Method for improving bacteriostatic activity of lactobacillus metabolite

Abstract

The invention discloses a method for improving bacteriostatic activity of lactic acid bacteria metabolites, and relates to the technical field of lactic acid bacteria metabolism. The method is used for accurately judging factors influencing the bacteriostatic activity of the lactic acid bacteria metabolites. Comprising the following steps: preparing a lactobacillus culture medium and a reagent; selecting a test tool; pretreating the lactic acid bacteria sample; carrying out an antibacterial test; and analyzing a test result. According to the method, the growth and metabolism of the lactic acid bacteria can be remarkably optimized by accurately controlling the culture conditions of the lactic acid bacteria, such as temperature, time and culture medium components, so that the bacteriostatic activity of metabolites of the lactic acid bacteria is improved, and the lactic acid bacteria with high-yield bacteriostatic activity can be quickly and effectively screened out by means of morphological observation, gene screening and the like; by carrying out acid-base treatment, heat treatment and enzyme treatment on the lactic acid bacteria metabolite, the stability of the lactic acid bacteria metabolite under different conditions can be evaluated, and the treatment conditions are further optimized to improve the antibacterial activity of the lactic acid bacteria metabolite.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid bacteria metabolism, and particularly to a method for improving the antibacterial activity of lactic acid bacteria metabolites. Background Art

[0002] As a type of probiotic widely present in nature, lactic acid bacteria have attracted much attention due to their beneficial effects on human health. Metabolites produced by lactic acid bacteria through fermentation, such as lactic acid, acetic acid, bacteriocins, etc., not only have flavoring and preservation functions, but more importantly, have significant antibacterial activity and can inhibit a variety of pathogenic bacteria. Therefore, lactic acid bacteria and their metabolites have broad application prospects in the fields of food preservation, medical and health care, agricultural biological control, etc. However, the antibacterial activity of lactic acid bacteria is affected by various factors, such as bacterial species, culture conditions, types and contents of metabolites, etc. In order to make full use of the antibacterial activity of lactic acid bacteria and improve its effect in practical applications, it is necessary to conduct in-depth research on lactic acid bacteria and optimize their culture conditions and metabolite extraction processes. However, although the antibacterial activity of lactic acid bacteria and their metabolites has been widely studied and applied, there are still some challenges and problems to be solved;

[0003] After retrieval, a patent with the Chinese patent application number 201510204732.7 discloses a fermentation method for improving the antibacterial activity of lactic acid bacteria metabolites. 1 g / L - 1.8 g / L of pepsin is added to the fermentation medium; lactic acid bacteria are fermented and cultured in this medium;

[0004] The above-mentioned fermentation method for improving the antibacterial activity of lactic acid bacteria metabolites has the following deficiencies: during the experiment on how to improve the antibacterial activity of lactic acid bacteria metabolites, only the addition of pepsin is considered to determine the experimental items, resulting in fewer experimental items and lacking persuasiveness, and it cannot be ensured that no other factors will affect the antibacterial activity of lactic acid bacteria metabolites. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose a method for improving the antibacterial activity of lactic acid bacteria metabolites.

[0006] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0007] A method for improving the antibacterial activity of lactic acid bacteria metabolites includes the following steps:

[0008] S1: Prepare lactic acid bacteria culture medium and reagents;

[0009] S2: Select experimental utensils;

[0010] S3: Pretreat lactic acid bacteria samples;

[0011] S4: Conduct an antibacterial test;

[0012] S5: Analyze the test results.

[0013] Preferably, the lactic acid bacteria culture medium is a solid medium, and the test utensils include a refrigerated refrigerator, a centrifuge, a constant temperature incubator, an electronic scale, a microscope, and a steam sterilizer.

[0014] Furthermore, the pretreatment steps include:

[0015] Anaerobically culture the solid medium in a constant temperature incubator at 36°C for 48 hours;

[0016] Dilute the bacterial solution with sterile normal saline and use ion chromatography and reverse-phase liquid chromatography separation techniques;

[0017] Screen for target lactic acid bacteria by the streak plate method;

[0018] Measure the absorbance value of the fermentation broth every 2 hours and plot a growth curve;

[0019] Measure the pH value of the fermentation broth every 2 hours.

[0020] Furthermore, the observation and screening of the lactic acid bacteria morphology are carried out by inoculating and purifying the lactic acid bacteria onto a solid medium by streak plate inoculation, observing the colony characteristics after anaerobic culture; designing and screening specific primers, extracting the genomic DNA of the lactic acid bacteria for PCR amplification, and confirming the target band by agarose gel electrophoresis; applying high-throughput screening technology combined with automation, fluorescence labeling, microfluidics, and bioinformatics analysis to screen for the optimal strain; establishing a database by combining the colony morphology and gene screening results, using machine learning to predict the optimal strain; conducting antibacterial activity tests, whole-genome sequencing, RNA sequencing, and metabolomics analysis on the screened strains to optimize the screening accuracy.

[0021] As a further scheme of the present invention: The antibacterial test includes:

[0022] Inoculate Escherichia coli into a common solid medium for culture and prepare a bacterial suspension;

[0023] Take the activated lactic acid bacteria and inoculate them into the medium, and centrifuge and filter after anaerobic culture;

[0024] Wash and dilute the precipitate after centrifugation with normal saline;

[0025] Perform heat treatment, acid-base treatment, and enzyme treatment on the metabolites.

[0026] As a still further scheme of the present invention: The treatment of the metabolites includes:

[0027] Treat at 100°C for 30 minutes;

[0028] Adjust the pH to 2.0, 3.0, 4.0, and 5.0 with 1 mol / L HCl;

[0029] Incubate with catalase, proteinase K, pepsin, and trypsin at 36 °C for 2 hours.

[0030] On the basis of the above-mentioned protocol: The antibacterial test uses the Oxford cup method to measure the size of the inhibition zone, and the indicator bacteria are Bacillus cereus and Escherichia coli (10 5 CFU / mL).

[0031] On the basis of the above-mentioned protocol: The analysis of the test results includes:

[0032] Extract the genomic DNA of lactic acid bacteria: Use the thermal lysis method to disrupt the cell structure by high temperature and purify the DNA;

[0033] Homology analysis: Use the BLAST tool to align the extracted DNA sequences and evaluate the homology.

[0034] On the basis of the above-mentioned protocol: The DNA extraction steps include:

[0035] Collect the lactic acid bacteria culture and separate the cells by centrifugation;

[0036] Place the cells in a high-temperature environment for thermal lysis to release DNA;

[0037] Remove cell debris and impurities by centrifugation to purify the DNA.

[0038] On the basis of the above-mentioned protocol: The homology analysis steps include:

[0039] Sequence the genomic DNA of the extracted lactic acid bacteria;

[0040] Submit the sequence to the BLAST database and select appropriate alignment parameters for alignment;

[0041] Analyze the alignment results and evaluate the sequence homology.

[0042] The beneficial effects of the present invention are:

[0043] 1. A method for improving the antibacterial activity of lactic acid bacteria metabolites. By precisely controlling the culture conditions of lactic acid bacteria, such as temperature, time, and medium components, the growth and metabolism of lactic acid bacteria can be significantly optimized, thereby improving the antibacterial activity of their metabolites.

[0044] 2. A method for improving the antibacterial activity of lactic acid bacteria metabolites. By means of morphological observation and gene screening, etc., lactic acid bacteria with high antibacterial activity can be quickly and effectively screened out.

[0045] 3. A method for improving the antibacterial activity of lactic acid bacteria metabolites. By performing acid-base treatment, heat treatment, and enzyme treatment on lactic acid bacteria metabolites, their stability under different conditions can be evaluated, and the treatment conditions can be further optimized to improve their antibacterial activity.

[0046] 4. A method for improving the antibacterial activity of lactic acid bacteria metabolites. By deeply studying the antibacterial mechanism and mode of action of lactic acid bacteria metabolites, more efficient and environmentally friendly antibacterial agents can be developed to meet the growing antibacterial needs. By means such as optimizing culture conditions, screening high-yield strains, and treating metabolites, the potential value of lactic acid bacteria can be fully utilized. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a schematic flow chart of a method for improving the antibacterial activity of lactic acid bacteria metabolites proposed by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] The technical solutions of this patent will be further described in detail below in conjunction with the specific embodiments.

[0049] The embodiments of this patent are described in detail below. The examples of the embodiments are shown in the drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout. The embodiments described by referring to the drawings below are exemplary and are only used to explain this patent and should not be construed as a limitation of this patent.

[0050] Example 1:

[0051] A method for improving the antibacterial activity of lactic acid bacteria metabolites, as Figure 1 shown, includes the following steps:

[0052] S1: Prepare the lactic acid bacteria culture medium and reagents

[0053] Prepare the lactic acid bacteria culture medium and reagents for improving antibacterial activity. The test culture medium is a solid culture medium.

[0054] S2: Select the test utensils

[0055] Select the required test utensils, including a refrigerated refrigerator, a centrifuge, a constant temperature incubator, an electronic scale, a microscope, and a steam sterilizer.

[0056] S3: Pretreat the lactic acid bacteria sample

[0057] Pretreat the prepared lactic acid bacteria sample. The specific steps are as follows:

[0058] S31: Culture the lactic acid bacteria

[0059] The lactic acid bacteria are cultured using a solid medium. The solid medium is placed in an anaerobic incubator at 36°C for 48 hours and then an antibacterial test is carried out.

[0060] S32: Purification and isolation of lactic acid bacteria

[0061] The enriched bacterial liquid is diluted with sterile normal saline; the solid medium at about 45°C is poured into a petri dish. After the medium cools and solidifies, 1 mL of the bacterial suspension with dilution factors of 10 -4 、10 -5 is respectively pipetted into the petri dish and transferred to an incubator at 36°C for 12 hours; ion chromatography and reverse-phase liquid chromatography separation techniques are used during the purification process;

[0062] S33: Observation and screening of the morphology of lactic acid bacteria

[0063] The purified lactic acid bacteria are inoculated onto a solid medium by the streak plate method. After anaerobic culture, the colony characteristics are observed and recorded. Specific primers are designed and screened. Genomic DNA is extracted from the lactic acid bacteria for PCR amplification and screening. Agarose gel electrophoresis analysis is used to confirm the target band. Preferably, the colony-PCR method can be used, combined with electrophoresis analysis to screen out the lactic acid bacteria colonies containing the target gene. High-throughput screening technology uses an automated system and fluorescently labeled probes to achieve batch amplification and real-time detection. Combining microfluidic technology and bioinformatics analysis, the optimal strains are screened. During the screening process, combining colony morphological characteristics and gene screening results, a strain database is established, and machine learning algorithms are used for prediction. Then, the antibacterial activity test and whole-genome sequencing verification are carried out on the screened lactic acid bacteria. Combining RNA sequencing and metabolomics analysis, the accuracy of the screening results is optimized;

[0064] S34: Determination of the growth curve

[0065] The pre-activated lactic acid solution is added to the culture medium at an inoculation amount of 3%. It is statically cultured at 36°C for 48 hours; the fermentation broth is taken every 2 hours, and its absorbance is measured at a wavelength of 500 nm. The measurement is carried out in parallel 3 times, and the growth curves of each strain are plotted;

[0066] S35: Determination of the pH value

[0067] The pre-activated lactic acid bacteria are inoculated into a liquid medium at an inoculation amount of 3%; the pH value of the fermentation broth is measured every 2 hours, and the measurement is carried out in parallel 3 times;

[0068] Antibacterial test

[0069] The antibacterial test includes the following steps:

[0070] 1: Preparation of the indicator bacterial suspension

[0071] Inoculate Escherichia coli into a common solid medium and culture it at 36°C for 24 hours; after centrifugation, prepare a bacterial suspension, and measure the bacterial content of the bacterial suspension using the plate counting method; take the bacterial liquid with a concentration of 10 5 CFU / mL as the indicator bacterial suspension;

[0072] 2: Preparation of lactic acid bacteria metabolites

[0073] Take 0.5 mL of activated lactic acid bacteria and inoculate it into 100 mL of their respective media, and culture it anaerobically for 48 hours; centrifuge at 3000 rpm, take the supernatant and filter it through a filter, and collect the filtrate as the lactic acid bacteria metabolites;

[0074] 3: Preparation of lactic acid bacteria cells

[0075] Wash the precipitate after the above centrifugation 3 times with physiological saline, and then dilute it with physiological saline to the original bacterial liquid concentration;

[0076] 4: Treatment of lactic acid bacteria metabolites

[0077] Treat the metabolites at 100°C for 30 minutes, and then conduct an antibacterial test; adjust the pH of the metabolites to 2.0, 3.0, 4.0, and 5.0 respectively with 1 mol / L HCl; incubate the metabolites with catalase (0.5 mg / mL), proteinase K (1 mg / mL), pepsin (1 mg / mL), and trypsin (1 mg / mL) at 36°C for 2 hours for antibacterial tests;

[0078] 5: Antibacterial test of lactic acid bacteria metabolites

[0079] Spread 0.1 mL of the indicator bacterial culture on the plate and let it dry naturally for 30 minutes; place the Oxford cups evenly on the plate, 3 for each plate, and finally fill them with the test solution; culture at 36°C for 12 - 15 hours and measure the antibacterial diameter; perform 3 replicates for each sample and take the average value; use the Oxford cup method to measure the size of the antibacterial zone, and the indicator bacteria are Bacillus cereus and Escherichia coli (10 5 CFU / mL);

[0080] 6: Antibacterial effect test after acid-base treatment of metabolites

[0081] Adjust the pH value of the metabolites to 2.0, 3.0, 4.0, and 5.0 respectively with 1 mol / L NaOH and HCl for antibacterial tests; measure the size of the antibacterial zone using the method mentioned in step 5, and the indicator bacterium is Bacillus cereus; adjust the pH value of the liquid medium to 2.0, 3.0, 4.0, and 5.0 with lactic acid solution and 1 mol / L NaOH for control tests;

[0082] 7: Antibacterial effect after heat treatment of metabolites

[0083] The antibacterial test was carried out by treating the metabolite in a boiling water bath for 25 minutes; the untreated metabolite was used as the control group;

[0084] 8: Antibacterial effect after enzymatic treatment of the metabolite

[0085] Take 10 mL of the metabolite, and adjust the pH value of the metabolite to the optimal range of each enzyme with 1 mol / L NaOH and HCl respectively; add Proteinase K (0.5 mg / mL), incubate in a water bath at 36 °C for 2 hours, then adjust the pH value of the metabolite to the initial value, and carry out the antibacterial test; the metabolite without enzymatic treatment was used as the control group;

[0086] S4: Analyze the test results

[0087] Analyze the test results, and the specific steps are as follows:

[0088] S41: Extract DNA

[0089] The genomic DNA of Lactobacillus was extracted by the thermal lysis method, and the specific steps include:

[0090] Take an appropriate amount of Lactobacillus culture, and collect the cells by methods such as centrifugation;

[0091] Place the collected cells in a high-temperature environment and heat for a period of time to destroy the cell structure and release DNA;

[0092] After thermal lysis, remove cell debris and impurities by methods such as centrifugation, and collect the DNA in the supernatant;

[0093] According to the experimental requirements, further purify the DNA by methods such as column purification and ethanol precipitation to remove residual impurities such as proteins and RNAs.

[0094] S42: Homology analysis

[0095] Use the search tool technology based on the local alignment algorithm of nucleic acids (such as BLAST) for homology analysis, and the specific steps include:

[0096] Sequence the extracted genomic DNA of Lactobacillus to obtain its nucleic acid sequence;

[0097] Select a suitable BLAST database according to the analysis requirements;

[0098] Submit the sequence to be analyzed to the BLAST server, select appropriate alignment parameters (such as alignment type, expectation value, etc.), and run BLAST analysis;

[0099] Analyze the BLAST results, find the sequences homologous to the sequence to be analyzed, and evaluate the degree and significance of homology.

[0100] As described above, the above is a preferred specific embodiment of the present invention. The protection scope of the present invention is not limited thereto. Any person skilled in the art, within the technical scope disclosed by the present invention, in combination with the prior art or common knowledge of the public, any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A method for improving the antibacterial activity of lactic acid bacteria metabolites, characterized in that: The following steps are involved: S1: Preparation of lactic acid bacteria culture medium and reagents; S2: Select test equipment; S3: pre-treating the lactic acid bacteria sample; S4: Conduct antibacterial test; S5: Analyze the test results.

2. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 1, characterized in that: The lactic acid bacteria culture medium is a solid culture medium, and the test tools include a refrigerator, a centrifuge, a constant temperature incubator, an electronic scale, a microscope and a steam sterilizer.

3. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 2, characterized in that: The pre-processing step comprises: The solid culture medium was placed in a 36°C constant temperature incubator for anaerobically cultured for 48 hours; The bacterial solution was diluted with sterile saline and separated by ion chromatography and reversed phase liquid chromatography; Screening of target lactic acid bacteria by plate streak method; The absorbance of the fermentation liquid was measured every 2 hours and a growth curve was drawn; The pH value of the fermentation broth was measured every 2 hours.

4. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 3, characterized in that: Observation and screening of lactic acid bacteria morphology: Purified lactic acid bacteria were inoculated into solid culture medium by streaking on plates, and the characteristics of colonies were observed after anaerobic culture. Design and screen specific primers, extract lactic acid bacteria genomic DNA for PCR amplification, and confirm the target bands by agarose gel electrophoresis; use high-throughput screening technology combined with automation, fluorescent labeling, microfluidics and bioinformatics analysis to screen the optimal strain; establish a database based on colony morphology and gene screening results, and use machine learning to predict the optimal strain; Antibacterial activity tests, whole genome sequencing, RNA sequencing and metabolomics analysis were performed on the screened strains to optimize the screening accuracy.

5. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 4, characterized in that: The antibacterial test includes: Inoculate Escherichia coli into a common solid culture medium to prepare a bacterial suspension; Take the activated lactic acid bacteria and inoculate them into the culture medium, culture them anaerobically and then centrifuge and filter them; The centrifuged precipitate was washed and diluted with saline; The metabolites were subjected to heat treatment, acid-base treatment and enzyme treatment.

6. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 5, characterized in that: Processing of the metabolites includes: Treat at 100°C for 30 minutes; Adjust the pH to 2.0, 3.0, 4.0, and 5.0 with 1 mol / L HCl; Incubate with catalase, proteinase K, pepsin, and trypsin at 36°C for 2 hours.

7. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 6, characterized in that: The antibacterial test used the Oxford cup method to determine the size of the inhibition zone, and the indicator bacteria were Bacillus cereus and Escherichia coli (10 5 CFU / mL).

8. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 7, characterized in that: The test result analysis includes: Extraction of lactic acid bacteria genomic DNA: thermal lysis method is used to destroy the cell structure and purify DNA through high temperature; Homology analysis: The extracted DNA sequences were compared using the BLAST tool to assess homology.

9. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 8, characterized in that: The DNA extraction step comprises: Collect the lactic acid bacteria culture and separate the bacteria by centrifugation; The bacteria are placed in a high temperature environment for thermal lysis to release DNA; Cell debris and impurities are removed by centrifugation and the DNA is purified.

10. A method for improving the antibacterial activity of lactic acid bacteria metabolites according to claim 9, characterized in that: The homology analysis step comprises: Sequencing the extracted lactic acid bacteria genomic DNA; Submit the sequence to the BLAST database and select appropriate alignment parameters for alignment; Analyze the alignment results and assess sequence homology.

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