Lactobacillus plantarum-sourced mixture with bacteriostatic and anti-inflammatory activity as well as preparation method and application of lactobacillus plantarum-sourced mixture

Abstract

The invention discloses a lactobacillus plantarum-sourced mixture with bacteriostatic and anti-inflammatory activity as well as a preparation method and application of the lactobacillus plantarum-sourced mixture. The core functional components comprise 5 '-methyl thioadenosine, 3-hydroxymyristic acid, tranexamic acid, indole-3-lactic acid, 3-phenyl lactic acid, ((R)-1-carboxyethyl)-L-phenylalanine hydrochloride, L-tyrosyl-L-proline, isopentenyl phosphate, 6-aminododecanedioic acid, N-acetylthreonine, 2-methyl-3-oxopropionic acid, 2, 4-dihydroxy-2, 4, 6-triazole-3-carboxylic acid, 2, 4, 6-triazole-3-carboxylic acid, 2, 4, 6-triazole-3-carboxylic acid, 2, 4, 6-triazole-3-carboxylic acid, 2, 4, 6-triazole-3-carboxylic acid, 2, 4-triazole-3- The compound is prepared from 2, 4-dimethyl quinoline and N-acetylalanine. The invention has the advantages that: the compound has broad-spectrum bactericidal activity and can be used for preparing antibacterial drugs, food additives, pesticides and the like.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and more particularly to Lactobacillus plantarum and its culture products. Background Technology

[0002] Lactobacillus plantarum, a native resident bacterium of the human gut and oral cavity, plays a vital role in human health by inhibiting the growth of harmful microorganisms and maintaining the balance of the microbiome. In recent years, research on Lactobacillus plantarum and its antibacterial substances has become a hot topic. Some studies have optimized specific culture media and fermentation conditions to increase the growth rate and metabolite yield of Lactobacillus plantarum, and then combined this with separation and purification techniques to obtain components with antibacterial activity. These studies provide a theoretical basis and experimental evidence for the development of novel antimicrobial drugs and food preservatives.

[0003] However, existing technologies have some problems, such as:

[0004] Limited antibacterial activity: Most studies only improve the growth and metabolite yield of Lactobacillus plantarum by optimizing the culture medium composition and fermentation conditions, but the improvement on the performance of the strain itself is limited and it is difficult to meet the requirements of practical applications for efficient antibacterial and anti-inflammatory effects.

[0005] Narrow antibacterial spectrum: Most studies only target specific harmful bacteria, and there is insufficient research on its broad-spectrum antibacterial effect against a variety of harmful bacteria, which limits the application of Lactobacillus plantarum in complex microbial environments.

[0006] Insufficient research on anti-inflammatory effects: While some studies have explored the anti-inflammatory potential of probiotics, most remain at the preliminary cell experiment stage, lacking in-depth research on specific anti-inflammatory mechanisms and failing to fully consider the synergistic effects of various active ingredients in the supernatant of *Lactobacillus plantarum* fermentation broth. For example, some studies have only verified the inhibitory effect of certain probiotic fermentation supernatants on specific inflammation models, but the specific active ingredients in these supernatants and their mechanisms of action remain unclear.

[0007] In summary, there is still room for improvement in the existing technologies to enhance the antibacterial activity of Lactobacillus plantarum and broaden its antibacterial spectrum. Summary of the Invention

[0008] The purpose of this invention is to provide a mixture of Lactobacillus plantarum with antibacterial and anti-inflammatory activities, its preparation method and application, so as to solve the problems of low antibacterial activity and narrow antibacterial spectrum of Lactobacillus plantarum in the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention also provides a method for preparing a mixture with antibacterial and anti-inflammatory activity derived from *Lactobacillus plantarum*, the process comprising:

[0011] Resuscitation and primary culture: Lactobacillus plantarum strain M3 was inoculated into MRS medium and cultured.

[0012] Expanded culture: The bacterial culture after the initial culture was inoculated into fresh MRS medium for expanded culture;

[0013] Obtaining fermentation supernatant: After expanding the bacterial culture, remove the bacterial cells and collect the supernatant, which is a mixture with antibacterial and anti-inflammatory activities derived from Lactobacillus plantarum.

[0014] Furthermore, the culture conditions for the resuscitation and primary culture are 12-16 hours in an anaerobic environment at 37 ℃.

[0015] Furthermore, during the expanded culture process, the inoculum volume is 1% v / v, and the culture conditions are continued culture in an anaerobic environment at 37 ℃ for 12-16 hours.

[0016] Furthermore, the centrifugation parameters for obtaining the fermentation supernatant are centrifugation at 4000-5000 rpm for 10-15 minutes.

[0017] A mixture derived from *Lactobacillus plantarum* with antibacterial and anti-inflammatory activities, the core active ingredients of which include: 5'-methylthioadenosine, 3-hydroxymyristic acid, tranexamic acid, indole-3-lactic acid, 3-phenyllactic acid, ((R)-1-carboxyethyl)-L-phenylalanine hydrochloride, L-tyrosyl-L-proline, isopentenyl phosphate, 6-aminododecanoic acid, N-acetylthreonine, 2-methyl-3-oxopropionic acid, 2,4-dimethylquinoline, and N-acetylalanine.

[0018] Even better, its core active ingredients also include a variety of peptides with different sequences.

[0019] More preferably, the various polypeptide sequences include polypeptides with sequences as shown in SEQ ID NO. 1~15.

[0020] The present invention also provides the application of the mixture of Lactobacillus plantarum-derived bacteria with antibacterial and anti-inflammatory activities in the preparation of antibacterial drugs, food additives or pesticides.

[0021] More preferably, the antimicrobial drug is a drug that is effective against Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Aspergillus fumigatus, or Basilella marneffei.

[0022] The advantages of this invention are: It employs a simple culture and separation process to obtain a mixture of *Lactobacillus plantarum* sources with high antibacterial activity and a broad antibacterial spectrum. This mixture exhibits high antibacterial activity against multidrug-resistant bacteria such as *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, *Klebsiella pneumoniae*, and *Staphylococcus aureus*, and can be used to prepare antibacterial drugs, food additives, pesticides, etc. The preparation method eliminates the need for complex separation and purification steps, reducing production costs and improving production efficiency, which is beneficial for large-scale industrial production and overcomes the shortcomings of cumbersome preparation processes in existing technologies. Attached Figure Description

[0023] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0024] Figure 1 This is a comparison of the results of peptide omics analysis of the fermentation supernatant of Lactobacillus plantarum strain M3 and wild-type Lactobacillus plantarum used in this invention.

[0025] Figure 2 This is a comparison of the metabolomics analysis results of the fermentation supernatant of Lactobacillus plantarum strain M3 and wild-type Lactobacillus plantarum used in this invention.

[0026] Figure 2-1 yes Figure 2 One of the partial schematic diagrams;

[0027] Figure 2-2 yes Figure 2 Partial diagram two;

[0028] Figure 2-3 yes Figure 2 Partial diagram three;

[0029] Figure 3 These are photographs showing the results of co-culturing different bacteria with the fermentation supernatant of the *Lactobacillus plantarum* strain of this invention;

[0030] Figure 4 These are photographs showing the results of co-culturing fermentation supernatants of different fungi with the *Lactobacillus plantarum* strain of this invention;

[0031] Figure 5 This is a diagram showing the anti-inflammatory results of the fermentation supernatant of the *Lactobacillus plantarum* strain of this invention in an A549+PBMC pneumonia cell model induced by LPS. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0033] The *Lactobacillus plantarum* strain M3 used in this invention was developed through directed evolution technology. A large number of *Lactobacillus plantarum* mutants were constructed by ARTP mutagenesis of strain 123. Microfluidic high-throughput screening was then used to select mutants with higher antibacterial activity. After multiple rounds of mutagenesis and screening, a *Lactobacillus plantarum* mutant strain M3 was finally obtained, exhibiting significantly enhanced antibacterial and anti-inflammatory activity in the fermentation broth supernatant. This strain was previously reported in this article: Screening antimicrobial peptides and probiotics using multiple deep learning and directed evolution strategies. Acta Pharm Sin B. 2024 Aug;14(8):3476-3492. doi: 10.1016 / j.apsb.2024.05.003. Epub 2024 May 10. PMID: 39234615; PMCID:PMC11372459.

[0034] 1. Cultivation method and preparation of fermentation broth supernatant

[0035] Resuscitation and primary culture: The Lactobacillus plantarum strain M3 was removed from the glycerol tube stored at -80 ℃, inoculated into MRS medium, and cultured in an anaerobic incubator at 37 ℃ for 12-16 hours to fully revive it and carry out primary culture to restore the activity and stability of the strain.

[0036] Expanded culture: The bacterial culture after the initial culture was inoculated into fresh MRS medium at an inoculation rate of 1% (v / v) and cultured in an anaerobic incubator at 37 ℃ for 12-16 hours to expand the culture, so as to increase the number of bacteria and the accumulation of metabolites, and provide sufficient raw materials for the preparation of supernatant of fermentation broth.

[0037] Obtaining the fermentation broth supernatant: Centrifuge the expanded culture at 4000-5000 rpm for 10-15 minutes to remove the bacterial cells and collect the supernatant, which is the fermentation broth supernatant with antibacterial and anti-inflammatory activity. This supernatant is rich in various active ingredients.

[0038] 2. Composition and mechanism of action of fermentation broth supernatant

[0039] Compositional analysis: such as Figure 1-2As shown, through peptidomics and metabolomics analysis, it was found that the concentrations of some metabolites and peptides in the supernatant (QW) of *Lactobacillus plantarum* M3 fermentation broth were significantly higher than those in the wild-type *Lactobacillus plantarum* fermentation supernatant (WT) (red indicates relatively higher concentrations). These substances may be the core active ingredients with antibacterial and anti-inflammatory activities. Specifically, these include lactic acid and its derivatives, other short-chain fatty acids, amino acids, small molecule antimicrobial peptides, and some active ingredients related to anti-inflammatory effects: 5'-methylthioadenosine, 3-hydroxymyristic acid, tranexamic acid, indole-3-lactic acid, 3-phenyllactic acid, ((R)-1-carboxyethyl)-L-phenylalanine hydrochloride, L-tyrosyl-L-proline, isopentenyl phosphate, 6-aminododecanoic acid, N-acetylthreonine, 2-methyl-3-oxopropionic acid, 2,4-dimethylquinoline, N-acetylanine, and peptides with sequences as shown in SEQ ID NO. 1~15.

[0040] SEQ ID NO.1:AAAGLLFAGVLNANA;

[0041] SEQ ID NO.2:AGMLVTSQAVANA;

[0042] SEQ ID NO.3:AKTNPKTYQPALNKSLVKSSDPTKV;

[0043] SEQ ID NO.4:ALAGMLVTSQAVANA;

[0044] SEQ ID NO.5:ALALFAISTTVANA;

[0045] SEQ ID NO.6:AMVATLGAAVPVTGITANA;

[0046] SEQ ID NO.7:ATASLFAIGATAQA;

[0047] SEQ ID NO.8:ATTGAIALGATAAKA;

[0048] SEQ ID NO.9:AVGLLLATGQAANA;

[0049] SEQ ID NO.10: LAGMVTSQAVANA;

[0050] SEQ ID NO.11:LSTALLPMLSGKA;

[0051] SEQ ID NO.12:NYTVESTTTTLNLFQEKKLDLTQL;

[0052] SEQ ID NO.13:SAILTATYIPTSRASA;

[0053] SEQ ID NO.14:SSLATTGAIALGATAAKA;

[0054] SEQ ID NO.15:VDFNVPIKDG.

[0055] Antimicrobial mechanism of action: These metabolites and peptides exert their antimicrobial effects through multiple mechanisms. For example, organic acids such as lactic acid can lower the pH of the environment, disrupt the cell membrane structure of harmful bacteria, and inhibit their growth and reproduction; antimicrobial peptides can specifically bind to receptors on the surface of harmful bacterial cells, forming pores, causing leakage of cell contents, and ultimately killing the harmful bacteria.

[0056] Anti-inflammatory mechanism: The anti-inflammatory active ingredients in the fermentation broth supernatant exert their anti-inflammatory effects by regulating the activity of immune cells and the expression of inflammatory factors. For example, certain polysaccharides can inhibit the aggregation and activation of inflammatory cells and reduce the release of inflammatory mediators; specific vitamins can regulate intracellular signaling pathways and reduce the intensity of the inflammatory response, thereby effectively alleviating inflammation at the cellular level.

[0057] 3. Killing effect on multidrug-resistant bacteria and fungi

[0058] For Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Aspergillus fumigatus, and Bassilago farfara, corresponding control and experimental groups were set up respectively. The experimental group used a culture medium containing 5% v / v Lactobacillus plantarum fermentation broth supernatant. The results are as follows: Figure 3 As shown, the supernatant of 0.5% v / v *Lactobacillus plantarum* fermentation broth completely inhibited the growth of multidrug-resistant bacteria such as *Staphylococcus aureus*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, and *Streptococcus pneumoniae*. Furthermore, as... Figure 4 As shown, the supernatant of the 5% v / v Lactobacillus plantarum fermentation broth also exhibited good inhibitory effects against fungi such as Aspergillus fumigatus and Bassilago farfara, significantly reducing fungal growth and reproduction. These results indicate that the fermentation broth supernatant of the present invention possesses broad-spectrum antibacterial activity, demonstrating significant killing effects against multidrug-resistant bacteria and fungi, effectively combating various drug-resistant pathogens, and providing strong support for the development of novel antibacterial drugs.

[0059] 4. Anti-inflammatory effect in a lipopolysaccharide (LPS)-induced alveolar epithelial cell A549+ PBMC pneumonia cell model.

[0060] The fermentation supernatant of Lactobacillus plantarum M3 refers to the culture medium of Lactobacillus plantarum M3.

[0061] A lung cell model of A549+PBMC pneumonia was established using lipopolysaccharide (LPS). Several groups were then established: A549 group, A549+LPS group, A549+PBMC+LPS group, A549+PBMC+LPS group + 1% Lactobacillus plantarum M3 culture medium group, A549+PBMC+LPS group + 5% Lactobacillus plantarum M3 culture medium group, and A549+PBMC+LPS group + 10% Lactobacillus plantarum M3 culture medium group. After culturing each group according to the requirements, the experimental results are as follows: Figure 5 As shown, after adding fermentation supernatant to the culture medium, compared with the A549+PBMC+LPS group, the viability of A549+PBMC pneumonia cells was maintained, and the concentrations of inflammatory factors IL-6, IL-1β, and TNF-α were significantly decreased. In the LPS-induced A549+PBMC pneumonia cell model, the fermentation supernatant of this invention exhibited a significant anti-inflammatory effect.

[0062] The above data indicate that the fermentation broth supernatant of the present invention not only has antibacterial activity, but can also significantly reduce inflammatory response, and has potential clinical application value.

[0063] 5. Application areas of fermentation broth supernatant

[0064] Food Preservation and Safety: The fermentation broth supernatant of this invention can be applied to the field of food preservation, extending the shelf life of food and improving its safety by inhibiting the growth of spoilage and pathogenic bacteria. Simultaneously, its anti-inflammatory effect helps reduce inflammatory responses that may occur during food processing and storage, providing a possibility for developing food additives with dual functions.

[0065] Medical and Health Care: In the medical field, the supernatant of this fermentation broth can be used to prepare antibacterial drugs, wound dressings, and oral care products, effectively inhibiting pathogenic bacterial infection, promoting wound healing, and maintaining the balance of the oral microecology. Its anti-inflammatory properties also make it potentially valuable in the treatment of inflammatory diseases, such as in the development of anti-inflammatory drugs and skin care products.

[0066] Agricultural planting and plant protection: In addition, it can be applied in agricultural planting as a biological pesticide or plant growth regulator to prevent plant diseases, reduce the use of chemical pesticides, and promote sustainable agricultural development. Simultaneously, its anti-inflammatory properties help improve the soil microecological environment and promote healthy plant growth.

[0067] The preparation process of this invention is simple: it adopts a simple culture and separation process, without the need for complicated separation and purification steps, which reduces production costs, improves production efficiency, and is conducive to large-scale industrial production, overcoming the shortcomings of the complicated preparation process in the prior art.

[0068] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing a mixture of *Lactobacillus plantarum* with antibacterial and anti-inflammatory activities, characterized in that, The process includes: Resuscitation and primary culture: Lactobacillus plantarum strain M3 was inoculated into MRS medium and cultured. Expanded culture: The bacterial culture after the initial culture was inoculated into fresh MRS medium for expanded culture; Obtaining fermentation supernatant: After expanding the bacterial culture, remove the bacterial cells and collect the supernatant, which is a mixture with antibacterial and anti-inflammatory activities derived from Lactobacillus plantarum.

2. The method for preparing a mixture of *Lactobacillus plantarum*-derived bacteria with antibacterial and anti-inflammatory activity according to claim 1, characterized in that, The culture conditions for the resuscitation and primary culture were 12-16 hours in an anaerobic environment at 37 ℃.

3. The method for preparing a mixture of *Lactobacillus plantarum*-derived bacteria with antibacterial and anti-inflammatory activity according to claim 1, characterized in that, During the expanded culture process, the inoculum volume was 1% v / v, and the culture conditions were to continue culturing in an anaerobic environment at 37 ℃ for 12-16 hours.

4. The method for preparing a mixture with antibacterial and anti-inflammatory activity derived from *Lactobacillus plantarum* according to claim 1, characterized in that, The centrifugation parameters for obtaining the fermentation supernatant are 4000-5000 rpm for 10-15 minutes.

5. A mixture of *Lactobacillus plantarum*-derived bacteria with antibacterial and anti-inflammatory activity prepared by the method described in any one of claims 1-4, characterized in that... Its core active ingredients include: 5'-methylthioadenosine, 3-hydroxymyristic acid, tranexamic acid, indole-3-lactic acid, 3-phenyllactic acid, ((R)-1-carboxyethyl)-L-phenylalanine hydrochloride, L-tyrosyl-L-proline, isopentenyl phosphate, 6-aminododecanoic acid, N-acetylthreonine, 2-methyl-3-oxopropionic acid, 2,4-dimethylquinoline, and N-acetylalanine.

6. The mixture with antibacterial and anti-inflammatory activity derived from *Lactobacillus plantarum* according to claim 5, characterized in that, Its core active ingredients also include various polypeptides with different sequences.

7. The mixture with antibacterial and anti-inflammatory activity derived from *Lactobacillus plantarum* according to claim 6, characterized in that, The various polypeptides with different sequences include polypeptides with sequences as shown in SEQ ID NO.1~15.

8. The use of a mixture of Lactobacillus plantarum-derived substances with antibacterial and anti-inflammatory activity as described in claim 5 or 6 in the preparation of antimicrobial drugs, food additives or pesticides.

9. The application according to claim 7, characterized in that, The antibacterial drug is a drug that is effective against Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Aspergillus fumigatus, or Basilella marneffei.

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