Milk-derived pseudomonas putrefaciens typing method based on pulsed field gel electrophoresis

Abstract

The invention discloses a milk-derived putrefying pseudomonas typing method based on pulsed field gel electrophoresis, and aims to observe the growth characteristics of bacteria by improving a culture medium on the basis that putrefying pseudomonas can produce protease or lipase so as to enable a dairy product to be putrefied. Screening of pseudomonas putrefaciens capable of generating protease and lipase can be visually completed, and further incision enzyme screening and typing are carried out. The method has the advantages of being convenient to operate, simple in process and sensitive in result, and by means of the pulsed field gel electrophoresis standardization method and analysis process, determination of pseudomonas putrescens and subsequent pulsed field gel electrophoresis experiments and software analysis clustering of results can be visually completed; the method can be applied to establishment of a pulsed field gel electrophoresis standardization technology.

Description

Technical Field

[0001] This invention relates to the field of microbial classification and detection technology, and in particular to a method for typing *Pseudomonas lactis* based on pulsed-field gel electrophoresis. Background Technology

[0002] Dairy products play a vital role in providing the world with sufficient protein and other nutrients and have long been considered an essential component of a balanced diet. The dairy industry, due to its enormous market size, diversified products, and ever-growing consumer demand, has become a global pillar industry and is highly valued by countries and regions. Therefore, improving the quality and efficiency of the dairy industry has become a key approach to enhancing competitiveness, promoting high-quality development, and meeting consumer needs.

[0003] Raw milk is the source material for all dairy products. Improving the quality and ensuring the safety of raw milk are crucial goals for enhancing the quality and competitiveness of dairy products. A major factor affecting raw milk quality is bacterial contamination, particularly psychrophilic bacteria. This is because the pre-cooling treatment after harvesting and the cold chain transportation process allow psychrophilic bacteria to grow and become the dominant flora, thus harming dairy product quality. Psychrophilic bacteria in raw milk mainly include *Pseudomonas*, *Acinetobacter*, *Serratia*, *Chlorobacterium*, *Clostridium*, *Lactobacillus*, *Flavobacterium*, *Micrococcus*, *Corynebacterium*, *Bacillus*, and *Enterobacter*. The composition of these psychrophilic bacteria depends on various factors, such as season, storage conditions, geographical region, and pasture management level. Based on the physiological characteristics of the bacteria themselves, the harm of psychrophilic bacteria to dairy product quality varies. Current research suggests that *Pseudomonas*, among psychrophilic bacteria, poses a serious quality hazard and should be prevented and controlled.

[0004] The primary hazard of Pseudomonas aeruginosa to dairy products is its putrefactive nature. The extracellular degradative enzymes secreted by Pseudomonas aeruginosa remain active even after heat processing of raw milk, continuously decomposing proteins and fats in dairy products. This can lead to quality degradation during storage, such as whey separation, fat floating, and the development of off-flavors (e.g., rotten, cheese, soapy smells). The persistent action of psychrophilic bacteria accelerates the spoilage process, shortening shelf life. This results in decreased sales, increased return rates, damage to brand reputation, and economic losses for businesses.

[0005] To control Pseudomonas contamination in dairy products, current methods include CO2 treatment of raw milk to eliminate Pseudomonas, or sampling and isolating bacteria during raw milk harvesting and processing to trace contamination pathways. However, these studies typically focus only on Pseudomonas. The composition of psychrophilic bacteria in raw milk depends on various factors such as season, storage conditions, geographical region, and pasture management. The harmful effects of Pseudomonas in raw milk from different origins and even in subsequent processed products are not limited to Pseudomonas. Furthermore, the lack of typing techniques for tracing contamination pathways makes it difficult to identify key strains and trace their entire contamination route, thus hindering the control of high-risk strains.

[0006] Molecular typing of key bacteria is a prerequisite for identifying target bacteria, tracing contamination pathways, and achieving targeted prevention and control. Pulsed-field gel electrophoresis (PFGE) is an advanced molecular biology technique that uses specific restriction endonucleases to cut the chromosomal DNA of bacteria and other microorganisms, generating high-resolution DNA fingerprints for molecular typing. This technique has become the "gold standard" for bacterial molecular typing and is widely used in epidemic control, disease tracing, and medical research. The selection of endonucleases is based on their specificity, activity, and stability to ensure cutting efficiency and the accuracy of experimental results. By optimizing reaction conditions and strictly controlling experimental procedures, PFGE provides strong support for bacterial typing and disease control. Currently, PFGE typing techniques for Pseudomonas aeruginosa only cover a standard method for Pseudomonas aeruginosa; other Pseudomonas species have not yet been addressed. Due to the influence of season, storage conditions, geographical region, and pasture management level on the composition of Pseudomonas in raw milk and subsequent processed dairy products, directly using standard protocols for Pseudomonas aeruginosa or directly selecting its restriction enzymes for typing is insufficient to accommodate other bacteria in the Pseudomonas genus. The diverse Pseudomonas species in dairy products require diverse standard methods for pulsed-field gel electrophoresis typing. In method implementation, species-specific restriction enzyme screening and validation methods are a primary prerequisite for developing standard methods.

[0007] Therefore, there is an urgent need to analyze the contamination pathways at the strain level based on typing technology. This field requires a simple, intuitive, and sensitive method to screen and validate endonuclease suitable for pulsed-field gel electrophoresis typing of different Pseudomonas species, screen and validate the optimal electrophoresis conditions suitable for enzyme digestion samples, and improve subsequent analysis procedures for the establishment of standardized pulsed-field gel electrophoresis technology. Summary of the Invention

[0008] The purpose of this invention is to provide a method for typing *Pseudomonas lactis* based on pulsed-field gel electrophoresis, which has the advantages of convenient operation, simple process, and sensitive results.

[0009] The technical solution adopted by this invention to solve its technical problem is: A method for typing *Pseudomonas lactis* based on pulsed-field gel electrophoresis includes the following steps: (1) Identification of putrefactive Pseudomonas: Inoculate the bacteria to be tested onto the selection medium for protease-producing bacteria and observe whether a hydrolysis zone is produced. The production of a hydrolysis zone indicates the production of protease. The bacteria to be tested were inoculated onto a lipase-producing bacteria selection medium and cultured. The state around the bacterial colonies was observed. If a halo formed around the colonies, it indicated that lipase was produced. Pseudomonas species that produce either protease or lipase are classified as putrefactive Pseudomonas species. (2) Endonuclease screening for pulsed-field gel electrophoresis typing S1: Identify the putrefactive Pseudomonas species selected in step (1) to obtain species information; S2: Based on the species information of the selected bacteria, download the whole genome sequence from the NCBI database to complete the preparations before screening for enzyme digestion targets; S3: Use SnapGene software to import the bacterial whole genome sequence, select the "Enzyme" option, and create an endonuclease alignment database for PFGE endonuclease screening; S4: Statistically analyze the results in the enzyme toolbar at the bottom right of the output results to check the number of sites for different endonucleases. If the number of sites shows "Too many sites to display", it means that the enzyme has too many sites. Screen out these endonucleases and keep other endonucleases with clear and countable cleavage sites for the next step of verification. If the number of sites shows "Too many sites to display", it means that the enzyme has too many sites, which will cause the genome to be excised into multiple small fragments that are difficult to distinguish using pulsed field gel electrophoresis. Therefore, screen out these endonucleases.

[0010] S5: Prepare bacterial gel blocks of the screened putrefactive Pseudomonas bacteria. Use the single enzyme selected in S4 to perform single enzyme digestion within the gel block. After digestion, perform PFGE detection. After completion, place the intact gel block in staining solution for staining. After staining, decolorize and then use a gel imaging system to image and verify the screening results of the restriction enzyme. Repeat this step while maintaining the same parameters and using different enzymes. S6: Based on the results of S5, screen for restriction enzymes with uniform and clear digestion bands. An ideal restriction enzyme will result in uniform and clear digestion of the target bacterial DNA, which can be well separated in pulsed-field gel electrophoresis. If no bands are observed after bacterial degradation, and the results remain the same after repeated experiments, it indicates that the digested fragments are too small, below the discrimination threshold of pulsed-field gel electrophoresis. If the digested fragments are clear but not uniform, showing a clumping pattern, it indicates that the fragments digested by this restriction enzyme tend to be uniform, making it difficult for pulsed-field gel electrophoresis to effectively distinguish them. Based on the above screening principles, the optimal restriction enzyme is screened and validated.

[0011] S7: After S6 is completed, further screening is conducted to determine the optimal conditions for pulsed field gel electrophoresis, and then standardized typing and molecular typing analysis are performed on the isolated milk-derived Pseudomonas putrefactive bacteria.

[0012] This invention uses the ability of putrefactive Pseudomonas to produce proteases or lipases, thereby causing spoilage of dairy products, as the basis for detection. By improving the culture medium and observing the bacterial growth characteristics, the screening of putrefactive Pseudomonas to produce proteases and lipases can be completed intuitively.

[0013] The complete process of this invention includes screening putrefactive bacteria based on their physiological and biochemical characteristics, and selecting alternative methods for mining restriction enzymes using public databases and software. This invention also summarizes commonly used PFGE typing libraries for restriction enzyme screening tasks, and provides experimental verification procedures and corresponding standards based on software screening. Furthermore, this invention provides screening verification methods and standardized analytical procedures for optimal electrophoresis conditions.

[0014] After screening for putrefactive Pseudomonas, bacterial species identification was performed to obtain species information. Based on the complete genome sequence of this bacterium in the NCBI database, restriction enzyme screening and validation were performed using pulsed-field gel electrophoresis typing technology.

[0015] In step (1), the culture conditions for culturing on the selection medium for protease-producing bacteria and the selection medium for lipase-producing bacteria are: 28-37℃ temperature and 50%-70% humidity for 24-48 hours.

[0016] In step (1), the screening medium for protease-producing bacteria consists of the following composition by mass percentage: 6-8% skim milk powder, 1.5% agar, and the remainder deionized water.

[0017] In step (1), the composition of the lipase-producing bacteria screening medium is: 1L of basal medium plus 30 mL of lipase substrate; The composition of 1L of basal culture medium is: 10-12g casein hydrolysate, 5-7.5g yeast extract, 0.15g Spirit Blue, 17g agar, and the remainder being deionized water; The lipase substrate consisted of 1 ml Tween 80, 100 ml olive oil, and 400 mL deionized water. The basal culture medium was sterilized at 121°C for 15 min, cooled to 60°C, and then 30 mL of the lipase substrate was added.

[0018] In S3, the selected enzymes are commonly used PFGE enzymes: ApaI, SgrAI, XbaI, Fsel, NheI, AsCI, KpnI, NotI, Smal, BInI, MluI, SfiI, and SpeI, totaling 13 enzymes, to explore the target sites of these 13 enzymes on the bacterial genome.

[0019] In S5, the intact gel block was stained with 0.5 μL / mL Gel Red staining solution for 30 min, and then destained with deionized water for 30 min.

[0020] In S5, the specific parameters for the instrument detection are as follows: the pulse angle is set to 60°, the running temperature is set to 14℃, the initial switching time for electrophoresis is set to 1 s, the final switching time is 25 s, and the running time for all pulsed field gel electrophoresis is set to 18 h; Salmonella H9812 digested with XbaI enzyme is used as the marker for instrument detection.

[0021] In S5, bacterial blocks of the screened putrefactive Pseudomonas were prepared in accordance with the "Technical Manual of the National Pathogenic Bacteria Identification Network (2020 Edition)".

[0022] In S7, the method for determining the optimal conditions for pulsed-field gel electrophoresis (PGFES) was as follows: Three sets of electrophoresis conditions were set for the same target bacterial gel blocks for testing: ① Initial switching time was set to 2 s, and final switching time to 30 s; ② Initial switching time was set to 2.2 s, and final switching time to 38.5 s; ③ Initial switching time was set to 2 s, and final switching time to 40 s. The pulse angle for all PGFES gel electrophoresis was set to 60°, the operating temperature to 14℃, and the operating time to 19 h. The optimal conditions were then determined based on the separation results. Ideally, PGFES gel electrophoresis should result in uniform, clear, and well-separated bands of target bacterial DNA, with good differentiation between different bacterial strains. If the bacterial bands are clear but uneven, large molecular weight bands are too far from the wells, and small molecular weight bands show significant aggregation, it indicates that the pulse time for PGFES gel electrophoresis is too long and should be reduced. If the enzyme digestion bands are clear and uniformly separated, but show aggregation at the top, it indicates that the electrophoresis time is too short and should be extended. Based on the above screening principles, the optimal conditions for pulsed field gel electrophoresis were screened and validated.

[0023] In S7, molecular typing analysis was performed using BioNumerics software for typing and cluster analysis. The PFGE diagram of Salmonella H9812 XbaⅠ restriction enzyme digestion, a standard strain internationally recognized, was used as a calibrator. The cluster similarity coefficient was selected as Dice, and the cluster analysis method was UPGAMA. The tolerance for band position differences and the optimization degree were adjusted according to clustering requirements, with a default setting of 1.5%, adjustable within the range of 0.5% to 3%. Based on the above clustering parameters, typing and cluster analysis of milk-derived putrefactive Pseudomonas aeruginosa was completed.

[0024] The beneficial effects of the present invention are: the method of the present invention has the advantages of convenient operation, simple process and sensitive results. Using the pulsed field gel electrophoresis standardization method and analysis process of the present invention, the identification of putrefactive Pseudomonas can be completed intuitively, as well as the subsequent pulsed field gel electrophoresis experiment and the software analysis and clustering of the results. It can be applied to the establishment of pulsed field gel electrophoresis standardization technology. Attached Figure Description

[0025] Figure 1 This is a visual representation of positive bacterial strains in protease-producing and lipase-producing selection media; the illustration shows that bacteria that can produce protein hydrolysis zones are protease-positive bacteria; and the bacterial strains that form halos around their colonies in the lipase-producing selection media are lipase-positive bacteria.

[0026] Figure 2 This is a diagram illustrating the use of SnapGene software and the principles of result screening. The imported information is the complete genome sequence of *Pseudomonas* downloaded from the NCBI database, with potential target mining based on an endonuclease alignment database. For this bacterium, the possible candidate endonucleases are SpeI and XbaI.

[0027] Figure 3 This is a diagram showing the actual validation results of software-based endonuclease screening for *Pseudomonas aeruginosa* from dairy products; the optimal endonuclease screening was achieved.

[0028] Figure 4 This is an optimized screening diagram of the best pulsed conditions for Pseudomonas aeruginosa, a type of bacteria that causes spoilage in dairy products; it also demonstrates the determination of the optimal conditions for pulsed-field gel electrophoresis of Pseudomonas aeruginosa.

[0029] Figure 5 This is a graph showing the PFGE typing results of 19 strains of Pseudomonas lactis verified by the method of this invention in actual sample testing.

[0030] Figure 6 This is a graph showing the PFGE typing results of 20 strains of Pseudomonas lactis verified by the method of this invention in actual sample testing.

[0031] Figure 7 This is a graph showing the PFGE typing results of 18 strains of Pseudomonas lactis verified by the method of this invention in actual sample testing.

[0032] Figure 8 This is a graph showing the results of a cluster analysis of 57 strains of Pseudomonas lactis using BioNumerics software.

[0033] Figure 9 This is a PFGE typing map of 57 strains of Pseudomonas lactis and a graph showing the number of strains in each typing. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0035] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0036] Example 1 The protease-producing screening medium consists of 6% skim milk powder and 1.5% agar (by weight), with the remainder being deionized water. To prevent the skim milk powder from denaturing and forming flocculent precipitates after high-temperature sterilization, the skim milk powder solution and agar solution should be sterilized separately during medium preparation. For example, in 1L of medium, add 60g of skim milk powder to 500mL of deionized water, sterilize at 100℃ for 3 minutes, and then cool to room temperature. Repeat this sterilization process 3-4 times. Then, place the skim milk powder solution in a 55℃ water bath until the medium is ready. Add 15g of agar to 500mL of deionized water, sterilize at 121℃ for 15 minutes, and then place in a 55℃ water bath until the medium is ready. When preparing the medium, mix the two solutions thoroughly and pour the protease-producing screening medium into the container for screening protease-producing bacteria.

[0037] Lipase production screening medium: 1L of basal medium with 30 mL of lipase substrate added; The 1L basal culture medium formula consists of 10g casein hydrolysate (Oxoid™, model LP0041B), 5g yeast extract, 0.15g Spirit Blue, and 17g agar. After sterilization at 121℃ for 15 min and cooling to 60℃, 30 mL of lipase substrate (a mixture of 1 mL Tween 80, 100 mL olive oil, and 400 mL deionized water) is added.

[0038] Example 2 The difference between this embodiment and Embodiment 1 is that: The protease production screening medium consisted of 8% skim milk powder and 1.5% agar by weight, with the remainder being deionized water.

[0039] The 1L basal culture medium formula consists of 12g casein hydrolysate (Oxoid™, model LP0041B), 7.5g yeast extract, 0.15g Spirit Blue, and 17g agar.

[0040] Example 3 This embodiment utilizes the physiological characteristics of Pseudomonas to produce lipases and proteases to screen for the putrefactivity of purified Pseudomonas isolated from dairy products. Protein-producing and lipid-producing selection media were selected as the culture media, and after overnight incubation, the turbidity was adjusted to 0.5 McFarland (approximately 10⁻⁶ bacterial colonies).8 The study used pure cultures of Pseudomonas aeruginosa (CFU / mL) (Pseudomonas aeruginosa was isolated from raw milk samples collected in June 2024 from 40 dairy farms in Jinan, Qingdao, Tai'an, Linyi, and Dongying cities in Shandong Province). Changes in culture plates incubated at 28°C and 70% humidity for 48 hours were observed. Figure 1 The differences in the width of the hydrolysis zones of the three Pseudomonas strains were shown, with the precipitation zones gradually widening from left to right, indicating an increasing protein degradation capacity. Spirit Blue was added to the lipase selection medium; when a strain could degrade fats in the culture medium, a halo would form around the colony. All three screened Pseudomonas strains were verified to produce both lipase and protease, thus being selected as putrefactive Pseudomonas.

[0041] Example 4 The difference between this embodiment and Embodiment 3 is that the changes in the culture medium plates cultured at 37°C and 50% humidity for 24 hours were observed.

[0042] Example 5 This embodiment describes a software-based initial screening of Pseudomonas strains selected in Example 3 using endonuclease inhibitors. Complete sequences of 48 Pseudomonas strains were downloaded from the NCBI database, including ATCC standard strains, CICC preserved strains, and food-derived isolates. The downloaded full genome sequences of these 48 strains were imported into SnapGene software. Enzyme target selection was performed based on a library of 13 commonly used endonucleases for PFGE typing (ApaI, SgrAI, XbaI, Fsel, NheI, AsCI, KpnI, NotI, Smal, BInI, MluI, SfiI, SpeI). Figure 2 This study demonstrates the differences in predicted restriction enzyme sites across the entire genome of different strains of the same Pseudomonas species. One strain showed a suitable number of SpeI, XbaI, and BlnI restriction sites, while another strain had a suitable number of SpeI and XbaI restriction sites, but the number of BlnI restriction sites was too high to be displayed. This indicates that BlnI digestion of this strain would yield multiple small base fragments below the minimum threshold range (20.5 kb) for pulsed-field gel electrophoresis. Based on the above procedures and preliminary screening principles for restriction enzymes, the likely candidate restriction enzymes for Pseudomonas are SpeI and XbaI.

[0043] Example 6 This embodiment verifies the use of the restriction enzymes screened in Example 5 on three Pseudomonas strains selected in Example 3. To comprehensively explain why the restriction enzyme screening based on the same bacteria in Example 5 uses intersection rather than union, the restriction enzyme BlnI, which was screened out in Example 5, was used as a control group. Bacterial blocks of the screened putrefactive Pseudomonas strains were prepared using the "National Pathogenic Bacteria Identification Network Technical Manual (2020 Edition)". Enzyme digestion within the blocks was performed according to the enzyme digestion system and digestion time parameters of the screened enzymes. Simultaneously, a standard Marker bacterial block H9812 was prepared for pulsed-field gel electrophoresis. XbaI digestion was used as the Marker for pulsed-field gel electrophoresis to determine the size of the fragments cleaved by the restriction enzymes. The specific parameters for the instrumental detection were as follows: pulse angles were all set to 60°, operating temperature was all set to 14℃, initial switch time was set to 1 s, final switch time was set to 25 s, and the total run time for pulsed-field gel electrophoresis was set to 18 h. After completion, the intact gel blocks were stained with 0.5 μL / mL Gel Red staining solution for 30 min, followed by destaining with deionized water for 30 min, and then the gel imaging system was used to examine the screening results of the restriction enzymes. This step was repeated with different enzymes while maintaining the same parameters. Figure 3 The results visually demonstrate the digestion effects of different restriction enzymes on *Pseudomonas*. In the gel digested with BlnI, the bands of two bacterial strains were not visible, exhibiting a completely diffuse state. This indicates that these two *P.* strains have too many BlnI cleavage sites across their genomes, resulting in multiple small fragments that cannot be distinguished by pulsed-field gel electrophoresis (PGFES). In the gel digested with XbaI, all bacterial bands were clearly visible, but they exhibited a strong aggregation. While different strains could be largely distinguished, this enzyme is not a preferred restriction enzyme for *P.*, but rather a candidate enzyme. Further optimization of the specific PGFES parameters is needed. In the gel digested with SpeI, all bacterial bands were clearly visible with good separation and uniform band distribution. SpeI can be considered a preferred restriction enzyme for *P.*, facilitating the establishment of standardized PGFES techniques.

[0044] Example 7 This embodiment optimizes the screening of optimal endonucleases selected in Example 6 using pulsed-field gel electrophoresis (PGFES). Bacterial blocks of the screened putrefactive *Pseudomonas* strain were prepared using the *National Pathogenic Bacteria Identification Network Technical Manual (2020 Edition)*. The enzyme digestion system and corresponding parameters for digestion time were determined according to Example 6 to complete the digestion within the gel blocks. Simultaneously, a standard marker bacterial block (H9812) for PGFES was prepared and digested with XbaI as the marker for PGFES to determine the size of the fragments cleaved by the endonuclease. Three sets of PGFES conditions were set for validation: ① Initial switching time set to 2 s, final switching time to 30 s; ② Initial switching time set to 2.2 s, final switching time to 38.5 s; ③ Initial switching time set to 2 s, final switching time to 40 s. The pulse angle for all PGFES was set to 60°, the running temperature to 14℃, and the running time to 19 h. After pulsed-field gel electrophoresis, the gel blocks that have completed electrophoresis are stained and imaged. Figure 4 The results visually demonstrate the electrophoretic effects of the same Pseudomonas strains and the same restriction enzyme digestion under different pulsed-field gel electrophoresis. When the initial switching time was set to 2 s and the final switching time to 30 s, the bands were clearly separated but not uniformly, and the large molecular bands were too close to the sample wells. Extending the pulse time resolved this issue, resulting in clearly and uniformly separated bands, with good separation of different strain types. Therefore, an initial switching time of 2.2 s and a final switching time of 38.5 s were selected as the optimal standard procedure.

[0045] Example 8 This example demonstrates the screening of putrefactive strains of *Pseudomonas* isolated from milk using the method described in Example 3, identifying a total of 57 putrefactive *P.* strains. The optimal restriction enzymes selected in Examples 5-6 and the optimal pulsed-field gel electrophoresis (PFGE) conditions in Example 7 were used for PFGE typing experiments. The PFGE results for all 57 milk-derived putrefactive *P.* strains showed clear and distinct banding, indicating good separation. Figures 5-7 The PFGE typing results of 57 strains of Pseudomonas lactis were presented, which confirmed the practical application effect of the standardized method for dairy-derived putrefactive Pseudomonas established in this invention. The results show that the standardized method of this invention is applicable to the standardized typing process of dairy-derived putrefactive Pseudomonas.

[0046] Example 9 This embodiment describes cluster analysis of the PFGE results of 57 *Pseudomonas lactis* strains from Experiment 5. The BioNumerics software was used for typing and cluster analysis. The PFGE image of *Salmonella* H9812 XbaⅠ enzyme digestion, a standard strain internationally recognized for clustering, was used as a control marker. The UPGAMA cluster analysis method was employed. The similarity between the PFGE band patterns of the 57 *Pseudomonas lactis* strains was measured using the Dice coefficient. In the BioNumerics software, the bandmatching tolerance and optimization were adjusted according to clustering requirements, with a default setting of 1.5%, adjustable within the range of 0.5% to 3%. A total of 12 PFGE band patterns were formed, with a clearly dominant band pattern and a similarity of 82%–100%. The overall band pattern similarity was high, indicating close phylogenetic relationships among the strains. This completed the standardized workflow verification for the typing and cluster analysis of *Pseudomonas lactis* of this invention. Figures 8-9 The results show that the pulsed-field gel electrophoresis typing standardization method for *Pseudomonas lactis* of the present invention can complete the experimental operation and subsequent analysis of actual commercial samples, and has application value.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for typing *Pseudomonas lactis* based on pulsed-field gel electrophoresis, characterized in that, Includes the following steps: (1) Identification of putrefactive Pseudomonas: Inoculate the bacteria to be tested onto the selection medium for protease-producing bacteria and observe whether a hydrolysis zone is produced. The production of a hydrolysis zone indicates the production of protease. Inoculate the bacteria to be tested onto a lipase-producing bacterial selection medium and observe the bacterial colony morphology on the medium. If a halo forms around the colony, it indicates that lipase is produced. Pseudomonas species that produce either protease or lipase are classified as putrefactive Pseudomonas species. (2) Endonuclease screening for pulsed-field gel electrophoresis typing S1: Identify the putrefactive Pseudomonas species selected in step (1) to obtain species information; S2: Based on the species information of the selected bacteria, download the whole genome sequence from the NCBI database to complete the preparations before screening for enzyme digestion targets; S3: Use SnapGene software to import the bacterial whole genome sequence, select the "Enzyme" option, and create an endonuclease alignment database for PFGE endonuclease screening; S4: Statistically analyze the results in the enzyme toolbar in the lower right corner of the output results and check the number of sites for different endonucleases. If the number of sites shows "Too many sites to display", it means that the enzyme has too many sites. Screen out these endonucleases and keep other endonucleases with clear and countable cleavage sites for the next step of verification. S5: Prepare bacterial gel blocks of the screened putrefactive Pseudomonas bacteria. Use the single enzyme selected in S4 to perform single enzyme digestion within the gel block. After digestion, perform PFGE detection. After completion, place the intact gel block in staining solution for staining. After staining, decolorize and then use a gel imaging system to image and verify the screening results of the restriction enzyme. Repeat this step while maintaining the same parameters and using different enzymes. S6: Based on the results of S5, screen for restriction enzymes with uniform and clear digestion bands; S7: After S6 is completed, further screening is conducted to determine the optimal conditions for pulsed field gel electrophoresis, and then standardized typing and molecular typing analysis are performed on the isolated milk-derived Pseudomonas putrefactive bacteria.

2. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In step (1), the culture conditions for culturing on the selection medium for protease-producing bacteria and the selection medium for lipase-producing bacteria are: 28-37℃ temperature and 50%-70% humidity for 24-48 hours.

3. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In step (1), the screening medium for protease-producing bacteria consists of the following composition by mass percentage: 6-8% skim milk powder, 1.5% agar, and the remainder deionized water.

4. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In step (1), the composition of the lipase-producing bacteria screening medium is: 1L of basal medium plus 30 mL of lipase substrate; The composition of 1L of basal culture medium is: 10-12g casein hydrolysate, 5-7.5g yeast extract, 0.15g alcohol-soluble blue, 17g agar, and the remainder being deionized water; The lipase substrate composition is: 1 ml Tween 80, 100 ml olive oil, and 400 mL deionized water.

5. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In S3, the selected enzymes are commonly used PFGE enzymes: ApaI, SgrAI, XbaI, Fsel, NheI, AsCI, KpnI, NotI, Smal, BInI, MluI, SfiI, and SpeI, totaling 13 enzymes, to explore the target sites of these 13 enzymes on the bacterial genome.

6. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In S5, the intact gel block was stained with 0.5 μL / mL Gel Red staining solution for 30 min, and then destained with deionized water for 30 min.

7. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In S5, the specific parameters for the instrument detection are as follows: the pulse angle is set to 60°, the running temperature is set to 14℃, the initial switching time for electrophoresis is set to 1s, the final switching time is 25s, and the running time for all pulsed field gel electrophoresis is set to 18h; Salmonella H9812 digested with XbaI enzyme is used as the marker for instrument detection.

8. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In S7, the method for determining the optimal conditions for pulsed-field gel electrophoresis was as follows: three sets of electrophoresis conditions were set for gel blocks of the same target bacteria to complete the instrument detection: ① the initial switching time of electrophoresis was set to 2 s, and the final switching time was 30 s; ② the initial switching time of electrophoresis was set to 2.2 s, and the final switching time was 38.5 s; ③ the initial switching time of electrophoresis was set to 2 s, and the final switching time was 40 s; the pulse angle of all pulsed-field gel electrophoresis was set to 60°, the running temperature was set to 14℃, and the running time was set to 19 h; the optimal conditions were selected based on the separation results.

9. The method for typing *Pseudomonas lactis* according to claim 1, characterized in that, In S7, molecular typing analysis was performed using BioNumerics software for typing and cluster analysis; the PFGE diagram of Salmonella H9812 XbaⅠ restriction enzyme digestion was used as a control maker for calibration, Dice was selected for cluster similarity coefficient, and UPGAMA was used for cluster analysis.

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