1.State Key Laboratory of Food Science and Technology, Nanchang University;2.College of Food Science &3.Technology, Nanchang University;4.Jiangxi Province Key Laboratory of Food Allergy;5.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易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，有关于含牛乳过敏原的高脂肪型复杂基质脱脂方法仍鲜有研究。以含有牛乳过敏原的巧克力为原料，分别测定了正己烷、异丙醇、乙酸乙酯和脂肪酶脱脂处理后高脂肪型复杂基质的脱脂率和蛋白损失率，比较了样品中蛋白结构、粒度、脂肪分布和微观结构的变化，同时利用间接ELISA评估样品中主要牛乳过敏原的溶出率。研究结果表明，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，其中正己烷和乙酸乙酯组变化较小。样品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样品的脂肪分布更为均一且液滴变小，样品表面形貌由致密光滑块状变为疏松多孔结构，而异丙醇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溶出率提升了61.53%，α-乳白蛋白和β-乳球蛋白在正己烷组中的溶出浓度最高，分别为0.73 mg/mL和1.89 mg/mL。综上所述，正己烷和乙酸乙酯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，针对不同的过敏原应选用其特定的脱脂方法。
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 decreasing methods for allergens in high-fat complex matrices was rarely reported. Chocolates containing cow’s milk allergens were used in this study 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 contents of major cow’s milk allergens were obtained by indirect ELISA to evaluate their dissolution rates. 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%). Among the different treatments, 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 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 the samples was more uniform and the fat droplets became smaller. The microstructure of the samples 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 dissolution rate of casein in the ethyl acetate group increased by 61.53%, and the highest dissolution concentrations of α-lactalbumin and β-lactoglobulin were observed in the n-hexane group, which were 0.73 mg/mL and 1.89 mg/mL, respectively. In summary, n-hexane and ethyl acetate degreasing treatments can significantly improve the extraction effect of cow’s milk allergens in high-fat complex matrices, and specific degreasing methods should be selected for different allergens.