Effect of Temperature Cycling on the Production of Aflatoxin

Transcription

1 APPLIE AN ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p Copyright 1976 Amerin Soiety for Miroiology Vol. 32, No. 3 Printed in U.S.A. Effet of Temperture Cyling on the Prodution of Afltoxin y Aspergillis prsitius1 H. KENT STUTZ2 AN PAUL H. KRUMPERMAN* eprtment offood Siene nd Tehnology, Oregon Stte University, Corvllis, Oregon Reeived for pulition 3 April 1976 Aspergillus prsitius (NRRL 2999) ws grown under yling temperture onditions on rie nd nutmet sustrtes. Under onditions of diurnl nd noturnl time-temperture sequening, totl het input is n importnt ftor of toxin prodution. When expressed in degree hours per dy, therml input eomes more definitive nd provides finite numer, whih n e relted to oservle hnges in the ulture suh s sporultion nd toxin iosynthesis. Three well-defined levels of response were oserved in reltion to het input: no growth ws deteted t therml inputs of less thn 28 degree hours/dy; myelil growth s well s opious mounts of n ornge pigment were oserved t therml inputs etween 28 nd 27 degree hours/dy; sporultion nd fltoxin iosynthesis ourred ove 27 degree hours/dy. Between the optimum nd minimum therml input, yling tempertures signifintly redued the period of the trophophse over ultures reeiving equl het input t onstnt rte. Cyling tempertures t the low nd high extremes of the temperture rnge hd little or no effet upon the growth pttern of the ulture. Regrdless of how temperture ws mnipulted, these responses were onsistent with the het input reeived y the ulture. A. prsitius did not ompete well when mixed with nturl fungl isoltes from nutmets nd ws esily overgrown y the wild isoltes even t reltively high therml input nd when present in superior numers. This ftor nd het input generlly elow tht required for toxin iogenesis t hrvest time pper to e two signifint ftors tht limit ourrene of fltoxin on nut rops of the Willmette Vlley. These ftors re likely to hve signifine for other rops grown nd hrvested under similr irumstnes. We oserved tht when onditions re fvorle, wlnut nd filert nutmets will support hevy growth of Aspergillus prsitius nd prodution of fltoxins. Yet extensive testing of ulled nutmets for fltoxins over severl sesons did not revel their presene in nut rops grown in Oregon nd Wshington. Severl environmentl ftors might e responsile for this nomly, ut dt on the nturl ourrene of fltoxins in griulturl ommodities indite tht temperture is likely to e most importnt. iener nd vis (1) showed A. prsitius strin NRRL 2999 to produe the highest levels of toxin etween 25 nd 3 C, with signifint derese ourring t 35 C nd only slight to tre mounts t 4 C. Shroeder nd Hein (9) found the lower temperture limit of toxin prodution to e 1 C. Optiml nd miniml tem- I Tehnil pper no. 4251, Oregon Agriulturl Experiment Sttion. 2 Present ddress: eprtment of Food Siene nd Tehnology, University of Msshusetts, Amherst, MA 12. pertures of toxin prodution for this strin re similr to those reported for strins ofa. flvus (2, 4, 7, 9, 12). With the exeption of work done y Shroeder nd Hein (1), studies on the reltionship of fltoxin prodution nd temperture hve een onduted t onstnt tempertures. Wheres these studies hve provided onsiderle nd signifint informtion, fltoxin genertion in nturl environment is likely to our under onditions of vrying temperture. Shroeder nd Hein simulted onditions tht might e found in the nturl environment y yling tempertures one over 24-h period. The progrmmed temperture hnge provided n verge temperture of 25 C/24 h. Sine the experiment ws designed to provide n verge temperture of 25, the exposure time t the lower temperture extreme ws, y neessity, of short durtion. In the high-temperture rnge, growth nd toxin prodution delined rpidly, wheres yling tempertures t the lower rnge hd little effet upon either my- 327 ownloded from on July 16, 8 y guest

2 328 STUTZ AN KRUMPERMAN elil growth or toxin prodution. These results re not totlly unexpeted sine the verge temperture for eh 24-h yle ws 25 C, well ove the point where temperture eomes limiting. Under onditions of diurnl nd noturnl time-temperture sequening, the time ulture is exposed to given temperture is n importnt ftor for toxin prodution. The reltionship etween the two n etter e expressed in terms of totl het input during the period of the yle. Therml input is more definitive nd provides finite numer, whih n e relted to oservle hnges in the ulture suh s sporultion nd toxin iosynthesis. The study presented in this pper ws designed to hrterize the effets of het input upon the funtions of A. prsitius nd determine the miniml therml input required for fltoxin prodution. Other fungi tht our nturlly nd in signifint numers on filerts nd wlnut mets were lso studied to determine the effets they might hve upon the development of A. prsitius when therml input eomes limiting. MATERIALS AN METHOS Culture. A strin of A. prsitius (NRRL 2999) ws seleted for this study euse of its prolifi myelil growth nd prodution of fltoxins t ool (1 to 13 C) refrigertion tempertures (12). The stok ulture ws mintined on Czpek gr nd stored t 5 C. Spores of the stok ulture were trnsferred to fresh Czpek gr nd inuted t 28 C for 5 dys efore use. To provide uniform inoulum throughout the study tht ould esily e quntitted, spore suspension ws prepred from spores eseptilly wshed from gr with sterile.1 M phosphte uffer, olleted on sterile filter pper, nd dried in vuum desitor. The dried spores were uniformly dispersed in dried, sterile, powdered ellulose. Wild strins of Peniillium hrysogenum, Aspergillis niger, Aspergillis flvipes, Aspergillis fumigtus, nd Rhizopus sp. isolted from the nturl flor of moldy filert nd wlnut mets were lso used in this study. These ultures were hndled in the mnner desried for A. prsitius. Medi. Polished rie ws used s sustrte for determining the influene of therml yling on growth nd toxin prodution. The medium ws prepred y mixing 1 g of rie with 4 ml of wter. Steriliztion ws done y utolving for 15 min t 1 C (15 l/in2). Wlnut nd filert sustrtes were prepred y dding 1 prt wter to 1 prt hopped nut kernel. These sustrtes were sterilized in the sme wy s the rie medium. Growth onditions. A growth hmer ws onstruted y modifying household refrigertion unit. Two 1,-W heting elements were pled in the refrigerted hmer; one ws designed to override the ooling unit nd provide ontrolled temper- APPL. ENVIRON. MICROBIOL. ture rise, nd the other ws designed to e overridden y the ooling unit nd provided ontrolled temperture deline. iurnl nd noturnl time sequening ws ontrolled y 24-h loks. Temperture during the stedy stte of the yle ws ontrolled within + C. The time nd temperture of eh yle ws ontinuously reorded to provide dily reord. iurnl nd noturnl time sequening ws lso omplished y using series of inutors operting t fixed tempertures. Cyling ws exeuted y mnully shuttling ultures etween inutors t 12-h intervls. Moisture ws not limiting in either method of inution. All smples were held t the indited temperture-time sequene until sporultion ws oserved. At this point ultures were ssyed for the presene of fltoxins. The tempertures in the growth hmer were djusted to provide 8.5 C differentil etween the high nd low temperture. The rnge ws from to C. The time required for the temperture to rise nd fll ws ontrolled to spn 3-h period. The temperture in the shuttle system lso rnged from to C; however, rtes of temperture rise nd fll were not ontrolled nd were different for eh progrm. The time for the temperture to go from one extreme to the other required from 1 to 2.5 h, depending upon the temperture spred of the progrm. Therml input. Therml input under yling onditions of tempertures ws lulted y modified method desried y Seton (11) nd used y gronomists to shedule plntings nd predit hrvest time of rops. To determine the het units reeived y ulture eh 24 h (one temperture yle), it ws neessry to divide the yle into periods of onstnt temperture nd periods of temperture hnge. The totl het input for eh period ws lulted y multiplying the temperture y the time of eh period. The men verge temperture ws used for the period of hnging temperture. The sum of these periods divided y 24 expressed het units s degree dys. Toxin determintion. Assys for fltoxin were mde upon eh sustrte t the time inution ws terminted. The presene or sene of toxins in the hloroform extrt ws onfirmed y thin-lyer hromtogrphy ording to the method of Pons et l. (6). When nutmets were the sustrte, the proedure ws modified to seprte oils nd other lipid mterils from the smple efore thin-lyer hromtogrphy y pssing the extrt through sili gel olumn ording to the proedure of Eppley et l. (5). RESULTS AN ISCUSSION Three well-defined levels of response were oserved in reltion to the mount of therml input reeived y the strin of A. prsitius used in this study (Tle 1). No growth ws deteted t therml input of less thn 28 degree hours/dy. Between 28 nd 27 degree hours/dy, myelil growth ws oserved s ownloded from on July 16, 8 y guest

3 VOL. 32, 1976 AFLATOXIN PROUCTION BY A. PARASITICUS 329 TABLE 1. Progrm Temperture progrms simulting diurnl nd noturnl temperture yling nd therml input required for fltoxin iogenesis Temp ( C) Temp differene ys to fltoxin egree hours/ Level of redy sponse Mximum Minimum ( C) iogenesis I A II A III A IV A V A VI A VII A VIII A Response:, Sporultion, fltoxin;, myelil d mesurle growth response. NA, No fltoxins NA, NA, NA, NA, NA, NA, NA, 68 NA, 14 NA, well s iogenesis of opious mounts of yellow pigment, yellow in olor in erly stges of growth nd turning deep ornge s the ulture ged. Aove 27 degree hours/dy, ultures onsistently sporulted nd produed fltoxin. Regrdless of how temperture ws mnipulted, these responses were onsistent with the het input reeived y the ulture. In the study onduted y Shroeder nd Hein (1), miniml temperture yling ws djusted to provide n verge of 25 C. Therml input t this verge temperture pprohed 6 degree hours/dy, nd exposure time t the lower rnge of tempertures ws of short durtion. It is ovious tht therml input ws well ove the point of eing limiting, sine temperture yling hd no signifint effet on toxin prodution. Tle 1 lso shows time lpse to toxin iogenesis to e dependent upon the rte of het input. Figure 1 is grphi omprison of three temperture progrms hving totl het input ove 27 degree dys. Line A of Fig. 1 is plot of the A portion of eh temperture prolevelopment, ornge pigment, no spores, no toxin;, no grm indited in Tle 1 nd represents the effets of different levels of therml input upon sporultion nd toxin iogenesis when het is pplied t onstnt temperture. Lines II nd III re plots of temperture progrms II nd III in their entirety nd represent the effets of different levels of therml input upon sporultion nd toxin iogenesis when het is pplied y yling temperture onditions. As the therml input per 24 h pprohed the optimum (772 degree hours/dy) yling temperture hd little effet upon the time required for sporultion nd toxin iogenesis. This onfirms the oservtion y Shroeder nd Hein. It ws lso oserved tht temperture yling hd little effet upon the time required for sporultion nd toxin iogenesis s the het input pprohed the minimum requirement. Between the optimum nd minimum therml input, yling temperture notly dereses time for sporultion nd toxin iogenesis. Fungi frequently isolted in lrge numers from nturlly ourring moldy filert nd wlnut mets were A. niger, A. flvipes, P. hry- ownloded from on July 16, 8 y guest

4 33 STUTZ AN KRUMPERMAN APPL. ENVIRON. MICROBIOL. A Cyled Temperture O Cyled Temperture II * Constnt Temperture A eoee Septemer is the most likely time of the yer when onditions might fvor toxin iogenesis, ut most of the rop is still tree ome nd moisture is usully limiting. Most of oth nut rops drop to the ground in Otoer, whih is oinidentl with the strt of sesonl rinfll. The wether reords (Tle 2) showed signifint numer of dys in Otoer of eh yer with het input ove 27 degree hours/dy; however, few dys ove this level were onseutive nd the therml input ws insuffiient in ll ut 1 yer to provide onditions fvorle for toxin prodution. Conditions re even less fvorle in Novemer, when the lst of the rops re hrvested. In summry, het input generlly elow tht required for toxin iogenesis t time of hrvest nd the nonompetitive nture of A. prsitius in the presene of the nturl miroflor, prtiulrly t ooler tempertures, pper to e two signifint ftors tht hve limited the ourrene of fltoxins on the two mjor nut rops of the Willmette Vlley. These ftors would likely hve signifine for other rops grown nd hrvested under similr irumstnes. Temperture yling signifintly redued the period of the trophophse nd elerted the tropho-idiophse trnsition over those ul hi hi soo 4* 3-5 lo Is TIME (AYS) FIG. 1. Effets of yled nd onstnt tempertures upon the time required for A. prsitius to sporulte nd produe fltoxins. sogeum, nd Rhizopus sp. These fungi were sujeted to the sme temperture progrmming s A. prsitius. Sporultion time for these fungi nd A. prsitius is ompred in Fig. 2. All wild isoltes were shown to hve greter tolernes to old nd grew more vigorously t lower therml rnges. It is signifint to note tht ll wild isoltes not only sporulted onsiderly elow the therml input required for A. prsitius to sporulte, ut lso well elow the therml input required for A. prsitius to grow. Mixtures ofa. prsitius with individul isoltes inoulted upon rie nd nutmets demonstrted tht A. prsitius did not ompete well nd ws esily overgrown y the wild isoltes even t reltively high therml het input nd when present in superior numers. Afltoxin ws not deteted in these sustrtes. Meteorologil reords representing the previling wether environment where nut rops re ultured in the Willmette Vlley of Oregon were ompiled over 1-yer period nd onverted to degree hours per dy for the ritil months of Septemer, Otoer, nd Novemer. t from these ompiltions re reorded in Tle 2. Conseutive dys tht equled or exeeded 24 degree hours/dy were lso reorded. ownloded from on July 16, 8 y guest

5 VOL. 32, 1976 AFLATOXIN PROUCTION BY A. PARASITICUS 331 * P.hrysogenum A. niger A A.flovipes O Rhizopus sp, * A. prsitius 49 3 U'. w w TIME (AYS) FIG. 2. Comprison of sporultion times of severl fungi to A. temperture. TABLE 2. prsitius under onditions of yling ys per month nd onseutive dys where degree hours per dy exeeded 27 in the Willmette Vlley, Oregon Septemer Otoer Novemer Yer ys Conseutive tive re- ys Conseutive dy tive re- ys Conseu- Conseumonth dy pttern quired mper pttem quired per tive dy t ivqired dys" dys dy (1) (2)4(2) (24) (1) (4) (1)6(1)4(3) (1)1(6) (1) (17)2(4) (1)7(2) (5)3(2)2(1) (1) (1)11(7) (1)4(2)4(5) Numers in prentheses indite intervening dys elow 27 degree dys. Required dys for iogenesis of fltoxin ws determined on the sis of the verge degree hours ove 27 reeived during onseutive dys indited. 3 ownloded from on July 16, 8 y guest tures reeiving equl het input t onstnt rte. The elertion is prtiulrly notle over the mid-region of the temperture rnge (19.5 to 25 C). Cyling t the high nd low extreme of the temperture rnge hd little or no effet. The lk of distint idiophse is hrteristi of ultures inuted t less thn 27 degree hours/dy. The mehnism y whih the ulture mkes the tropho-idiophse trnsition is pprently repressed y low het input nd fltoxins, the seondry metolites of the idiophse, re not formed sine trnsition to the idiophse is never mde. The mehnism y

6 332 STUTZ AN KRUMPERMAN whih the pigment is formed is tivted in response to limited or suoptiml growth imposed y limited het nd is, in sense, seondry metolite of the trophophse. Two other ornge pigments hve reently een desried: versionl ette produed y A. flvus inhiited in fltoxin iosynthesis y dihlorvos (3, 13) nd mutnt strin of A. prsitius, impired in fltoxin iosynthesis (8). The pigment formed y A. prsitius repressed y low het input ppers to hve physil hrteristis different from those desried for either verufin or versionl ette. LITERATURE CITE 1. iener, V. L., nd N.. vis Afltoxin prodution y isoltes ofaspergillus flvus. Phytopthology 56: iener, V. L., nd N.. vis Reltionship of environment to growth nd fltoxin prodution y Aspergillus flvus in stored penuts. J. Al. Ad. Si. 37: onkersloot, J. A., R. I. Mteles, nd S. S. Yng Isoltion of Averufin from mutnt of Aspergillus prsitius impired in fltoxin iosynthesis. Biohem. Biophys. Res. Commun. 47: Eldridge,. W Influene of temperture on fltoxin prodution y Aspergillus flvus in soyens. J. Al. Ad. Si. 37:191. APPL. ENVIRON. MICROBIOL. 5. Eppley, R. M., L. Stoloff, nd A.. Cmpell Collortive study of "A verstile proedure for ssy of fltoxins in penut produts," inluding preprtory seprtion nd onfirmtion of identity. J. Asso. Off. Anl. Chem. 51: Pons, A. W., Jr., J. A. Roertson, nd L. A. Goldltt Ojetive fluorometri mesurement of fltoxin on TLC pltes. J. Am. Oil Chem. So. 43: Shindler, A. F., J. Plmer, nd W. V. Eisenerg Afltoxin prodution y Aspergillus flvus s relted to vrious tempertures. Appl. Miroiol. 15: Shroeder, H. W., R. J. Cole, R.. Grigsy, nd H. Hein, Jr Inhiition of fltoxin prodution nd tenttive identifition of n fltoxin intermedite "versionl ette" from tretment with dihlorvos. Appl. Miroiol. 27: Shroeder, H. W., nd H. Hein, Jr Afltoxin: prodution of the toxins in vitro in reltion to tempertures. Appl. Miroiol. 15: Shroeder, H. W., nd H. Hein, Jr Effet of diurnl temperture yles on the prodution of fltoxin. Appl. Miroiol. 16: Seten, H. L Sheduling plntings nd prediting hrvest mturities for proessing vegetles. Food Tehnol. 9: Vn Wleek, W., T. Cldemenos, nd F. S. Thther Influene of refrigertion on fltoxin prodution y strins of Aspergillus flvus. Cn. J. Miroiol. 15: Yo, R. C., nd. P. H. Hsieh Step of dihlorvos inhiition in the pthwy of fltoxin iosynthesis. Appl. Miroiol. 28: ownloded from on July 16, 8 y guest