Global population growth has made food safety a priority, yet traditional chemical preservatives raise allergy, resistance, and carcinogenicity concerns^[1,2]. Lysozyme and antimicrobial peptides are natural, safe alternatives whose combination can reduce chemical use while enhancing shelf life and freshness^[3–5]. We screened two lysozymes and 74 antimicrobial peptides, retaining one lysozyme and nine peptides devoid of hemolytic activity, toxicity, or long half-life. The lysozyme and antimicrobial peptide genes were then cloned into expression vectors and transformed into Escherichia coli DH5α and Pichia pastoris GS115 respectively. Recombinant protein expression was carried out in Pichia pastoris to construct the genetic system of lysozyme and antimicrobial peptides. Finally, the antibacterial effect of lysozyme and antimicrobial peptides was verified by qualitative characterization and judgment, as well as quantitative characterization and quantitative analysis.

Our design consists of two main cycles:

Cycle 1: Construction and verification of plasmid containing lysozyme

Cycle 2: Construction and Verification of Plasmids Containing Antimicrobial Peptides

Test and learn

Generally speaking, our research is mainly divided into two cycles, the first cycle is mainly to construct lysozyme protein needed for the experiment, and the second cycle is mainly to construct antibacterial peptide protein needed for the experiment. After the plasmid was successfully constructed, we detected the protein expression of antimicrobial peptides and lysozyme by SDS-PAGE. MIC, MBC, Time-Kill curve, Microscopic observation and other methods were used to determine the antibacterial effects of antibacterial peptides and lysozyme.

Design: Lysozyme is a natural protein with low toxicity, and egg white lysozyme has been approved to be used in food industry. Easy to produce, lysozyme gene has been widely studied and applied, with mature expression and purification process, especially suitable for large-scale production by engineering microorganisms. In this experiment, we chose lysozyme from human. We chose this gene because HLYZ has broad-spectrum antibacterial activity. HLYZ can effectively inhibit many kinds of bacteria, especially gram-positive bacteria, by hydrolyzing peptidoglycan in bacterial cell wall. Therefore, HLYZ is likely to play an important role in food preservation.

Figure 1. The plasmid maps. pPICZα A/hLYZ.

Build:

After confirming the successful amplification of DNA sequence(Figure 2A), we further connected the gene into plasmid. The ligated plasmid was transformed into Escherichia coli by chemical transformation method and was preliminarily screened by antibiotics (Figure 2B). Subsequently, the single colony which resistant to antibiotics was selected, and the plasmid construction was confirmed by DNA sequencing (Figure 2C). Furthermore, we extracted the plasmid from E. coli and transformed it into Pichia pastoris(Figure 2D), and the construction of the strain was verified by colony PCR(Figure 2E).

Figure 2. Verification of strain construction. A: The result of target gene amplification; B: Transformation into DH5α after ligation.C: DNA sequencing after plasmid transformation into Escherichia coli. D: Successful transformation of correct plasmid into Pichia pastoris GS115.E: Colony PCR results.

Design:

Antimicrobial peptides, as natural preservatives, have strong antibacterial activity, low toxicity and innocuity, showing great application potential and market prospects in prolonging the shelf life of food, reducing the use of chemical preservatives and ensuring food safety. With the continuous development of technology, the application of antimicrobial peptides in food preservation will be further expanded, providing more green and healthy food preservation schemes. Compared with lysozyme, the reported antimicrobial peptides are richer and more diverse. Therefore, in order to find effective and safe antimicrobial peptides, we found 74 possible antimicrobial peptide molecules by consulting the literature, and eliminated the antimicrobial peptides with hemolysis risk and long intestinal half-life by online tools (HemoPI2 and ToxinPred).Finally,8 candidates were selected: Cecropin A, Melittin, Buforin II, LL-37, Plectasin, Hepcidin, Piscidin-1, and Lactoferricin. We also design a antisepsis mix protein, which connecting lysosome and 9 AMPs by flexible linker (GGGGS).

Figure 3. Construction results of plasmid pPICZα A/antisepsis mix, pPICZα A/Hepcidin, pPICZα A/Buforin II, pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin, pPICZα A/Plectasin, pPICZα A/Lactoferricin.

Build:

After confirming the successful amplification of DNA sequence(Figure 2A), we further connected the gene into plasmid. The ligated plasmid was transformed into Escherichia coli by chemical transformation method and was preliminarily screened by antibiotics (Figure 4A). Subsequently, the single colony which resistant to antibiotics was selected, and the plasmid construction was confirmed by DNA sequencing (Figure 4B). Furthermore, we extracted the plasmid from E. coli and transformed it into Pichia pastoris(Figure 4C), and the construction of the strain was verified by colony PCR(Figure 4D).

Figure 4 Verification of strain construction. A: Transformation into DH5α after ligation.B: DNA sequencing after plasmid transformation into Escherichia coli.C: Successful transformation of correct plasmid into Pichia pastoris GS115.D: Colony PCR results.

1. Protein expression

1.1 SDS-PAGE

Pichia pastoris GS115 strains carrying the correct plasmid were cultured individually at 30℃ and induced protein expression with 10 g/L methanol, and the proteins were purified using His-Tag affinity chromatography. SDS-PAGE was used to verify the protein expression of the target gene. As shown in Figure 5, different bands corresponding to the expected molecular weight were clearly observed in the purified components, confirming the accurate expression of each protein.

Figure 5. The SDS-PAGE results.

1.2 Growth curve

The expression of protein may affect the growth level of the strain. Therefore, in order to determine whether the protein expression of antimicrobial peptides and lysozyme affects the normal growth of the strain, we tested the growth curve of the strain with different antimicrobial peptide protein expressions. As shown in Figure 6, all engineered strains exhibited good growth kinetics under the test conditions. This result shows that yeast strains can grow normally and express the required antimicrobial peptides and lysozyme proteins under this culture condition.

Figure 6. Growth curves of 10 recombinant strains.

2. Functional Test

2.1 Minimum Inhibitory Concentration (MIC) determination

The minimum antibacterial concentration (MIC) of biological preservatives was determined by Broth microdilution method to evaluate their antibacterial activity. Single colonies of the test strains E. coli and Bacillus subtilis were inoculated into LB medium for overnight culture, and the resulting suspension was adjusted to 10⁶ CFU mL^-1 in fresh medium. Dispense 100 μL of the purified protein, from 128 to 2 μg·mL^-1, into a 96-well microplate. Subsequently, 100 μL of standardized bacterial suspension was added to each well and the plates were incubated at 37℃ for 24 h. Wells containing only sterile LB medium can serve as negative controls, while wells containing bacteria serve as positive controls to confirm bacterial viability.

The MIC₅₀ of the purified protein is shown in Table 1. From MIC results, we can see that Plectasin has the strongest antibacterial effect on both Escherichia coli and Bacillus subtilis. When the concentration of Plectasin is 4 μg/ml, 50% bacteria can be killed. The MIC₅₀ values of all antimicrobial peptides are below 16 μg/ml, which indicates that all antimicrobial peptides involved in the experiment have certain antibacterial activity. However, the MIC₅₀ value of lysozyme is higher, this means the bacteriostatic effect of lysozyme is slightly weak, but the MIC₅₀ value is also below 32 μg/ml.Thus lysozyme also has certain bacteriostatic effect.

Table 1. The Inhibitory concentration curve results.

Drug	MIC50
	E. coli	Bacillus subtilis
Cecropin A	8 μg/ml	16 μg/ml
Melittin	8 μg/ml	16 μg/ml
LL-37	8 μg/ml	4 μg/ml
Plectasin	4 μg/ml	4 μg/ml
Piscidin-1	8 μg/ml	4 μg/ml
Hepcidin	8 μg/ml	16 μg/ml
Lactoferricin	8 μg/ml	16 μg/ml
Buforin II	8 μg/ml	16 μg/ml
HLYZ	16 μg/ml	32 μg/ml
Antisepsis mix	16 μg/ml	16 μg/ml

2.2 Time-Kill curve assay

The growth of bacteria at different time points was monitored by optical density method (OD₆₀₀). By drawing the growth curve of bacteria treated with antimicrobial peptides, we can know the inhibitory effect of antimicrobial peptides on bacteria and how long it takes for bacteria to recover their growth ability after antimicrobial peptides treatment. Equal volumes of biopreservatives and bacterial suspensions were mixed and incubated at 30℃ for 24 h, and absorbance was measured at 0, 2, 4, 6, 8 and 24 h to determine the growth curve of bacteria under the condition of adding different biopreservatives.

As shown in Figure 7, the addition of biological preservatives inhibited the growth of bacteria to varying degrees. From the curve results, the inhibitory effect of some antibacterial peptides will gradually weaken with the increase of time. In the experiment of inhibiting Escherichia coli, four antimicrobial peptides(Cecropin A, Lactoferricin, LL-37 and Plectasin) showed strong activity. With the increase of culture time, the growth of bacteria was still inhibited to a great extent. In order to explore whether there is a combined effect between antibacterial peptides and lysozyme, we mixed all antibacterial peptides with lysozyme and determined the antibacterial activity of the mixed samples. The results show that the mixed samples have certain antibacterial activity, but the combined effect is not significant, which may be because we mixed all antibacterial peptides, some of which have poor antibacterial activity or antagonistic effect, resulting in poor mixed effect. Later, we will consider the combination of two, so as to find the best combination scheme with antibacterial effect. Compared with Escherichia coli, most antimicrobial peptides have better antibacterial effect on Bacillus subtilis. Notably, Plectasin showed the most significant inhibitory effects against E. coli and Bacillus subtilis.

Figure 7. Time-Kill curve. A: E. coli; B: Bacillus subtilis.

2.3 Laser Scanning Confocal Microscope observation

The bacterial suspension was incubated with different biopreservatives for a total of 10 h at 37℃, followed by double staining of live and dead cells using Calcein-AM/PI. Calcein-AM is hydrolyzed by intracellular esterase after entering the cell to produce a strong anionic green fluorescence product, thereby labeling the living cells with green fluorescence. Propidium iodide (PI) cannot cross the cell membrane of living cells and only enters when the membrane is damaged; It inserts into the DNA, producing a red fluorescence that labels dead cells. This differential staining protocol labels both live and dead cells in all samples, and their visualization under confocal microscopy provides a rapid quantitative assessment of the antimicrobial efficacy of bio preservatives.

As shown in Figure 8, all tested bio preservatives exerted bactericidal activity against both Escherichia coli and Bacillus subtilis, although the extent of killing varied markedly among treatments. Notably, exposure to Plectasin or the LL-37 resulted in a pronounced reduction in B. subtilis viability, indicating potent bactericidal efficacy against this species. Conversely, Cecropin A left a substantially higher residual viable count of B. subtilis, revealing a comparatively weak activity against this bacterium. In contrast, Plectasin was less effective against E. coli than against B. subtilis, whereas LL-37 displayed superior bactericidal performance against E. coli relative to its effect on B. subtilis. Through the observation under confocal microscope, we confirmed that our antimicrobial peptide protein has certain killing ability to bacteria. Moreover, our lysozyme also has a certain bactericidal effect on Bacillus subtilis. Through the screening of the above experiments, we determined that Plectasin and LL-37 are the best antibacterial peptides we have obtained at present, and further we want to observe the morphology of the treated bacteria by scanning electron microscope.

Figure 8. Laser Scanning Confocal Microscope results. Laser Scanning Confocal Microscope results. A: Bacillus subtilis; B: E. coli; C: Bacterial mortality.

2.4 Morphological observation of scanning electron microscope

Further, we want to observe the morphology of bacteria treated with antibacterial peptides by scanning electron microscope. We chose two antibacterial peptides (Plectasin and LL-37) which had the most remarkable effect in the previous antibacterial experiment and chose AMP as the positive control. By scanning electron microscope, we can see that compared with the bacteria not treated with antimicrobial peptides, the surfaces of Escherichia coli and Bacillus subtilis were damaged to varying degrees after treated with antimicrobial peptides, which means that our antimicrobial peptides may damage the cell wall or cell membrane structure of bacteria, thus causing bacterial death. The specific bactericidal mechanism of antimicrobial peptides needs further analysis.

Figure 9. Scanning electron microscope observation results. A: E. coli; B: Bacillus subtilis.

1. In this study, lysozyme and antimicrobial peptides were successfully expressed, but yields remained modest, most likely due to suboptimal promoter selection and inadequate codon optimization. Therefore, we intend to implement codon-promoter joint coordination, co-express chaperones, and employ a high-density fermentation strategy to significantly improve production titers and biological activity.

2. Functional assays indicate limited antimicrobial potency of the resulting bio preservatives, suggesting that the expression environment may impair the conformation or stability of the fusion protein. Therefore, in the future, we will use multiple expression systems in combination: try to compare different hosts to determine the system that best preserves the natural fold and maximizes antimicrobial efficacy.

3. We also need to obtain more proteins, so as to repeat the experiment many times to obtain more accurate data. Therefore, increasing protein production is the most important thing that we need to optimize and improve in the next step. At present, our experiment only found that our antibacterial peptide can inhibit the growth of bacteria, and the specific mechanism has not been resolved. Further, we may analyze the bactericidal mechanism of this antibacterial peptide.

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