Analyze the degradation products of Nisin

Nisin is a class of natural antibacterial peptides produced by Lactococcus lactis, featuring advantages such as high efficiency, safety, and no residue. It is widely used in food preservation, pharmaceuticals, and other fields. However, during production, processing, storage, and application, nisin is prone to degradation under the influence of environmental factors (e.g., temperature, pH, enzymes, oxidative conditions). Its degradation products are not only related to the maintenance of antibacterial activity but may also affect food quality and safety. Therefore, systematic analysis of its degradation products holds significant theoretical and practical value.

I. Structural Characteristics and Degradation Triggers

The molecular structure of nisin is the core basis for its degradation behavior. A mature Nisin molecule consists of 34 amino acid residues, containing special thioether ring structures such as lanthionine (Lan) and β-methyllanthionine (MeLan), as well as unsaturated amino acid residues like dehydroalanine (Dha) and dehydrobutyrine (Dhb). These structures endow nisin with a stable conformation and antibacterial activity but also make it sensitive to the external environment—thioether bonds, peptide bonds, and unsaturated amino acid residues have low chemical stability and are prone to cleavage or modification under specific conditions, becoming the main "targets" for degradation.

There are four main triggers for nisin degradation:

Enzymatic hydrolysis: Proteases (e.g., trypsin, pepsin, peptidases secreted by microorganisms) and esterases in food systems or processing environments can specifically hydrolyze peptide bonds in Nisin molecules, especially in regions rich in hydrophilic amino acids (e.g., serine, threonine).

Chemical degradation: Acidic or alkaline conditions disrupt the stability of peptide bonds (acidic conditions tend to cause acid hydrolysis, while alkaline conditions induce alkaline hydrolysis). Oxidative environments (e.g., hydrogen peroxide, oxygen in food) oxidize methionine residues (Nisin contains 1 methionine residue) or cause cleavage of thioether bonds.

Thermal degradation: High-temperature processing (e.g., can sterilization, baking) breaks secondary bonds (hydrogen bonds, hydrophobic interactions) inside nisin molecules, leading to peptide bond hydrolysis and ring structure opening. The degradation rate accelerates significantly when the temperature exceeds 121℃.

Storage degradation: During long-term storage, light and humidity changes slowly accelerate the oxidation and hydrolysis of nisin. Particularly in neutral to slightly alkaline environments (pH 7.0–8.0), the degradation rate is much higher than in acidic environments (pH 2.0–5.0).

II. Main Degradation Pathways and Corresponding Products

The types of nisin degradation products are directly related to their degradation pathways. Products generated under different triggers differ significantly in molecular size, structural characteristics, and activity.

1. Enzymatic Degradation Products

Enzymatic hydrolysis is the most common degradation pathway of nisin in food systems. Different enzymes act on different sites, resulting in "fragmented" products.

Protease-mediated degradation: Endopeptidases such as trypsin mainly act on peptide bonds near arginine and lysine residues in Nisin molecules (e.g., the peptide bond between lysine at position 29 and alanine at position 30), cleaving the complete 34-amino-acid peptide chain into small peptide fragments composed of 2–10 amino acid residues (e.g., Nisin 1–29 peptide, nisin 30–34 peptide). Most of these fragments retain part of the thioether ring structure, but their antibacterial activity decreases significantly (usually only 10%–30% of the original molecule) due to damaged molecular conformation. If enzymatic hydrolysis proceeds further (e.g., under the action of exopeptidases), small peptide fragments are gradually hydrolyzed into free amino acids (e.g., alanine, leucine, lanthionine), and the products completely lose antibacterial activity.

Specific degradation by lactic acid bacteria peptidases: During food fermentation, peptidases secreted by Lactococcus lactis itself preferentially act on the N-terminus or C-terminus of nisin, producing "truncated nisin derivatives" (e.g., Nisin A1–32 peptide, Nisin Z2–34 peptide). Although these products retain most of the ring structure, the loss of terminal amino acids reduces their binding ability to receptor proteins on bacterial cell membranes, resulting in activity of approximately 50%–70% of the original molecule.

2. Chemical Degradation Products

Chemical degradation is centered on "structural modification" and "bond cleavage," and the products exhibit both fragmented and chemically modified characteristics.

Acid-base hydrolysis products: Under acidic conditions (e.g., the organic acid environment in food), the unsaturated bonds of Dha and Dhb in Nisin molecules easily undergo addition reactions, generating hydroxylated derivatives (e.g., Dha converted to serine). Meanwhile, some peptide bonds undergo slow hydrolysis, producing short peptide fragments with hydroxyl modifications. Under alkaline conditions (e.g., pH＞8.0), thioether bonds are prone to cleavage, and ring structures open to form linear peptide fragments; at the same time, amide bonds are hydrolyzed to generate carboxylated peptide fragments (e.g., glutamine residues converted to glutamic acid). Due to the destruction of ring structures, these products almost completely lose antibacterial activity.

Oxidative degradation products: Under the action of oxygen, hydrogen peroxide, or oxidants in food, the methionine residue (at position 21) in nisin molecules is preferentially oxidized to methionine sulfoxide, forming "oxidized nisin." This product still retains the complete ring structure and peptide chain, but the changed polarity of methionine sulfoxide affects its interaction with bacterial cell membranes, reducing its activity to 40%–60% of the original molecule. If oxidation intensifies, thioether bonds are oxidized to sulfoxides or sulfones, leading to the disintegration of ring structures and further degradation into small peptides with oxidative modifications and free amino acids.

3. Thermal Degradation Products

The degradation of nisin at high temperatures is "complex," involving peptide bond hydrolysis, ring structure destruction, and amino acid decomposition simultaneously.

At medium to low temperatures (80–100℃, e.g., pasteurization), mild cleavage of thioether bonds in ring structures mainly occurs, generating a small amount of ring-opened peptide fragments, with an activity retention rate of approximately 60%–80%.

At high temperatures (121–140℃, e.g., high-temperature and high-pressure sterilization), extensive peptide bond hydrolysis occurs, producing short peptides of 1–5 amino acid residues (e.g., dipeptides, tripeptides). Meanwhile, unsaturated amino acids such as Dha and Dhb undergo decarboxylation and isomerization reactions, generating volatile nitrogen-containing compounds (e.g., ammonia, amines) and organic acids (e.g., acrylic acid). Some amino acids (e.g., tryptophan, tyrosine) also undergo carbonization, producing small amounts of dark-colored degradation products (e.g., melanoidins). Among these thermal degradation products, short peptides have no antibacterial activity, and volatile substances may affect food flavor (e.g., producing a slight fishy smell).

4. Storage Degradation Products

Degradation during long-term storage (e.g., within the food shelf life) is mainly dominated by "slow oxidation and hydrolysis," with products primarily being low-activity derivatives and trace fragments.

Light (especially ultraviolet light) accelerates the oxidation of nisin, generating methionine sulfoxide-type nisin. High humidity promotes peptide bond hydrolysis, producing a small amount of small-molecule peptides. The total amount of storage degradation products is usually low (accounting for 5%–20% of the initial nisin content) but accumulates gradually with prolonged storage, leading to a slow decline in the antibacterial and preservative effects of food.

III. Analytical Techniques and Methods for Degradation Products

The analysis of nisin degradation products involves three steps: "separation and purification, structural identification, and activity detection." Common technical methods need to consider the molecular size, chemical properties, and activity differences of the products.

1. Separation and Purification Techniques

First, degradation products must be separated from complex systems (e.g., food matrices, fermentation broths), with the core being fractional separation based on differences in molecular weight and polarity of the products:

Ultrafiltration: Using ultrafiltration membranes with different molecular weight cutoffs (e.g., 1 kDa, 3 kDa), degradation products are divided into "macromolecular fragments (>3 kDa, e.g., truncated nisin)," "small-molecule peptides (1–3 kDa)," and "free amino acids (<1 kDa)," realizing preliminary fractionation by molecular weight.

High-performance liquid chromatography (HPLC): Using a reversed-phase chromatographic column (e.g., C18 column) with water-acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase, degradation products with different polarities (e.g., oxidized Nisin, ring-opened peptide fragments, free amino acids) are separated by adjusting gradient elution conditions. The retention time difference between intact nisin and main degradation products (e.g., methionine sulfoxide-type Nisin) can reach 1–3 minutes, enabling quantitative analysis.

Capillary electrophoresis (CE): Utilizing the charge differences of degradation products (e.g., carboxylated peptide fragments are negatively charged, free amino acids are positively charged), efficient separation is achieved in a buffer solution. This method is particularly suitable for charged degradation products generated under acidic or alkaline conditions.

2. Structural Identification Techniques

The molecular structure and modification characteristics of degradation products are analyzed using various spectroscopic and mass spectrometric techniques:

Mass spectrometry (MS): Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) can quickly determine the molecular weight of degradation products, preliminarily judging whether they are fragmented (e.g., a molecular weight 200–500 Da smaller than that of original nisin indicates short peptide fragments) or chemically modified (e.g., a molecular weight increase of 16 Da suggests oxidative modification). Tandem mass spectrometry (MS/MS) analyzes the amino acid sequence of peptide fragments through fragment ion peaks, clarifying enzymatic cleavage sites (e.g., the presence of an Arg-Pro structure in fragment ions indicates trypsin action) or chemical modification sites (e.g., the appearance of sulfoxide characteristic fragment peaks at methionine residues).

Nuclear magnetic resonance (NMR): ¹H-NMR and ¹³C-NMR can identify special structures in degradation products through characteristic chemical shifts (e.g., -CH₂-SH groups after thioether bond cleavage, -SO- groups of methionine sulfoxide), confirming whether ring structures are opened and whether hydroxylation or carboxylation modifications exist.

Fourier-transform infrared spectroscopy (FT-IR): Changes in functional groups are determined through variations in characteristic absorption peaks. For example, the appearance of an absorption peak at 1050 cm⁻¹ indicates the presence of sulfoxide groups (oxidation products), and enhanced absorption at 1720 cm⁻¹ suggests an increase in carboxyl content (acid-base hydrolysis products).

3. Activity and Safety Detection

The activity and safety of degradation products are the core of application-oriented analysis:

Antibacterial activity detection: Using the agar diffusion method or microbroth dilution method, with Staphylococcus aureus (a Nisin-sensitive strain) as the indicator bacterium, the antibacterial activity of degradation products is determined by measuring the diameter of the inhibition zone or the minimum inhibitory concentration (MIC). If the diameter of the inhibition zone is less than 1/3 of that of original Nisin, or the MIC value increases by more than 3 times, it indicates a significant decrease in product activity.

Safety assessment: High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is used to detect toxic degradation products (e.g., excessive amines, carbonized products). Combined with cytotoxicity tests (e.g., CCK-8 assay to detect effects on intestinal epithelial cells), the potential safety of degradation products to the human body is evaluated.

Food quality impact analysis: Volatile substances in degradation products are detected (e.g., analyzed by gas chromatography-mass spectrometry (GC-MS)) to determine whether off-flavors are produced. Texture analyzers and colorimeters are used to assess the impact of degradation products on food texture (e.g., hardness, elasticity) and color.

IV. Application Significance and Prospects of Degradation Product Analysis

The analysis of nisin degradation products not only provides a basis for optimizing its application conditions but also promotes its efficient utilization in the food and pharmaceutical fields.

From an application perspective, by analyzing the types and contents of degradation products under different processing conditions (e.g., sterilization temperature, pH), the "optimal application parameters" of nisin can be determined—for example, in acidic foods (e.g., pickles, fruit juices), nisin degrades slowly, allowing for reduced addition; in high-temperature processed foods (e.g., cans), stabilizers (e.g., EDTA, citric acid) need to be compounded to inhibit thermal degradation and reduce activity loss. Meanwhile, "low-activity short peptides" and "free amino acids" among degradation products can be used as natural nutritional components in food (e.g., amino acid fortifiers), realizing "secondary utilization of degradation products" and improving resource utilization efficiency.

From a research perspective, future efforts should focus on three directions:

Developing "real-time online analysis technologies" (e.g., in-situ Raman spectroscopy, micro-HPLC) to realize dynamic monitoring of nisin degradation during food processing.

In-depth analysis of the interaction between degradation products and food matrices (e.g., proteins, fats) to clarify the impact of matrices on degradation pathways.

Exploring "functional re-excavation" of degradation products—for example, although some truncated nisin fragments have reduced antibacterial activity, they may have new functions such as antioxidant activity and intestinal flora regulation, providing possibilities for expanding their application scenarios (e.g., as functional food additives).