Our project uses synthetic biology to assemble plasmids carrying lysozyme and antimicrobial peptide genes in vitro, and then transform them into chassis strains Pichia to establish a dedicated genetic expression platform. Subsequently, an integrated set of qualitative and quantitative assays was used to verify antimicrobial efficacy. Four key measurements were designed throughout the engineering cycle:
(1)	Minimum inhibitory concentration (MIC) to quantify antimicrobial potency. 
(2)	Time-kill curve analysis to monitor bacterial growth kinetics under the challenge of biological preservatives. 
(3)	Laser Scanning Confocal Microscope was used for live/dead staining to determine the bactericidal effect of biological preservatives.  
(4)	Scanning electron microscopy (SEM) can observe morphological changes of bacterial cells under the action of biological preservatives.  
These experiments are crucial for us to test the antibacterial effect of biopreservatives, MIC needs to measure the optical density value of bacteria after the action of biopreservatives to quantify their antibacterial ability, and the Time-kill curve needs to be measured by optical densitome (OD600) to monitor the growth of bacteria at different time points after the action of biological preservatives, Scanning electron microscope is an important means to observe the morphological changes of bacteria after antibacterial peptide treatment and Laser Scanning Confocal Microscope needs to observe the number of living and dead cells to determine the bactericidal effect of biological preservatives.
Accordingly, the measurement protocol is as follows:
(1)	MIC: OD₆₀₀ values recorded across a concentration gradient to generate dose-response curves.
(2)	Time-kill curves: OD₆₀₀ readings at defined time intervals to construct bacterial survival kinetics.
(3)	Laser Scanning Confocal Microscope: dual-fluorescence images (green/red) discriminating live from dead cells to evaluate bactericidal activity.
(4)	SEM: high-resolution micrographs capturing surface morphology changes.
      

Measurement Background
The rapid emergence of antibiotic resistance has made accurate determination of antibacterial activity essential for infection diagnosis, drug discovery, and food-safety assessment. The Minimum Inhibitory Concentration (MIC) is the internationally accepted in-vitro reference, guiding dose selection, resistance surveillance, and development of natural food preservatives [1].
Measurement Principles
MIC is defined as the lowest concentration of an antimicrobial agent that completely inhibits visible bacterial growth after 16-24 h incubation under standardized conditions.
Measurement Protocols
Materials:
(1)	Bacterial suspension (106 CFU mL-1)(E. coli and Bacillus subtilis)
(2)	LB culture medium
(3)	Sterile water 
(4)	Incubator
(5)	purified protein, from 128 to 2 μg·mL-1
(6)	Microplate reader，Steriled 96-well microplate
(7)	Centrifuge tubes
(8)	Ampicillin
Procedures:
(1)	Prepare standard target protein solution:
We uniformly configured the purified protein solution to a standard concentration of 128 μg/mL and diluted 128 μg/mL of lysozyme and antimicrobial peptide protein solution with sterile water to obtain six standard samples at concentrations of 128, 64, 32, 16, 8, 4, and 2 μg/mL, respectively.
(1)	Add 100 μL each diluted sample and equal volume bacterial suspension to the 96-well microplate
(2)	Wells containing only sterile LB medium can serve as negative controls, while wells containing bacteria serve as positive controls, At the same time, we use AMP with concentration of 100 μg·mL-1 as control.
(3)	After the bacteria and the antimicrobial peptide are fully mixed, We put the culture plate in a constant temperature incubator and cultured it at 37°C for 24 hours.
(4)	Measure the absorbance of each well at 600 nm wavelength using microplate reader, At the same time, by observing and reading the concentration of antimicrobial peptides when the turbidity of bacteria decreases by 50%, it is regarded as the MIC50 value of antimicrobial peptides to bacteria
      

Table 1. The Inhibitory concentration curve results.

The MIC₅₀ of the purified protein is shown in Table 1. The bactericidal ability of antimicrobial peptides to bacteria was determined by gradient dilution method. The higher the concentration of antimicrobial peptides, the stronger the bactericidal ability. Based on the current standard reading scheme of MIC, we take MIC₅₀ value as an important index to measure the antibacterial activity of antibacterial peptides. MIC₅₀ refers to the minimum inhibitory concentration that inhibits 50% of the strains. That is to say, the lowest drug concentration when 50% of the tested strains are inhibited from growing in the experiment. MIC₅₀ is a reference index to measure the antibacterial effect of drugs, which is helpful to understand the sensitivity of drugs to specific strains. 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.

Measurement Background
The quantitative time-kill curve dynamically monitors live bacterial counts over time, providing a direct measurement of kill rate, concentration dependence, making it a cornerstone of pharmacodynamic studies.
Measurement Principles
Biological preservatives can play a certain role in inhibiting the growth of bacteria. The growth rate of bacteria slowed down after the addition of biological preservatives, and the stronger the antibacterial effect of biological preservatives, the more obvious the inhibition of bacterial growth, and the OD600 value decreased significantly or did not increase.
Measurement Protocols
Materials:
(1)	Bacterial suspension (106 CFU mL-1) ( E. coli and Bacillus subtilis)
(2)	LB culture medium
(3)	Sterile water 
(4)	Incubator
(5)	purified protein (128 μg·mL-1)
(6)	Microplate reader
(7)	Steriled 96-well microplate
(8)	Centrifuge tubes
Procedures:
(1)	Prepare standard target protein solution:
We uniformly configured the purified protein solution to a standard concentration of 128 μg/mL.
(1)	Add 500 μL each protein solution and equal volume bacterial suspension to the centrifuge tubes.
(2)	Tubes containing only sterile LB medium can serve as negative controls, while tubes containing bacteria serve as positive controls, At the same time, we use AMP with concentration of 100 μg·mL-1 as control.
(3)	Incubate at 37°C for 24 h.
(4)	Samples were taken into 96-well plates at time points 0, 2, 4, 6, 8, and 24 h and measure the absorbance of each well at 600 nm wavelength using microplate reader.
(5)	Curves are plotted with Origin showing the change in bacterial absorption values over time (Figure 1)
(6)	The curves are fitted with logistic functions.
      

Figure 1. Time-Kill curve. A: E. coli; B: Bacillus subtilis.

In this experiment, we co-incubate antibacterial peptides with bacteria in LB liquid, and measure the absorbance of the partially co-incubated liquid at regular intervals, and draw the growth curve of bacteria, so as to preliminarily judge whether the antibacterial effect of antibacterial peptides changes with time.

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. Notably, Plectasin showed the most significant inhibitory effects against E. coli and Bacillus subtilis.

Measurement Background
Laser scanning confocal microscopy (LSCM) is a modern biomedical imaging instrument, which is based on fluorescence microscopy imaging by adding a laser scanning device to excite fluorescent probes using ultraviolet light or visible light. The instrument uses computer, laser, and image processing techniques to obtain fluorescence images of microstructures inside cells or tissues, as well as to observe physiological signals and changes in cell morphology at the subcellular level. As the most advanced molecular cell biology analytical instrument, this instrument is mainly used to observe the biological changes of living cell structure and specific molecules and ions, quantitative analysis and real-time quantitative determination [3-5].
Measurement Principles
We used CaIcen-AM/PI to double stain live and dead cells. Calcein-AM penetrates intact cell membranes. After entering the living cells, they are hydrolyzed by esterase to produce a strongly negatively charged green fluorescent product, which gives the living cells a green color, propidium iodide (PI) cannot enter living cells with intact membranes. When cells die under the action of biological preservatives, PI enters the cell and binds to DNA, producing red fluorescence that gives dead cells a red color. By fluorescence staining, Live and dead cells are labeled in different samples and viewed under Laser scanning confocal microscopy, enabling rapid assessment of the antimicrobial effects of biopreservatives.
Measurement Protocols
Materials:
(1)	Bacterial suspension (106 CFU mL-1)( E. coli and Bacillus subtilis)
(2)	LB culture medium
(3)	Sterile water 
(4)	Incubator
(5)	purified protein
(6)	Calcein-AM/PI Live/Dead Cell Double Staining Kit
(7)	Slides
(8)	Coverslip
(9)	StedyconGallery software
(10)	Confocal microscope
Procedures:
(1)	Cell preparation
After mixing 100μL bacteria（106 CFU mL-1） and antimicrobial peptides(128 μg/mL), they were placed in a 96-well plate and incubated at 37°C for 12 hours. Harvest and centrifuge the cells, then resuspend in 1× Assay Buffer.
(1)	Calcein-AM staining (live-cell marker)
Add 1–2 µL Calcein-AM(2mM) stock per 1 mL of cell suspension, mix gently, and incubate at 37 °C in the dark for 20–25 min.
(1)	PI staining (dead-cell marker)
Supplement the same tube with 3-5 µL PI(1.5 mM) stock, mix, and incubate at room temperature in the dark for 5 min.
(1)	Washing and slide preparation
Centrifuge at 450×g for 5 min to remove excess dye, wash once with 1×PBS, and resuspend. Spot 3-5 µL of the suspension onto a slide and cover with a coverslip.
(1)	Fluorescence detection
Observe under a fluorescence or laser-scanning confocal microscope. Excitation at 490 ± 10 nm simultaneously reveals live cells (yellow-green fluorescence) and dead cells (red). 545 nm excitation visualizes only the red PI-positive dead cells. Complete microscopic observation within 30min as far as possible.
      

Figure 3. Laser Scanning Confocal Microscope results. A: E. coli; B: Bacillus subtilis.

In order to further determine the antibacterial effect of antibacterial peptides. After co-incubation of antimicrobial peptides with bacteria, we dyed the bacteria with live-dead dyes and observed the experimental results under confocal microscope. If the death rate of bacteria is high, most bacteria will emit red fluorescence under the microscope, and if most bacteria are alive, they will emit green fluorescence. In this way, we can directly observe the killing effect of antimicrobial peptides on bacteria.

With fluorescence staining, we can label live and dead cells in different samples, so that the number of live/dead cells can be clearly observed under confocal microscopy. As shown in Figure 3, 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.

Measurement Background
Scanning electron microscope (SEM) is an observation method between transmission electron microscope and optical microscope. It uses a narrow focused high-energy electron beam to scan the sample, and through the interaction between the beam and the substance, it stimulates various physical information, and collects, amplifies and re-images this information to achieve the purpose of characterizing the micro-morphology of the substance. The resolution of the new scanning electron microscope can reach 1 nm; The magnification can reach 300,000 times or more and can be continuously adjusted; And the depth of field is large, the visual field is large, and the imaging stereoscopic effect is good. Through scanning electron microscope, we can observe the surface of bacteria in detail to understand the bactericidal mechanism of antibacterial peptides
Measurement Principles
The bacteria treated with antimicrobial peptides or lysozyme were fixed by paraformaldehyde and glutaraldehyde, and the morphology of the bacteria was observed under scanning electron microscope.
Measurement Protocols
Materials:
Escherichia coli K-12
Bacillus subtilis
LB culture medium
Petri dishes
Microcentrifuge tubes
Pipette tips
Sterile water 
Incubator
Ampicillin
PBS buffer
Scanning electron microscope and related reagents
Procedures:
1.Cell preparation：After mixing 100 μL bacteria（106 CFU mL-1） and antimicrobial peptides(128 μg/mL), they were placed in a 96-well plate and incubated at 37°C for 12 hours. Harvest and centrifuge the cells, then resuspend in PBS Buffer. Take 3 μL of the strain and drop it on the cell slide.Sampling: Generally, the sample size is not more than 5mm x 5mm. Determine the target of observing the sample to avoid pulling and squeezing the sample during sampling. 
2. Cleaning: remove impurities from the observation surface and fully expose the observation surface without damaging it. 
3. Fixation: Generally, it is better to fix glutaraldehyde and acid at 4℃.
Glutaraldehyde and acid can be fixed separately or double-fixed with "glutaraldehyde-osmium acid". After fixing for 1-3 hours, the fixed solution is sucked out, 0,1M of cool acid buffer wave (PH=7.2) is added, and the solution is rinsed for 3-4 times for 1 hour. 
4. Dehydration: Suck out the buffer wave from the vial, add ethanol to dehydrate step by step (the ethanol concentration is 30%-50%-70%6-80%-90%-100% at a time), and the dehydration time depends on the sample size, generally 15-30 minutes. Add the 1:1 mixture of isoamyl acetate and ethanol, soak for 10-20min and shake properly, discard the mixture, add pure isoamyl acetate and soak for 10-20min and shake properly.
5. Drying: Drying is a key link in the biological preparation of scanning electron microscope. It is necessary to reduce the distortion of surface morphology caused by water evaporation surface as much as possible during the drying process, and it is necessary to ensure thorough drying. Commonly used methods include natural drying, dry drying, critical point drying, freeze drying and vacuum drying. Different drying methods will be selected for different biological samples.
6. Sample loading: the surface is plated with gold and observed by scanning electron microscope.
      

Figure 4. Scanning electron microscope observation results. A: E. coli; B: Bacillus subtilis.

Compared with confocal microscopes, the sensitivity and resolution of scanning electron microscope are significantly higher. After co-incubation of antimicrobial peptides with bacteria, we can see more clearly whether the cell structure of bacteria has been damaged by scanning electron microscopy. Through morphological observation, we can further confirm whether antibacterial peptides have bactericidal effect.

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.

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