Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to the study of bacteriocins

Bacteriocins produced by lactic acid bacteria have the potential for use as natural preservatives in minimally processed foods. However, sensitive detection methods are needed to understand their role in the enhanced safety and/or storage life of foods and to clarify their fate in foods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was examined as a method of detection of bacteriocins from crude biological samples. After minimal sample clean-up to remove natural contaminants that interfere with MS analysis, nisin A, pediocin AcH/PA-1, brochocins A and B and enterocins A and B were detected in culture supernatants using MALDI-TOF MS. This technology was then applied to determine the fate of nisin in fresh meat. Nisin is widely used in the food industry; however, it is ineffective for use in raw meat products. Fresh and cooked meat samples were held in a nisin solution for 1 hr, vacuum packaged and stored overnight at 4°C. The extracts of the meat were analyzed by antibacterial assays and by MALDI-TOF MS. Antibacterial activity was recovered from the cooked meat extracts; whereas no activity was recovered from the raw meat extracts. MALDI-TOF MS results for the cooked meat extract showed a signal at the molecular weight of nisin; whereas only a signal that was 307 Da greater than the mass of nisin was observed for the raw meat extracts. These results suggested that nisin was inactivated in raw meat by an enzymatic reaction with glutathione, a low molecular weight (307 Da) thiol compound that is widely distributed in plant and animal tissues. To further examine this theory, soluble glutathione S-transferase was isolated from fresh beef by affinity chromatography. The in vitro reaction between nisin and glutathione, catalyzed by the purified enzyme, was then studied. The products of the reaction were analyzed for antibacterial activity and by MALDI-TOF MS. Results showed that after reaction with glutathione, nisin lost its antibacterial activity. MALDI-TOF MS results showed that the reaction could result in the addition of up to three glutathione molecules to one nisin molecule. Glutathione assays with nisin variants and fragments confirmed that the dehydroalanine residues of nisin were two sites of addition for glutathione. It is speculated that dehydrobutyrine is the third site of addition of glutathione to nisin. These results provided the first scientific evidence for the method of inactivation of nisin in fresh meat.