1.State Key Laboratory of Food Science and Technology, Nanchang University;2.College of Food Science &Technology, Nanchang University;3.Jiangxi Province Key Laboratory of Food Allergy;4.Sino German Joint Research Institute, Nanchang University
脂肪作为食品的常见成分，会与牛乳过敏原蛋白之间发生相互作用从而影响过敏原蛋白的提取效果和检测准确性。然而，有关高脂肪型复杂基质的脱脂方法仍鲜有研究。以含有牛乳过敏原的巧克力模拟高脂肪型复杂基质，分别测定了正己烷、异丙醇、乙酸乙酯和脂肪酶脱脂处理后模拟基质的脱脂率和蛋白损失率，比较了脱脂前后蛋白结构、粒度、脂肪分布和微观结构的变化，同时利用间接ELISA测定脱脂前后从模拟基质中提取主要牛乳过敏原的浓度。结果表明：正己烷的脱脂率最高且蛋白损失率最低，分别为87.87%和6.86%，脱脂会改变蛋白质空间结构，整体趋势由紧凑变为松散，其中正己烷组和乙酸乙酯组的变化较小。粒径在脱脂后略微降低，但异丙醇处理会使蛋白聚集成较大的颗粒，粒径由297.9 nm升高至445.1 nm。正己烷和乙酸乙酯脱脂后模拟基质的脂肪分布更为均一且液滴变小，表面形貌由致密光滑的块状变为疏松多孔结构，而异丙醇组和脂肪酶组仍然存在部分结块。间接ELISA结果表明：酪蛋白在乙酸乙酯组中的提取率提升了61.53%，α-乳白蛋白和β-乳球蛋白在正己烷处理后的提取浓度最高，分别为0.73 mg·mL-1和1.89 mg·mL-1。正己烷脱脂处理可以明显改善高脂肪复杂基质中过敏原的提取效果，希望研究结果可为实现高脂肪型复杂基质食物中过敏原的准确检测提供参考。
As a common food component, fat could interact with cow’s milk allergens, and thereby affecting the extraction effect and detection accuracy of allergens. However, research on degreasing methods for allergens in high-fat complex matrices was rarely reported. Chocolates containing cow’s milk allergens were used to simulate the high-fat complex matrix and decreased by n-hexane, isopropanol, ethyl acetate and lipase. The degreasing rate and protein loss rate were determined, the changes of protein structure, particle size, fat distribution and microstructure were compared, and the concentration of major milk allergens extracted from simulated matrix before and after degreasing were determined by indirect ELISA. The results showed that the sample treated with n-hexane had the highest degreasing rate (87.87%), and the lowest protein loss rate (6.86%). Degreasing would change the spatial structure of proteins, with the overall structure changing from compact to loose, and there is no obvious change in protein in the n-hexane and ethyl acetate treatment groups. The particle size of decreased slightly after degreasing, but the isopropanol treatment caused the protein to aggregate into larger particles, and the particle size increased from 297.9 nm to 445.1 nm. After degreasing by n-hexane and ethyl acetate, the fat distribution in simulated matrix was more uniform and the fat droplets became smaller. The microstructure of the simulated matrix changed from a dense and smooth clump to an incompact and porous structure, while the isopropanol and lipase groups still showed partial agglomeration. The indirect ELISA results showed that the extraction rate of casein in the ethyl acetate group increased by 61.53%, and the concentrations of α-lactalbumin and β-lactoglobulin were highest in the n-hexane group, which were 0.73 mg·mL-1 and 1.89 mg·mL-1, respectively. N-hexane degreasing treatments can significantly improve the extraction effect of cow’s milk allergens in high-fat complex matrices, hoping that the results can provide reference for the accurate detection of allergens in high-fat complex matrix foods.