten strains belonging to section Circumdati were ran­domly chosen for further characterization (see the next section).

Our preliminary study showed that section Circum­

dati (identified as A. ochraceus) was detected in 13% of beans in 13 samples from the same region in year 2,000 (unpublished data). Other reports also proved that sec­tion Circumdati was occasionally found in Vietnamese coffee beans3l. These and our present results suggest that Vietnamese coffee is commonly infected with a variety of ochratoxigenic fungi including sections of Nigri and Circumdati.

Species identification of the strains by DNA

analyses

Species identifications of yellow aspergilli (section Circumdati) and A. carbonarius were done by sequenc­ing of the ~-tubulin 13

'·25

) and cytb genes23l. The ten yel­low aspergilli strains were identified by the ~-tubulin gene analysis with the reference strains of A. westerdi­

jkiae (IFM 60979), A. melleus (IFM 60743) and A. ochra­

ceus (IFM 60744). The gene analysis showed that all the yellow aspergilli strains were A. westerdijkiae except one A. melleus strain (Supplementary Table 1). The cytb

gene sequencing23l also showed that the aspergilli strains were clustered into 2 clades, i.e. A0-4 and A0-3, corresponding to A. westerdijkiae and A. mel/ues,

respectively (Supplementary Table 1). A. ochraceus strain (A0-2) was not found in this study. Eleven of the

4 HASHIMOTO eta!. JSM Mycotoxins

Table1 Detection of putative sections Nigri (black aspergilli) and Circumdati (yellow aspergilli) from Vietnamese coffee sample beans, and contents of OTA and FB2 as analyzed by LC-MS/MS.

Fungi detected (%)a Contents in coffee beansb

Cultivars of beans Sample No. section Nigri section Circumdati

A. niger A. carbonarius

1 24 6

2 26 4

3 52 0

4 40 4

5 82 2

6 10 0

7 96 2

8 82 2

9 88 2

10 54 12

11 56 6

12 12 0

13 6 0

14 100 2

15 78 8

16 100 2

Average 56.6 3.3

a Based on coffee beans b Limits of detection: 0.5 119/kg, Limits of quantification: l!Jg/kg c Not detected

A. carbonarius strains, presumptively identified by mor­phology, were also examined. The ~-tubulin gene analysis13l indicated that all of the strains tested were A. carbonarius (Supplementary Table 2). On the other hand, the cytb gene analysis showed that 5 of them were aligned in the clade of A. carbonarius, AN-D-4, as previously reported 12l; however, the PCR amplification was not successful in the remaining 6 strains (Supple­mentary Table 2). The cytb primers might not be matched for some strains of A. carbonarius.

Our previous report20l indicated that strains of A. niger complex were clustered into 3 clades on the basis of the cytb sequences, i.e. AN-D-5-l, AN-D-7-1, and AN-D-9-112l, which corresponds to A. niger, A. tubin­gensis, and Aspergillus luchuensis, respectively. In this study, presumptive A. niger strains randomly chosen were subject to the cytb gene analysis (Supplementary Table 3). The sequencing showed that a majority of strains (41 strains out of 49 strains) were aligned to AN­D-5-1, i.e. A. niger, which is basically in agreement with the previous report on Thai beans4l. We also found A. luchuensis (6 strains), a black Koji mold that is used to ferment Awamori (a kind of spirit of Japan) or other alcoholic beverages in East Asian countries 18l·19l, and A. tubingensis (2 strains). Occurrence of A. luchuensis in coffee beans indicated that the fungus might also be distributed in agricultural products of Southeast Asian countries.

4

26

0

2

0

0

0

0

6

0

0

4

0

0

2

2

2.9

OTA FB2

-c Robusta

Robusta

Robusta

Robusta

Robusta

Robusta

Robusta

Robusta

Robusta

6.3 49 Robusta

Robusta

Robusta

Robusta

55 Arabica

Arabic a

2.3 Arabica

4.3 52

Mycotoxin production of identified strains

All strains of section Circumdati (A. westerdijkiae and A. melleus) (Supplementary Table 1) and A. carbon­arius (Supplementary Table2) identified in this study produced OTA in barley grains in the range of 0.2-41.3 !Jg/g (10.3 !Jg/ml on average) and 4.0- 67.7 !Jg/g (29.8 !Jg/ml on average), respectively. In agreement with our result, A. westerdijkiae and A. carbonarius were previ­ously reported as consistent OTA producers 17l·21 l. Rela­tively small amount of ochratoxin B (OTB) was produced by several strains of A. westerdijkiae (Supplementary Table 1). In section Circumdati, A. ochraceus was previ­ously described as the major fungus responsible for OTA contamination in coffee beans2l. However, in recent literatures4l·26l, A. westerdijkiae rather than A. ochraceus is reported as the primary causative fungus of OTA contamination in food and feed.

All A. niger strains, except one, failed to produce OTA; the one strain formed 2.7 !Jg/g of OTA along with relatively small amount (1.5 flg/g) of OTB in barley grains (Supplementary Table 3). Previous reports showed that 7-9% of A. niger strains were OTA producers1l,2l, Sl,6l. Although A. niger is the most predominant in mycobiota of coffee beans, the ochratoxigenic species seems to be less problematic because of infrequent presence as OTA producer in the beans. On the other hand, A. carbonarius and section Circumdati seemed to play an important role in OTA contamination in agree-

Vol65, No. l, l - 6 (2015)

ment with a previous report3l. The results indicated that the major sources of OTA contamination in Vietnamese coffee beans were not A. niger, but A. westerdijkiae and A. carbonarius.

With regard to fumonisins, no typical fumonisin­producing species including Fusarium proliferatum was detected in the beans tested. The forty-nine represen­tative strains of A. niger were examined for the fumoni­sin production. While none of the A. tubingensis (AN-D-7-1) and A. /uchuensis (AN-D-9-1) strains produced the mycotoxin, 90 % of A. niger strains (37 strains) pro­duced detectable amount of FB2, and one strain (No. 37) produced both OTA and FB2 (Supplementary Table 3). These results are essentially in agreement with the result of mycotoxin analysis of Thai coffee beans by Noonim and co-workers, who reported that 76.5% of A.

niger strains were FB2 producers9l; among the A. niger strains, 11.7% produced both OTA and FB2. In addition, occurrence of FB2, OTA, and co-occurrence of FB2 and OTA, were also reported in 81%, 17%, and 10% of the industrial A. niger strains, respectively22l.

In accordance with recent reports9l,nl, we also found putative FB4 in the culture of the A. niger strains by LC-MS/MS analysis although structural conforma­tion could not be done due to the unavailability of the FB4 standard (Supplementary Table 3).

Analysis of mycotoxin in coffee beans

LC-MS/MS analysis demonstrated contamination of OTA in 2 samples (No. 10 and No. 16) with concen­trations of 6.3 and 2.3 ~g/kg, respectively (Table 1). A.

carbonarius and/or A. westerdijkiae were isolated from both samples. Previous reports2l showed that OTA was detected from green coffee beans in a wide concentra­tion range of 0.2 - 360 1-1g/kg in commercial raw coffee. The incidence of OTA contamination in the beans was relatively low in this study.

FB2 was found in 2 of the samples (No. 10 and No. 14) with concentrations of 49 and 55 ~g/kg, respec­tively (Table 1). Although the number of the samples was limited, the levels of FB2 contamination in Viet­namese coffee beans were generally higher than those previously reported with Thai coffee beans9l. The mycobiota of the coffee beans may thus be affected by climate conditions from year to year17l or manner of processing in each country.

Co-contamination of OTA and FB2 in Vietnamese coffee beans was reported for the first time in this study. These mycotoxins may exhibit synergistic toxicity in living tissue of animal and human although the effect has not been investigated in details. The European Commission set regulation for ochratoxin ((EC) No.123/ 2005) that a maximum limit was 5 ~g/kg in the case of the roasted coffee and ground coffee, and 10 ~g/kg in soluble coffee. For fumonisins, the Codex Alimentarius Commission (Geneva, 2014) has set the maximum levels of total fumonisin (FB1, FB2) at 4 mg/kg in raw maize

5

and 2 mg/kg in maize product. Our study demonstrated that the mycotoxin con­

tamination in coffee beans should be controlled for reducing human health risk because coffee beans are likely to be infected with the mycotoxigenic fungi at high incidences.

Acknowledgements

This work was supported by The Tojyuro Iijima Foundation for Food Science and Technology. We would like to express our gratitude for their financial support. We would also like to extend our special thanks to Dr. Nguyen Tho and Mr. Masahiro Saga. We additionally thank the Western Highland Agricultural and Forestry Science Institute.

Supplementary Materials

Supplementary materials may be found in the online version of this article:

Supplementary Table 1 Species identification of puta­tive section Circumdati (yellow aspergilli) strains by ~­tubulin and cytb gene analyses, and ochratoxin produc­tion of the strains

Supplementary Table 2 Species identification of puta­tive A. carbonarius strains by ~-tubulin and cytb gene analyses, and ochratoxin production of the strains

Supplementary Table 3 Species identification of puta­tive A. niger strains by cytb gene analysis, and myco­toxin production of the strains

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ベトナム産コーヒー豆から分離されたAspergル'Ssections Nigri及び。'Cumdaliのカビ毒産生性とコーヒー豆のカビ毒汚染

橋本ルイコl，中川博之2，陰地義樹3，浅野勝佳3，横山耕治4，高橋治男5

1千葉県衛生研究所(干260-8715 千葉県千葉市中央区仁戸名町666-2)2農業・食品産業技術総合研究機構食品総合研究所(干305-8642 茨城県つくば市観音台2・1・12)3奈良県保健研究センター(干633-0062 奈良県桜井市粟殿1000)4千葉大学真菌医学研究センター(干260-8673 千葉県千葉市中央区亥鼻1-8-1)5国立医薬品食品衛生研究所(〒158-8501 東京都世田谷区上用賀1-18-1)

本邦がその多くを輸入しているベトナム産コーヒー豆について，カピ毒産生菌及びカピ毒による汚染の調査を行った

採取した16サンプルからは，Aspergillus niger (種複合体，汚染粒率56_6%)， Aspergillus cαrbonαrius (3_3%) ，

Aspergillus section Circumd，αti (2_9%)を含む複数のオクラトキシンA(OTA)産生菌が検出された.形態により

A nigerと関連菌種及び、sectionCircumdαtiと同定された株のうちの一部について， sチュープリン及びミトコンドリア

チトクロームb遺伝子を用いて菌種を同定し，カピ毒産生性を調べた.豆から分離された主要な菌はA nigerで、あっ

た SectionCircumdαtiの分離株はAspergillusωesterdijkiaeあるいはAspergillusmelleusと同定され，Aspergillus

ochrαceusは認められなかった LC-MSIMSによりそれらの分離株のカピ毒産生性を分析した結果， Acαrbonαrius及び、

A westerdijkiaeで、大量のOTAの産生が見られたが， A nigerで、はOTAの産生は少量で、あったことから，豆のOTA汚染の

原因はA nigerよりAcαrbonαrius及びA westerdijkiaeで、あると考えられた.また， A niger株におけるフモニシンB2

(FB2)の産生率は90%と高率であった. 2つのサンプルからはそれぞれOTA(2_3μglkg)あるいはFB2 (55μg/kg)が，

1つのサンプルからは両方のカピ毒が検出された (6_3μglkgOTA， 49μg/kg FB2) .これらの結果は，ベトナム産コー

ヒー豆にはOTA産生菌とFB2産生菌が一般的に存在しており，まれにそれらの菌によるカピ毒汚染が発生していることを

示している.

キーワード:アスペルギルスセクションサーカムダティ;アスペルギルスセクションニグリ;オクラトキシン;フモニシン;

ベトナムコーヒー;ミトコンドリアチトクロームb遺伝子