Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant β2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two β2-agonists were; IC50 salbutamol = ∼0.310 μg/ml, IC50 clenbuterol = ∼0.076 μg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 μg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 μg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.
Edible bird's nest (EBN) is widely consumed as a delicacy and traditional medicine amongst the Chinese. In the present study, for the first time, the antioxidant properties of an EBN pepsin-trypsin hydrolysate of the swiftlet species Aerodramus fuciphagus and its ultrafiltration fractions were investigated. Thirteen peptides with molecular weights between 514.29 and 954.52 Da were identified in the EBN fraction with the use of mass spectrometry. Two novel pentapeptides Pro-Phe-His-Pro-Tyr and Leu-Leu-Gly-Asp-Pro, corresponding to f134-138 and f164-168 of cytochrome b of A. fuciphagus, indicated the highest ORAC values of 14.95 and 14.32 μM of TE μM(-1) peptide, respectively. Both purified peptides showed resistance against simulated gastrointestinal proteases. In addition, both peptides had no in vitro cytotoxicity on human lung MRC-5 cells and prevented human liver carcinoma HepG2 cellular damage caused by hydroxyl radicals. Therefore, it is suggested that EBN protein hydrolysates are a good source of natural antioxidants and could be applied as nutraceutical compounds.
The physicochemical properties and sensory analysis of duck meatballs containing duck meat surimi-like material during frozen storage were evaluated. Properties of meatballs containing duck surimi-like material prepared by acid solubilization (ACDS), alkaline solubilization (ALDS), and conventional processing (CDS) as well as duck mince (as the control, CON) were compared. ACDS had significantly higher (P < 0.05) moisture and protein content and lower fat content compared with CON. The thiobarbituric acid-reactive substances (TBARS) value of all samples increased as the storage time increased up to week 8 (P < 0.05), but thereafter it decreased in most of the samples. ACDS and ALDS had significantly higher TBARS values (P < 0.05), and these values remained higher than those of the other samples throughout the frozen storage period. Addition of surimi-like material to the meatballs had significant effects (P < 0.05) on springiness, gumminess, and chewiness values of all samples. Ingredients and frozen storage affected most sensory attributes in samples significantly (P<0.05). No significant increase in growth of organisms occurred during 12-wk frozen storage The results indicate that acid-alkaline solubilization methods improve both physicochemical and sensory properties of duck meatballs containing duck surimi-like material. Thus, these techniques should be applicable to product development of duck surimi-like material.