With the increasing global population, food safety has gradually become the focus of global attention[1]. Food preservation and preservative play a crucial role in the food industry. Although traditional chemical preservatives are effective in extending the shelf life of food, their widespread use poses many health risks, including allergic reactions, drug resistance issues, and potential carcinogenicity[2]. Therefore, the development of natural, green and harmless preservatives has become an important trend in the development of the food industry.
As natural antibacterial ingredients, lysozyme and antimicrobial peptides have become hot topics in food preservative research in recent years due to their excellent antibacterial properties and good safety[3,4,5]. The combination of the two can not only enhance the antibacterial effect, but also reduce the dependence on traditional chemical preservatives, further improving food safety and freshness.
The main goal of this project is to develop a novel food preservative technology to improve the shelf life and safety of food through the combination of lysozyme and antimicrobial peptides. We first screened from 2 lysozymes[6,7] and 74 antimicrobial peptides[8-81], eliminated antimicrobial peptides with hemolytic risk, toxicity, and long intestinal half-life, and finally screened out 1 lysozyme and 9 antimicrobial peptides. The lysozyme and antimicrobial peptide genes were then cloned into expression vectors and transformed into Escherichia coli DH5α and Pichia pastoris GS115 respectively. We expressed antimicrobial peptides and lysozyme proteins by Pichia pastoris and separated and purified these proteins by affinity chromatography column. Finally, the antibacterial effect of lysozyme and antimicrobial peptides was verified by qualitative characterization and judgment, as well as quantitative characterization and quantitative analysis.
We obtained target genes for constructing 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/hLYZ, pPICZα A/Lactoferricin using PCR technology, as shown in Figure 1A. We obtained fragments of approximately 1600bp, 80bp, 80bp, 80bp, 130bp, 130bp, 90bp, 130bp, 460bp, 160bp in length, which are consistent with our expected sizes.
Subsequently, we selected EcoRI and NotI restriction sites for enzymatic digestion of the target genes and vector fragment (as shown in Figure 1B). We obtained fragments of approximately 3600bp in length, which are consistent with our expected sizes.
Figure 1. Gel electrophoresis validation. A: PCR results; B: vector enzyme digestion results.
2.2 Fragment ligation and transformationThe digested PCR fragment and vector were ligated using T4 DNA ligase and then we transformed them into Escherichia coli DH5α, as shown in Figure 2A.The results preliminarily proved that the strain construction was successful. In order to further confirm the result, we sequenced the DNA(Figure 2C). The results of DNA sequencing are completely consistent with expectations, demonstrating that all ten target gene fragments were precisely integrated into pPICZα A.
Compared with bacteria, yeast is a eukaryote, which is more closely related to human beings, and the protein expressed by yeast may be safer for human beings. Therefore, we chose Pichia pastoris as the strain for protein expression. The verified plasmids were then extracted from E. coli and transformed into Pichia pastoris GS115 (Figure 2D). Further, in order to verify the correctness of the strain construction, we selected Single colony from the plate for colony PCR and observed the results by gel electrophoresis. Consistent with expectations, all candidates are the same size as expected (Figure 2B). In order to determine whether antibacterial peptides and lysozyme can be expressed normally, we want to induce the strain and verify whether the protein can be expressed normally after being transformed into yeast by SDS-PAGE.
Figure 2. 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/hLYZ, pPICZα A/Lactoferricin. A: Transformation into DH5α after ligation; B: Colony PCR results; C: Sequencing results; D: Successful transformation of correct plasmid into Pichia pastoris GS115.
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 3, different bands corresponding to the expected molecular weight were clearly observed in the purified components, confirming the accurate expression of each protein.
Figure 3. The SDS-PAGE results.
3.2 Growth curveThe 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 4, 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 4. Growth curves of 10 recombinant strains.
We hope that through our experiments, antibacterial peptides with the strongest bactericidal effect can be screened, so as to be combined with lysozyme to achieve the purpose of food preservation. First, we tested the minimum inhibitory concentration of each antimicrobial peptide. 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 |
Based on MIC results, we initially found that some antimicrobial peptides have good antibacterial effects, such as LL-37 and Piscidin-1, among which Plectasin has the best antibacterial effects. In order to further verify this result and find the most suitable antibacterial peptide, we tested the bactericidal effect of antibacterial peptide after treatment for different times. Based on the experimental results of MIC, we found that some bacteria could not be killed even if the concentration of antimicrobial peptides had risen to the highest concentration (128μg/ml). Therefore, in order to ensure that antibacterial peptides can kill most bacteria, in the subsequent experiments, we all use antibacterial peptides with a concentration of 128μg/ml for experiments. 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 bio preservatives 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 bio preservatives.
As shown in Figure 5, 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 5. Time-Kill curve. A: E. coli; B: Bacillus subtilis.
4.3 Laser Scanning Confocal Microscope observationThe bacterial suspension was incubated with different bio preservatives 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 6, 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 6. Laser Scanning Confocal Microscope results. A: Bacillus subtilis; B: E. coli; C: Bacterial mortality.
4.4 Morphological observation of scanning electron microscopeFurther, 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 7. Scanning electron microscope observation results. A: E. coli; B: Bacillus subtilis.
Through the above experiments, we determined that antibacterial peptides and lysozyme played an important role in inhibiting bacterial growth, suggesting that antibacterial peptides and lysozyme may play a key role in preventing food spoilage, and they can be used as important components of food preservatives. At the same time, there are still some shortcomings in our experiments. Due to time constraints, some of our experiments have not been done perfectly. First of all, due to the limitation of our protein purification process at present, the purity of antibacterial peptides we obtained is not enough, so the protein concentration used in our experiment may be inconsistent with the actual antibacterial concentration. In order to obtain more accurate results, we need to optimize the protein purification process, so as to obtain higher purity protein.Secondly,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. Finally, we also need to obtain more proteins, so as to repeat the experiment many times to obtain more accurate data. In the follow-up experiments, we will try to combine our antimicrobial peptides with lysozyme to explore whether the combination of them can have better bactericidal effects.
In the future, the work will be carried out simultaneously at three levels: improving expression efficiency, ensuring biosecurity, and promoting industrial implementation: the yield and activity of lysozyme and antimicrobial peptides will be significantly improved through codon and promoter collaborative optimization, the introduction of molecular chaperones, and the optimization of fermentation processes, and the synergistic antimicrobial spectrum of the two against foodborne pathogens will be systematically evaluated. Construct suicide switches that can induce suicide switches, carry out environmental release risk assessment, immunogenicity and long-term impact research on intestinal flora to ensure that GMO hosts and products comply with food safety regulations of various countries. On this basis, a low-cost continuous purification process and a GMP-level quality control system are established to complete scale-up verification from laboratory to industrial scale and realize commercial application.
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