Antifungal activity and identification of bioactive peptide from Etawa crossbreed goat (Capra hircus) milk protein hydrolyzed using trypsin enzyme

Abstract

There had been a growing interest in biopreservation in recent years due to the increasing demand for food products that were free of synthetic preservatives and therefore safer to consume. This made it necessary to explore natural bioactive with antifungal properties that can inhibit microbial spoilage and extend shelf life. Etawa crossbreed goat milk (Capra hircus) peptide fraction has antifungal activity. Therefore, this study aimed to determine the peptide with antifungal activity resulting from the casein and whey proteins of goat milk hydrolysis by trypsin. The hydrolyzate was fractionated using a Strong Cationic Exchange (SCX) catridge and the antifungal activity test was performed against Aspergillus sp. using the disc diffusion method. The most active peptide fraction with the highest antifungal activity was identified using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Based on the results, the ratio of the enzyme substrates used to hydrolyze casein and whey proteins is 1:10; 1:20; 1:30 and 1:40. The greatest degree of hydrolysis of casein and whey proteins was obtained at a 1:40 enzyme-substrate ratio of 66.8% and 75.4%, respectively. The analysis of Sodium Dodecylsulfate - Polyacrylamide Gel Electrophoresis (SDS PAGE) showed that goat milk casein contained αS1-casein and β-casein, while the whey proteins contained beta-lactoglobulin and α-lactalbumins. The casein and whey protein hydrolysates were fractionated using an SCX with pH variations of 3–9. The Goat Milk Casein (GMC) 5, 6, 7 dan 8 peptide fractions inhibited Aspergillus sp, and the most active was a fraction from GMC6 with an inhibition zone of 26.95 ± 0.63 mm and Minimum Inhibitory Concentration (MIC) value of 31.25 μg/mL. Furthermore, the morphology of Aspergillus sp. mycelia after treatment with peptide fraction showed that the mycelia were wrinkled and withered. The fractions with the highest antifungal activity contained 4 peptide sequences, namely YNVPQLEIVPK, KENNINELSK, GLSPEVPNENLLR, and YLGYLEQLLK. These results showed that peptide from goat milk had potential applications as a natural antifungal compound.