Supercritical CO2 treatment reduces the antigenicity of buttermilk β-lactoglobulin and its inflammatory response in Caco-2 cells

Abstract

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 CO₂ (ScCO₂) 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 CO₂ 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 ScCO₂ 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 ScCO₂. 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.