ࡱ>  *bjbjUU 4??0y$(fyyyyppp'''''''$=*,J'ppppp'yy(###p yy'#p'###y@SB"#'(0(#)-B")-##)-%pp#ppppp''#ppp(pppp)-ppppppppp :MOGUNOST BRZE IDENTIFIKACIJE KVASACA I PLIJESNI FTIR SPEKTROMETRIJOM Manuela Zadravec, Mario Mitak Hrvatski veterinarski institut, Laboratorij za mikrobiologiju hrane za ~ivotinje, Zagreb Sa~etak Prehrambena industrija, zakonodavstvo i patoloaka stanja bolesti i ~ivotinja zahtjevaju brzu i pouzdan u metodu za identifikaciju plijesni i kvasaca. Za tradicinalnu identifikaciju potrebno je znanje i iskustvo stru njaka. Molekularna identifikacija zahtjeva laboratorijske vjeatine, a i cijena metode nije zanemariva. FTIR (Fourier Transform Infrared) spektroskopija je brza i efikasna obe avajua metoda koja zahtjeva minimalnu pripremu uzorka, za analizu nisu potrebni regensi i relativno je jeftina, a bazira se na usporedbi karakteriti nih spektara (specifi nih otisaka). FTIR ima mogu nost da postane rutinska metoda za identifikaciju kvasaca i plijesni u prehrambenoj industriji i dijagnostici. U Laboratoriju za mikrobiologiju hrane Hrvatskog veterinarskog instituta zapo elo se s kreiranjem baze specifi nih ostisaka kvasaca i plijesni. Baza za sada broji skroman broj karakteristi nih otisaka kvasaca i plijesni, meutim, o ekuje se da bi u skoro vrijeme baza mogla podr~avati brzu identifikaciju kvasaca i plijesni izoliranih iz hrane, hrane za ~ivotinje i razli itih patoloaki promjenjenih organa ~ivotinja. Uvod Kvasci i plijesni vrlo su va~ni mikroorganizmi koji imaju veliku ulogu u prehrambenoj industriji (pekarski proizvodi, pivo, mlije ni proizvodi, suhomesnati i fermentirani proizvodi) te u proizvodnji lijekova, meutim mogu biti i uzro nici kvarenja hrane (Filtenborg i sur. 1996), tvoriti mikotoksine (Perai i sur. 2011) i uzrokovati bolesti ljudi i ~ivotinja (Einsele i sur. 1997). Tradicionalna identifikacija kvasaca i plijesni zahtjevna je i dugotrajna (Pitt i Hocking 2009), a zasniva se na morfoloakim karakteristikama plijsni i kvasaca. Za treadicinalnu identifikaciju potrebno je znanje i iskustvo stru njaka. Biokemijskim nizovima mogua je samo identifikaciju kvasaca, meutim, samo za klini ki zna ajne vrste (Wenning i sur. 2002). U novije vrijeme identifikaciji kvasaca i plijesni izvodi se molekularnim metodama, kao to je sekvenciranje ITS (internal transcribed spacer) regije ribosomske RNA (Schoch i sur. 2012) kao zlatnog standarda, ili nekih drugi geni od interesa (npr tubulin) (Lecellier i sur. 2014). Za molekularnu identifikaciju potrebne su zahtjevne laboratorijske vjetine, a i cijena metode nije zanemariva (Nilsson i sur. 2009). Meutim, sama molekularna metoda ne daje potpuno to ne rezultate za identifikaciju plijesni (Hall i sur. 2004) te ju je potrebno kombinirati s morfoloakim karakterstikma plijesni. Prehrambena industrija, zakonodavstvo i patoloaka stanja bolesti ljudi i ~ivotinja zahtijevaju brzu i pouzdan u metodu za identifikaciju plijesni i kvasaca. FTIR (Fourier Transform Infrared) spektroskopija je brza i efikasna obe avajua metoda koja zahtjeva minimalnu pripremu uzorka, za analizu nisu potrebni reganasi i relativno je jefina. Jedna od prednosti FTIR spektroskopije, kao analiti ke metode, je mogunost dobivanja karakteristi nog spektra za slo~ene strukture kao ato su i mikroorganizmi. Stoga se FTIR tehnka koristi u raznim podru jima kontrole kvalitete, a ujedno i za identifikaciju mikroorganizama, meu kojima su i kvasci i plijesni (Beekes i sur 2007). FTIR Infracrvena spektroskopija s Fourierovom transformacijom instrumentalna je metoda za detekciju, odnosno identifikaciju funkcionalnih skupina prisutnih u molekuli. U FTIR-u se koriste IR (infracrvene) zrake koje ili prolaze kroz uzorak ili ih sam uzorak apsorbira na osnovu ega se posljedi no odreuje struktura molekula tj. molekularni sastav promatranog medija. Infracrveno podru je elektromagnetskog spektra prote~e se od vidljive svjetlosti do mikrovalova (slika 1).  Slika 1: Spektar elektromagnetskog zra enja FTIR ureaj se sastoji od izvora zraka infracevene svijetlosti koje prolaze kroz uzorak ili se reflektiraju od povraine uzorka, te dolaze do detektora koji mjeri signal te se rezultat prikazuje na monitoru ra unala (slika 2) (Santos i sur. 2010).  Slika 2: Spektrometar FTIR spektri organskih spojeva imaju jako slo~ene i detaljne spektre, dok su anorganski spojevi naj eae puno jednostavniji. Kemijska struktura i trodimenzionlna orijentcija molekula odgovorni su za generiranje razli ite IR apsorpcije te posljedi nog kreiranja  specifi nog otiska (finger print) odreenog spoja ili mikroorganizma. IR spektri su jako specifi ni za svaku vrstu, jer prikazuju potpunu kemijski strukturu stanice kao ato su lipidi, proteini, nukleinske kiseline i polisaharidi te predstavlja specifi an otisak za ispitivani bioloaki materijal (slika 3) (Mardar i sur. 2006).  Slika 3: Specifi ni otisak Candide albicans dobiven FTIR-om Mogunost dobivanja specifi nih otiska pomou FTIR spektroskopije koristi se za identifikaciju nepoznatih mikroorganizama uz pomo baze podataka (Ngo-Thi i sur. 2003). Dobivanje specifi nof otiska i kreiranje baze podataka Kako bi znali od koje je to no plijesni ili kvasca pojedini specifi an otisak, naprije je potrebno to no identificirati plijesan ili kvasac pomou kombinacije molekularne metode i morfoloakih karakteristika ispitivanog mikroorganizma. Za molekularnu identifikaciju koriste se univerzalne ili specifi ne po etnice te se rezultati uspreuju i dopunjuju karakteristikama poraslih kolonija i mikroskopskih struktira plijesni i kvasaca poraslih na specifi nim hranjivim podlogama. Tada se pristupa FTIR tehnici dobivanja karakteristi nog spektra. Dobiveni spektar usporeuje se sa spektrima u bazi. Ako u bazi postoji takav ili sli an spektar, mogue je identificirati mikroorganizam. Stoga, identifikacija direktno ovisi o raznolikoau baze, koja se mo~e stalno nadopunjavati (Kummerle i sur. 1998). Statisti ki gledano, referentna baza spektara je krucijalna za pouzdanu identifikaciju mikroorganizama. U Laboratoriju za mikrobiologiju hrane Hrvatskog veterinarskog instituta zapo elo se s kreiranjem baze podataka. Baza za sada broji skroman broj karakteristi nih otisaka kvasaca i plijesni, a sadr~i kvasce i plijesni koji su izolirani s tradiocionalnih fermetiranih mesnih proizvoda i ~itarica. S obzirom da arhivirana zbirka izolata kvasaca i plijesni Laboratorija sadr~i na desetke joa neidentificirane vrste kvasaca i plijesni, a broj izolata razli itog podrijetla raste (hrana, hrana za ~ivotinje, ~itarice, izolati iz razli itih patoloaki promjenjenih organa ~ivotinja:z | J ` ~ 2 D p r x ssdUsUUFhh?lCJOJQJaJhh_ZCJOJQJaJhhBRNCJOJQJaJhhuqCJOJQJaJhhCbCJOJQJaJhh]4CJOJQJaJhhCb5CJOJQJaJhhhE6CJOJQJaJhhoCJOJQJaJhh5CJOJQJaJhho5CJOJQJaJhh[h5CJOJQJaJz | NP\h h$l$$$ ^gdl $dha$gdldhgds $dha$gd=2$dh`a$gd $dha$gds $dha$gdCb $dha$gdYgdo$a$gdo$(4HNP\ DVXz(ӵӵӵӵӦxl]N]N]N]hh nCJOJQJaJhhJRCJOJQJaJhCJOJQJaJhhCJOJQJaJhhx CJOJQJaJhho5CJOJQJaJhhuqCJOJQJaJhhoCJOJQJaJhh?lCJOJQJaJhhYCJOJQJaJhhCbCJOJQJaJhh@dQCJOJQJaJ(<HXr ,.Xntv gstӵӦӗӗyj[LhhXhCJOJQJaJhh;CJOJQJaJhhxCJOJQJaJhhx CJOJQJaJhhzCJOJQJaJhhzCJOJQJaJhhhYmHnHtHu$jޝhhYUmHnHtHuhYCJOJQJaJhsCJOJQJaJhhfBCJOJQJaJhh(CJOJQJaJhh.CJOJQJaJhhlCJOJQJaJhDCJOJQJaJhlCJOJQJaJ hhD$jhhLMUmHnHtHuhhDCJOJQJaJhh7 CJOJQJaJ$&&&&&++,,,b-d--4SS(S W W$WdhgdDLgd_pgdDL $dha$gd=2 d`gdY $da$gd>dhgds $dha$gdY&&' '"'&'('0'2'J'X'Z'^'''''( (()+R+f++++,,ӵӦĦĈ|p`|Th^CJOJQJaJjh>CJOJQJUaJh['CJOJQJaJh>CJOJQJaJhhaBCJOJQJaJhh nCJOJQJaJhh@dQCJOJQJaJhhTCJOJQJaJhh['CJOJQJaJhhHBACJOJQJaJhhFkOCJOJQJaJhhYCJOJQJaJ,,2-^-`-b-d--.. .".v000 11011r233ĵxiZiiiK<hhG<CJOJQJaJhhfCJOJQJaJhhCJOJQJaJhhCCJOJQJaJhh['CJOJQJaJhhACJOJQJaJhh(+CJOJQJaJhh_p5CJOJQJaJhhDLCJOJQJaJhhHBACJOJQJaJhh$cCJOJQJaJhh nCJOJQJaJhh>CJOJQJaJ334444~55.6>6X6J8L8949@9T9RSSS(S(UUUĵ||zn_O@hhXCJOJQJaJhhf5CJOJQJaJhhdjCJOJQJaJhLMCJOJQJaJUhhoCJOJQJaJhdjCJOJQJaJhhUE<CJOJQJaJhh@]CJOJQJaJhh_pCJOJQJaJhhLMCJOJQJaJhhfCJOJQJaJhhG<CJOJQJaJhh%f/CJOJQJaJ), o ekuje se da bi u skoro vrijeme baza mogla podr~avati brzu identifikaciju kvasaca i plijesni karakteristi nih za hrvatsko podru je. Zaklju ak FTIR ima mogu nost da postane rutinska metoda za identifikaciju kvasaca i plijesni u prehrambenoj industriji i zdravstvenim centrima. Usporeujui druge fizikalno-kemijske metode FTIR oprema nije toliko skupa. Meutim, za stvaranje referentne baze podataka koja je krucijalna za preciznu identifikaciju potrebno je ulo~iti mnogo truda i vremena s obzirom da je naprije potrebno sve plijsni i kvasce to no identifucirati molekularnim i klasi nim metodama prije arhiviranja karakteristi nog otiska. Literatura: BEEKES, M., P. LASCH, D. NAUMANN (2007): Analytical applications of Fourier transform-infrared (FT-IR) spectroscopy in microbiology and prion research. Vet. Microbiol. 123, 305-319.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Einsele%20H%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" EINSELE, H., H.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Hebart%20H%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" HEBART , G.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Roller%20G%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" ROLLER , J.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=L%C3%B6ffler%20J%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" LFFLER , I.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Rothenhofer%20I%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" ROTHENHOFER, C. A.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=M%C3%BCller%20CA%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" MLLER, R. A.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Bowden%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" BOWDEN, J.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=van%20Burik%20J%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" VAN BURIK, D.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Engelhard%20D%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" ENGELHARD, L.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Kanz%20L%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" KANZ, U.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/?term=Schumacher%20U%5BAuthor%5D&cauthor=true&cauthor_uid=9163443" SCHUMACHER (1997): Detection and identification of fungal pathogens in blood by using molecular probes.  HYPERLINK "https://www.ncbi.nlm.nih.gov/pubmed/9163443" \o "Journal of clinical microbiology." J Clin Microbiol. 35, 1353-60. FILTENBRG, O., J.C.FRISVAD, U. THRANE (1996): Moulds in food spoilage. Int J Food Microbiol. 33, 85-102. HALL, L., S. WOHLFIEL, G. ROBERTS (2004): Expirience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequecing Kit for Identification of Filamentous Fungi Encountered in the Clinical Laboratory. J. Clin. Microbiol. 42, 622-626. KUMMERLE, M., S. SCHERER, H. SEILER (1998): Rapid and reliable identification of food-borne yeasts by Fourier-transform infrared spectroscopy. Appl. Environ. Microbiol. 64, 2207-2214. LECELLIER, A., J. MOUNIER, V. GAYDOU, L. CASTREC, G. BARBIER, W. ABLAIN, M. MANFAIT, D. TOUBAS, G. D. SOCKALINGUM (2014): Differentiation and identification of filamentous fungi by high-throughput FTIR spectroscopic analysis of mycelia. Int. J. Food Microbiol. 168 169, 32 41. MARDER, L., V. A. CORBELLINI, M. F. FERRO, M. L. SCROFERNEKER, R. C. S. SCHNEIDER (2006): Quantitative analysis of total mycotoxins in metabolic extracts of four strains of Bipolaris sorokiniana (Helminthosporium sativum). Process Biochem. 41, 177-180. NGO-THI, N.A., C. KIRSCHNER, D. NAUMANN (2003): Characterization and identification of microorganisms by FT-IR microspectrometry. J. Mol. Struct. 661-662, 371-380. NILSSON, R.H., M. RYBERG, K. ABARENKOV, E. SJOKVIST, E. KRISTIANSSON (2009): The ITS region as a target for characterization of fungal communities using emerging sequencing technologies. FEMS Microbiol. Lett. 296, 97 101. PER`I, N., J. PLEADIN, A. VULI, M. ZADRAVEC, M. MITAK (2011): Mikotoksini u ~itaricama i hrani ~ivotinjskog podrijetla.Veterinarska stanica : znanstveno-stru ni veterinarski asopis 42, 335-345. PITT, J. I., A. D. HOCKING (2009): Fungi and Food Spoilage. Springer Science, New York, USA. SANTOS, C., M. E. FRAGA, Z. KOZAKIEWICZ, N. LIMA (2010): Fourier transform infrared as a powerful technique for the identification and characterization of filamentous fungi and yeasts. Res. Microbiol. 161, 168 175. SCHOCH, C.L., K. A. SEIFERT, S. HUHNDORF, V. ROBERT, J. L. SPOUGE, C. A. LEVESQUE, W. CHEN (2012): Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc. Natl. Acad. Sci. U. S. A. 109, 6241 6246. WENNING, M., H. SEILER, S. SCHERER (2002): FTIR microspectroscopy, a novel and rapid tool for identification if yeasts. Appl. Environ. Microbiol. 68, 4717-4721. Rapid identification of yeasts and molds by FTIR spectrometry Abstract The food industry and pathogen microorganisms require fast and reliable methods for the identification of molds and yeasts. Knowledge and expertise of experts are needed for traditional identification. Molecular identification requires laboratory skills, and the cost of the method is not negligible. FTIR (Fourier Transform Infrared) spectroscopy is a fast and efficient promising method that requires minimal sample preparation, no reagents are required for analysis and is relatively inexpensive. The method is based on the comparison of characteristic spectra (finger prints). FTIR has the potential to become a routine method for the identification of yeasts and molds in the food industry and health centers. The Laboratory of Microbiology of food at Croatian Veterinary Institute is started to create database of yeasts and molds finger prints. The base for now has a modest number of characteristic finger prints of yeast and mold, however, it is expected that the base could soon support rapid identification of yeasts and molds isolated from food, feed and various pathologically changed animal organs. 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