engleski

Biochemical indicators of wheat seeds infestation by Sitophilus granarius

Sitophilus granarius is one of the most destructive pests of stored wheat grains. Immature stages of this insect develops inside kernels causing weight and nutritional loss, fungal growth and subsequent mycotoxins accumulation leading to impracticability of using infested grains in food and feed production. Although Croatian and EU Regulative prohibits the use of infested grains in food production, production of infestation-contaminated cereal products cannot be avoided since detection of the internal grain infestation could be easily misinterpreted due to imperfection of the most worldwide methods used, basically relying on visual detection and counting. Since advanced methods for detection of internal grain infestation, such as X-ray analysis and computer tomography are quite expensive, there is a need for the development of alternative methods of detection, among which biochemical analysis could be valuable tool. The objective of this research was to examine whether the oxidoreductive or proteolytic enzyme activities and differences in electrophoretic protein pattern can be used for detection of internal wheat seed infestation by Sitophilus granarius. Infested and non-infested wheat seed samples, and Sitophilus granarius adults were examined for activities of glutathione reductase (GR), catalase (CAT), guaiacol peroxidase (POX), polyphenol oxidase (PPO), lipooxygenase (LOX), aminopeptidases preferentially splitting N-terminal amino acids of arginine and phenylalanine (Arg-AP, Phe-AP), carboxypeptidase II (CP-II) and aspartic protease, as well as for differences in protein pattern of sequentially extracted proteins (50 mM Tris-HCl buffer pH 8.0 ; 55 % (w/v) 2-propanol containing 0.5 % (w/v) DTT ; 3 M urea containing 1 % (v/v) Nonidet NP-40 and 0.5 % (w/v) DTT) separated by Native PAGE, SDSPAGE and IEF with pH 3.5-9.5 gradient. Enzyme activity differences between non-infested and infested wheat seeds indicate that CPII (91 % activity decrease), CAT (172 % activity increase), and PPO (146 % activity increase) could be used as potential indicators of wheat seed infestation by Sitophilus granarius. Electrophoretic analysis revealed SDS-PAGE of proteins extracted with (a) 50 mM Tris-HCl buffer pH 8.0, or (b) 2-propanol containing 0.5 % (w/v) DTT, and IEF with pH 3.5-9.5 gradient of proteins extracted with 2-propanol containing 0.5 % (w/v) DTT as potential electrophoretic methods for detection of internal wheat seed infestation.

wheat seeds; oxidoreductases; peptidases; protein electrophoresis; Sitophilus granarius

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