The potential of the breakdown products of casein by Lactococcus lactis strain 146 as an inhibitory therapeutic agent(s) forMRSA

Abstract number: P1087

Al-Mahrous M., Alqamber M., Burnie J., Upton M., Tagg J.

Objective: lactococci possess a proteolytic system that can release free amino acids, peptides and oligopeptides from casein (milk protein). The proteolysis involves the action of cell wall-associated peptidases (CWAP) and subsequent hydrolysis is carried out by several enzymes found in the cell envelope; which can eventually be taken up by the bacteria. This fermentation process results in milk diaries flavour and proteinacious end-products. The objective of the current study was to investigate the end-products of casein degradation by Lactococcus lactis strain 146, as inhibitory agents for MRSA.

Methods:

1Investigation of the end-products of L. lactis strain 146 was performed using plate-diffusion method from casein-containing minimum essential media (MEM).

2Casein-free MEM was used as a negative control for the inhibitory end-products, on which alternative growth factors were included.

3Purification and/or concentration of the end-products in broth supernatants was carried out using ammonium-sulphate precipitation, XAD-2 resin separation, cation-exchange; then C18 reverse-phase chromatography.

4MALDI TOF/TOF mass-spectrometry (MS) was used for mass analysis.

5A range of published and/or designed primers was used for PCR of gene(s) responsible for the synthesis of CWAP in strain 146.

6The gene was cloned using T-tailed vector; and then sequenced.

Results: Supernatant from casein-containing media displayed activity against MRSA, but not the casein-free media. The reversed-phase HPLC profile of the processed active fractions revealed several peptide species (Fig. 1). In addition, determining the mass of the peptides with MS showed that they were seized in a window of 0.9 and 5 kD. Among the tested primers, BG95/146CEP–invlwoer1; BG97/146CEP–invlwoer1; BG95/4CA showed positivity with strain 146 on PCR. However, cloning was successful; the sequence data of the vector still needs further analysis.

Conclusion: The effect of the breakdown-products of casein by L. lactis strain 146 against MRSA suggests the potency of these peptides as future therapeutic agents for treating highly drug-resistant Staphylococcus aureus, on which the cloned vector or the sue of strain 146 can be a powerful biological tool for the breakdown of casein by its CWAP. Up to our knowledge, this is the first study that discusses casein breakdown products by L. lactis and their activity against staphylococci.

Figure 1. Detection of peptide species of casein breakdown products by Lactococcus lactis strain 146 using mass spectrometry. Resulting amino acids were detected on a time-of-flight (TOF) mass spectrometer with matrix-assisted-laser-desorption ionisation (MALDI).