BO Y TE TRUONG DALHOC DUQC HA NOL HOANG THI THANH HUYEN NGHIEN CUU HE VI NAM TREN CAC VI THUOC SINH DIA VA SAI HO NAM DANG LUU HANH O CAC HIEU THUOC DONG DUQGC THUOC DIA BAN HA NOI KHOA LUAN TOT NGHIEP DUQC Si Nguoi hudng din: ThS. Tran Trinh Céng Noi thue hign : Bé m6n Vi sinh — Sinh hoe Trung Dai hoe Durge Ha NGi Thoi gian thue hi¢m: = Tar 10/2009 dén 04/2010 HA NOI- 2010LOI CAM ON Vii tang bids on sin sde, 161 xin ehdn thanh eam on thi ThS Tren Trinh Céng—Gidng vién bd mdn Vi sinh-Sinh hae, truémg Dai hoc Duge Ha Ni, nguuii da trive tiép Inedmg atin va tdn tinh chi bao toi nhiing kién hike qu bau trang qué irink thue hién va hon tharh kheod luge, Téi xin giet loi cam on tét tép thé can b6 BG mén Vi sink—Sinh hoe, tmudng Dai hoe Due Ha Noi, nhitng ngwii da nhiés tink gitip dé tao moi diéu kién cho tdi thye hién dé tai. Nahar dip nay, toi cing xin giti loi cam en tdi Ban gin higu nha trong cig ton thé ede théy co wudng Bai hoe Duge Ha Noi da wang be kien hire, gitip da tdi rang suéz thoi gian hoe wap tai iewéng. Cudi cing, téi xin bay té long biét on dét vei gia dih, ban be da Tuan gitip db, déng vién tdi trong sudt thd gian qua. Ha NGi, ngdy 18 thang OS nd 2019, Sinh vin Hoang Thi Thank HuyéaMUC LUC BAT VAN BE sesiscencscsereectinentn CHUONG 1: TONG QUAN. Ll 1.1.1, Nganh ndm tiép hyp — Zygomycota 1 1.1.3. Nhém dim bat ton — Deuteromyeetes. Cac nhom nam thuong giip trén cdc san pham cé ngu Neanh nam thi — Ascomycota 1.2. Tinh hinh aghién ecu mie d6 nhiém nam mée wén duge ligu & Viet CHUNG 2; BOL TUQNG WA PHUONG PHAP NGHIEN CUU 2.1, Nguyén vat ligu, thiét bi .. 2.1.1. Bai turgng nghién etru.. 2.1.2, Moi truéng phan lap va xdc dinh nam mac... 2.1.3. Thiét bj thi nghiém 2.2. Noi dung nghién cia 2.3. Phuong phap nghign ctr. 2.3.1. Phuong phap lay mau ..... 2.3.2. Phuong phdp xée dinh him dt duge lt ..c.eccecnnce 2.3.3. Phuong phap phan lap nim m6e ......2--essceseesetsineesiennanete Dl 2.3.4. Phuong phap phan loai nam méc CHUNG 3: THC NGHIEM, KET Qua VA BAN LUAN...0...seecseneis23 3.1. Hé vi nm wén vj thude sinh di 3.2. Hé vindm trén vj thuée sai hé nam KET LUAN VA KIEN NGIKY HIEU VIET TAT Aspergillus flaves — ; A. flavus Penicillium citrinum : P. citrirum Rhizopus stolonifer +R. stolonifer AEPA : Agpergilltes flavus and parasitions Agar CMA : Com Meal Agar CREA : Creatine Sucrose Agar CYA :Czapek Yeast Agar DGIs : Dichloran 18% Glycerol agar HA Infusion Agar MEA Malt Extract Agar 4%. PDA : Potato Detrose Agar DBYN IV : Duge dién Vigt Nam IV DK : Duong kinh KL : Khuan lac DANH MUC CAC BANG Bang ‘NOi dung ‘Trang Bang 1.1 | Cac chi, lodi vi sO ching ndm phin lap duge tir cdc mau 24 cba vi thude sinh dia nghién cima Bang 1.2 | Cac loai va sé lueng ching phin lp duge tir 10 mau cua 26 vi thudc sinh dja nghién citu Bang 1.3 | Cac chi, lodi va s6 ching nam phan lip duge tir cdc mau 32 cla vj thude sai hé nam nghién ciru Bang 1.4 | Cac loai va sé Luang. ching phan lap duge tir 10 mau cla 34 vi thuse sai hd nam nghien ctuDANH MUC CAC ANH Anh Noi dung, Trang | i Z, 1 Anh 11 | Bac diém hinh thai cha chi Rhizopus 4 Anh 1.2| Cau trie sinh conidi eta chi Aspergilles 9 Anh 13 | Cau trae co quan sinh conidi va conidi cua chi Pericifiium | 14 Anh 3.1) Loai Aspergillus niger vhidn teen vi thude sinh dia 28 Anh 32| Loai Penicifliunt cftrinune nhiem teén vj thude sinh dja 29 Anh 33 | Loi Aspergillus «amarii nhiém trén vj thude sinh dja 30 Anh 3.4| Loai Aspergillus fuunigatus nhiém trén vj thu6e sai ho nam | 35 Anh 3.5 | Lodi Rhizopus stolonifer nhiém wén vj thude sai hd nam 36 Anh 1 | Vi thuéc sinh dia truéc khi phan lap | Anh 2 | ¥i thuée sai hé nam trude khi phan ipDAT VAN DE Vigt Nam [i mé@t nude e6 nén y hoe od truyén phat tien tir Mu. Ngay nay bén canh nény hoc hign dai, nén y hoe cé tmyén ching ta van ngdy cdng phat trién manh m@ véi nhing tinh ning uu wigt eta né. Thude y hoe co truyén va ede ché phim déng duge da s-ed nguén gée thye vat [12]. Thue 1 cde phuong phap, phuong tién che bién, bdo quin duge ligu con nghéo nan lac hau, chit yéu 1a phoi séy theo kinh nghiém, Diéu do lam cho ede duge ligu rat dé bj ndm mde xdm nhiém va phat trién, nat 1a trong dieu kign khi hau nhiét déi néng dm & nude ta. Khi bi nhiém nam mée, ede sin phim niy bi gidm chat ligng nhu gidin ham luomg hoat chat, vitamin, lipid, tinh bét, bin mau, sinh mii vi khé chin... hon nfa cén bi nhiém céc déc 16 nam (myeatoxin), Mysotoxin la sin phim chuyén hoa thi cap doc hai cia nam. Hign nay da c6 trén 200 mycotoxin duge con ngudi phat hign va mghién ciru [23]. Trong sé nay cé nhiéu chat déc hai doi vai con nguai va déng vat khi hit phai, &n phai hofe tip xtic qua da. Cie dée 6 nam nay gay ra nhiéu loai bénh khée nhau cho con ngwéi vi déng vat goi chung 1A cde bénh do de té nam (mycotoxicoses), voi céc tae dOng tir cp tinh dén man tinh, c6 thé dan dén quai thai, ung thir... [4], [20]. Trong céc mycotoxin, dang lua y nat la aflataxin- dgc t6 e6 kha nang gay ung thu gan [6], [15]. Mfc di ta khéng thé phi nhn vai tr to lon eta hé vi nm trong d6i séng hing ngay (1am tueng, ché bién phomat) cling nhu trong nén kinh té (san xudt khdng sinh, vitamin, axid hiru co), nhung nhing tic hai ma ching mang lai rit Ién, Nam [a nhom vi sinh véit e6 anh hudng quan trong d6i vei hd lugng va dé an toan ctia duge ligu. Tuy nhién déi vei mai loai duge ligu, ede logi ndm khdée nhau od vai tro, doh hudng khée nhau. Do véy vide didu tra hg vinam dic trmg trén timg co chat la mét burée quan trong, mang tinh quyéty dinh trong vigc tim ra cac bién phdp hitu higu dé phong trinh céc tac hai néu tren. Voi muc dich, ¥ nghia do ching toi tién anh thye hign dé tai; “Neghién circu hg vi nine irén cae vi thude sinh dia va sai ho nant dang fu hinh & cde hiéu thudc ding duge thebe dja ban Ha Npi” vei hat myc 1, Phan lap va phan loai céc ching ndm nhiém trén vi thude sinh dia dén. cap chi & loai, 2, Phan lip va phan logi cdc ching ndm nhiém wén vj thude sai hd nam d&n cap chi & loai.(CHUGNG 1: TONG QUAN 1.1. Cae nhém nim thuong gap trén cde sin phim c6 ngudn goc thyre vat Cac co chat ed ngudn géc thye vat nhu cdc loai hat leong thye, co chat thyre vat chét ..., thuémg co mgt cic nhém nm quan trong nhu nganh nam tiép hep (Zygemycota), nednh nim thi (Ascampeota) va nhém nam bat toan (Fungi Imperfecti hay Deuteromycetes). Sau day ching t6i xin trinh bay mgt sé nét chinh cha cdc nhém nam ké trén. 1.1.1. Ngan nim tiép hop — Zygomycota LL1.1. Cae die tink quam trong Bay la nhém nam phé bién trén long thie, diac thio. Nhém nay cé hé soi nim céng bio, ede vieh ngin chi duge hinh thanh dé phan chia cdc té chite dae biét nhur nang bao tr (sporangium). Bao tr tiép hop (zygospore) duge hinh thanh tir ede hé soi hitu thy, ¢é vach day, chic Sinh san vé tinh bing cdc bao tir mang (sporangiospore), due ndi sinh trong cde nang hinh cdu hoge hinh qua 1é e6 hoac khéng c6 bong hogc phan trung tam bén trong nang (try nang-colummella), ti¢p néi bei mot cudng nang (sporangiphore). Hoge cdc bao tir VG tinh duge sinh ra trong ede nang bio tir chudi (ching duge phan ra thinh mét hang cdc bao tt chudi-merospore), nang bio tit chudi khong oé try nang. Sporangiole (cde nang bao tir thuéng ¢é hinh cau, véi mét hoe mot s6 it bdo ur vi try mang bj tiéu bién) bat gp & cdc ho Thamnidiaceae, Cunninghamellaceae va Cheanephoraceae. Mot sb chi cb cdu tric dae trmg apophysis (phan phng lén cia cudng nang, ngay sit bén dui nang). Sq! bé (stolon) cé thé xudit hi8n, IA ede soi dic biét nim trén bé mit thach vA sink ede cudng nang. Cée soi bd nay dink vao eo chat bing cae soi giéng nhu ré goi la ré gid-rhizoid [Anh 1.1]. Mét sé loai sinh ede bao ti thanh day (chlammydospore), hoac cdc bio tr thanh mang (oidium) thuongcé hinh cau, ching 6 thé dirge sinh G cudi soi, xen ké, don doc hode thanh chudi, Cac bao tir nang, bae tr thank day, bao tir thanh mong néy mam sé tao thanh hé sgi mei. Sinh san hitu tinh bang sy hoa rn cdc nang giao tir da hin, don bi (gametagium) tao ra m6t bao tit tiép hop (zygospore) thanh day, mau vanp, nau hode den, thurémg duge bao phi bi mOt lép cfc gai nhon va cae cau trie 18i ra khac, Hai phan soi ¢ hai dau két thie cia bao tir durge goi ld cudng nodin (suspensor). Cac cudng nodin ndy 6 thé giéng nhau hoge khéng gidng nhau wé kich thuée va hinh dang. Béi khi cuéng nofin hinh thanh cc phan phy hay mau (nhu & in6t s6 loai cia chi Absicia). Hiu hét cdc thanh wign cia nhom nay la eac sinh vat hogi sinh, tay nhién mot sé loai thuimg gay hai cdc loai nam khac, gay cae bénh Gnguii va thyre wit. M6t sé loai cha Mucor va Rhizopus cé vai tro quan trong trong qué trinh Ién men thye phim va san xudt cdc acid hitu co, nhung cing gay tic hai nh lam théi nhiin céc Joai rau, qua chin sau thu heach. Bénh do nam Mucorales gip chi yeu 6 cde: chi Rhizopus, Rhizomucor, Cuninghamella [24]. sporanguim > & iy ay * 98 : porangiophore — stolon word Anh 1.1; Bae didm hinh théi cita chi Rhizopus Reé gia (rhizoid), sai ba (stolon) wa nang bio tir (sporangium)1.1.1.2. Nudi cdy dé nhin dign Cac ching cua nhém nam nay thudng duge nudi cay trén mdi trong MEA (Malt Extract Agar 4%) trong cic dia Petri nhya. Déi vai chi Mucor, viée nudi cdy nhu vay sé tao ra su phat trién khong bj xdo tn do phat trién qué mire (Schipper, 1973), Nhigt d6 nudi edy thay di tir 20°C déi vai chi Mucor, dén 20-30°C ddi vii cdc chi Rhizopus va Syncephalastrum va x4p xi 36°C ddi vii chi dbsidia, Tat ca cdc loai duge nudi cay trong moi trutmg wi trong vong mét tulin, Dé tao digu kign cho sur phat trign bao tir tiép hep, méi tring vi nhiét d} thudmg duge luin phién. Hai ching (+ va -) duge edy trong pharm vi 0,5-1 em étrung tim dia petri. Cac bao ut tiép hop s@ phat trién a viing 6 sw tidp niie ghép dai. 1.1.1.3. Khod phén logi m6t 56 chi pho bién trén ede logi hat long the: Hesseltine va Ellis (1973) chia ho Marcoraccae thanh 20 chi trong dé chi Rhizopms va chi Mucor la quan trong nhat. ta. Bio tir nang due hinh thanh trong cde nang chudi bao tr merosporangium bao phil xung quanh mét bong (phdn cudi phéng lén ctia gid nang-sporangiophore) .... eis _Syncephatasirum 1b. Bao tirnang duge hinh thanh trong cdc nang hinh cdu hodc qua 1é 06 16i Nang-ColuMmMel a os ..s0s 0000 2a. Cie nang va gid nang co mau t6i, gid mang hau nhu khdng phan nhanh, thuéng xuat hién thanh nhém. Burong kinh (DK) nang thay doi tir $0- 360 um. Bao tir thudng eb soc... » Rhizopus 2b. Cic nang va gid nang khGng cé mau hoje chi co mau nhat, thing phan nhénh nhiéu, DK ede nang khéng bao git vugt qué 100 um. Bio 3 tir thurimg khéng €6 soe. 3a. Che nang hinh qua 1é di mét apophysis dic tamg, DK 10-40 jum (ede bsidia nang cudi cling 66 BK tai 80 pm).3b. Cac nang hinh cau khong 66 apophysis, hau hét 6 duvmg kinh lim hon 40 um... viVFucor 1.1.2. Nganh mam tdi— Ascomycota 1.1.21. Cac die tink quan trong Hé soi eta nam e6 vach nin va don béi. Sinh san hitu tinh bing cac tdi bao tir (ascus), thudng duge hinh thanh bing vige tiép hyp cla hai nhan tir hai tii giao ti khéic nhau (giao tit dye-antheridium, giao ti edi-ascogonium). Thuing <6 8 (ddi khi 2 hoje 4) bao tir tii duge hinh thanh trong cde thi sau qua trinh gidm phn. Céc tdi bao tir thuémg dirge bao boe trong thé qua tii (ascomata hay ascoearp). Cée thé qua ndy xudt hign dom dge hode két ty lai thanh cum trong hoae trén mét dém (stroma), céu tao bi hé soi dinh duéng. Hinh thai eta cic thé qua cé ¥ nghia quan trong trong viée phan logi. Ching thurong 6 dang nhw eé< (apothecia), dang edu hode gan cau, khéng c6 lé mé (thé qua kin-cleistothecia), ho&e ede thé qua hinh binh {perithecia), Cac thi bao tir cing 6 thé phat trién trong cic 6 cla ede lép dém (stromata), hode moe lén tir ede cau trie gidng géi dem (pseudothecia). Trang thdi sink tii bao wr (teleomerph) ihudng duge kem theo mot ho&e nhiéu cae trang thai sinh sin v6 tinh (anamorph). Da sé ndm bat toan Derteromyeetes (Fungi imperfecti) 08 trang thai anamorph trong cde chu ki) adag oba tila til. Tat ca cdc chi ndm ii duoc néi dén & day déu thude bé Euroriales, cic thé qua tii ed dic trumg la cde tii bao ti nho inh cau hoge gin cau, chia cae bio tir tdi (ascospore) ed céiu wtic bé mit da dang. Dang vé tinh dirge mé 88 & day thude cde chi Penicillium, Paecilomyces, Basipetospora va Aspergillus. 1.1.2.2. Nudd cay dé nbdin dién Cée ching phai duge mudi edy trén cic méi trudng thich hap. Cac chi Byssochlam)s, Monascus, Emericella, Talaramyces va Neosartorya hinhthanh bao tir tat nhat én ede moi tung MEA, OA 6 24-30°C. Cac ching cla chi Eurorfum phat tién cham wén cdc moi truéng nay va tao ra cae conidiophore khéng dién hinh, cdc thé qui [ép. D8 Eurosium hinh thinh bao tir toi wu, phai nudi cay trén cde mdi trmg MEA, Czapek bé sung thém 20- 40% duréng kinh hode 10-30% NaCl. Bé phan lp nén ding méi traong DGIS (Dichloran 18% Glycerol agar). Hau hét ede loai ndm tli due mdi téi day chin sau 10-14 ngay. Luu y ring m6t sé loai ndm tii cia c&e chi nhu Tataromyces va Neosartorya, dang v0 Unh dOi khi kho tim thdy va ching chi xudt hign twén méi trudng gid hoge khi nudi cdy én méi trudng nghéo nhu Potato Carrot hoac HA (Infusion Agar}. 1.1.2.3. Khod phd logi la. Thé qua thi co cuéng dic trung, cudng edu tao béi mét soi don. Dang vd tink anamorph la Baisipetospora fanascus 1b, Thé qua tai khong cé cudng, dang v6 tinh | Penicillium, Paccilomyces hode Aspergillus a ie? 2a. Thé qua tii khéng e vach dic trig. Dang v6 tinh 1a Paccilomyces .. Byscochlamys 2b. Thé qua tdi cd mét ldp vch do. Dang vé tinh la chi Aspergillus hode ud Dang vé tinh [8 chi Penicillium. Thé qua tii co lap v6 boc dac tring, Penicillium... 3a, mau vang di khi chuyén thanh mau hoi dé . Tataromyces 3b. Anamorph la Aspergillus. The qua tii co cae té bao Hiille, hoje co | dén 2 1ép thanh bao boc . A 4a. Vach eda thé qua tdi durge bao quanh béi ede té be Hille od thanh day, Anamorph la nhém \i A. nidulans: Emericella 4b. Vich eda thé qua tdi khong durge bao quanh béi cdc té bio Hille.......5Sa. Thé qua tii mau vang, vach tii dugc cdu tao boi mot lép té bio det. Anamorph la nhom lai A, 2faziciis wom veo MrO THEE Sb. Thé qua tui od mau kem, vach tii duge ciu tgo boi hai lap té bao det, Anamorph la nhoém Joai A. fumigatus .,...... seosneeeses Neosertor yar 1.1.3. Nhom nim bit toan — Deuteromycetes Deweromycetes hay nim bat ton bao gbm cae tac nban ly nhiém thye phém quan wong, nhiéu loai co thé sink céc chat chuyén hed doc. Lop nay gém cde dang chi va loai chi cé wang thai sinh sin v6 tinh (trang thai conidi hay ve tinh) 1a da biét. Sau day ching t6i xin trinh bay dac tinh ctia 2 chi quan trong la Aspergillus va Penietllitun, LL.3.1. Chi Aspergillus Micheli ex Fries Chi Aspergilfus do Micheli mé ta nim 1729, sau dé nim 1832, Fries chp nhgn chi nam nay cia Micheli va thee ludt quée 1é vé danh phdp thye vat, chi Aspergillus chinh thire mang, tén Aspergitlus Micheli ex Fries [26]. Cée dgic tink quan trong Khuan lac thurémg phat trién nhanh, cd mau trang, ving, nau vang, ndu téi mau den, hodc nga mau xanh Id ey, chil yéu duge cau tao bai mét lap diy ctta cdc conidiophore thing dimg. Conidiophore (thurong khéng phan nhanh) dirge céu tao boi mot cudng (stipe) khéng phan nhanh, voi phan dinh phong to goi la bong (vesicle). ‘The binh (phialide) duge sinh tryc tiép trén bong durge goi la the binh mét lop (uniseriate), howe trén eudng thé binh (metulae) | thé binh hai lép (biseriate), Bong, thé binh, cuéng thé binh (néu cé mat) va conidi hinh thank m6t khdi conidi (conidial head). Conidi & dang chudi khé ¢6 thé hinh thanh dang edt (columnar) heac dang phan ky (radiate). Conidi (conidium) chi gim mot té bao, bé mat nhiin hoge xd xi, cé gai, khéng mau hode cé mau.9 Mat 6 loai cé the sinh té bao Hille (HUlle cell) cé thanh nhin, day don déc hogc thanh chudi _ cae hach ndm (sclerotia) la cde khdi soi thuémg cé hinh cau cimg, Cac trang, thai hitu tinh: Eurotfum, Emericella, Neosartorya va cae chi khade. Cic loai cla chi Aspergillus 14 nhimg tac nhan lay nhiém pho bién trén cde co chat khae nhau, dic biét Ia trén ede loai hat long thue, cdc loai co chat chét co nguén goc thy vat [21]. O cac ving nhiét doi (nhu Vigt Nam) va can nhigt déi, sy xudt hién etia ching phd bién hon rit nhigu so v6i cdc loai cha chi Penicillium. Mgt s6 lodi da thu hit durge sit quan tim die bigt do ching la nhtig:tie: aida aly bak # neni vA dong Wee node Kad ping sind cae chk chuyén hod thir cép déc.Thé nhiém nam duge chi ¥ nhiéu nhat 1a u nam phéi do Aspergillus. Lodi nbit la Aspergillus furigats nén cin c6 bign phap phong tranh hit phai bdo te. Mt s6 loai khde c6 tim quan trong trong vige Ién men cae sin phim truyén théng phuong déng, hode cde tng dung trong céng nghé sin xuit acid hitu co holic ede enzym [21], [26] ‘Cau tie sinh conidi cla chi AspergillusVige phan logi chi Aspergillus chia yéu dya tren cde die diém hinh thai, co bin dys tron cfu trie khdi eonidi (conidial head), miu sic conidi. Mau sc Khun Ige cic loai cia chi Aspergillus do miu conidi quyét dinh. Raper & Fennell (1963) da chia chi nay thanh 18 nhém (group) va da chap nan 132 lodi vai 18 thir, Hign nay s4 lweng cic loai cua chi di duge phat hign lén 16 trén 200 loai, trong dé véi khoang irén 70 loai IA cdc trang thai hiru tinh. Raper& Fennell (1965) 44 chia nho chi Aspergilfus thanh ¢de nhom (group). Su phan chia dudi chi nay khong 6 vj tri trong ma danh phap thuc vit quoc 1, boi vay Gams & ¢Ong su (1986) da thay cdc don vj “group” bang cic “Subgenus” va “Section”. Trong pham vi céng trinh nay, ching t6i chi gidi thigu ngn gon va dura vio khod phin loai cdc lodi phé bién nhat, die biét 1a cde Joai thudng nhiém trén luong thye, san pham c6 nguén géc thye vat. Do vay dé co thé nbn dién chinh xée cde ching quan trong edn tham khéo thém cde tai ligu khac (Raper & Fennell 1965, Samson 1992, 1994a &b). Nhu da ndi & trén, cdc lodi cla chi Asporgillus 14 nhting tae nhfin Ity nhiém rét phé bign, do vay khéng cd mét anh sich cy thé cde cor chat ma mBi mGt loai duege phiin lp. Cae dit idu we phan bé ctia nhiéu loai 48 duge Domsch va eGng su ligt ké 1993, Cic chi dan dé nhan dién cdc loai Aspergilfus chi yéu dua tén cdc dic tinh hinh thdi khuan lac 48 duge nhieu téc gia cng bo nhu Pitt & Hocking (1985), Klich & Pitt (1988a), Tzean & cOng sy (1990). Nudi edly d8 abgin dign: Cie ching duoc cay tai 3 diém trén moi trending Czapek va MEA 2% va 016 25°C, Déi véi cdc loai nam wa kh6 nhu 4. penicilloides, mgt s6 loai khac ‘va trang thai Euroriwn c6 thé sir dung cic méi truémg Czapek va MEA véi 20-40% duémg kinh. Nuéi cdy cdc ching tai 3 diém trén cde m6i trudng, sit dung dia Petri thug tinh tét hon ede dfa Petri bang chat déo, Hau hét ede loaihinh thanh bao tir trong vong 7 ngay. Mau va edu trie ca khéi conidi (dang cét hodc phan ky) dug quan sit tt hat bing kink hién vi phan tich. Khod phan logi céc loai cia chi Aspergiflus throng gap ten lneng thyc va ede sin pham cé ngudn géc thye vat khée: La. 1b. 2a, 2b. 3a. 3b. 4a. 4b. 5a. Sb. 6a. 6b. 7a. 7b. 8a Khun lac (KL) cé mau trang, den, vang, nau hod xam....... KL ngaé mau xanh 14 cay... Khéi conidi mau tring, wet Khdi conidi mau vang, mét sé nga mau nau hode den... Khéi conidi e6 mau nau téi dén mau den... . Awniger Khéi conidi khéng cé mau néu, ti hove mau den nhung cd mau liu, 3 nn vang hofie nga MAU MA ee tees Kh6i conidi dang c6t thuémg cé mau néu qué dén mau néu shi nhat. A, terreus ae Khéi conidi mau éliu dén nau sang, cudng khéng mau hode. vang nhat Khoi conidi khéng od dang edt mau vang hode nau... thutmg sinh té bao Hille... A ASTUS Khdi conidi khéng e@ mau Oliu dén nau sdng, stipe khong mau hodc vang nhgt khong sinh té bao Hillle.........cc0seseeseeseoee 6 Khél conidi ed mau vang rong, conidi nhin dén rép min...4. achraceus Khoi conidi mau nau vang, conidi khéng ohdin hay edp min. Conidi duge 06 diém 16 net bing cic mun céc va mau nbd, véch ngoai va trong eG thé phan bigt TO... eres A, tamnarié Conidi hau hét rap, wach trong va ngoai khong thé phan bigt duge... «A, wentii Conidiophore mau nau dién hinh, cé mat t8 bao Hiille va dang hiru tinh Rpm Oa ioe icaseeneaeidmocactinesentae nae A, nidutans:8b. Conidiophore khéng co mau nau diém hink, khéng cé dang hiu tinh Erxramter icetlel os sssscsssssanessovenseessancervenieanivann sa 9a. KL tén Capek hoc MEA hdu nhir bi gidi han (dong kinh KL thudng < 1,5 cm trong | tudin).....-..-... aa RC AD 9b. Khuin lac phat trién nhanh hon, dung kinh > 1,5 em... it 10a. KL c6 mau thay déi, khdi conidi 2 ting, ddi khi c6 té bao Hille .A.versicolor 10b. KL mau xanh la cay xam, Khoi conidi 1 tang, tren MEA, Czapek phat trign rit gigi han, nghéo bao wi, trén cae mdi tredng nude hoat tinh thip, phat trién té1 hon, 16 bao: Hille khdng cé. .. A. peniciltoides Ila, Trén mdi trudng nudi cdy gid bolic cde méi rubng nude hoat tinh thip, 6 giai doan hin tinh Evrotiwn mau vang A, ghoucus 1b, Khéng c6 dang hitu tinh Etter mau van 12a. Khéi conidi mau xanh la c@y-vang tGi mau xanb 1a edy-vang 126, Khéi conidi xanh da tréi téi mau xanh La edy ti 13a. Khéi conidi chi yéu | ting, conidi mau xanh Id edy-viing t6i, e6 gai rd sane. parasitions dk sive flavus 13b. Khoi conidi | hoc 2 tang... 14a. Conidi c6 gai nho, xamh ld cay vang.. 1db, Conidi rip hodc nhin khéng cé guy lvat, mau 6! 15a, Khéi conidi hinh edt, bong hinh chuy rong, conidi rap 1 5b, Khéi conidi khOng co dang ot, bong hinh chuy hep, thanh ahin........ oe creed, Clawanns1.1.3.2, Chi Penicillium Link ex Fries Chi Penieiffium thuée ho Monifiaceae, b@ Moniliales, \ép nim bat todn, trong hé thang phan loai hinh thai Saccardo 1886. Cic dae tinh quan trong Khudn lac thudng phat trién nhanh, thudng nga mau xanh ld cay, 461 Khi cd mau tring, chi yéu edu tgo boi mat |ép day cac conidiophore. Conidiophore co thé moe lén tir eo chat, tir ede sof Khi sinh, tr cde soi bé lan trén mat thach, hoae cdc bd soi thang dimg chat hay long. Conidiophore khéng mau thanh nhin hay rap, ching co thé don 4c hode két thinh bd, cdu tg0 gém mét stipe don va duge két thie hoe bang mét ving xofin cla cdc thé binh (phialide), d’y Ko cdu aac chdi 1 vong (monoverticillate) hofe bang, cdc chéi da vong, cd cdc nhanh va metulae (cdc nhénh gin dean cudi mang mét vOng phialide). Tat ca cac té bao gita metulac va stipe dugs xem nhu la cde nhanh. Kiéu phan nhanh: cé thé m@t giai dogn phan nhanh (biverticillate), 2 giai dogn phan nhanh (terverticillate) hoge 3 (quaterverticillate) 163 nhiéu hon ede giai doan phin nhanh. Céc thé binh thiréng cé hinh chai, véi phan day hinh try va phan cé dc trung, ho’ic dang mée, phan day hep nhieu hay it, thon nh6 tdi mét dinh kha nhon (acerose), Conidi sip xép thinh dang chudi dai, khd, phan ly hay dang cét, cé dang hinh cau, elip, hinh try hoge hinh thoi, khéng mau, hodc hei xanh 1a cay, thinh nhin hoge rap. Mat s6 loai tao hach nam (sclerotia), Trang, thai hitu tinh: Expenicéifiwr, Talaromyees, Hamigera va cae chi khae. Nhidu loai ca chi Peniciffium la nhimg tac nhén lay nhiém phd bién trén nhiéu cw chat kha nhau va déng thoi tiém an kha nang sinh doc t6 nam méc, Do vay vide aban diém chinh xdc li van dé quan trong khi nghién ctrukhi ning lay nhiém do chi Penicillium. Vige nhan dign cic loai cla Penicilliwe d8 due Raper va Thom (1949) thuc hign, chit yéu dya trén co sé cdc dac diem nudi cay, nhumg ede die tinh nay cOing da durge chtmg minh 1a kha dé thay di. C6 thé nhain dién duge phan lén cdc ching phin lap dén cap loai néu ede loai nay duge mudi cay G cdc diéu kién chuan cia méi truong, nhiét d6 va dugc nhjn dign sau mGt thdi gian tuong doi ngan la thich hyp nhat. Duémg kinh khudn lac duge xde dinh dé dang va cung cap cac thong tin cé gid tri. Céc dic tinh hinh thai thudin ching ¢6 thé durgc diing cho vige phan loai, ngoai ra cdc dic diém nhu ty 1 phat trién, nude hoat tinh & che mdi trudmg wa ohigt 4 khéc nhau efing hé tre cho vige xéc djnh loai (Pitt, 1979), Viée sit dung cdc dit ligu ve cde chat chuyén hoa thit cap cling cho thay ¢6 pid tr] cho viée nbn dign (Frisvad, 1981, 1985; Frisvad va Filtenbord, 1983; Lund & Frisvad, 1994...). Nhiéu mé ta chi tiét va cde khoa phan logi cia Penicillin cing d& duc céng bd (Pitt 1985). MOt sd loai tao ede chat tiét va mai sé gidip nhin ra vj tri phan loai cia chung, nhung mot diéu can uu y 1a hit phai bao ti va cde chat bay hoi co thé bi stress, pay anh hudng téi sire khoé. Tuy nhién gy bénh o ngwoi chi mot loai duy nhat cia chi gay bénh co hoi la P. marneffei [4], [27]. wy Anh 1,3: Cau tao co quan sinh conidi va conidi cia chi PerticilliumNudi cay dé nhan diga: Samson va Pitt (1985) 4¢ xudt nén cay cae chting @ 3 diém wen moi truéng CYA (Czapek Yeast Agar) va MEA (2%) 0 @ 25°C, toi hoac sing. Tuy nhién nudi cfy trén méi tru’ng Czapek ciing cho két qua hep ly. Mot diém luu ¥ ring sy phat trién khéng dién hinh va cée chat mau ctia cdc loai Penicillium én CYA va Czapek co thé xuat D n, throng Ja-do thiéu cc vét kim loai nang, dic bigt la déng, Kho khiin 6 thé duge giai quyét khi bo sung thém dung, djch déng hoge kém. Cc dia Petri thuy tinh cho két qua tt hon so voi cdc dia Petri nhua, khi nudi cay bing cde chiing khé (bao tirkhé), Hau hét céc loai hinh thanh bao tit sau 5-7 ngay. Mgt sé loai thude ahém Torverticillate Penicillium, khi sir dyng m6i tiring CREA (Creatine Sucrose Agar) cing rat higu qua trong vige phan biét cdc loai co méi quan hé rat than thuge. Cae loai Penicilliem Terverticillate thuge P. aurantiogrisetm/ verrucosum complex li rit kh xdc dinh khi chi dya trén co sir cdc clu trie hinh thai ciia ching. Khéa phan loai duéi day sé tro gidp bd sung cho sir nhan dién Loai. Khod phan togi cdc loai phé bién cla chi Peniciftiten: la. Khuan lac mau trang (hoge xanh la céy xdm nhat). puenaetad Sd Ib. KL nga mau xanh Id cay, d6i khi hé sgi khi sinh mau vang .. 2a. Conidiophore c6 stipe rap sr. P. camemtberti 2b. Conidiophore cé stipe nhan.... nalgiovense 3n, KL trén méi truéng Czapek phat trién va hinh thimh bao tir nghéo nin, conidiophore ngin véi phialide lén dic tamg (dai 12-20 jum, conidi hinh elip dén hinh try [4 cn nguyén gay thai lui qua ho cam chanh...... P. digitatum 3b. KL trén Czapek phat trién va hinh thanh conidi phong phu, conidiophore véi stipe dai dc trrmg va phialide bé hon (5-12 pm)...4a. 4b. Sb. Ga. 6b. Fa. 7b. 8a, 8b. 9a. 9b. 10a, 101 = Ta, = 12a. 12b. Conidiophore mot vong (don gin, khong phin nhénh)......P. géabream Conidiophore phan nhanh ......... ag Phialide hinh kim, conidiophore chi yéu két thie bing vong metulae va phialide (biverticillate) di khi cé chdi 3 wing (terverticillate)..........6 Phialice hinh el 8 i, conidiophore 2 vang, 3 vong dén 4 véng... Khuin lac phat trién nhanh, dung kinh lon hon 1,5 cm trong | tudn trén moi truémg MEA. . P: funicelosume KL bj gidi han, DK < 1,5 em trong | tudn trén méi trong MEA........7 Conidi rp, hinh elip. cieetneunenenn Pe rtagetlostim Conidi aban hode tap thidng 6 hinh thoi........... a P. veriabile Conidiophore chit yeu 2 vOmg ...-sccssescsssesccetenneeneseetioneceiensernaneeneeedl Conidiophore 3-4 vang Khutin Ige bj gidi han trén MEA, hau hét ¢@ DK < 1,5 cm treng I tan, mat trai co mau vang....... P. citrinum Khun lac ¢6 DK > 1,5 cm trén MEA trong 1 tuan, mat trai xanb La cay ti C61 dem Mhatescssenssseecas ses see ce aee P, cOnlophilin Conidiophore co stipe nhan trén Czapek & MEA; déi khi 66 thanh rap trén MEA... wd Conidiophore cé stipe hoi rip t6i rp nhiéu hogc gd ghé (myn cée)..15 Conidiophore lin, cdc stipe rng 46 pm két day chic, khutin lac hau hét bj gidi han _.P, brevicopactum . Conidiophore véi cée stipe rong 2,54 cm, khudn lee phat trién trong di nhanh... cole Khuin lee dang nhung, thrdng c6 giot tiét va mat teai mau vang, conidi 06 dang hinh cau t6t elip ....cscseeecceseeeesnnseeeeneneeene P. chnysogensum Khuan lac ¢6 conidi két cum, khong ¢6 giot tiét mau vang, conidi hink panied eliptél plivtepewcec sce15a, 15b. Ia, 16b, 17a. 17b, 18a, 18b. 19a, 19b. . Thé binh da sé-cO chiéu dai > 6,5 ym, conidiophore 3 vong... . Khudn lac eo DK 2-2,5 cm sau 14 ngay, cor . Thé binh nein, chiéu dai < 6.5 wm, conidiophore thudng co céu trac 4 soe Pe griseofulvin dd yong (quaterverticillate).. . Khuin lace cé DK 4-5 cm sau 14 ngay, conidi hinh cdu dén elip, tac nhan gay thoi qua tao... oP. expanscun c6 dang hinh elip dén hinh wy, tac nhdin gay thi qua hg cam chanbh.weciseesn Ps italien P. echifulatcon wd Con Con 6 gai (echinulate)... hin dén hei rép (rap min)... stipe gé ghé myn ede, conidi hink edu BK 4-5 um, Conidiophore khudn lac dang nhung, khéng (90 mdi, mat trdi khudn Ige xanh 14 edy tdi digo hinh... P. roqueforti Conidiophere véi stipe rap nhung khong xb xi dang myn coc, conidi hinh cdu téi elip, PK 3-4,5 pm, khudn Ige két cum, thirmg yo mii, wd? mit trai khong ed mau xanh Id edy téi Khuan lac véi hé soi mau vang, giot tiét mau vang eam nit... -P. hirsutum Khuian lac khéng cé hé soi mau vang va giot tiét mau vang cam néu 78 KL trén cde mdi turing théng dung Czapek & MEA , 6 25°C, 7 ngay ¢6 BK < 10mm, mau vang-xanh 1a cay nhat, conidi co PK (2,5)2,8- 3,2(3,5) pm, it khi lon hon kich thud trén.... uP, verrucos tint KL én Czapek & MEA 6 25°C, 7 ngay, DK > 10mm... wD Conidi cé DK tuong d6i bé (2,5)2,8-3,2(3,5), hinh elip ¢@ chicu dai tei 3,5(4,0) pm; phat triém yéu trén méi trudng CREA, tao acid, khong tgo Conidi tong déi lén cb BK 3-4(5)am, dang hinh clip od chiéu dai tei a sel 4,5-6 jum: phat trién tot wén CREA, thurong tg0 04 base. VIA Nia} gar20a. KL mau xanh la cay xaim trén MEA, Conidi hinh gan cau 167 elip, phat trign nghéo trén CREA jesse P, auraniiogriseun 20b, KL ¢é mau xanh ld ciy-da troi tr¢n MEA, téc 49 phat trién nhanb, hinh thanh nhiéu bao tir trén ede mdi trudng, conidi hinh cdu, elip rong, phat trign trén mdi trong CREA... _P. polonieum 21a. Ving sinh conidi xanh ld cdy 10] ti xanh 14 cay xam nhat, cac ching m6i phan lap hinh thanh cac v6 cimg sau 10 ngay... P, crestosurrt 21b. Céc ving sinh conidi ngé mau xanh Mi cy hoge xanh 1a cay-da trai wen 22a. Cac viing sinh eonidi cé mau xanh 1d edy-xanh da troi t6i hog xanh 14 khae nhau, khuan lac khéng hink thanh vé cimg.. cay t6i 6 cde ching mei phan lp, mat trai trén Czapek khong mau, vang nhat hoc nau nhat, conidi hinh cau hodc gan cau........P. sofifean 22b. Cae ving sinh conidi 6 céc ching méi phan lip cé mau xanh LA cdy- xanh da tdi nhat, viang-xanh 1d cay hoge xanh ld cy xdm nha; mat trai KL wén Czapek khong mau hoc yang nhat. Conidi gin cdu ii clip P. commune 1.2. Tinh hinh nghién ctru mire 43 nhiém nim mie trén duge ligu & Vist Nam Do sir phat trién etia nén y hoe cé truyén ngay cang lan manh da lam cho khdi lugng va ching loai dirge ligu, dac biét 1a cdc duge liéu cé nguén géc duge thao ngay cang ting ed vé lugng tigu ding, bao quan. Thye 18 ela céng téc thu hoach, bao quan va tiéu ding cho thay cde logi duge li¢u rir dé bj nhiém nam moe. Tuy nhién ede céng tink nghién etm vé mite d§ nhiém nam méc va doc té nim tén durge ligu edn rat it. Mic dau vay eting di cd mt sé ong trinh dang chi ¥. Dé la céng trinh nghién ctu vé hé nam mde die true trén duce ligu @ kho cua tde gia Nguyén Thi Sinh nim 1984 [15]. Mat s6 dan ligu quan trong gitp dinh huéng cho c4¢ céng tink nghién ciru tiép theo vé ndm mde vamycotoxin trén duce ligu ma tac gia da cing bé li: hé vi ndm dc trung trén duge ligu bae quan trong 6 noi bat 1a sy eG mat cde loai cba 2 chi Aspergflius va Penicillium, dic bigt 1a sy xuit hign cia loai A. flavus trén 80% duge ligu nghién edru. Ngoai ra tac gid cling cho biet 4 am tuong di toi thiéu can thiet cho sy phat trign cita hé vi nam duge ligu o kho 1a 75%, tir 65-85% cde loa thude chi Aspergillus chiém wu thé vé sé lvgng ching. Voi RH > 90% cde loai thuge chi Penicillium sé chiém uu thé vé sd lurong ching. Céng trinh nghién cin gain day (1/2003) eda tie gid Trin Trinh Céng [8] nghién cia vé mite 46 nhiém nim méc va aflatoxin B, trén mét sd vi thude dong duge dang luu hanh trén dja ban Ha NOI, Két qua cia céng trinh da cung cdp cho ching ta nhimg bing chimg cy thé vé mdi de doa tiémn tang cia nam méc va déc t6 aflatoxin, mhAt Ii cdc vi thude ©6 nguén gée qua va hat. Trong sé 6/15 vj thudc c6 ngudn gée qua, hat ma tac gid nghién ctu da phat hign thay 4 vj thudéc 1a nguén co chat rit phus hop cho cdc loai nam sinh déc t6 phat trién. Vj thuée dau tién 1a hat sen, mét loai hat duge ding phé bién tir lau doi di bj nhiém Lodi A. flavus trung binh téi 40% trong cic mau tic gid nghién eu. Dae biét hon tic gia d2 phat hién thay 4/20 mau hat sen lay 6 cdc thuéc déng duge va cae chg thye pham bi nhiém aflatoxin By véi ham lugng dao dng 17,5-434 ppb (ng/g). Trong khi dé gidi han ti da eta déc t6 nay trén Iuong thye, thye phdm ma nganh y 18 cho phép chi [A 10 ppb. Hai vi thude khae trong s6 6 vi thude e6 ngudn gée qua, hat 1a nga vi ur va phd cé chi di bi nhiém loai A. niger véi ty 18 In luot 1a 91 va 30,6%, Day 1a mét loai tuy khéng phai la loai nguy hiém nhu A. flavus hay A, parasiticus nhumg cling cé kha ning sinh aflatoxin. Vi thude cuéi cing trong sé 6 vi thudc trén la ba tir nhan cing cho thay day 1A co chat dae trung cla mot Loai nao dé thuge chi Peniciffitem, chi nd ma theo céc nha nghign ciu doc té nim trén thé gidi 14 cd kha nting sinh nhiéu mycotoxin cé hai cho ngwdi va dong vat [6], [15]CHUONG 2: DOL TUQNG VA PHUONG PHAP NGHIEN CUU 2.1. Nguyén vat ligu, thict bi LAA. Déi ucong nghién civu 20 miu sinh dja va sai hd nam duge thu thip tit cde higu thude déng duge thudc dia ban Ha NGi (phé Lin Ong) chuyén vé phong thi nghiém, bio quan @ nhigt d6 phdng true khi phan lap va phan loai nim, 2.1.2. Méi micing phan lép va xéc dink mim méc - Mai 15g; dich chi - Méi truéng Czapek-Dox (g/l): Sacarose: 30g; NaNO): 3,0g; KCI: 0,5; KsHPO,: 1,0g; MgSO,.7H:0: 0,52; FeSQ,.7H:O: 0,01g; thach: 17,5g; nude cit vira di 100ml. Khir tring 6 121"C trong 15 phit, pH = 6,2 0,2 inring PDA (Potato Dextrose Agar) (g/l): Glucose: 20g; thach: khoai tay: it. Khirtring 6 121°C trong 15 phat. - Méi tring AFPA (Aspergillus flavus and parasiticus Agar): Pepione: 10g; cao ném men: 20g; Sit amoni citrat: 0,5g; chloramphenicol: O,lg; thach: 15g; nude vira dit [lit, Kht tring ¢ 121°C trong 15 phut 2.1.3. Thiét bj tht nghiém - Kinh hién vi Axiostar plus (Carl Zeiss- Die, gin may chup anh ky thuat s6 SONY-Nhat Ban). - Ta cy vd tring BIOAIR, Cing hoa lién bang Bite. - May do a6 dm Precisa (Thuy $¥) + Ni hip tit tring Hirayama Model HL3030 2.2. Noi dung nghién eiru Nghién céu hé vi ndm trén cdc vj thude sinh dja va sai hé nam dang liu hinh 6 cdc higu thuédc dng duge thuge dja ban Ha Ngi ~ Phin lap va phan logi ec ching nam nhiém trén vj thuoe sinh dja den cap chi & lodi.~ Phan lap wa phan logi ede ching den nbidm trén vj thude sai hd nam dén cap chi & loai 2.3. Phuong phap nghién cirw 2.3.1. Phuong phép léy mau Phuong phap léy mau dya trén co sé phrong phap ciia FAO (Food and Agriculture Organization). [23]. Mau duce ligu duce tn déu, chia theo phuong phip duong chéo, ky khodng 0,5-1kg lam mau dai di¢n. Mau dai lai due tron déu, lly mot phdn tu theo phuong php chia thee duimg chéo dé i nghien edu, 2.5.2. Phuong phdp xdc dink ham dine duge Iu Xde dinh biing may do d6 dm Preeisa: duge ligu duge nghién thé, duéng kinh khéng qua 3mm, sau do cho khoang 1g dirge ligu da nghién vo dia cin, diy nip efin ch’ én dinh va doc két qua, 2.3.3. Phuong phip phn lip ném mac Phuong phap phan ip nim mic trén duge ligu duge ap dung dua trén co sé cla phyong phip Samson va céng su [11], [13]. Cae mau durge ligu duge phan lap nam mdc bing phuong phap dat truc tiép wén méi truong PDA. Voi sinh dja khéi Iugng duge liéu nghién ctu la 40 gimdu. Voi vi thude sai ho nam khdi long durge ligu nghién cru la 40 g/mdu. Cae mau duge Ligu duge Khu trang h@ nam ben ngoai bang eich ngdm tong dung dich Natri hypoehlorid 1% msi pha trong 2 phiit. Sau d6 ria sach bing nude cat khit tring, Dé rio nude va dit nhanh ede mau duge ligu vao dia Petri d& ed mdi trudng PDA bang kep v6 tring, M@i dia dat 5-15 mau tuy timg vi. U o nhigt 46 25-30°C, sau 3-5 ngay tién hanh phan lip ede chung nim moc chuyén sang mdi trrdng Czapek-Dox, ti & 25°C, sau 3-5 ngay tién hinh cdc bude phan logi va két Luda, Trén co sé dé tink % mie 4 nhiém ede nhém nim, ede chi, lodi eda timg miu va cita time vi thude.2.3.4, Phicong pip phan logi nim mac ~ Phan loai cic chi nam thee kha phan loai cla Bamet va Hunter [19]. - Phan loai cac loai cua chi Aspergillus, Penicilfium va cac chi khac dua thee m6 ta chi tiét ea cdc lai, khod phin loai cia Raper & Fennell 1965 [25], Pitt & Hocking 1985 [24], Samson va céng su 1995 [26] va K.H. Domsech 1980 [27]. > Xée dinh hai loai 4. flavus, 4. parasiticus theo phuong phdp hoa sinh cila Pitt & Hocking [21]: cde chiing eta 2 loai A, flavus va A. parasiticus khi edy trén méi truréng AFPA va d & nhigt d$ 30°C sau 42-48h sé xudt hién mau yang cam sang mat trai khudn lac.23 CHUONG 3: THUC NGHIEM, KET QUA VA BAN LUAN 3.1. Hé vi ndim trén vi thude sinh dia Sinh dja (Radix Rehmanniae glutinosae) 1A ré ci phoi hay sAy khé cha cay Bia hoang (Relumannia glutinosa (Goertn.) Libosch.), ho Hoa mom ehd (Scrophulariaceae) [12], [18]. Yéu cdu vé ham 4m duge ligu cia DDVN IV (2009) 1a thap hon 16% [5]. ‘Thanh phn hoa hoc [3], [12], [18] © Iridoid glycosid: trong dé catalpol, rehmanin A,B, C, D e6 tic dung ha dudng huyét. + Phenethyl alcohol glycosid: trong da 4 chit 2'-O-acetyl-aceosid (1), jionosid C (2), jionasid D (3), isoacetosid (4) da thir tic dung sinh hos, Cae chat (1), (2), (4) Ge ché S-lipoxygenase voi IC la 10°M. Cac chat (1), (2), (3) tre ché aldose reductase vai ICsyLd 107-10°M. © Carbohydrat: gém D-glucose, D-fructose, sucrose, mannotriose, raffinose, vesbascose, D-manitol, trong dé chi yéu IA stachyoze véi ham long 48,3% so véi duee liu kha. * Ngoai ra cin cé ede thanh phan khéc: acid amin, ester cia acid béo, acid carboxylic, hop chat sterol. ‘Theo Y hoc c@ truyén, sinh dia c vi ngot, dang, tinh han, quy vao kinh tm, can, than, tiéu trudng. Sinh dia cé tie dung thanh nhiét Iuong huyét, dung am sinh tin dich, chi khat. Chi tri cde chimg: huydt nhiét gy néng sét, tn dich Kh6. kidt, Indi dd tam phién, tao két, mat ngi, tang chay mau, thd huyét, bang huyét, kinh nguyét khéng déu, d6ng thai, tiéu ra mau, tiéu dong (2), [12], (18).24 Két qua vé mite d6 nhiém nm mée trén 10 mau ciia vi thude sinh dia duge trinh bay bang 3.1 ya 3.2. Qua ket qua cia bang 3.1 va 3.2 ching t6i thay mét sé diém dang chi ¥ véhé vi ndm xudt hign trén vj thudc may Bang 3.1: Cac chi, loai ve 6 ching nam phan lap dirge tir cde miei eta vi thude sinh dia nghién esu | ;| Hamam], 5 ; Bjadiém lay | Cach ché _. | Téngsdching | 86 86 TT dugeligul mau bien nam nhiém chi | lodi (my 1 | 36LanGne | Phoikhd | 13,22 14 g 5 2 | 24LanOng | Phoikho 1637 4 a | = 3 | WLanOng | Phoikhe | 17,83 41 im 4 4 | 35LanOng | Phoikhé | 14,16 19 4 5 5 | I7LanOng | Phoikho | 16,09 37 3 | 6 | 69ALa Ong | Phoikho | 12,07 9 2 4 7 | S3banOng | Phoikhs | 10,94 19 2 3 & 30-LanOng | Phoikhé 13,75 25 2 4 9 | 37LanGng | Phoikhé | 1001 10 q 4 10 | 55 LanGng | Phoikhé | 949 7 4 5 Tong 255 emilee - Ham am cua cde mau dure ligu dao dong tir 9,49-17,37%, mge di mot s6 miu dat tiéu ehudn ham am theo Duge dién Vigt Nam 1V (<16%) nhung tit cd ede mau déu bi nhiém nim méc. Biéu nay cé thé do cée mau cia vj thuée nghién ciru 4a bi nhi&m nam trude thu heach hoge giai dogn thu hogch. Mit khée, v6i co chat duge ligu thich hyp, é him ém dat tigu chudn sinh dja vn c6 thé bj nhiém ném mdéc. Sinh dja véi thanh phin co chat la acid amin, ester cila acid béo, earbohydrat néa dé nhiém nam moe.- Tong so ching mam phan lip dirge trén méi truong PDA | 255 chang, thudc 12 loai ctia 7 chi. Trong dé mau e666 ching phan lap duge cao nhat la 24. LO (54 chiing, 3 loai, 4 chi) v6i him 4m 16,37%. Mu bi nhiém thap nbat 1837 LO (10 ching, 4 loai, 4 chi) vai ham dm 9,49%. Diéu nay cho thay méi lién hé gitta méc 46 nhiém nam va ham 4m cia duoc ligu, Hau hét cdc miu cé ham Am cao c6 6 ching phin lip duge nhiéu hon = MOt s6 mau ca vj thude co ham am cao hon nhung c6 s6 chang nhiém phan lip duge thap hen. Diu nay 1a do cdc mau ctia vj thuée nghién edu tuy cling duge thy thap ¢ phd Lan Ong, nhung co nguon goc, xudt xir khée nhau, chju tic ding ca cic yéu té sinh thai khdc nhau (diéu kiga wéng, thu hoach, ché bién, bao quain,...) nén sé bj nhiém ndm 6 mire dé khde nhau, + Voi két qua chi tiét wir bang 3 nhit 1a thanh vién cla chi Aspergillus. Chi Aspergiffus 1d chi nam phd bién ho thay: Cac ching ndm thuéng gip nhit, bit gip hau hét wrén cdc miu nghién etru, Trong 11 leai phan lip duge 48 c6 $ Jodi thude chi Aspergilfus, véi ting sé ching nim phfn lip duge la 143/955, chiém $3,7%. Két qua nay phi hop vei céng bé cia nhi ule gid cho ring Aspergillus 1a tic nhin quan trong nhat gly hur héng cde sin phim ed nguén gée thye vat nhu long thye, dirge thio, = Trong 5 loai cita chi Aspergilds ma ching ti phan lap duge, 4. niger [anh 3.1] Hi loai phd bién nhét; cd téng sé ching phan lép dugc cao nha (76/255) chiém 29,8%, xudt hign 6/10 mau nghién ctu, véi sb ching dao déng tir 4-36 ching/mdu nghign ctru (40 gam), Day la lodi via co kha ning sinh dde t6 gay hai than ochratoxin A [20], vita c6 thé gfy cde bénh nam co hi G ngudi va dong val, gay hu hong thye pham. A. niger con lé loai ma theo mor s6 tac gia c6 lién quan dén kha ning sinh d9c 16 aflatoxin mac da khong dl 6b nguy co cao nhu 2 loai 4. flavus vA A. parasiticus [26]. Day 1a loai cin Juu ¥ trong qué trinh bao quan duge Lidu may.26 Bang 3.2: Cac lodi vaind tugng ching phan lap dueae tie 10 mdte ctia vE thude sinh dia mghién citi 36] | oa; | 7 [oa] ss] so) a7] os eal a ..| aan ChB AoaI | 4 | xo] o | 18 | 10 | 10 | 0) wo] v0 | ca} Tone] THR Asperglflas - | 142 Aniger 7 [36 5 9/15 4| 76 [29.8% 4 frig 2; 9/2 Fla a1 | 16,1% tama 6|é6]1 1 14 15.5% A flaws 1 2 3 | 12% Exaow E | 3| 7 | 2% Ahizopus |G 4a[5][6|2 )s [4] 32 [135% siolanifior | Abita 5 a a TT | 43% conmatyera_| fe Syncephatastrum | 2 2 4 1,6% Mees ala 2 9 | 35% Eurostem N 3/ 3 | 12% herbariorum = Penicillium a oe 4/20 4 55 | 21,6% chieinum: si saoelientd _ TB 14] 54] 51/19/37] 9 [19] 95) 10/17] 235 | 100% > Xép vj tri thir 2 chia chi Aspergifius va thir 3 trong tng sd 11 lod phan lap duge ld loi A. fumigatus, xuat hig 5/10 mau, voi sé ching dao 6ng tit 2-20 ching/mau, tong sO ching phin lap duoc 1A 41/255, chiém 16,1%. Day 1d loai nam tuy khéng ¢6 kha nding sinh d§c té nguy hiém song | tae nhan gay ede bénh nam co hdi 6 nguéi va dong vat phd bién nhat trén thé gidi, cn phong tranh hit phai bao tr trong qua trinh liép xtic, nghién ctu. - Loai 4. flavecs chi phan lap duge 3 ching, chiém ty 1é 1,2%, xudt hign 2/10 mau, cho thay kha nding abiém dgc té nguy hiém aflatoxin la khéng cao.= Chi Penicillium xép vj ti thir 2 voi loai Penicilliam citrinum [Anh 3.2] ¢O so chiing phin lap duge 55/255,chiém ty 1g 21,6%, xuat hign 6/10 mau nghién cir, 1a loai chiém ty Lé cao Goi vdi vj thude sinh dja, Loai P.eitintwn c6 kha nang sinh citrinin-ddc té gay hai thin & ngubi va vat nudi [20]. Day la chi nam ¢6 kha ning sinh nhiéu mycotoxin eb hgi cho ngudi va dong vat, [am hong duge ligu, gay ra cdc bénh ndm co hi, cdn hu ¥ eng qua trinh bio quan [6], [15]. - Nginh ndm tiép hop véi 4 loai phan lép duge Rhizopus stofonifer, Syncephalastrum racemosum, Mucor, Absicia corymbifera co sb ching nhiém 1a $6/255, chiém ty 1¢ 22%. Day eiing li nhém nam eiin ru ¥ vi ching gay hogi sinh manh lam héng duge liéu, ding thoi gay ede bénh ndm ky) sinh co hoi zygomycetes (zygomycosis), dic big déi vdi ngudi mac bénh tiéu during, suy giam mién dich [4] ~ Ngoai ra, ehiing ti cn phan Ip duoc 3 loai ndm khde Id: A. tamarit {Anh 3.3] chiém 5,5% (14/255), 4. aculeates chiém 2,7%7/255, Eurasian herbariorum chiérn 1.2% (3/255).28 Cc D Anh 3.1: Lodi Aspergillus niger nhiém trén vj thudc sinh dia A: Khuan lac nam nhiém trén vj thudc sinh dja (mdi truang PDA) B, C; Conidi va cu trac sinh conidi ca loai D: conidi cia loai29 A B e D Anh 3.2: Loai Penicillium citrinum nhiém trén vj thuoc sinh dja A: Khudn lac nam nhiém trén vj thude sinh dia (méi trudng PDA) B, C, D: Conidi va edu tric sinh conidi eta loai (cdu tric chéi 2 ting)30 B D Anh 3.3: Loai A. tanrarii ohiém trén vi thudc sinh dia A: Khun lac nam nhiém trén vi thude sinh dia (mdi trrimg PDA) B, C, D: Conidi va edu trie sinh conidi eta lodi31 3.2. Hé vi ndm trén yj thuée sai hd nam Sai ho nam (Radix Pluchea pieropodae) |a ré phoi séy khé cla edy sai ho nam (Pluchea pteropoda Hemsl.), hg Cie (cAsteraceae) [12], [18]. Yéu cdu vé ham dm durge ligu cia DBVN IV (2009) la thip hon 12% [5]. Thanh phan hod hoe: Cac nghién eu gain day trén cay sai he nam da phan Idp duge mét sé titerpenoid (boemery axetat, hop-17(21)-en-3p-yl axetat, hop-17(21)-en-22-ol-3f-yl axetat), nhém hgp chat sterol (P-sitosteral, stigmasterol, 3-O-P-+D-glucopyranosyl-fi-sitosterol), tinh dau (cht yéu cde hop chit sesquiterpen), hyp chat thiophen, acid carboxylic (acid trianeontancie). Ngodii ra e6 phan img duong tinh véi flavonoid, saponin, coumarin [7], [17]. Theo ¥ hee cé truyén, sii hd nam eé vj ding, tinh hoi hin, quy vio 4 kinh: can, dom, tam bao, tam tiéu. Thude co tae dung phat van phong nhiét, loi tiéu, kinh. Cho tri cac chimg phong nhiét phat s6t, hoi rét, nhire dau, khat murée, tire ngye, khé chju, Igi tiéu nhe, an than, giam dau, rdi Joan kinh nguyét [2], [18]. Két qua nghién ecu hé vi ndm trén 10 mu eda vi thude sii hd nam duoc trinh bay & bang 3.3 va 3.4. Qua két qua o bang 3.3 va bang 3.4 cho thay: ~ Ham dim cia ce mau duge ligu dae dong tir 9,36-15,94%, mae di mOt sé miu dat tiéu chudn ham 4m theo Duge dién Vigt Nam IW (<12%6) nhumg tit ca déu bi nhiém nam méc. + Téng sé ching nim phan lap duge trén mdi truong PDA 1a 206 ching, thuge 12 loai eta 7 chi, Trong 7 chi nim phan Kip-dugc ¢6 2 chi nam bat tan (Aspergillus, Penicillium), 4 chi ndm tiép hep (Rhizopus, Absidta, Syncephalastrum, Mucor), 1 chi nfm tii (Neosartorya). Trong dé miu di phan lip duge ed sé ching, lodi va chi cao nhit 14 30 LG (34 ching, 7 Lodi, § chi) voi ham am 15,04%, Mau bj nhiém thap nhat la 17 LG (10 ching, 3 loai,32 2 chi) vai ham dm 9.36%, Cling nhir ede duge 1igu ndi chung, théng throng. cdc miu cd ham am cao ¢6 s6 ching phan lip due nhiéu hon. - Khée véi vi thuée sinh dja, sai hé nam co 36 Iugng ching phan lap durge thap hon. Chi Peniciliun voi loai Penicillium oxalicum xudt hign 2/10 mau yéi sé chiing phin lap duge 3/206 chiém 1,5%. Do dé kha nang nhiém Gc to cia chi Penicilfium la khéng dang ké. Do cdc yéu té thu hoach, ché bién, bio quan, thinh phan co chat cia timg vj thuée khdc nhau nén cé sir Khe nhau gitta sai hd nam va sinh dja vé sé lugng ching, vé tinh dic trung ciia chi. Sai hd nam cé thinh phan oo chat 1a tinh dau va ester cila acid béo cling thich hop cho sy phat trién cia ndm mde. Bang 3.3: Cae chi, lodi va sé ching nim phair ldp diege tie ecie mei ctza vj WitiGe sai ha nam nghién citw Bia diém ly | Cach ché Mili) “They a so | so ee mau bién save: | sehog af chi | loai ligu (%) nhiém 1 | 36LanOng | Phoikhé | 10,73 15 3 3 2 | 24LanOng | Phoikhs | 981 | 14 3 3 [a | f0LanGng | Phoikhé | 14,31 20 4 [4 | 4 | 35LanGng | Phoikhé | 11,98 19 me 4 5 | 17LanGng | Phoikhé | 9,36 10 2 3 6 | 69A Lan Ong | Phoikhé | 10,57 2 3 4 7 $3 Lan Ong Phoi kh 14,71 32 | @ § [8 | 30LanGng | Phoikhe | 15.94 34 5 7 9 | 37LanOng | Phoikho | 12.23 | 28 4 6 10 | 55LanOng | Phoikhe | 12,98 Hi a7 5 Tong a33 - Gidng véi sinh dja, chi Aspergifins efing suit hién phd bién nhat vei téng sé ching nam phan lap duge la 136/206, chiém 66,0%. Biéu nay cho thay sur lay nbiém phd a chi nim may trén cac co chat durge thio sau thu hogeh. - Trong 6 loai ctta chi Aspergillus, c6 2 lodi due ching tdi phan lip vei ty 18 cao mhat [a 4. niger, A. fumigatus. Ngoai ra ching ti cén phan lp dirge cic lodi khdc cha chi nay vGi oF 1G thép 1a A. parasiticus, A. tamari, A. foeniels, A, ustusy Trong dé 4. niger xép vj tri ther I: xuat hign 5/10 mau nghién ctu, voi s6 ching dao déng tir 3-21 ching/mau nghién citu (40g mu duge ligu) 06 tong s6 ching phan lip duge cao what (55/206) chiém 26,7%. Xép vj tri thir2 trong chi Aspergillus 1a loai A. fumigatus [Anh 3.4] xudt hign 6/10 mau nghign etm, véi sé ching phan lap duge 1 40/206, chiém 19.4%. Nin da néi @ trén, A.niger la loai vita ¢6 kha nang sinh dge té hai thin ochratoxin A, vita e6 thé edy cde bénh nam co héi, gay hur hong thye pham [17], [20]. Con A fumigatus 1a loai tuy khéng sinh déc té nguy hiém song Hi the nhan gly ede bénh ndm co hdi. - Lodi cé kha mang sinh dé té aflatoxin 1a A. parasiricus xuat hién 2/10 mau véi s6 ching phn lap dug 3/206 chiém 1,5%. Do dé it o6 kha nding lay nhiém aflatoxin. - Nginh nam tiép hgp cé mit 4 loai Rhizopus stolenifer, Absidia corymbifera, Syneephalasirum racemosum, Mueor eb sé ching nhiém 1a 66/206, chiém ty 1¢ 31%. Bay ciing 1a nhom nam can duge quan tim. Dién hinh la Rhizopus stolonifer [Anh 3.3], xép vi ti thir 3 trong 12 leai vei sb chiing phin Ifip durge 30/206 chidm 14,6%, Nhu trén, day 1a nhém nim hoai sinh manh, la méi nguy ca cao gay ra cde bénh nd eo hi (zygomycosis) & ede doi trong bi suy gidm mign dich va bénh tigu dudmg.34 - Ndi tii (Neosartorya fischer’) ty 66 xuat hién song véi ty lé thap 1,5% vei sé ching 3/206. Bang 3.4: Cec lodi va 30 lneong chiing phan lp duege tir 10. mdusdé he nam tha thdp tit cétc hig thude déng diege thude dia ban Her Ni ean, *)™)] ©] |) lon] 5] 0] 37] ]oq -_ Chi&oal (15) 19 | uo {uo | 16 | uo | ve | vo | co | uo | Teme) TH Aspergillus 136 Separate 4 1] 3 | 15% aoe ul afi7}3|3| ss [267% Agnigcen | 3 10 6 a/ Mét sé mdu cia 2 vi thude tuy him Am dat tiéu chuiin DD'VN LV song tit ca cde miu nghién ciru déu bj nhiém nam moc. Théng thuong mau cé ham dm exo e6 sé ching phan ip duge nhieu hon. ® Da co-255 ching nim phan lap duoc tr 10 mau vi thuée sinh dia nghién ctru ca 11 loai thude 7 chi, wén mdi trudmg PDA, Voi 10 mau ota vj thudc sai hd nam s6 ching phan lap duge la 206 thuge 12 leai etia 7 chi. > Cie chi va loai phan lap duge tr 2 vj thude thuge 2 nhém nam quan trong [i ndm bat toan-Dewteromyeetes (dién hinh 1a chi Aspergillus) va nganh nim tiép hop (dién hinh la chi Rafzepus), Nganh nam wii ty co xuat hign song vai tin sé rat thap. > Vai vj thude sinh dja, chi Aspergillus chiém vj tri s6 1 (55.7%) ci vé s6 loa, 3d ching bat gdp trén co chat nay. Trong dé 4. niger xudt hién voi ty 18 cao nat chiém 29,8% (76/255) 1a loai sinh déc t6 gay hai than ‘Ochratoxin A, gay cde bénh nim co hGi. A. fumigatus xudt hién vei ty 16 16,1%. Nigoai ra cé m@t s6 loai khac chiém ty 18 thap nhu A. flavus, A. aculeatus, A. tamarii, Tiép theo 1a chi Penicillium xép vi tri thir 2 vei Lodi Penicilliem citrimem chiém 21,6%, day la chi kha dic trmg cua vi thudc, Neanh nam tiép hop ed mat 4 loai thude 4 chi la Rhizopus stolonifer, dbsidia eorymbifera, Syncephalastram racemasum, Mucor sp., trong d6 di@n hinh la R stolonifer chiém 12,5%. Nganh nam tii c6 mat 1 loai 1a Ewrosiem Aerbartorum,30 > Wi thude sai hé nam, cing nhwr vj thude sinh dja, chi Aspergillus 1a chi én nhat ca vé sé lugng loai va sé ching chiém 66% (136/206). Cac loai phé bién mht véi_ ty 18 cao nhat bao gém 4. niger chiém 26,7%, A. firmigatus chiém 19.4%. Ngodi ra c6 mét sé loai khdc chiém ty 1é thap nhu A, A. foenivis. Nganh nim tiép hop co mat 4 parasiticus, A. ustus, A. tamar loai thuge 4 chi, dién hinh mhat la chi Rhizopus xép vi ti thir 2 vei loai RK. stolonifer chiém 14,6%, Khac vii sinh dja, chi Penicillin tuy o6 swat hign song t¥ 1 thap véi loai 1a P. exalicum chiém 1,5%. Nganh nam tai 66 mit 1 loai Neosartorva fischeri chiéna ty 1é thip 1,5% * MOts6 kin nghi 1. Can mo rong pham vi nghién ctu ca vé khu vye dia ly va sé luong mu thu thap dé 6 két lua chinh xde hon vé mit dé nhiém nam méc wen cae vj thude 2. Can tiép tue nghién ci vé mtte 46 nhiém etia lodi 4. niger véi dge t6 gay hai than cép ochratoxin A. va loai Penicillin eitrinum cing, cé nguy eo xudt hién dge 06 citrinin gay hai thén cin phai luu y. 3, Véi yj thude sai hd nam cing vép me nghién eiw thém vé mire dp nhiém lod 4. niger véi déc 16 gay hai thin ochratoxin A va loai A. flenigatus thutng aay cde bank ridin cot igi 4, Can cé cae bién php thich hgp gidm sit cée giai doan tude thu hoach, thu heach, ché bién va bae quén cde loai direc thao n6i chung va 2 i thude sinh dia va sti hd nam néi rigng 48 gidm 16% thidu mire dQ nhiém nam mée. §. Cdn khuyén edo kip thai téi nevrdi tiéu dling, cdc nha nudi tréng, kinh doanh va ché bién duoe liéu vé nguy eo nhiém nam mée, dée t6 nam va cde bign phap phong tranh.TALLIEU THAM KHAO Tiéng Viet 1. 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Nguy€n Thj Chung, Lé Vin Hac, Nguyén Xuan Diing (2007), “Acid x triancontanoic lén dau tign duge phan lip tir cy sdihé nam (Pluchea pleropoda Hemsl.) 6 Dién Chau, Ngh¢ An”, Tap chi duye hoe, 47374), tr 30-31. Trin Trinh Céng (1/2003), Nghién cia mice dé ahidin nd moe va: kha oo néng sink Aflatoxin By cua hom Aspergillus flavus wén mot svi thude Aéng diege dang lew hénh trén dia ban Ha Nei, Luan van tat nghiép Thge si dure hoe, Bai hge Dusge Ha Néi, tr. 42-52.9, Nguyén Dinh Hoa (2006), Nehién cin mnie dd nhiém ndm mde tren mét 56 vi thude dong dirge dang lieu Frank trén dia ddn ede tinh Ha Tay, Bde Ninh vet Hai Dicong, Khoa luan tat nghigp duge si, Dai hoe Duyge Ha NOI, tr. 15-34. 10. TranThi Huyén (2008), Nghién ctu hé vi ndm trén ede vj thude ngwu tat va déing sdm dang fiw hanh G- cde hig tinede déng duc thudc cia ben Ha , Khoa luan tit nghigp duge si, Dai hoc Duge Ha Ndi, tr, 18-32 11. Nguyén Hong Lién (2008), Nghién cit mize dé nhiém ede loci thuge chi ivi thudc dong duoc dang Iu hank o dia ben Hai Noi, Khoa luan t6t nghiép duge si, Dai hee Duge Ha Ndi, tr, 20-35, 12. D3 Tat Loi (1990), Ning edy thude va wi thude Vist Nam, Nha Xuat ban Khoa hoc va Kf thudt, Ha Néi, tr. 633-634, 838-841 Penicillium trén 13. Tran Thj Nguyén (2009), Nghién etn hg vi ndim trén cde wf thudc chi thyre va bd ni nkrdn dang lw hinh 6 céc hidu thudc déng deroc thuce aia ban Ha N6?, Khoa ludn tét nghiép duge si, Dai hoc Duge Ha NGi, tr. 22-36, 14. Tran Anh Phusng (2007), Nghiém cir mire dé nhiém 2 lodi Aspergillus Slavus link ex fr. A. parasiticus speare va Aflatoxin trén mat 56 mau hat sen dang hu hanh ot ede higu thude ding duge thuge dia bin Ha Néi, Khod lun wt nghiép duge st, Dai hoc Duge Ha NOi, tr. 22-37. 15. Nguyén Thi Sinh (1984), Gép phein nghién ert hé vi ndm de trung mén chege liéu & kho, Luan an Pho tién si duge hoc, Dai hee Duge Ha Noi, Ha N@i, tr. 78-80. 16. Phiing Ké Togi (2004), Nghién cite mize df whim ede lodi Aspergillus flavus link ex fr. A. parasiticus speare va Aflatoxin By irén hat sen caia um6t s6 higu thude & Ha N6i, Thi Bink, Thanh Hod vai Nghé An, Khwa ludin tt nghigp duge si, Dai hoe Dirge Ha NGi, tr. 21-3617, Doan Thanh Tudng (2001), Nghién citu mot 36 drink phn hod hoe cia sai hd nam (Pluichea pteropoda Hemsi.) va cay ciic tam (Pluchea indica (L.) Less) Viet Nam, Ludn an Tién st hod hoc, Trudmg Dai hoe su pham Ha Nai, Ha Noi, tr. 68-82. 18. Vign duge ligu (2004), Cay thude va dong wat fam thude & Viée Nam, Nhat xudt ban Khoa hoc va kj thudt, Ha Noi, Tap 1, tr. 774-779, tap II, tr, 666-668. Tiéng Anh 19. Barnett H.L and Hunter B.B (1972), [ustrated General Of Imperfect Fungi, Bugress Publising Company, Mimenosia USA, pp. 23-24. 20. Champ BR et al. (1991), Fungi and mycotoxins in stored products, pp. 24-34. 21. Domsch K.H, Gams W., Anderson T. (1980), Compendia of soif fungi, Acadermic Press. 22. Heathcote JG, and Hibbert J.R. (1978), “Aflatoxins:; Chemisal and biological aspect", Elsevier, New York, pp. 173-186. 23. Pitt JL (1991), Methods for isolation, emumeration and identification of Fungi. Leeture note. BIOTROP fourth training Course on pests of siored products, Bogor Indonesia, pp. 38-42. 24. Pitt J.I. and Hocking A.D. (1985), Fungi and food spoflage, Academic press Sydney, pp. 29-51 25. Roper K.B and Fennell D.] (1965), Genus Aspergilins, Baltimo, USA, pp. 58-62. 26. Robert A. Samson et al (1995), Introduction to food- borne Fungi, pp. 52-83. 27. Samson R.A, & Frisvad J.C. (2004), Penicillium subgenus Penicillium: mew taxonmic schemes and mycotoxins ard other extrolites, CBS, Netherlands. 28, Wogan G.N (1973), Aflatoxinn cancernogennesis, Academic press, New York, pp. 309-344.PHU LUC Phu lye 1: Sinh dia Olga - 35 Lin Ong Ban THUD BA prt oobi 2508. mms TTA DD 091440 1 Anb 1: Vi thudc sinh dja truéc khi phan KipPhu luc 2: Sai ho nam ‘tints SeeaFate aime, Anh 2: Vi thade sai hd nam true khi phan lap