García-Cano Israel, Yeh Po-Wei, Rocha-Mendoza Diana, Jiménez-Flores Rafael
Department of Food Science and Technology, Parker Food Science & Technology Building, The Ohio State University, Columbus 43210.
JDS Commun. 2020 Dec 11;2(1):1-6. doi: 10.3168/jdsc.2020-0028. eCollection 2021 Jan.
β-Lactoglobulin (β-LG) is believed to be a common allergen in bovine milk. Buttermilk (BM) powder has abundant contents of milk fat globule membrane and phospholipid, both of which have been demonstrated to have positive effects on brain and cognitive development during early infancy. This study focused on modifying β-LG in BM via supercritical CO (ScCO) treatment to modify its reactivity to antibodies and thus reduce its antigenicity. Buttermilk powder was treated in a supercritical fluid extraction system with food-grade CO at 100, 150, 200, 250, 350, and 400 bar at 2 temperatures, 50 and 75°C. All analyses were completed in a 10% BM suspension (wt/vol). The BM proteins were examined using sodium dodecyl sulfate (SDS)-PAGE, Western blot, ELISA, and periodic acid staining methods. Semi-purified β-LG was used to evaluate the cytotoxicity, viability, and inflammatory response in the Caco-2 cell line by means of the lactate dehydrogenase assay, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay, and IL-8 production, respectively. The SDS-PAGE showed that the signal intensity of β-LG bands was reduced by up to 50% after being processed at 250 bar and 75°C for 30 min. Lighter and more diffuse signals were found by Western blot, indicating modification of the protein structure. The ELISA demonstrated that ScCO treatment could significantly change β-LG antigenicity in BM. Sugar moieties in bands corresponding to β-LG were revealed by periodic acid staining, indicating glycosylation only in samples treated with ScCO. Caco-2 cells treated with whey proteins had high viability, 24.9% lower inflammation, and no evidence of cytotoxicity compared with untreated cultures. These results showed that reduced antigenicity of β-LG was caused by lactosylation, which has been reported as a possible pathway to reduce the allergenicity in foods. The denaturation of β-LG by supercritical fluid processing is a promising way to address milk allergy, which remains a problem requiring more attention and further research.
β-乳球蛋白（β-LG）被认为是牛乳中的一种常见过敏原。酪乳（BM）粉富含乳脂肪球膜和磷脂，这两者均已被证明对婴儿早期的大脑和认知发育具有积极作用。本研究聚焦于通过超临界CO₂（ScCO₂）处理来修饰BM中的β-LG，以改变其与抗体的反应性，从而降低其抗原性。酪乳粉在超临界流体萃取系统中，于50℃和75℃这两个温度下，分别在100、150、200、250、350和400巴的压力下，用食品级CO₂进行处理。所有分析均在10%的BM悬浮液（重量/体积）中完成。使用十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳、蛋白质印迹法、酶联免疫吸附测定（ELISA）和高碘酸染色法对BM蛋白进行检测。分别通过乳酸脱氢酶测定、MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑] 测定以及IL-8产生，利用半纯化的β-LG评估Caco-2细胞系中的细胞毒性、活力和炎症反应。SDS-聚丙烯酰胺凝胶电泳显示，在250巴和75℃下处理30分钟后，β-LG条带的信号强度降低了多达50%。蛋白质印迹法发现信号更浅且更弥散，表明蛋白质结构发生了修饰。ELISA表明，ScCO₂处理可显著改变BM中β-LG的抗原性。高碘酸染色揭示了与β-LG相对应的条带中的糖部分，表明仅在经ScCO₂处理的样品中发生了糖基化。与未处理的培养物相比，用乳清蛋白处理的Caco-2细胞具有较高的活力、炎症降低24.9%且无细胞毒性迹象。这些结果表明，β-LG抗原性的降低是由乳糖基化引起的，乳糖基化已被报道为降低食品中过敏原性的一种可能途径。通过超临界流体处理使β-LG变性是解决牛奶过敏问题的一种有前景的方法，牛奶过敏仍是一个需要更多关注和进一步研究的问题。
