Metabolomics research on Nisin

Metabolomics, leveraging high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) technologies, can precisely capture characteristic metabolites generated by Nisin degradation in food. Combined with differential metaboliteanalysis of differential metabolites and validation of enzyme activity correlations, it clearly reveals that Nisin decomposes primarily through three core pathways: enzymatic hydrolysis, oxidation, and thermal degradation. Different food matrices influence the priority and rate of these pathways. The specific research conclusions and details are as follows:

Enzymatic Hydrolysis Pathway: Peptide Bond Cleavage Mediated by Microbial and Food Endogenous Proteases

This is the dominant degradation pathway of Nisin in microbe-containing or protease-rich foods. Metabolomic studies have identified various characteristic truncated peptide products and clarified the mechanisms of key degrading enzymes:

Microbial Protease Degradation

In systems containing Lactococcus lactis (e.g., dairy products, fermented foods), Nisin resistance protein (NSR) expressed by Nisin-tolerant strains acts as the key degrading enzyme. Mass spectrometry (MS) detection shows that NSR specifically cleaves 6 amino acid residues from the C-terminus of Nisin, generating the truncated product Nisin1-28. This product exhibits significantly reduced affinity for cell membranes, with antibacterial activity decreased by 100-fold. Additionally, metabolomic analysis during the late fermentation stage of Nisin by Hubei University of Technology revealed that the expression of intracellular peptidase genes (pepO, pepT, etc.) in L. lactis is upregulated by 12%–24%. These peptidases decompose Nisin into small peptides as nitrogen reserves to meet the subsequent nitrogen demands of bacterial growth.

Food Endogenous Protease Degradation

Chymotrypsin, peptidases, and other enzymes present in meat and dairy products randomly cleave Nisin’s peptide bonds. Metabolomic detection indicates that such degradation produces characteristic fragments like Nisin1-32, which lose the critical C-terminal active region, resulting in a significant attenuation of antibacterial activity. In simulated human digestive tract systems, metabolomic data further confirm that Nisin is ultimately fully hydrolyzed into amino acids by proteases, verifying its non-residual safety after ingestion.

Oxidative Degradation Pathway: Oxidative Modifications and Product Formation During Food Storage

This pathway is particularly prominent in foods containing oxygen, additives, or fruit/vegetable components. Metabolomics clarifies the mechanism of structural damage to Nisin by capturing oxidation-specific products:

Oxidation Characteristics of Specific Variants

HRMS (Orbitrap) analysis of metabolomes of Nisin A and Z in yogurt drinks shows that Nisin Z is more susceptible to oxidative degradation, with its characteristic metabolite being Nisin Z+O (oxidized modified form). In fruit-flavored yogurt drinks, unstable polyphenols and other antioxidants in fruit/vegetable components easily induce oxidative stress in the system, significantly accelerating the oxidative degradation rate of Nisin Z—even reducing its parent ion peak below the detection limit during the shelf life.

Oxidative Damage to Active Groups

Special residues in Nisin, such as dehydroalanine, are targets of oxidative attack. NMR analysis reveals that oxidation causes cleavage of the thioether bond in the lanthionine ring, generating degradation products like des-delta ala5-nisin1-32. Although these products retain partial spatial conformation, the loss of dehydroalanine residues prevents binding to lipid Ⅱ on bacterial cell membranes, resulting in complete loss of antibacterial activity.

Thermal Degradation Pathway: Ring Structure Destruction and Fragmentation During Thermal Processing

Although Nisin has strong heat resistance, it undergoes heat-induced degradation in the processing of high-temperature-sterilized foods. Metabolomics reconstructs its degradation patterns by comparing metabolic profiles before and after thermal processing:

Characteristic Degradation Products and Structural Changes

Nisin’s activity depends on the stable structure of its 5 lanthionine rings. Thermal processing causes ring opening or fracture. NMR detection shows that thermal degradation produces linear peptide fragments without intact rings, such as truncated peptides lacking specific lanthionine rings. These fragments exhibit unique molecular weight peaks in metabolic profiles, serving as markers of Nisin inactivation during thermal processing.

Influence of Food Matrix on Thermal Degradation

Metabolomic data indicate that food fat content exacerbates Nisin’s thermal degradation. For example, in high-fat milk containing 12.9% fat, Nisin activity decreases by over 88% after thermal processing, whereas in skim milk, activity decreases by only 33%. This is because fat molecules form complexes with Nisin, altering its thermal stability and making peptide bonds and ring structures more susceptible to destruction during heating.

Additionally, metabolomic studies have found that food matrix factors such as pH and additives regulate the direction of degradation pathways. For instance, low pH environments inhibit peptidase activity, delaying enzymatic hydrolysis; adding the non-ionic emulsifier Tween 80 reduces Nisin’s binding to fat, lowering the rates of thermal and oxidative degradation. These findings provide important metabolomic evidence for extending Nisin’s antibacterial validity period by optimizing food formulations (e.g., adjusting pH, adding suitable emulsifiers) and processing techniques (e.g., controlling sterilization temperature).