Manufacture of a purified peptide fraction with antifungal activity from whey protein hydrolysate

Abstract

Fungal contamination is a persistent challenge in cheese industry. Strategies used to prevent mold contamination include the use of preservatives (natamycin). However, consumers are demanding clean label food products. Bioactive peptides (BP) are small protein fractions with several biological activities, like antimicrobial. We hypothesized a BP fraction with antifungal activity (AnA) can be made from hydrolysis of whey proteins. Hence, this study aimed to produce a BP fraction with AnA from whey protein hydrolysates (WPH) for potential use in cheese. Preliminary WPH (n = 3) were made at laboratory scale (2 L) from whey protein isolate solutions (5% wt/vol protein) using 2% (wt/wt on a protein basis) of commercial plant-based enzymes (bromelain, B; ficin, F; papain, P; and thistle enzyme, T) at 50°C up to 8 h at pH values 5.0 and 7.0. Enzymatic hydrolysis was stopped by heating WPH (90°C, 20 min). The degree of hydrolysis (DH) was impacted by enzyme, hydrolysis time and pH (P < 0.05) with varying levels at 8 h of hydrolysis for B (63%–88%), F (2%–22%), P (63%–100%) and T (<5%), which were in agreement with degradation of individual proteins evaluated by SDS-PAGE and peptide profile assessed by UPLC. WPH yielding improved AnA against Penicillum commune (PC, the most common mold found in cheese; B and P hydrolyzed for 8 h and pH 7.0 and 5.0, respectively), by estimating the minimum inhibitory concentration of <3 kDa fractions, were selected for 400-L pilot-scale manufacture, followed 3 kDa ultrafiltration and spray drying of the permeate to obtain purified BP fractions (n = 3). BP fractions were mixed (0–2% wt/wt) with 1 mo Cheddar cheese shreds, inoculated with PC and incubated for 10 d at 12°C. AnA was measured by the delay in visible PC growth, BP treatments delayed the onset of visible PC in cheeses by 2 d versus the control. AnA could be related to mineral binding properties (50% of Fe+2 and Zn+2 chelation ranging between 43 and 75 and 28 to 52 mg peptide powder/mL, respectively) due to the presence of BP and free amino acids (Asp, Gly, Glu, His) with chelation properties, as evaluated by LC-MS and UPLC, respectively. These findings highlight that WPH contains AnA BP, aiding their potential application as a potential clean-label solution for cheese.