Interconversion of Aflatoxin B1 and Aflatoxicol by Several Fungi

Transcription

1 PPLIED ND ENVIRONMENTL MICROBIOLOGY, My 199, p Vol. 56, No /9/ $2./ Copyright C) 199, merin Soiety for Mirobiology Interonversion of fltoxin B1 nd fltoxiol by Severl Fungi MITSUO NKZTO,1* STOSHI MOROZUMI,1 KZUO SITO,' KENJI FUJINUM,l TICHIRO NISHIM,l ND NOBUHIKO KSI2 Deprtment of Food Hygiene nd Nutrition, The Tokyo Metropolitn Reserh Lbortory of Publi Helth, Shinjuku-ku, Tokyo 169,1 nd Deprtment of Mirobil Chemistry, Shool of Phrmeutil Sienes, Show University, Shingw-ku Tokyo 142,2 Jpn Reeived 5 Deember 1989/epted 24 Februry 199 Four fungl strins, nmely, spergiuus niger, Eurotium herbriorum, Rhizopus sp., nd non-fltoxin (F)-produing spergillus flvus, whih ould onvert F-B1 to fltoxiol (FL), ould lso reonvert FL to F-B1. The interonversion of F-B1 to FL nd of FL to F-B1 ws sertined to our during prolifertion of the fungi. These retions were distintly observed in ell-free systems obtined from disrupted myeli of. flvus nd the Rhizopus sp., but they were not observed in ulture filtrtes from intt (nondisrupted) myeli of the sme strins. The interonversion tivities of F-B1 nd FL were not observed when the ell-free systems were preheted t 1 C. These findings strongly suggest tht the interonversion of F-B1 nd FL is medited by intrellulr enzymes of. flvus nd the Rhizopus sp. In ddition, the isomeriztion of FL- to FL-B observed in ulture medium ws lso found to our by the lowering of the ulture ph. fltoxiol (FL) is formed by redution of the ylopentenone rbonyl of fltoxin (F)-B, nd hs two types of stereoisomers, FL- nd FL-B, determined by the stereoonfigurtion of the OH group in the ylopentenol ring (2) (Fig. 1). FL is well known (1) s min in vitro metbolite produed from F-B, by redutse in the superntnt of liver homogentes of severl vin nd mmmlin speies. It hs lso been reported tht FL is produed from the biologil redution of F-B, by miroorgnisms suh s Rhizopus spp. (3), Dtylium dendroides (4-6), bsidi repens (5), Muor griseo-ynus (5), spergillus niger (8, 9), Muor mbiguus (8, 9), Trihoderm viride (8, 9), Streptoous ltis (1), nd Tetrhymen pyriformis (14). These fts suggest the possibility of deteting FL in foods nd feeds ontminted with F-Bl. Sito et l., this lbortory (16), reported the simultneous ontmintion of ommeril pisthio nuts nd orn with FL nd F-B, in 1984, in the first report on nturl ontmintion of food with FL. To eluidte the use of nturl FL ontmintion, we reported the bilities of vrious speies of non-f-blproduing fungi whih were isolted from FL-ontminted orn to onvert F-B, to FL. s result, it ws found (11) tht. niger, Eurotium herbriorum, the Rhizopus sp., et., hd firly potent onversion bilities. It ws lso found (12) tht severl strins of F-Bl-produing spergillus flvus isolted from FL-ontminted orn showed firly high FL-produing bility. s mentioned bove, the onversion of F-B, to FL by miroorgnisms hs lredy been demonstrted, but little is known bout the reverse onversion, nmely, tht from FL to F-Bl. This pper dels with the metboli interonversion of FL nd F-B, by non-f-produing fungi isolted from FL-ontminted orn. * Corresponding uthor. in 1465 MTERILS ND METHODS Orgnisms. The fungl strins used in this study were the following:. niger C-41-i, E. herbriorum C-41-f, Rhizopus sp. C-4-m, nd non-f-produing. flvus C-4-d, whih were isolted from FL-ontminted orn (11). Culture medium nd regents. Low-slt (SL) medium (13) ws used to study the bility of the fungi to onvert from FL to F-Bl. The medium onsisted of the following omponents: surose, 85 g; sprgine, 1 g; (NH4)2SO4, 3.5 g; KH2PO4, 75 mg; MgSO4 7H2, 35 mg; CCl2-2H2, 75 mg; ZnSO4-7H2, 1 mg; MnCl2 4H2, 5 mg; (NH4)6Mo724-4H2O, 2 mg; N2B47, 2 mg; FeSO4.7H2, 2 mg; nd distilled wter, 1, ml. FL-, FL-B, nd F groups, i.e., B1, B2, B2, Gl, G2, Ml, M2, Pl, nd Ql, were obtined from Mkor Chemils Ltd. (Jeruslem, Isrel). It ws onfirmed by high-performne liquid hromtogrphy (HPLC) tht FL- did not ontin FL-B nd tht FL-B did not ontin FL-. Inoultion nd inubtion. Fungl strins were inoulted previously on potto dextrose gr slnts (Eiken Chemil Co., Ltd., Tokyo, Jpn) nd ultivted for 7 dys t 25 C until they were well sporulted. The spores were hrvested in.1% Tween 8 solution to mke spore suspension ontining 14 spores per ml. Smples (1,ul) of the spore suspension were inoulted onto 1 ml of SL ulture medium ontining FL- nd FL-B (2, ng/ml eh), nd the medium ws ultured for 6 nd 1 dys t 25 C under sttionry onditions. fter the inubtion, FL metbolites were investigted. To study the time ourse of onversion from FL to F-Bl, eh strin of fungus ws ultured for 2 to 2 dys, under the sme onditions. Weighing of myelil mt nd mesurement of ph. fter inubtion, the ulture ws filtered through glss filter. The fungl mt ws dried t 5 C for 48 h nd then stored over sili gel for 1 week. The weight of the dried fungl mt ws then determined. The ph of the ulture filtrte ws mesured with ph meter. Extrtion of FLs nd Fs nd preprtion of test solution. Chloroform (1 ml) ws dded to the whole ulture. Downloded from on Jnury 24, 219 by guest

2 1466 NKZTO ET L. PPL. ENVIRON. MICROBIOL. fltoxiol fltoxin C H 3 O H B1 fltoxiol C H 3 FIG. 1. Strutures of F-B1, FL-, nd FL-B. The mixture ws homogenized for 1 min nd entrifuged to seprte the hloroform lyer. The hloroform lyer ws dehydrted with nhydrous sodium sulfte nd filtered through filter pper. The solvent ws evported under redued pressure. The residue ws dissolved with n pproprite mount of hloroform nd used s the test solution for thin-lyer hromtogrphy (TLC). fter the detetion of FLs nd Fs by TLC, onstnt quntity of the TLC test solution ws seprted into n FL frtion nd n F-B1 frtion by olumn hromtogrphy (15) nd eh frtion ws used in the ssy. The test solution ws pplied onto the olumn ontining 1 g of sili gel. The olumn ws wshed with 15 ml of n-hexne, nd then FL ws eluted with 18 ml of ethyl ether. Then, F-B1 ws eluted with mixed solution of methnol-hloroform (3:97). The solvent ws evported from the respetive elutes. The residue ws dissolved in n pproprite mount of hloroform to mke the test solution for HPLC. F-B2 ws diretly nlyzed by using the filtrte from nother ulture by HPLC. TLC. Qulittive nlysis of the F groups, i.e., B1, B2, G1, G2, M1, M2, P1, Q1, nd FLs ws rried out by TLC using preprtive Kieselgel 6 pltes (gel thikness,.25 mm; E. Merk G, Drmstdt, Federl Republi of Germny) under the development onditions desribed by Sito et l. (15). HPLC. The HPLC system (Shimdzu, Kyoto, Jpn) ws omposed of n LC-S pump, n RF-53 fluoresene detetor, nd C-R3 dt proessing unit. HPLC onditions for the determintion of F-B1 were s follows: olumn, Finepk SIL (prtile size, 5 plm; 4.6-mm inside dimeter by 25-m length; Jso, Tokyo, Jpn); mobile phse, toluene-ethyl ette-formi id-methnol (712:6: 16:28); flow rte, 1.2 ml/min; nd detetion, fluoresene (exittion, 365 nm; emission, 425 nm). The determintion of FL- nd FL-B ws s desribed in previous report (11). HPLC onditions for the determintion of F-B2 were s follows: olumn, Finepk SIL C18 (prtile size, 1,um; 4.6-mm inside dimeter by 25-m length); mobile phse, methnol-etonitrile-wter (2:2:6); flow rte, 1. ml/ min; detetion, fluoresene (exittion, 37 nm; emission, 435 nm). Confirmtion of F-B1, F-B2, nd FL. Confirmtion of F-B1 ws rried out by the method of Hghighi et l. (7). Nmely, F-B1 ws onverted to F-B2 by tretment with trifluoroeti id nd F-B2 ws nlyzed by HPLC. Confirmtion of FL nd F-B2 ws rried out by reding exittion nd emission spetr of the peks seprted by HPLC. The spetr were ompred with those of stndrd substnes. Preprtion of ell-free system. Eh strin of Rhizopus sp. nd non-f-produing. flvus ws inoulted onto 5 ml of SL ulture medium nd ultured for 4 nd 6 dys, respetively. fter the inubtion, the ultures were seprted into fungl myelil mt nd ulture fluid. The ulture fluid ws filtered through membrne filter (pore size,.2,um; Gelmn Sienes, In., nn rbor, Mih.). The filtrte ws sved for mesurement of the bilities to onvert F-B1 to FL nd FL to F-B1. The myelil mt ws rinsed three times with distilled wter nd lyophilized. Freeze-dried myelium (1 g) ws ground with 2 g of glss beds (dimeter, 15 to 21,um) nd mixed with 2 ml of old.5 M phosphte buffer (ph 7.2). The mixture ws vibrted for 1 min in n ultrsoni bth filled with ie wter (model UT24; Shrp, Tokyo, Jpn) nd then filtered through Toyo no. 5 filter pper. The filtrte ws entrifuged t 15, x g for 2 min with high-speed refrigerted entrifuge (model CX- 25; Tomy Seiko, Tokyo, Jpn). The superntnt ws used s the ell-free system nd ws employed in the onversion ssy. The protein ontent in the ell-free system ws determined by biinhonini id protein ssy regent (Piere Chemil Co., Rokford, Ill.). ssy of interonversion tivity of ell-free system nd ulture filtrte. The ell-free system nd ulture filtrte (1 ml eh) were pipetted into 2-ml test tube. The retion ws strted by dding 2,ul of F-B1 or FL in N,Ndimethylformmide (2 ppm) to the test tube. The retion mixture ws inubted t 3 C in wter bth. fter 1 to 18 min of inubtion, 1 ml of the retion mixture prepred from the ell-free system ws extrted with 5-ml portions of hloroform. F-B1 nd FL in the hloroform solution were hromtogrphed by HPLC. In the se of the retion mixture prepred from ulture filtrtes, fter 1 to 72 h of inubtion, 1 ml of the retion mixture ws pled in test tube nd then treted s mentioned bove. RESULTS Conversion of FL- nd FL-B to F-Bl. F-B1 ws deteted in the ultures of ll four speies,. niger, E. herbriorum, the Rhizopus sp., nd. flvus. F-B2 ws deteted only in the. niger ulture. Other F groups, i.e., B2, G1, G2, M1, M2, P1, nd Q1 were not deteted in ny ultures (Tble 1). F-B1 ws deteted in ll ultures to whih FL- nd FL-B were dded. In both ultures inoulted with the sme strin of fungus, the residul FL ws lmost the sme nd the produed F-B1 ws lso nerly the sme. When. flvus nd E. herbriorum were used s inoul, the mounts of F-B1 in ultures inubted for 1 dys were greter thn those in ultures inubted for 6 dys. In. niger nd the Rhizopus sp., lrger mounts of F-B1 were found in the 6-dy ultures thn in the 1-dy ultures. In ll ultures, F-B1 inresed with derese in FL. This finding suggested tht F-B1 ws produed by the onversion of dded FL. In ll of the ultures, both FL- nd FL-B were observed. FL-B ws deteted in the ultures supplemented with FL-, nd FL- ws deteted in the ultures supplemented with FL-B. The rtio of FL- to FL-B ws nerly the sme in ll of the ultures. On the other hnd, similr results were lso observed in the ontrol medium whih ws not inoulted with fungus. This finding suggested tht the interonversion between FL- nd FL-B ourred independently of fungl metboli tivity. Time ourse of onversion of FL to F-B,. In the exper- Downloded from on Jnury 24, 219 by guest

3 VOL. 56, 199 TBLE 1. Residul mounts of FL- nd FL-B nd deteted mounts of F-B1 nd F-B2 in ulture medium inoulted with four speies of fungi FusInu- Initil onn Conn deteted Fungus btion ofgfls nll time (gm) FL F (dys) B B B1 B2 spergillus niger 6 2, C-41-i 1 2, , , spergillus flvus 6 2, ND C-4-d 1 2, ND 6 2, ND 1 2, ND Eurotium herbri- 6 2, ND orum C-41-f 1 2, ND 6 2, ND 1 2, ND Rhizopus sp. 6 2, ND C-4-m 1 2, ND 6 2, ND 1 2, ND None (ontrol) 6 2, ND ND 1 2, ND ND 6 2, ND ND 1 2, ND ND ND, Not deteted. iments mentioned bove, F-B1 inresed more in the 1-dy ultures thn in the 6-dy ultures with. flvus nd E. herbriorum. On the ontrry, F-B1 deresed in 1-dy ultures with. niger nd Rhizopus sp. In ultures with. niger, F-B2 ws deteted. Considering these fts, we investigted the time ourses of growth of the fungl myelil mt, the hnge in the ph of ulture medium, nd the prodution of F-B1 nd F-B2. Sine lmost the sme levels of F-B1 were onverted from either FL- or FL-B, only FL- ws used in the following experiment. Eh of five tubes from the respetive strins ws ultured, nd two of them were used for the mesurement of ph nd the weighing of myeli nd three of them were used for the ssying of F nd FL. The results re summrized in Fig. 2. FL is shown s the sum of FL- nd FL-B. In the. niger ulture, FL deresed rpidly nd ws srely deteted on dy 2. F-B1 ws deteted on nd fter dy 2, nd it rehed mximum level of 87 ng/ml on dy 4. This finding suggests tht 4% or more of the dded FL ws onverted to F-B1 on dy 4. fter dy 4, the mount of F-B1 deresed rpidly. fter the pperne of F-B1, F-B2 ws deteted, showing mximum level on dy 7. In this se, the drop in ph of the ulture filtrte ws mrked, ompred with the ulture filtrtes inoulted with other fungi, rehing ph of 2 on dy 7 of ulturing; even on dy 2, the ph ws still 2.9. In the Rhizopus sp. ulture, bout hlf the dded FL ws onverted to F-B1 despite only slight growth of the myeli (11 mg) on dy 2. F-B1 showed mximum on dy 2. fterwrds, it deresed rpidly nd disppered ompletely from the ulture on dy 2. F-B2 ws not deteted in the Rhizopus sp. ulture. In the non-f-produing. flvus ulture, FL deresed slowly, ompred with. niger nd the Rhizopus sp. F-B1 umulted in ultures without showing rpid dereses in the ses of. niger nd the Rhizopus sp. INTERCONVERSION OF F-B1 ND FL 1467 These tendenies were observed in the se of E. herbriorum. In ny se, the sum of the residul mount of FL nd the deteted mount of F-B1 ws bout equl to the mount of FL dded t the initil stge of inubtion: however, fterwrds, the sum of FL nd F-B1 deresed with time. These findings suggest tht both F-B1 nd FL were further metbolized to unknown substnes by the fungi. This experiment ws repeted one more, nd the reproduibility ws stisftory. Interonversion of F-B1 nd FL in ell-free system nd ulture filtrte. The bility to interonvert F-B1 nd FL ws not observed in ulture filtrtes prepred from. flvus nd the Rhizopus sp. ultures; however, in the ell-free systems prepred from these fungi, it ws lerly observed (Fig. 3). The ell-free system prepred from. flvus onverted bout 13% of FL- to F-B, nd bout 5% of F-B1 to FL- during 18-min inubtion. On the other hnd, the ell-free system prepred from the Rhizopus sp. ws better ble to onvert FL- to F-B1 nd F-B1 to FL- thn ws. flvus. It ws pble of onverting 85% of FL- to F-B1 during 18-min inubtion, nd it onverted 43% of F-B1 to FL- in 6-min inubtion. fter 6 min, FL- deresed rpidly nd ws reonverted to F-B1. In ny se, the sum of FL nd F-B1 ws pproximtely equl to the mount of dded substne. No FL-B ws deteted in ny of the ell-free systems. This result suggests tht the FL isomer produed from F-B1 by the miroorgnism ws only FL-. No interonversion tivity of F-B1 or FL- ws observed when the ell-free system ws preheted t 1 C for 5 min. Isomeriztion between FL- nd FL-B. From the results of the experiment mentioned bove, it ws found tht isomeriztion between FL- nd FL-B ourred in ulture medium ontining no fungl inoul nd tht the FL level in the medium deresed with time. This tendeny of isomeriztion ws similr to the isomeriztion of FL in the ultures inoulted with fungi. However, only the FL- isomer ws found when FL ws onverted from F-B1 by the ell-free systems from the two strins of the Rhizopus sp. nd. flvus. ordingly, it ws thought tht the isomeriztion of FL ws used by environmentl ftors, suh s the ingredients of the ulture medium or hnges in the ph of the ulture medium. Consequently, the uses of isomeriztion of FL- to FL-B nd of LF-B to FL- were investigted. Solutions of individul ingredients of SL medium were prepred with distilled wter so s to hve onentrtions orresponding to those of omponent ingredients of the SL medium, nd FL- nd FL-B were dded to these solutions. The solutions were inubted t 25 C for 5 dys in drk room, nd FL- nd FL-B were ssyed. Corresponding isomers of FL were deteted in ll solutions in whih the ph ws less thn 5.5, suh s in solutions of sprgine, mmonium sulfte, nd potssium dihydrogen phosphte. The lower the ph ws, the more mrked ws the isomeriztion observed. From this experiment, it ws supposed tht the hydrogen ion onentrtion in the ulture medium ffeted the isomeriztions of FL- to FL-B nd of FL-B to FL-. Therefore, buffer solutions rnging from ph 2.2 to 8. were prepred by using solutions of.1 M itri id nd.2 M sodium phosphte dibsi. FL- ws dded to these Downloded from on Jnury 24, 219 by guest

4 1468 NKZTO ET L. PPL. ENVIRON. MICROBIOL. B o :5 2, CO C ;-I 5 C x e o li 11 ob.5 l;- x 4i.T 'O 1; u x 4..T r6.l w 4i 4-) Inubtion time (dys) 3 ji,' do 1; U 4 3 ^r 2 ' 1 _ 9 - x 9 ; 7 E 7 O 5 s x 3 I E I o 9 - O _ Inubtion time (dys) Inubtion time (dys) Time ourse of fungl growth, ph of ulture filtrtes, nd deteted mount of FL, F-B1, nd F-B2 in the ultures inoulted Inubtion time (dys) FIG. 2. with four fungl strins. (). niger; (B) Rhizopus sp.; (C). flvus; (D) E. herbriorum. Symbols:, FL; *, F-B1; El, F-B2;, myelil weight;, ph. The vlues of myelil weight nd ph re mens of two determintions, nd the vlues of Fs nd FL re mens of three determintions. buffer solutions to mke solution of 2, ng/ml, nd fter 3 nd 24 h nd 3, 6, 1, nd 15 dys, the quntities of FL- nd FL-B were exmined t eh ph level. The results re shown in Fig. 4. It ws onfirmed tht the lower the ph ws, the more mrked ws the isomeriztion of FL. t the sme time, it ws proved tht the lower the ph ws, the more mrkedly FL itself deresed. No F-B1 ws deteted t ll under these onditions. The hloroform extrts of the buffer solutions were nlyzed by norml-phse HPLC, nd two new peks (retention times, 17 nd 19 min, respetively) ppered fter the FL- nd FL-B peks (retention times, 8.5 nd 9.8 min, respetively) on hromtogrms. Consequently, it ws onjetured tht FL ws onverted to substne with greter polrity thn FL. t ph 7. or 8., no isomeriztion ourred nd no derese in FL ws observed t ll. The new substne ws deteted only in the. niger ulture. DISCUSSION In this study, we found tht four orgnisms,. niger,. flvus, Rhizopus sp., nd E. herbriorum, whih hd the 4 3-2_ 2W- 4 3 s 2 Z bility to onvert F-B1 to FL, lso possessed the bility to reonvert FL to F-B1. In the onversion of F-B1 to FL, FL umulted in ultures nd rehed mximum level in the middle stge of inubtion (11). In the reverse retion from FL to F-B1, F-B1 umulted in the ultures ttined mximum level between the initil to middle stges when the fungi were proliferting. These observtions indite tht the reiprol retions, i.e., the redution from F-B1 to FL nd the oxidtion from FL to F-B1, our simultneously. These retions were distintly observed in the ell-free systems obtined from disrupted myeli of. flvus nd the Rhizopus sp.; however, they were not observed in the ulture filtrtes from nondisrupted myeli of the sme strins. ordingly, these retions re onsidered to be ontrolled by fungl enzymti systems. F-B2 ws lso found in the ulture inoulted with. niger. Tsubouhi et l. (17) stted tht F-B2 ws produed from F-B1 beuse of lower ph of the medium, used by orgni ids produed by. niger. In this experiment, F-B2 ws observed when the ph of ulture filtrtes 1 3, l = Downloded from on Jnury 24, 219 by guest

5 VOL. 56,199 INTERCONVERSION OF F-B1 ND FL , 2, B 3, 1,5 ox 2W1,. o 1, Inubtion time (min) Inubtion time (min) FIG. 3. Interonversion of F-B1 nd FL- by ell-free systems of the Rhizopus sp. nd non-f-produing. flvus. () Conentrtion of F-B1 in ell-free system when FL- ws dded; (B) onentrtion of FL- in ell-free system when F-B1 ws dded. Symbols:, Rhizopus sp.; *, non-f-produing. flvus. The protein onentrtions of the ell-free systems prepred from. flvus nd the Rhizopus sp. were 6.7 nd 5.8 mg/ml, respetively. deresed. It ws supposed tht F-B2 observed in this experiment ws lso produed from F-B1, whih ws onverted from FL, by lower medium ph due to orgni ids produed by. niger. This experiment showed tht FL ws onverted to ompound with higher polrity thn FL nd deresed not only s result of fungl metboli deomposition but lso by the tions of orgni ids whih umulted in the ulture medium. Beuse the tretment of F-B1 with wek id onverts it to F-B2, hemietl of F-B1, it ws presumed tht the more polr ompounds were hemietls onverted from FL- nd FL-B, respetively. It ws lso proved tht the onversions of FL- to FL-B nd of FL-B to FL- were produed by the 1 ;; 8-6 ph 2. 2 IC- 2-1 n - n 1D O u2 ph 3. ph 4. ph ph 5. ph 6. ph 7. ph 8. 4 :2 n nuzm %D m -z to UZ nr m 1D O I-n n %DO CM l _ C _ N C" _: fltoxiol : fltoxiol B FIG. 4. Conversion of FL- to FL-B nd derese of totl FLs in buffer solutions with the ph djusted to 2.2 to 8.. Eh buffer solution initilly ontined 2, ng of FL- per ml. d, Dys of inubtion. lowering of the ulture ph. It is thought tht the interonversion of FL- nd FL-B results from the id-tlyzed isomeriztion of n llyli lohol on the ylopentenol ring. In studies on the onversion of F-B1 to FL by fungi, reported by us (11), Cole et l. (3), nd Detroy nd Hesseltine (5), FL- ws found t first in the medium nd then FL-B ppered lter. On the bsis of these results, we onlude tht F-B1 is first onverted to FL- by fungi nd tht FL- is further onverted to FL-B by the tions of medium omponents or orgni ids produed by the fungi. LITERTURE CITED 1. Cmpbell, T. C., nd J. R. Hyes The role of fltoxin metbolism in its toxi lesion. Toxiol. ppl. Phrmol. 35: Cole, R. J., nd R. H. Cox The fltoxins, p In Hndbook of toxi fungl metbolites. demi Press, In., New York. 3. Cole, R. J., J. W. Kirksey, nd B. R. Blnkenship Conversion of fltoxin B1 to isomeri hydroxy ompounds by Rhizopus spp. J. gri. Food Chem. 2: Detroy, R. W., nd C. W. Hesseltine Isoltion nd biologil tivity of mirobil onversion produt of fltoxin B1. Nture (London) 219: Detroy, R. W., nd C. W.. Hesseltine Trnsformtion of fltoxin B1 by steroid-hydroxylting fungi. Cn. J. Mirobiol. 15: Detroy, R. W., nd C. W. Hesseltine fltoxiol: struture of new trnsformtion produt of fltoxin B1. Cn. J. Biohem. 48: Hghighi, B., C. Thorpe,. E. Pohlnd, nd R. Brnett Development of sensitive high-performne liquid hromtogrphi method for detetion of fltoxins in pisthio nuts. J. Chromtogr. 26: Mnn, R., nd H. J. Rehm Degrdtion produts from fltoxin B1 by Corynebterium rubrum, spergillus niger, Trihoderm viride nd Muor mbiguus. Eur. J. ppl. Mirobiol. 2: Mnn, R., nd H. J. Rehm Degrdtion of fltoxin B1 by vrious miroorgnisms. Z. Lebensm.-Unters.-Forsh. 163: Megll, S. E., nd M.. Mohrn Fte of fltoxin B1 in Downloded from on Jnury 24, 219 by guest

6 147 NKZTO ET L. fermented diry produts. Myopthologi 88: Nkzto, M., K. Sito, Y. Kikuhi,. Ibe, K. Fujinum, M. Nishijim, Y. Noi, T. Nishim, S. Morozumi, T. Wuke, nd H. Hitokoto Conversion of fltoxin B1 to fltoxiols by vrious fungi isolted from fltoxiols ontminting orn. J. Food Hyg. So. Jpn. 26: Nkzto, M., K. Sito, Y. Kikuhi,. Ibe, K. Fujinum, M. Nishijim, T. Nishim, S. Morozumi, T. Wuke, nd H. Hitokoto fltoxiol formtion by spergillusflvus nd. prsitius. J. Food Hyg. So. Jpn. 26: Reddy, T. V., L. Viswnthn, nd T.. Venkitsubrmnin High fltoxin prodution on hemilly defined medium. ppl. Mirobiol. 22: Robertson, J.., D. J. Teunisson, nd G. J. Boudreux PPL. ENVIRON. MICROBIOL. Isoltion nd struture of biologilly redued fltoxin B1. J. gri. Food Chem. 18: Sito, K., M. Nishijim, K. Ysud, H. Kmimur,. Ibe, T. Ngym, H. Ushiym, nd Y. Noi nlytil method for fltoxins nd fltoxiols in erels, nuts nd their produts. J. Food Hyg. So. Jpn. 25: Sito, K., M. Nishijim, K. Ysud, H. Kmimur,. Ibe, T. Ngym, H. Ushiym, nd Y. Noi Investigtion of the nturl ourrene of fltoxins nd fltoxiols in ommeril pisthio nuts, orns nd orn flours. J. Food Hyg. So. Jpn. 25: Tsubouhi, H., K. Ymmoto, K. Hisd, Y. Skbe, nd K. Tsuhihir Degrdtion of fltoxin B1 by spergillus niger. Pro. Jpn. sso. Myotoxiol. 12: Downloded from on Jnury 24, 219 by guest