The antimicrobial potential of peptides from whey proteins has been widely demonstrated. However, it is fundamental to design the best processing conditions able to form peptides with targeted antimicrobial functionalities. This study aimed to investigate the relationship between process parameters (i.e. enzyme and hydrolysis time) and antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella enterica subsp. enterica. The in vitro hydrolysis was performed with Alcalase 2.4 and Protamex for 30-480 min reaching a hydrolysis degree higher than 90% after 120 min. As hydrolysis time increased peptide molecular weight decreased, and a different molecular weight profile was observed depending on the enzyme. Alcalase 2.4 was more prone to form peptides with molecular weight <0.4 kDa (80%) compared to Protamex (61%) after 480 min. The antimicrobial activity increased as the peptide molecular weight in the mixture decreased. Alcalase-produced more active peptides against Salmonella regardless of the hydrolysis time. After 30 min, Protamex was more effective against Staph. aureus reducing its viability by 38.61% compared with Alcalase (18.94%). The same trend was maintained at 240 min. A strong linear correlation between the prevalence of peptides having MW < 0.4 kDa and the percentage of inhibition of each target pathogen was found. Results evidence the crucial role of process design to obtain peptides with targeted antimicrobial functionality, which could be potentially exploited to produce new functional ingredients. The latter may open new possibilities in the preservation of foods where the growth of alternative microorganisms could reduce the shelf-life.

The aim of the present study was to evaluate the ability of Lacticaseibacillus casei to prevent anomalous fermentation in Latteria-type semi-hard cheese. Four laboratory-scale cheeses were produced: two control cheeses and two sperimental ones obtained with the addition of L. casei LC4-P1 (a commercial strain) and a strain isolated from Grana Padano. Chemical, physical, and microbiological analysis were performed to evaluate the development of ripening and the kinetics of microbial growth. From a microbiological point of view, results excluded a negative influence of the bioprotective species on the indigenous microflora throughout ripening. On the other hand, both L. casei strains slowed down the proteolytic phenomena during ripening and the aromatic profile analysis highlighted the inefficacy of LC4-P1 to prevent butyric acid production, a typical indicator of late blowing defect.