Antimicrobial Activity of Trypsin and Pepsin Hydrolysates Derived From Acid-Precipitated Bovine Casein

Foodborne pathogens are a major concern to the food industry and consumers but they may be controlled with antimicrobials. Naturally occurring antimicrobials may be isolated from a variety of plant, animal and microbial sources. Previous studies have demonstrated that peptides isolated from enzyme hydrolyzed milk proteins may have in vivo and in vitro antimicrobial activity. Such compounds could be of use as inhibitors of foodborne pathogens. The objectives of this study were to determine the antimicrobial effectiveness against Salmonella Typhimurium and Listeria monocytogenes of digests of bovine acid-precipitated casein with the enzymes pepsin and trypsin and to determine if these peptides were effective in combination with ethylendiaminetetraacetic acid (EDTA) and sodium lactate against these foodborne pathogens.

Whole casein was precipitated from fresh, unpasteurized skimmed cow’s milk by addition of 2 N HCl. Precipitated casein was separated by centrifugation, washed and lyophilized. Rehydrated casein was hydrolyzed with either pepsin or trypsin and the reaction mixture was heated to inactivate each enzyme.

For method 1, solutions with hydrolyzed protein were dialyzed against water and freeze-dried. For method 2, 5.0% casein was dissolved in buffer and treated similarly to method 1, however the peptides that were created from enzymatic hydrolysis were separated by centrifugation after inactivation of the enzymes by heat and were not dialyzed against water. For both methods, hydrolysates created were adjusted to pH 7 and filter sterilized through a 0.45 mm membrane filter

The inhibitory effect of filtered pepsin and trypsin hydrolysates (0.5% and 1.0%) (method 1) and filtered supernate (pepsin and trypsin) (method 2) alone and in combination with EDTA and sodium lactate against four strains each of L. monocytogenes and S. Typhimurium DT104 was determined. Growth was monitored over 24 hours using a microbroth dilution assay for all hydrolysates. Growth curves were used to relate microtiter data to actual colony counts.

For method 1, pepsin hydrolysates were not very effective in inhibiting the growth of any of the four strains of S. Typhimurium or L. monocytogenes while trypsin hydrolysates were only slightly effective at extending the lag phase and/or reducing the final growth level of all four strains of L. monocytogenes. This ineffectiveness was most likely due to the loss of small molecular weight peptides during the dialysis step. The addition of EDTA had little effect in enhancing the inhibitory effect of pepsin or trypsin hydrolysates against either microorganism.

For method 2, trypsin hydrolysates were effective in extending the lag phase and/or reducing the final growth level of all four strains of L. monocytogenes tested; however, they were not effective against any of the strains of S. Typhimurium. Pepsin hydrolysate was not effective in extending the lag phase or reducing the final growth level in S. Typhimurium. However, pepsin did reduce the final growth level of one strain of L. monocytogenes, 101. Trypsin and pepsin hydrolysates derived from bovine milk in combination with EDTA and sodium lactate had antimicrobial activity against both L. monocytogenes and S. Typhimurium in tryptic soy broth (TSB). Trypsin hydrolysates also enhanced the antimicrobial activity of sodium lactate against Listeria monocytogenes.

The protein concentrations of pepsin and trypsin hydrolysates (before and after membrane filtration) prepared using method 2 was determined with three different protein assays, Bradford dye-binding, modified Lowry and UV 280 nm. For all three methods, non-filtered hydrolysates were higher in protein concentration than those that were filtered. Using the Bradford method, pepsin hydrolysates were higher in protein concentration than trypsin hydrolysates in both filtered and non-filtered samples. The opposite results were observed when using both modified Lowry method and UV 280 nm method to determine protein concentration. By examining the location of the peptide bond hydrolysis of the enzymes, it was possible to determine that small molecular weight peptides were created by the addition of trypsin and pepsin to bovine casein. Variation in number of amino acids as well as types of amino acids of peptides created during hydrolysis likely influenced the antimicrobial effectiveness of each hydrolysate.

Casein-derived peptides could provide an alternative or adjunct to antimicrobials currently used in foods. It is suggested that antimicrobial peptides can be created by enzymatic hydrolysis of casein with trypsin and these peptides have the potential to serve as antimicrobials in food systems. Further research needs to be conducted in enhancing the activity by concentrating the hydrolysates or isolating and characterizing those peptides with the greatest antimicrobial potential.