Lactobacillus alimentarius irradiated by laser and its application in controlling foodborne pathogenic bacteria

Abstract

This invention discloses a laser-irradiated *Lactobacillus amylovorus* and its application in controlling foodborne pathogens, belonging to the field of microbial technology. The *Lactobacillus amylovorus* LY2332 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37009. This invention induces metabolic remodeling in *Lactobacillus amylovorus* through laser irradiation, effectively enhancing its environmental adaptability and the antibacterial activity of its cell-free supernatant, and demonstrating good application results in the preservation of chilled meat. This invention not only clarifies that laser irradiation can enhance the antibacterial activity of cell-free supernatant of lactic acid bacteria, but also provides a new strategy for developing natural food control agents and lays a theoretical foundation for the industrial application of lactic acid bacteria in food preservation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a laser-irradiated Lactobacillus amyloliquefaciens and its application in controlling foodborne pathogens. Background Technology

[0002] Foodborne pathogen contamination is a significant factor affecting food safety and food quality stability. Fresh meat and other animal-derived foods are susceptible to exogenous microbial contamination during processing, transportation, and storage. Common representative foodborne pathogens include Yersinia enterocolitica, Salmonella enteritidis, and Listeria monocytogenes. These pathogens not only cause foodborne illnesses but also accelerate food spoilage, leading to shortened shelf life and increased economic losses. Therefore, developing safe, efficient, and applicable natural antimicrobial measures for practical food systems is an important research direction in the field of food safety control.

[0003] Lactic acid bacteria, due to their relatively good safety and abundant metabolites, have become an important microbial resource for the development of antimicrobial agents in natural foods. Among the many lactic acid bacteria, *Lactobacillus amyloliquefaciens* has gradually become an important subject in lactic acid bacteria research due to its strong carbon source utilization ability, environmental adaptability, and potential functional characteristics. Existing studies have shown that *Lactobacillus amyloliquefaciens* can be derived from fermented foods, animal intestines, and the natural environment, and has shown certain antimicrobial activity, stress resistance, and probiotic potential in some studies. However, the function of lactic acid bacteria exhibits significant strain specificity; strains from different sources often show significant differences in antimicrobial spectrum, gastrointestinal tolerance, safety, and host interaction capabilities.

[0004] Furthermore, compared to using live bacteria directly, cell-free supernatants of lactic acid bacteria have attracted widespread attention in food preservation and pathogen control due to their absence of live bacteria, relatively simple preparation, easier standardization of their action, and better processing adaptability and storage stability. However, the antibacterial strength and stability of cell-free supernatants from natural strains are often limited by the genetic background of the strains and culture conditions, making it difficult to meet the demands of higher-level food applications. Therefore, further enhancing the metabolic capacity and antibacterial activity of existing functional strains is an important way to improve the application value of lactic acid bacteria. Summary of the Invention

[0005] The purpose of this invention is to provide a laser-irradiated Lactobacillus amyloliquefaciens to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A laser-irradiated Lactobacillus amylovorus, wherein the Lactobacillus amylovorus is Lactobacillus amylovorus LY2332, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37009, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on December 17, 2025.

[0008] Another object of the present invention is to provide a cell-free supernatant of Lactobacillus amyloliquefaciens, which is obtained by fermentation culture of Lactobacillus amyloliquefaciens irradiated by the above-mentioned laser.

[0009] Furthermore, the method for preparing the cell-free supernatant includes the following steps:

[0010] The Lactobacillus amyloliquefaciens was activated and then prepared into a bacterial suspension;

[0011] After laser irradiation treatment of the bacterial suspension, fermentation culture is carried out to obtain fermentation broth;

[0012] After centrifuging the fermentation broth, the supernatant was filtered to obtain a cell-free supernatant.

[0013] Furthermore, the laser is a blue laser.

[0014] Furthermore, the wavelength of the blue laser is 450 nm.

[0015] Furthermore, the irradiation power of the blue laser is 30-240 mW, and the irradiation time is 20-100 min.

[0016] Another object of the present invention is to provide an application of the above-mentioned laser-irradiated Lactobacillus amyloliquefaciens or the cell-free supernatant of the above-mentioned Lactobacillus amyloliquefaciens for the control of foodborne pathogens.

[0017] Furthermore, the foodborne pathogens are one or more of Yersinia enterocolitica, Listeria monocytogenes, and Salmonella enteritidis.

[0018] Another object of the present invention is to provide the application of the above-mentioned laser-irradiated Lactobacillus amyloliquefaciens or the cell-free supernatant of the above-mentioned Lactobacillus amyloliquefaciens in the antibacterial and / or preservation of fresh meat.

[0019] This invention provides a laser-irradiated *Lactobacillus amyloliquefaciens* strain that exhibits high inhibitory activity against foodborne pathogens such as *Yersinia enterocolitica*, *Salmonella enteritidis*, and *Listeria monocytogenes*, significantly superior to the original strain (significantly increased inhibition zone diameter, significantly reduced minimum inhibitory concentration, demonstrating a stronger broad-spectrum antibacterial effect). This invention induces metabolic remodeling in *Lactobacillus amyloliquefaciens* through laser irradiation, effectively enhancing the antibacterial activity and environmental adaptability of *Lactobacillus amyloliquefaciens* and its cell-free supernatant, and demonstrating good application results in fresh meat preservation. This invention not only clarifies the mechanism by which laser irradiation enhances the antibacterial activity of lactic acid bacteria but also provides a new strategy for developing natural food preservatives and lays a theoretical foundation for the industrial application of lactic acid bacteria in food preservation. Attached Figure Description

[0020] Figure 1 The image shows the antibacterial effect of strain LY2332 against three indicator bacteria; in the image, A is Yersinia enterocolitica, B is Listeria monocytogenes, and C is Salmonella enteritidis.

[0021] Figure 2 The images show the colony morphology and Gram staining of strain LY2332; in the images, A is the Gram staining image and B is the colony morphology image.

[0022] Figure 3 This is a comparison chart of the diameter of the inhibition zone under different irradiation conditions.

[0023] Figure 4 This is a graph showing the results of the growth curve measurement.

[0024] Figure 5 The graph shows the results of the acid production curve determination.

[0025] Figure 6 The image shows the results of the bile salt tolerance test.

[0026] Figure 7 This is a diagram showing the results of a simulated gastrointestinal fluid experiment.

[0027] Figure 8 The figure shows the results of the acid tolerance test.

[0028] Figure 9 The image shows the inhibition zone of CFS after laser treatment; in the image, A represents Yersinia enterocolitica, B represents Listeria monocytogenes, and C represents Salmonella enteritidis.

[0029] Figure 10 The figure shows the effect of blue laser irradiation on the antibacterial ability of CFS; in the figure, A is Yersinia enterocolitica, B is Listeria monocytogenes, and C is Salmonella enteritidis.

[0030] Figure 11 The figure shows the stability test results of the CFS antibacterial ability of Lactobacillus amyloliquefaciens after laser irradiation.

[0031] Figure 12 This is an ion flow map in NEG mode.

[0032] Figure 13 This is an ion flow map in POS mode.

[0033] Figure 14 Principal component analysis diagram; in the diagram, A represents positive ions and B represents negative ions.

[0034] Figure 15 The diagram shows the PLS-DA discriminant analysis model; in the diagram, A represents positive ions and B represents negative ions.

[0035] Figure 16 This is a differential metabolic volcano diagram; in the diagram, A represents positive ions and B represents negative ions.

[0036] Figure 17 This is a classification diagram of KEGG in POS mode.

[0037] Figure 18 This is a classification diagram of KEGG under the neg pattern.

[0038] Figure 19 This graph shows the changes in the number of pathogenic bacteria in pork at different temperatures during storage.

[0039] Figure 20 This graph shows the pH changes of pork at different temperatures during storage.

[0040] Figure 21 This is a comparison chart of sensory evaluation scores for pork at different temperatures during storage.

[0041] In each figure, "Lactobacillus amylovorus LY2332" refers to the untreated group; "Lactobacillus amylovorus LY2332 with lg" refers to the laser-treated group. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] In one embodiment of the present invention, a laser-irradiated Lactobacillus amylovorus is provided, wherein the Lactobacillus amylovorus is Lactobacillus amylovorus LY2332, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37009, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, and deposited on December 17, 2025.

[0044] In another embodiment of the present invention, a cell-free supernatant (CFS) of *Lactobacillus amyloliquefaciens* is also provided, which is obtained by fermentation culture of *Lactobacillus amyloliquefaciens* irradiated by laser as described above. Specifically, the preparation method of *Lactobacillus amyloliquefaciens* CFS includes the following steps:

[0045] S1. After activating Lactobacillus amyloliquefaciens LY2332, a bacterial suspension was prepared.

[0046] S2. Irradiate the above bacterial suspension with a wavelength of 450 nm and an irradiation power of 30-240 mW for 20-100 min, and then carry out fermentation culture to obtain fermentation broth.

[0047] S3. After centrifuging the above fermentation broth, take the supernatant and filter it to obtain CFS.

[0048] In this embodiment of the invention, laser irradiation, as a physical regulation method, compared with traditional chemical induction or gene modification methods, not only avoids the interference and residual risks of chemical reagents on the metabolism of strains, but also regulates the metabolic process of lactic acid bacteria through gentle energy stimulation. It does not require complicated subsequent separation and purification steps, and has no significant inhibitory effect on the growth and reproduction of the strain itself. It can enhance the activity of strain metabolites while maintaining its environmental adaptability, providing a convenient and safe technical path for the efficient development of high-performance lactic acid bacteria, and has broad application prospects in the food industry.

[0049] Laser irradiation significantly accelerated the early acid production rate of *Lactobacillus amyloliquefaciens* strain LY2332, enabling it to lower environmental pH more rapidly, and significantly enhanced its environmental tolerance (e.g., acid and bile salt tolerance). LC-MS / MS non-targeted metabolomics analysis revealed a significant reconstruction of the metabolic profile of the CFS strain after laser irradiation, identifying numerous differential metabolites. These differential metabolites were primarily enriched in membrane lipid synthesis, amino acid metabolism, and secondary metabolite synthesis pathways. It is speculated that laser irradiation, by regulating these metabolic pathways, promoted the synthesis and accumulation of antibacterial-related metabolites, thereby enhancing the antibacterial activity of CFS. In a pork preservation model, under 4°C refrigeration conditions, the CFS strain after laser irradiation effectively inhibited the proliferation of pathogens in pork, delayed the increase in pork pH, and better maintained the sensory qualities of pork, such as color and odor, extending its shelf life. At 25°C, its antibacterial and preservation effects were also superior to the original CFS strain, effectively slowing down the rate of pork spoilage.

[0050] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, the materials, reagents and instruments involved are all commercially available products; unless otherwise specified, the experimental methods used are all conventional methods.

[0051] Example 1: This example provides a method for isolating and identifying Lactobacillus amyloliquefaciens LY2332, specifically including the following steps:

[0052] (1) Samples were collected, mainly bovine-related samples such as anal swabs, oral swabs and vaginal swabs. After collection, the samples were placed in sterile sampling tubes and transported to the laboratory at 4°C. Separation and processing were completed within 24 hours.

[0053] (2) The collected samples were appropriately diluted and spread onto MRS agar plates, and anaerobically cultured at 37°C for 48 hours. Single colonies with typical lactic acid bacteria colony characteristics were picked for purification and culture. The purified strains were stored at -80°C using the glycerol cryopreservation method. Among them, a total of 371 Gram-positive suspected lactic acid bacteria strains were isolated from the collected samples under anaerobic culture conditions on MRS plates. Preliminary observation showed that the colonies formed by these isolates on MRS medium were mostly milky white or light milky yellow, round or nearly round, with relatively neat edges and smooth and moist surfaces, exhibiting typical lactic acid bacteria colony characteristics.

[0054] (3) Yersinia enterocolitica CMCC52225, Listeria monocytogenes ATCC19115 and Salmonella enteritidis CMCC(B)50335 were inoculated at 1% in LB liquid medium and cultured at 37°C and 160 rpm for 14 h for later use.

[0055] (4) Using *Yersinia enterocolitica* CMCC52225, *Listeria monocytogenes* ATCC19115, and *Salmonella enteritidis* CMCC(B)50335 as indicator bacteria, the antibacterial activity of the isolated strains was determined by the agar diffusion method. The indicator bacteria were evenly spread on LB agar plates, and cells-free supernatant (CFS) of the test strains was added after perforation with a sterile punch. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured. Finally, a dominant strain exhibiting strong inhibitory activity against all three pathogens was selected and named LY2332. The LY2332 strain with significant antibacterial activity was further screened, and the results are as follows: Figure 1 As shown, the inhibition zone diameters of this strain against Yersinia enterocolitica, Listeria monocytogenes and Salmonella enteritidis were 19.80±0.26 mm, 17.03±0.15 mm and 18.77±0.15 mm, respectively.

[0056] (5) After screening, the LY2332 strain, which showed significant antibacterial activity, was cultured in MRS medium for 48 hours. Colony morphology and Gram staining were then performed. The results are as follows: Figure 2 As shown. Figure 2 As shown in A, strain LY2332 is Gram-positive, short rod-shaped with blunt ends, and is arranged singly, in pairs, or in short chains, without spores or obvious capsule structures. Figure 2 As shown in Figure B, the colonies of this strain are round, smooth and moist, with regular and slightly convex edges, and a diameter of approximately 1-3 mm. They are milky white or milky yellow. The results indicate that the colony characteristics and Gram staining of this strain are consistent with the morphological characteristics of lactic acid bacteria.

[0057] In addition, the physiological and biochemical characteristics of strain LY2332 were identified using the Lactic Acid Bacteria Biochemical Identification Strips (HBI, GB standard) from Qingdao Haibo Co., Ltd. The results are shown in Table 1 ("+" in the table represents positive). The strain was positive in 10 biochemical reactions, including esculin, cellobiose, mannitol and raffinose, indicating that the strain can metabolize a variety of carbohydrates.

[0058] Table 1

[0059]

[0060] (6) Molecular biological identification of strain LY2332: DNA from strain LY2332 was extracted using a rapid bacterial genomic DNA isolation kit and amplified by PCR using universal primers 27F and 1492R. The PCR products were sequenced by Shanghai Sangon Biotech. These sequences were analyzed using the BLAST tool in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and identified based on the highest similarity. Strain LY2332 was found to have 99.1% sequence similarity to *Lactobacillus amylovorus*. Based on the preceding growth characteristics and physiological and biochemical identification results, strain LY2332 can be preliminarily identified as Lactobacillus amylovorus, and is therefore named Lactobacillus amylovorus LY2332. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37009, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 17, 2025.

[0061] Example 2: This example is a process optimization experiment for laser irradiation of Lactobacillus amyloliquefaciens LY2332. A blue laser was used to irradiate the Lactobacillus amyloliquefaciens LY2332 bacterial suspension to screen for the optimal irradiation conditions, as detailed below:

[0062] Lactobacillus amyloliquefaciens LY2332 was activated and inoculated into MRS liquid medium. The culture was incubated at 37°C until the logarithmic growth phase. The culture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in sterile PBS buffer. The bacterial concentration was adjusted to 1.0 × 10⁻⁶. 6 CFU / mL, for later use.

[0063] Take 0.2 mL of the prepared bacterial suspension and add it to a sterile centrifuge tube. Set different laser irradiation powers (30, 60, 90, 120, 150, 180, 210, 240 mW) and different irradiation times (20, 40, 60, 80, 100 min), and use a blue laser with a wavelength of 450 nm to treat the bacterial suspensions in the above different groups.

[0064] After irradiation, the treated bacterial culture was inoculated into MRS liquid medium and incubated at 37°C for a certain period of time. The fermentation broth was then collected, centrifuged at 8000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain CFS. Using Yersinia enterocolitica as an indicator bacterium, the antibacterial activity of CFS in each treatment group was determined by agar diffusion method.

[0065] Using the size of the inhibition zone as an evaluation index, the CFS antibacterial effects obtained under different combinations of laser power and irradiation time were compared to screen out the optimal blue laser irradiation conditions for subsequent experimental research.

[0066] Experimental results are as follows Figure 3 As shown, different blue laser irradiation conditions had varying effects on the antibacterial activity of strain CFS. Under conditions of lower power or shorter irradiation time, the antibacterial activity of CFS produced by *Lactobacillus amyloliquefaciens* LY2332 was relatively weak. With increasing blue laser irradiation power and time, the diameter of the inhibition zone gradually increased, indicating that appropriate blue laser stimulation could enhance the antibacterial ability of strain CFS to some extent. Among all treatment combinations, irradiation with a 180 mW blue laser for 60 min resulted in the largest CFS inhibition zone diameter, exhibiting the strongest antibacterial effect. However, when the irradiation power or time was further increased, the inhibition zone diameter did not continue to increase significantly, and some treatment groups even showed a decreasing trend, indicating that excessively strong or prolonged irradiation was not conducive to improving the antibacterial ability of strain CFS. In summary, using the inhibition zone diameter as an evaluation index, 180 mW and 60 min were determined as the optimal irradiation conditions for blue laser treatment in this study and will be used for subsequent experimental research.

[0067] Example 3: This example describes the growth, acid production, and physiological characteristics of *Lactobacillus amyloliquefaciens* LY2332 irradiated by laser, as detailed below:

[0068] 1. Growth Curve Determination: *Lactobacillus amyloliquefaciens* LY2332 from both the laser-treated group (treated with blue laser at 180 mW for 60 min) and the untreated group (without blue laser treatment) were inoculated into fresh MRS liquid medium at the same inoculum size and cultured under identical conditions. Samples were taken every 2 h during culture, and the absorbance (OD) of the culture medium was measured at 600 nm. 600 The study used uninoculated MRS medium as a blank control. Three replicates were set up for each time point, and growth curves were plotted accordingly.

[0069] To evaluate the effect of laser irradiation on the growth characteristics of Lactobacillus amyloliquefaciens LY2332, the OD values ​​of the laser-treated and untreated groups were measured during the culture process. 600 Changes and results are shown below. Figure 4 The OD values ​​of the two strains in the early stage of culture (0-12 h) were... 600 The values ​​were all low, and then entered the logarithmic growth phase after about 12 hours, OD 600 The value increased rapidly. Compared with the untreated group, the laser-treated group showed a significantly higher OD value during the logarithmic growth phase. 600The overall values ​​were slightly higher. After 24 hours of cultivation, the growth rates of both groups of strains gradually slowed down and tended to stabilize in the later stages of cultivation (after approximately 30 hours). The OD values ​​of the two groups during the stabilization period were... 600 At similar levels, laser irradiation did not significantly affect the overall trend of the bacterial growth curve, but it did show a certain regulatory effect on the growth process in the early stages of growth.

[0070] 2. Acid production capacity determination: The acid production of the strain during culture was measured simultaneously with the growth curve. Samples were taken every 2 hours during the culture period to measure the pH of the culture medium and plot the acid production curve.

[0071] To investigate the effect of laser irradiation on the acid production characteristics of *Lactobacillus amyloliquefaciens* LY2332, the pH changes during cultivation were measured. The results are shown in [Figure number missing]. Figure 5 The initial pH values ​​of both strains were approximately 5.8, gradually decreasing with increasing culture time. Within the first 6 hours of culture, the pH values ​​of both groups decreased rapidly, with the laser-treated group showing a slightly greater decrease than the untreated group. After 6-10 hours of culture, the rate of pH decrease gradually slowed; towards the later stage of culture (approximately 12 hours later), the pH values ​​of both groups stabilized within the range of 3.5-3.6, and the differences between the groups gradually narrowed. Overall, laser irradiation did not alter the overall acid-producing trend of the strains, but it exhibited a faster acidification process in the early stages of culture.

[0072] 3. Bile salt tolerance test: Centrifuge the overnight cultured bacterial suspension at 8000 r / min for 10 min, wash twice with PBS, and adjust the bacterial suspension concentration to OD500 with buffer. 600 Approximately 1.0. Activated bacterial solution (1×10⁻⁶) 8 The bacterial culture (CFU / mL) was inoculated into MRS liquid medium containing 0.1%, 0.3%, 0.5%, and 1.0% bile salts, respectively, with the bile salt-free group as a control. After incubation at 37℃ for 3 h, 0.1 mL of the bacterial culture was spread onto MRS solid medium, and the number of viable bacteria was counted and the survival rate was calculated after incubation.

[0073] The results are as follows Figure 6 As shown, at bile salt concentrations of 0.1% and 0.3%, the survival rates of the laser-treated group were significantly higher than those of the untreated group (p<0.05). When the bile salt concentration increased to 0.5%, the survival rates of both groups decreased, but there was no significant difference between the treated and control groups (p>0.05). Notably, at a high bile salt concentration of 1.0%, although the survival rates of both groups dropped to extremely low levels, the survival rate of the laser-treated group was still significantly higher than that of the untreated group (p<0.05). These results indicate that blue laser irradiation treatment can significantly improve the tolerance of *Lactobacillus amyloliquefaciens* LY2332 at low to moderate bile salt concentrations and still exhibits a certain protective effect in high bile salt environments.

[0074] 4. Simulated Gastrointestinal Fluid Tolerance Experiment: Take 20 mL of 1 mol / L HCl, adjust the pH to 3.0 with NaOH solution, add pepsin to maintain a concentration of 10 g / L, sterilize through a 0.22 μm filter membrane, and store at 4℃ to obtain simulated gastric fluid. Take 1 mL of the bacterial suspension to be tested and inoculate it into 9 mL of simulated gastric fluid, incubate at 37℃ for 3 h, and then determine the viable bacterial count. Take 6.8 g of KH2PO4, dissolve it in water to 500 mL, and adjust the pH to 6.8 with NaOH solution; separately, take 10 g of trypsin, dissolve it in an appropriate amount of water, mix it with the previous solution, add water to make up to 1000 mL, sterilize through a 0.22 μm filter membrane, and store at 4℃ to obtain simulated intestinal fluid. Take 1 mL of the bacterial suspension treated with simulated gastric fluid and inoculate it into 9 mL of simulated intestinal fluid, incubate at 37℃ for 4 h, and then determine the viable bacterial count.

[0075] The survival rate is calculated using the formula: Survival rate (%) = n1 / n0 × 100%, where n0 is the number of viable bacteria before treatment and n1 is the number of viable bacteria after treatment.

[0076] The results are as follows Figure 7 As shown, in a simulated gastric juice (SGF) environment, the survival rates of both the original strain and the laser-treated strain decreased significantly, to approximately 30%-40%. The survival rate of the untreated group was slightly higher than that of the laser-treated group, but the overall difference was small, indicating that blue laser treatment had a limited effect on improving the strain's tolerance to simulated gastric juice. Under simulated intestinal juice (SIF) conditions, the survival rates of both groups of strains increased significantly, exceeding 160%, demonstrating good intestinal juice tolerance. In contrast, the survival rate of the laser-treated group was slightly higher than that of the untreated group, suggesting that blue laser treatment may have a slight enhancing effect on the strain's adaptability to the intestinal juice environment.

[0077] 5. Acid resistance test: The pH of MRS broth was adjusted to 1.0, 2.0, 3.0 and 4.0 using 1 mol / L HCl, with pH 6.0 as the control. 1 mL of overnight culture was inoculated into 7 mL of MRS broth at different pH values. After incubation at 37℃ for 3 h, the viable cell count was determined using the plate count method, and the survival rate was calculated.

[0078] The results are as follows Figure 8As shown, the survival rate test results under different pH conditions revealed that in strongly acidic environments of pH=1 and pH=2, the survival rates of both groups of strains were close to zero or remained at extremely low levels, with no significant difference between groups. As the pH value increased to 3, the survival rates of both groups significantly improved, and the survival rate of the laser-treated group was significantly higher than that of the untreated group (p<0.05). At pH=4, the survival rate of the laser-treated group further increased and was significantly higher than that of the untreated group (p<0.01). The results indicate that blue laser irradiation treatment under weakly acidic conditions of pH=3-4 can significantly enhance the acid tolerance of *Lactobacillus amyloliquefaciens* LY2332.

[0079] 6. Hydrophobicity determination: Activated Lactobacillus amyloliquefaciens LY2332 was inoculated into MRS liquid medium at 2% (v / v) and cultured at 37℃ for 24 h. The bacterial cells were collected by centrifugation at 8000 r / min for 10 min, washed twice with PBS, and the OD of the bacterial suspension was adjusted with PBS. 600 To a concentration of 0.6-0.7, mix 2 mL of bacterial suspension with 0.4 mL of xylene by vortexing for 120 s, incubate at 37℃ for 1 h, and then collect the aqueous phase for OD measurement. 600 .

[0080] Hydrophobicity is calculated using the formula: [(A0−A) / A0]×100%, where A0 is the absorbance before extraction and A is the absorbance after extraction.

[0081] The hydrophobicity of the cell surface of *Lactobacillus amyloliquefaciens* LY2332 after laser irradiation was (47.50±0.98)%, which was significantly higher than that of the untreated group (p<0.05). The results indicate that laser irradiation can enhance the hydrophobic properties of the bacterial surface, thereby improving its potential intestinal adhesion ability and colonization advantage.

[0082] 7. Self-aggregation assay: Activated Lactobacillus amyloliquefaciens LY2332 was inoculated into MRS broth at 2% (v / v) and cultured at 37℃ for 24 h. After centrifugation at 8000 r / min for 10 min, the bacterial cells were collected and washed three times with PBS. The OD of the bacterial suspension was then adjusted with PBS. 600 To a value of 0.5-0.6. The bacterial suspension was incubated at 37℃ for 4 h, and the OD value of the supernatant was measured. 600 .

[0083] The self-aggregation rate is calculated using the formula: [(A1−A2) / A1]×100%, where A1 is the initial OD of the bacterial suspension. 600 A2 is the OD of the upper bacterial solution after standing. 600 .

[0084] After laser irradiation treatment, the self-aggregation rate of *Lactobacillus amyloliquefaciens* LY2332 was (72.34±1.56)%, significantly higher than that of the untreated group (p<0.05). The results indicate that laser treatment can significantly enhance the self-aggregation ability of the strain, further improving its adaptability in the intestinal environment and its potential ability to form biological barriers.

[0085] Example 4: In this example, the antibacterial activity of CFS obtained by fermentation culture of Lactobacillus amyloliquefaciens LY2332 treated with blue laser (irradiation conditions: 180 mW, 60 min) and CFS obtained by fermentation culture of raw Lactobacillus amyloliquefaciens LY2332 without blue laser treatment were determined, as follows:

[0086] 1. Agar diffusion assay: The antibacterial activity of cell-free supernatant was evaluated using the agar diffusion method. The activated indicator bacteria were adjusted to approximately 1.0 × 10⁻⁶ mg / L with sterile physiological saline. 8 For CFU / mL, take 100 μL and spread it evenly on the surface of an LB agar plate. After the plate surface dries, use a sterile punch to make wells (8 mm diameter), remove the agar blocks from the wells, and add 100 μL of the CFS to be tested to each well. Use sterile MRS medium as a negative control. After incubating the plates at 4℃ for 1-2 h, invert them and incubate at 37℃ for 16-24 h, and measure the diameter of the inhibition zone. Set up 3 replicates for each group, and take the average value of the results.

[0087] The inhibitory effect of CFS of Lactobacillus amyloliquefaciens LY2332 on three foodborne pathogens was evaluated by agar diffusion method. The results are shown in Table 2 and 3. Figure 9 After irradiation, strain CFS showed significant inhibitory effects against *Yersinia enterocolitica*, *Listeria monocytogenes*, and *Salmonella enteritidis*, with inhibition zone diameters of 27.87±0.21 mm, 29.07±0.90 mm, and 26.40±0.66 mm, respectively. The inhibitory effect against *Listeria monocytogenes* was the strongest, followed by *Yersinia enterocolitica*, while the inhibitory effect against *Salmonella enteritidis* was relatively weak. The diameters of the inhibition zones for all three indicator bacteria increased, with increases of 40.76%, 70.70%, and 40.66% for *Yersinia enterocolitica*, *Listeria monocytogenes*, and *Salmonella enteritidis*, respectively.

[0088] Table 2

[0089]

[0090] 2. Determination of Minimum Inhibitory Concentration (MIC): The MIC of CFS was determined using the 96-well plate micro-broth two-fold dilution method. After culturing the activated pathogen to the logarithmic growth phase, the bacterial concentration was adjusted with LB medium to ensure a final bacterial concentration of approximately 1.0 × 10⁻⁶ in each well.5 CFU / mL. CFS was serially diluted twofold with LB medium to final concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. After adding the pathogen working solution, the cultures were incubated at 37°C for 16–24 h. An equal volume of MRS medium was added to the control wells instead of CFS, while the blank control wells contained only LB medium. OD was measured after the culture period. 600 , with OD 600 The lowest CFS concentration close to that of the blank control was used as the MIC. Each group had 3 parallel wells and was repeated 3 times.

[0091] Comparing the antibacterial effects of CFS in the laser-treated group (with lg CFS) and the untreated group, it can be seen that the overall inhibition rate of CFS against the three pathogens is improved after blue laser treatment, but the degree of enhancement varies depending on the target bacteria (e.g., Figure 10 (As shown). When targeting *Yersinia enterocolitica*, the inhibition rate of CFS in the laser-treated group was significantly higher than that in the untreated group at 50% and 12.5% ​​(p<0.05), and the difference was even more significant at 6.25% (p<0.001). When targeting *Listeria monocytogenes*, the CFS in the laser-treated group showed extremely significant differences at 50% (p<0.001), significant differences at 25% (p<0.05), and significant differences at 12.5% ​​and 3.125% (p<0.01). When targeting *Salmonella enteritidis*, the inhibition rate of CFS with lg CFS was significantly higher than that in the untreated group at 50%, 12.5%, and 6.25% (p<0.01). Overall, the antibacterial activity of CFS in the laser-treated group was enhanced against all three pathogens, with the most significant enhancement effect against *Listeria monocytogenes*.

[0092] 3. Antibacterial Activity Stability Assay: To evaluate the passage stability of the antibacterial activity of CFS derived from the laser-irradiated strain, the irradiated strain was continuously passaged in MRS liquid medium at a fixed inoculum size, and CFS samples were prepared at generations 1, 5, 10, 15, and 20. After centrifugation at 4°C to remove bacterial cells, the supernatant was collected and filtered through a 0.22 μm sterile filter. Using *Yersinia enterocolitica*, *Salmonella enteritidis*, and *Listeria monocytogenes* as indicator bacteria, the antibacterial activity of CFS at different generations was evaluated using the agar diffusion method.

[0093] The passage stability of the antibacterial activity of CFS after laser treatment was analyzed. CFS were prepared by continuously passaged the enhanced strain to generations 1, 5, 10, 15, and 20. The diameter of the inhibition zone against three indicator bacteria was determined using the agar diffusion method. The results are shown in [Figure number missing]. Figure 11The diameter of the inhibition zone against the three pathogens remained relatively stable across different passage numbers for the CFS strains. The inhibition zone against Listeria monocytogenes remained consistently within the range of approximately 28.9–29.2 mm, against Yersinia enterocolitica within approximately 27.9–28.1 mm, and against Salmonella enteritidis within approximately 26.2–26.5 mm, without any observed trend of continuous decrease with increasing passage number. These results indicate that the CFS antibacterial activity of the laser-enhanced strains remained well stable during continuous passage.

[0094] Example 5: This example is a non-targeted metabolomics analysis experiment. The bacterial culture, after reaching the logarithmic growth phase, was collected by centrifugation and the supernatant was gathered. 0.5 mL of the supernatant was taken and an equal volume of protein precipitant (methanol:acetonitrile:water = 2:1:1, v / v / v) was added. The mixture was vortexed, incubated at -20°C for 2 h, and then centrifuged at 4°C, 15000 × g for 15 min. Equal volumes of supernatant from each sample were mixed as the quality control sample (QC). The remaining samples were dried under nitrogen, reconstituted with 70 μL of 50% acetonitrile, centrifuged, and the supernatant was used for instrumental analysis.

[0095] 1. LC-MS / MS Detection: Metabolite detection was performed using a Thermo U3000 ultra-high performance liquid chromatography system coupled with a QExactive high-resolution mass spectrometer. Chromatographic separation was performed using a Kinetex XB-C18 column (100 mm × 2.1 mm, 2.6 μm), column temperature 40℃, flow rate 0.3 mL / min, injection volume 25 μL. The mobile phase consisted of an aqueous solution containing 0.1% formic acid (phase A) and an acetonitrile solution containing 0.1% formic acid (phase B). The gradient elution program was as follows: 0–12 min, phase B increased from 5% to 60%; 12–14 min, increased to 100%, and maintained until 16 min.

[0096] Mass spectrometry data were acquired in positive and negative ion switching modes. Parameter settings: sheath gas flow rate 35 arb, auxiliary gas flow rate 8 arb, spray voltage 3.5 kV, capillary temperature 275℃, heater temperature 350℃, S-lens RF level 50. Full MS resolution was set to 70000, dd-MS. 2 The resolution is set to 17500, and the first-level scan range is 80-900 m / z.

[0097] To analyze the stability of the metabolomics detection method, QC samples were set up during the analysis process, and detection sequences were periodically inserted. The total ion chromatogram (TIC) overlay results of the QC samples are shown below. Figure 12 and Figure 13The results showed that the ion current curves of samples from different batches were consistent under both positive and negative ion modes, the retention times of the main chromatographic peaks were stable, and no obvious signal drift was observed, indicating that the detection method has good stability and repeatability.

[0098] 2. Data Processing and Differential Metabolite Screening: Raw mass spectrometry data were processed in both positive and negative ion modes. Data preprocessing was performed on the IP4M platform, including peak extraction, peak alignment, denoising, deconvolution, and normalization. First, the stability of QC samples was assessed, and characteristic peaks with a coefficient of variation (CV) <30% were selected for subsequent analysis.

[0099] After log2 transformation and scaling of the screened metabolite feature matrix, principal component analysis (PCA) was performed in both positive and negative ion modes to assess overall metabolic differences and intragroup aggregation. Differential metabolite analysis combined univariate and multivariate statistical methods, with screening thresholds set as VIP≥1, FC≥1.2, or FC≤0.83, and p<0.05. Metabolite annotation was primarily based on the HMDB database, supplemented and validated using the KEGG and PubChem databases.

[0100] To comprehensively evaluate the differences in metabolic levels between the untreated and laser-treated groups, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed on the metabolomics data. The results are as follows: Figure 14 and Figure 15 As shown, under both positive and negative ion modes, the two groups of samples exhibited clear separation in the PCA score plots, with good sample aggregation within each group, indicating significant metabolic differences between the different treatment groups and good experimental reproducibility. PLS-DA further validated this trend, with both groups of samples showing clear separation under both ion modes. Model parameters show that R under positive ion mode... 2 and Q 2 The values ​​are 0.873 and 0.785 respectively, and 0.988 and 0.678 respectively in negative ion mode. 2 The p-values ​​were all 0.05, indicating that the model has good fitting and predictive capabilities.

[0101] Based on this, and combining the results of multivariate and univariate analyses, differentially expressed metabolites were identified using VIP≥1, fold-change≥1.2 or≤0.83, and p<0.05 as screening criteria. In positive ion mode, a total of 256 significantly differentially expressed ions were screened, of which 189 were annotated as known metabolites, including 67 upregulated metabolites and 122 downregulated metabolites. In negative ion mode, a total of 127 significantly differentially expressed ions were screened, of which 123 were annotated as known metabolites, including 67 upregulated metabolites and 56 downregulated metabolites.

[0102] To visually demonstrate the distribution characteristics of differentially metabolites, volcano diagrams were plotted under both positive and negative ion modes. Figure 16 The results showed that a large number of significantly upregulated and downregulated metabolites were present in both modes, with upregulated metabolites mainly distributed on the right side of the volcano plot and downregulated metabolites mainly distributed on the left side. Combined results from PCA, PLS-DA, and differential metabolite screening indicated a significant difference in metabolic profiles between the untreated group and the laser-treated group.

[0103] 3. Pathway analysis: To elucidate the potential biological processes and metabolic changes involved by differentially metabolites, the screened differentially metabolites were imported into the KEGG database for metabolic pathway annotation and enrichment analysis. Key metabolic pathways were screened based on pathway coverage and enrichment significance to explain the potential mechanisms of changes in the metabolic profile of cell-free supernatant before and after laser treatment.

[0104] Using VIP≥1 and p≤0.05 as screening criteria, 50 significantly differentially expressed metabolites were identified in both the control and blue laser treatment groups, with 26 upregulated and 24 downregulated. First, multiple phosphatidylglycerol (PG), phosphatidic acid (PA), and diacylglycerol (DG) membrane lipid molecules were significantly upregulated in the blue laser group, indicating that blue laser can act as an external physical stimulus to induce changes in cell membrane structure. Membrane lipid remodeling is usually an important regulatory mechanism for microorganisms to cope with stress, maintaining cell integrity by regulating membrane fluidity and stability. During this process, some lipids and fatty acid derivatives may be released into the extracellular environment, and lipid molecules have certain membrane interference activity, which can disrupt the cell membrane structure of pathogens, thereby enhancing the antibacterial effect. Therefore, the upregulation of membrane lipid metabolism may be an important material basis for the enhanced antibacterial ability of CFS by blue laser. Second, small peptide-related metabolites were significantly increased in the blue laser group, such as the enhanced expression of N-Formyl-Met-Leu-Phe, suggesting increased protein turnover and secretion of bioactive peptides. Lactic acid bacteria are known to produce a variety of antimicrobial peptides, which exert their antibacterial effects by disrupting the cell membrane of target bacteria, inhibiting cell wall synthesis, or interfering with intracellular metabolism. Analysis of the secondary biosynthetic clusters of *Lactobacillus amyloliquefaciens* revealed the presence of gene clusters for the synthesis of three classes of lanothioneins. Although metabolomics did not directly identify complete antimicrobial peptides, enhanced small peptide signals suggest that blue laser treatment may promote the generation or release of antimicrobial-related peptides, thereby enhancing the antimicrobial efficacy of CFS (Cellular Freezing). Furthermore, aromatic and phenolic metabolites (such as Phenol, N-Acetyl-L-phenylalanine, and N-Acetylserotonin) were significantly upregulated after blue laser treatment, indicating activation of aromatic amino acid metabolic pathways. Phenolic and aromatic derivatives possess certain antimicrobial activity, inhibiting pathogen growth by altering bacterial membrane permeability or interfering with metabolic enzyme activity. The decrease in some amino acids and metabolic intermediates in the blue laser group suggests substrate redistribution for membrane repair, antioxidant regulation, and the synthesis of antimicrobial-related substances.

[0105] KEGG annotation results are as follows Figure 17 and Figure 18 As shown, differentially metabolites were mainly distributed in metabolic pathways, followed by those related to genetic information processing, environmental information processing, cellular processes, and organismal systems. Among secondary pathways, metabolic pathways annotated with the most differentially metabolites, followed by secondary metabolite biosynthesis and microbial metabolism in various environments. Furthermore, differentially metabolites were also mainly enriched in pathways involving phenylalanine metabolism, tyrosine metabolism, tryptophan metabolism, arginine and proline metabolism, amino acid biosynthesis, linoleic acid metabolism, glycerophospholipid metabolism, aminoacyl-tRNA biosynthesis, and ABC transporters. The results indicate that metabolic changes in the strains after blue laser treatment were mainly concentrated in pathways related to amino acid metabolism, lipid metabolism, and secondary metabolite biosynthesis.

[0106] Example 6: To evaluate the CFS (Continuous Flavoring and Preservative Effects) of *Lactobacillus amyloliquefaciens* LY2332 on pork, a pathogen contamination model for pork was established, and changes in pathogen quantity, pH, and sensory quality during storage were measured, as follows:

[0107] 1. Establishment of a Pathogenic Bacterial Contamination Model in Pork: Fresh pork hind leg meat was selected, and after removing the fascia and visible fat, it was aseptically cut into small pieces of uniform size. Samples were routinely disinfected and washed with sterile saline before use. Representative strains commonly found in foodborne contamination (Salmonella enteritidis, Listeria monocytogenes, and Yersinia enterocolitica) were selected as the target pathogens to simulate potential exogenous contamination of pork during processing and storage. The concentration of the pathogen suspension was adjusted to approximately 10. 3 At an inoculation level of CFU / g, pork samples were in contact with the bacterial suspension according to a set method to complete the contamination. Excess bacterial suspension was removed and the samples were briefly drained to ensure uniform and stable contamination.

[0108] The experiment was divided into a blank control group (no pathogen inoculation, no CFS added), a pathogen control group (inoculated with pathogen, no CFS added), and a CFS treatment group (inoculated with pathogen and CFS added). In the CFS treatment group, CFS was sprayed evenly onto the sample surface at a rate of 1 mL per sample. After treatment, the samples were stored at 4℃ and 25℃, respectively, and samples were taken for testing at designated time points.

[0109] The experimental results are as follows: Under conditions of 25℃ and 4℃, the number of the three pathogens in each treatment group increased with the extension of storage time, such as... Figure 19 As shown in the figure, compared with the corresponding pathogen control group, the CFS treatment group showed lower colony levels at most time points. At 25℃, CFS showed a significant inhibitory effect on the three pathogens in the early and middle stages of storage (Day 1-Day 5), with the difference weakening in the later stages; at 4℃, the inhibitory effect of CFS was more stable, maintaining a low colony count throughout the storage period (p<0.05). The results indicate that the enhanced strain of CFS can inhibit the growth of pathogens in pork under both room temperature and refrigerated conditions, and the effect is more prolonged under refrigerated conditions.

[0110] 2. Determination of pathogen quantity and pH changes during storage: Take 2.5 g of sample, add 22.5 mL of phosphate buffer or sterile physiological saline to prepare a 1:10 homogenate, homogenize and then dilute it 10 times in a serial manner. The number of pathogens is determined by plate count method.

[0111] pH determination was performed according to GB / T5009.044-2003 and GB5009.237-2016. Take 1 g of sample, add 9 mL of sterile water for homogenization, and then measure the pH value of the homogenate using a pH meter.

[0112] The pH value of samples in all treatment groups increased with prolonged storage time, such as Figure 20 As shown in the figure, at 25℃, the pH of the control group and the pathogen group increased rapidly, with some samples exceeding 6.7 in the later stages of storage. In contrast, the pH increase rate in the CFS-treated group was significantly slower, and it was significantly lower than that in the corresponding pathogen group at most time points (p<0.05). At 4℃, the overall pH increase trend weakened, and the CFS-treated group maintained a lower level throughout the entire storage period. The results indicate that CFS treatment can delay the pH increase during pork storage.

[0113] 3. Sensory Evaluation: After the samples have returned to room temperature, three trained personnel will conduct a sensory evaluation. Evaluation indicators include color, odor, and texture, with each indicator scored on a scale of 1-5. The total score, combining the scores for color, odor, and texture, ranges from 0 to 15, with 13-15 indicating freshness, 8-12 indicating good quality, and 7 or below indicating significant deterioration.

[0114] The sensory evaluation results under the two storage conditions are shown in the figure. Figure 21 Sensory scores for all treatment groups decreased over time. Overall, the CFS treatment group showed higher sensory scores than the corresponding pathogen group at most time points. At 25℃, this difference was mainly observed in the early and middle stages of storage; at 4℃, the CFS treatment group maintained a higher score throughout the entire storage period (p<0.05). This indicates that CFS treatment helps maintain the sensory quality of pork, and its effect is more stable under refrigeration conditions.

[0115] In summary, this invention has screened and obtained a strain of *Lactobacillus amyloliquefaciens* LY2332 with good safety and broad-spectrum antibacterial activity. The cell-free supernatant derived from this strain showed significant inhibitory effects against various foodborne pathogens. Blue laser irradiation, without affecting the stable growth of the strain, enhanced its early metabolic activity, partial tolerance, and surface characteristics, and further improved the antibacterial ability of the cell-free supernatant through metabolic reprogramming. Furthermore, the cell-free supernatant enhanced by blue laser irradiation exhibited good pathogen control and biopreservation effects under both room temperature and refrigeration conditions. Overall, *Lactobacillus amyloliquefaciens* LY2332 and its blue laser-enhanced cell-free supernatant show good application potential in the development of natural food pathogen control agents.

[0116] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A laser-irradiated Lactobacillus amyloliquefaciens, characterized in that, The Lactobacillus amylovorus mentioned is Lactobacillus amylovorus LY2332, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37009.

2. A cell-free supernatant of Lactobacillus amyloliquefaciens, characterized in that, The cell-free supernatant was obtained by fermentation culture of Lactobacillus amyloliquefaciens irradiated by laser as described in claim 1.

3. The cell-free supernatant of *Lactobacillus amyloliquefaciens* according to claim 2, characterized in that, The method for preparing the cell-free supernatant includes the following steps: The Lactobacillus amyloliquefaciens was activated and then prepared into a bacterial suspension; After laser irradiation treatment of the bacterial suspension, fermentation culture is carried out to obtain fermentation broth; After centrifuging the fermentation broth, the supernatant was filtered to obtain a cell-free supernatant.

4. The cell-free supernatant of *Lactobacillus amyloliquefaciens* according to claim 3, characterized in that, The laser is a blue laser.

5. The cell-free supernatant of *Lactobacillus amyloliquefaciens* according to claim 4, characterized in that, The wavelength of the blue laser is 450 nm.

6. The cell-free supernatant of *Lactobacillus amyloliquefaciens* according to claim 4, characterized in that, The blue laser irradiation power is 30-240 mW, and the irradiation time is 20-100 min.

7. The application of laser-irradiated Lactobacillus amyloliquefaciens as described in claim 1 or cell-free supernatant of Lactobacillus amyloliquefaciens as described in any one of claims 2-6 for the control of foodborne pathogens.

8. The bacterial control application according to claim 7, wherein the foodborne pathogen is one or more of Yersinia enterocolitica, Listeria monocytogenes, and Salmonella enteritidis.

9. The application of a laser-irradiated Lactobacillus amyloliquefaciens as described in claim 1, or a cell-free supernatant of Lactobacillus amyloliquefaciens as described in any one of claims 2-6, in the inhibition of bacteria and / or preservation of fresh meat.

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