Pepsin

Commercial whey protein concentrate (WPC) was obtained from Kalleh Dairy Ltd. (Amol, Iran). WPC had 81.2 g/100 g protein (dry basis), 5.3 g/100 g fat, 6.6 g/100 g lactose, and less than 3.0 g/100 g ash. Medium‐chain triglycerides (MCT‐60 with predominantly MCT C8 [Caprylic acid; 58.6%] and C10 [Capric acid; 41.4%]) were purchased from Kamani Oil Industries Pvt. Ltd. (Mumbai, India). Curcumin (95% purity), pepsin (activity of more than 3000 units/mg), and pancreatin (200 units/mg) were obtained from Bio Basic Inc. (Canada). Tween‐80 and other chemicals used in this study were purchased from Merck (Darmstadt, Germany) and Sigma‐Aldrich. All chemicals were analytical grade.

Sensitivity to enzymes was checked by treatment with proteolytic enzymes: trypsin (BIO BASIC Canada INC) and pepsin and with lysozyme (BIO BASIC Canada INC); fifty microliters of the CFS was mixed with enzymes solutions to obtain final concentration and 1× reaction buffer. Incubation was held at 37°C for 1 h. After incubation, the enzymes were denatured by heating the samples at 80°C for 10 min, and the residual CFS activity was determined. The effect of pH on the CFS antimicrobial activity was determined by adjusting the pH of CFS using 1 N HCl, 1 N NaOH, and pH meter following that pH 2, pH 4, pH 6, pH 10, and pH 12 were used. After 2 h of incubation at room temperature, the samples were readjusted to pH 6.5 with sterile 1N HCl or 1 N NaOH, and the activity was determined. Staphylococcus aureus ATCC25923 was used as an indicator bacterium.