Suchergebnisse

Research background. The presence of insect fragments is one of the major constrains in stored food commodities and it causes considerable loss in the quality of the produce. The management of the pest is viewed as a huge challenge in foodprocessingindustry. Conventionally, the detection of Tribolium castenaum in the food processing industry is carried out by acid hydrolysis and staining methods that are time consuming and lack precision. Experimental approach. Considering the importance of a quick and effective method, a quantitative polymerase chain reaction (qPCR)-based approach was developed and elucidated in this study. The mitochondrial cytochrome oxidase I (mtCOI) gene was identified as a target due to its abundance in the pest. Specific primers were designed against the target gene by Primer Premier software and amplified in a qPCR. Results and conclusions. This method is capable of detecting all the ontogenic stages of T. castaneum in stored wheat flour. Earlier experiments had demonstrated that about 20 µg of DNA can be obtained from 2.2 mg of insects. To quantify the infestation levels, the cycle threshold (Ct) values obtained from known samples were subjected to regression analysis and expressed as adult equivalents. In the unknown samples, the infestation was calculated as 1.74 and 0.046 adult insects in 5 g of wheat flour. The maximum permissible limit of insect fragments in flour is 75 insect fragments or approx. 3 adults per 50 g of flour as per the US Food and Drug Administration (FDA). Hence, by adopting this new method, it is possible for the warehouse operators to arrive at a decision to proceed with efficient management practices where wheat flour is stored. Also, this method can be ratified by government agencies associated with international business to ascertain whether the wheat flour meets the standards set by the respective country before subjecting to foreign trade. Novelty and scientific contribution. This study is the first of its kind in the detection and quantification of T. castaneum in milled products. So far, only conventional methods have been employed to assess the presence of the pests and manual counting of fragments are practiced to quantify the infestation levels. The developed qPCR method is faster, reliable and can be employed in milling industries, bakery industries, food processing plants and foreign trade units for critical detection and quantification of T. castaneum pest infestation. ; Pozadina istraživanja. Prisutnost dijelova insekata u namirnicama jedno je od glavnih ograničenja pri njihovom skladištenju, te uzrokuje velike gubitke u kvaliteti proizvoda. Suzbijanje štetnika predstavlja velik izazov u prehrambenoj industriji. Tradicionalno se prisutnost kestenjastog brašnara (Tribolium castaneum) u prehrambenim proizvodima utvrđuje kiselinskom hidrolizom i metodom bojanja, koje su dugotrajne i neprecizne. Eksperimentalni pristup. Uzevši u obzir potrebu za primjenom brze i učinkovite metode određivanja prisutnosti T. castaeneum u brašnu, u radu je razvijena i opisana kvantitativna metoda koja se temelji na lančanoj reakciji polimerazom u stvarnom vremenu (qRT-PCR). Kao ciljni gen odabran je mitohondrijski gen za podjedinicu 1 citokrom c oksidaze (mtCOI) zbog njegove rasprostranjenosti u nametniku. Pomoću kompjuterskog softvera Primer Premier izrađene su početnice za umnožavanje DNA ciljane vrste metodom qRT-PCR. Rezultati i zaključci. Ovom se metodom može identificirati kestenjasti brašnar (T. castaneum) u svim fazama ontogeneze u uskladištenom brašnu. Prijašnji su eksperimenti pokazali da se iz 2,2 mg kukaca može izdvojiti otprilike 20 µg DNA. Za utvrđivanje opsega zaraze provedena je regresijska analiza broja ciklusa (Ct-vrijednost) potrebnih za određivanje poznatih uzoraka, a rezultati su izraženi kao broj odraslih kukaca. U nepoznatim je uzorcima izračunat stupanj zaraze od 1,74 odnosno 0,046 odraslih insekata u 5 g pšeničnog brašna. Prema regulativi Američke agencije za hranu i lijekove (engl. U.S. Food and Drug Administration (FDA)), najveći dopušteni broj dijelova insekata u brašnu je 75, odnosno otprilike 3 odrasla kukca u 50 kg brašna. Pomoću ove nove metode voditelji skladišta mogu donijeti odluku o tome gdje skladištiti brašno, te učinkovito provoditi plan suzbijanja ovog nametnika. Također, državne agencije koje posreduju u međunarodnom poslovanju mogu temeljem ove metode utvrditi zadovoljava li kakvoća proizvedenog pšeničnog brašna standarde zemalja kojima se ono isporučuje. Novina i znanstveni doprinos. Ovo je prvo istraživanje u kojem je utvrđena količina kestenjastog brašnara T. castaneum u mljevenim žitaricama. Dosad su korištene samo konvencionalne metode utvrđivanja prisustva nametnika u brašnu, a opseg zaraze je određivan ručnim brojanjem dijelova insekata. Nova qRT-PCR metoda je brža, pouzdanija i može se koristiti u mlinarskoj i pekarskoj industriji, pogonima za preradu hrane i pri međunarodnoj trgovini za utvrđivanje opsega zaraze kestenjastim brašnarom.