Leuconostoc mesenteroides and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to leuconostoc mesenteroides and application thereof. The Leuconostoc mesenteroides disclosed by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the Leuconostoc mesenteroides is CCTCC (China Center for Type Culture Collection) NO: M2024452. The fermentation product of the leuconostoc mesenteroides has the effect of inhibiting propionibacterium acnes, can effectively recover skin functions, improve skin micro-ecology and remove whelks, and components in the fermentation product of the leuconostoc mesenteroides are all natural components, are low in irritation and are not prone to causing side effects such as skin allergy.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Leuconostoc mesenteroides and applications thereof. Background Art

[0002] Acne is a common chronic inflammatory skin disease that primarily occurs on the cheeks, forehead, and jaw, and can also extend to the trunk, such as the chest, back, and shoulder blades. It manifests as comedones, papules, pustules, nodules, cysts, and scars, accompanied by seborrhea. The most susceptible population is adolescent boys and girls. The causes of acne are complex, including inflammatory reactions triggered by Propionibacterium acnes. The chemical ingredients in existing acne-removing products can also have side effects. For example, some organic acids can cause skin allergies and other side effects, failing to meet consumer demand. Summary of the Invention

[0003] In view of the shortcomings of the existing technology, the present invention aims to provide a strain of Leuconostoc mesenteroides and applications thereof.

[0004] The invention provides a strain of Leuconostoc mesenteroides. The Leuconostoc mesenteroides is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M2024452.

[0005] Another object of the present invention is to provide the use of the above-mentioned Leuconostoc mesenteroides fermentation product in inhibiting Propionibacterium acnes.

[0006] Furthermore, the application method is: contacting the fermentation product of Leuconostoc mesenteroides with Propionibacterium acnes.

[0007] Furthermore, the preparation method of the Leuconostoc mesenteroides fermentation product comprises: inoculating Leuconostoc mesenteroides into a fermentation medium containing protein powder and fermenting.

[0008] Furthermore, the protein powder is skim milk powder, whey protein or milk protein.

[0009] Furthermore, the fermentation medium also includes isomaltooligosaccharide, glucose, medium chain triglyceride and water.

[0010] Furthermore, the fermentation medium comprises, by weight, 1-5 parts of protein powder, 1-3 parts of isomaltooligosaccharide, 1-3 parts of glucose, 1-3 parts of medium-chain triglycerides and 100 parts of water.

[0011] Furthermore, the preparation method of the fermentation product of Leuconostoc mesenteroides is as follows: inoculating Leuconostoc mesenteroides into the fermentation medium, and adding Leuconostoc mesenteroides in an amount of 10 7 -109 CFU / mL, ferment at 25-37℃ for 24-48h, and vacuum freeze-dry after fermentation.

[0012] The beneficial effects of the present invention are:

[0013] The fermentation product of Leuconostoc mesenteroides of the present invention has the effect of inhibiting Propionibacterium acnes, can effectively restore skin function, improve skin microecology, and eliminate acne. Moreover, the ingredients in the fermentation product of Leuconostoc mesenteroides are all natural ingredients, low in irritation, and not likely to cause side effects such as skin allergies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention is attached Figure 4 width,

[0015] Figure 1 This is a macroscopic morphology of Leuconostoc mesenteroides;

[0016] Figure 2 This is a microscopic morphology of Leuconostoc mesenteroides;

[0017] Figure 3 This is a trend diagram of the expression levels of tryptophan metabolites in the fermentation product of Example 2;

[0018] Figure 4 This is a mean analysis chart of the effect of the fermentation product from Example 2 on neutrophils in specific areas of zebrafish. DETAILED DESCRIPTION

[0019] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0020] Example 1

[0021] Isolation of Leuconostoc mesenteroides

[0022] Take 100 μL of breast milk, add it to TSA medium, culture it at 37°C for 18 h, and pick single colonies.

[0023] Identification of Leuconostoc mesenteroides

[0024] Morphological identification

[0025] MRS medium, cultured at 37℃ for 24h, the macroscopic morphology was as Figure 1 As shown, the microscopic morphology Figure 2 shown.

[0026] from Figure 1 It can be seen that the colonies are white, round, with a moist surface, opaque and neat edges.

[0027] from Figure 2 It can be seen that the bacteria are ellipsoidal, 0.4-0.6μm×0.6-0.9μm, arranged singly, in pairs or in chains, and are Gram-positive.

[0028] Molecular biology identification

[0029] 1. Extraction of bacterial genomic DNA

[0030] Use 2 mL centrifuge tube to collect 1.0 × 10 9 Centrifuge the bacterial culture (OD600 of 1 mL of bacterial culture is 1-1.5) at 12,000 × g for 30 seconds, discard the supernatant, and resuspend the pellet in 150 μL of Buffer S containing RNase A.

[0031] Add 20 μL of lysozyme stock solution, mix well, and let stand at room temperature for 5 min;

[0032] Add 30 μL of 0.25 M EDTA (pH 8.0), mix well, and incubate on ice for 5 min;

[0033] Add 450 μL Buffer GA, vortex for 15 seconds, and incubate in a 65°C water bath for 10 minutes.

[0034] Add 400 μL of Buffer GB and 1 mL of Buffer DV (pre-cooled at 4°C), mix vigorously, and centrifuge at 12,000 × g for 2 min;

[0035] Discard the upper phase as much as possible, retain the interphase precipitate and lower phase, add 1 mL of 4°C pre-cooled Buffer DV, mix vigorously, and centrifuge at 12,000 × g for 2 min;

[0036] Discard the upper phase and transfer the lower phase to a filter (place the filter in a 2 mL centrifuge tube). Centrifuge at 12,000 × g for 1 min.

[0037] Discard the filter, add 400 μL of Buffer BV to the filtrate, and mix well;

[0038] Place the preparation tube in a 2 mL centrifuge tube, transfer the mixed solution from the previous step into the preparation tube, and centrifuge at 12000 × g for 1 min;

[0039] Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 500 μL Buffer W1, and centrifuge at 12,000 × g for 1 min.

[0040] Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL Buffer W2, and centrifuge at 12,000 × g for 1 min.

[0041] Wash once more with 700 μL Buffer W2 using the same method;

[0042] Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12000 × g for 1 min;

[0043] Place the prepared tube in another clean 1.5 mL centrifuge tube, add 100-200 μL of Eluent or deionized water to the center of the silica membrane, let it stand at room temperature for 1 minute, and centrifuge at 12,000 × g for 1 minute to elute the DNA.

[0044] 2. PCR amplification of bacterial genome

[0045] Table 1 Primer design

[0046] Primer name sequence 27F 5-AGAGTTTGATCCTGGCTCAG-3 1492R 5-GGTTACCTTGTTACGACTT-3

[0047] PCR amplification reaction system

[0048] Add the following ingredients to a 0.2 mL centrifuge tube:

[0049] Table 2 PCR amplification reaction system

[0050] Reagents volume Genomic DNA (20 ng / μL) 1.0μL <![CDATA[10×Buffer (containing 2.5 mM Mg 2+ )]]> 5.0μL Taq polymerase (5 U / μL) 1.0 μL dNTP (10mM) 1.0μL 27F primer (10 μM) 1.5 μL 1492R primer (10 μM) 1.5 μL <![CDATA[ddH2O]]> 39.0μL Total volume 50.0μL

[0051] Mix by flicking gently, centrifuge briefly to collect the droplets on the tube wall to the bottom of the tube, and perform PCR reaction on a PCR amplifier. After the reaction is completed, take 3 μL of PCR product for 1% agarose gel electrophoresis to confirm the PCR amplification fragment.

[0052] 3. Recovery of PCR Products

[0053] The PCR product was recovered using the AxyPrep DNA gel recovery kit. The specific operation was carried out according to the kit instructions.

[0054] 4. Sequence determination and analysis

[0055] The purified PCR products of each bacterial strain were taken and DNA sequencing was performed using a sequencer ABI3730-XL.

[0056] After sequencing by China Food and Fermentation Industry Research Institute Co., Ltd., the 16S rDNA of Leuconostoc mesenteroides is shown as SEQ ID NO. 1:

[0057]

[0058] 5. Sequence analysis

[0059] The spliced ​​sequence file was compared with the data in the NCBI 16S database using the NCBI Blast program, and the result was Leuconostoc mesenteroides.

[0060] The Leuconostoc mesenteroides subsp. cremoris provided by the present invention has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, on March 11, 2024, with the deposit number being CCTCC NO: M2024452.

[0061] Example 2

[0062] S1. Leuconostoc mesenteroides was added to TSB medium, cultured at 37°C for 24 hours, and centrifuged to obtain a precipitate. 4 parts of whey protein, 3 parts of oligosaccharides, 2 parts of glucose, and 2 parts of medium-chain triglycerides were added to 100 parts of water by weight to obtain a fermentation medium. The precipitate was added to the fermentation medium. The amount of Leuconostoc mesenteroides added was 10 8 CFU / mL, fermented at 37°C for 48 h to obtain the fermentation product of Leuconostoc mesenteroides;

[0063] S2. vacuum freeze-drying the fermentation product of Leuconostoc mesenteroides to obtain fermentation product powder.

[0064] Example 3

[0065] S1. Leuconostoc mesenteroides was added to TSB medium, cultured at 37°C for 24 hours, and centrifuged to obtain a precipitate. 4 parts of skim milk powder, 3 parts of oligosaccharides, 2 parts of glucose, and 2 parts of medium-chain triglycerides were added to 100 parts of water by weight to obtain a fermentation medium. The precipitate was added to the fermentation medium. The amount of Leuconostoc mesenteroides added was 10 8 CFU / mL, fermented at 37°C for 48 h to obtain the fermentation product of Leuconostoc mesenteroides;

[0066] S2. vacuum freeze-drying the fermentation product of Leuconostoc mesenteroides to obtain fermentation product powder.

[0067] Example 4

[0068] S1. Add Leuconostoc mesenteroides to TSB medium, culture at 37°C for 24 hours, centrifuge to obtain a precipitate, add 4 parts of milk protein, 3 parts of oligosaccharides, 2 parts of glucose and 2 parts of medium-chain triglycerides to 100 parts of water by weight to obtain a fermentation medium, add the precipitate to the fermentation medium, and add 10 parts of Leuconostoc mesenteroides to the fermentation medium. 8 CFU / mL, fermented at 37°C for 48 h to obtain the fermentation product of Leuconostoc mesenteroides;

[0069] S2. vacuum freeze-drying the fermentation product of Leuconostoc mesenteroides to obtain fermentation product powder.

[0070] The pH before and after fermentation of Example 2, Example 3, and Example 4 were recorded. The results are shown in Table 3:

[0071] Table 3

[0072] Example 2 Example 3 Example 4 pH before fermentation 5.4 5.4 5.4 pH after fermentation 4.1 4.4 4.8

[0073] From Examples 2 to 4 and Table 3, it can be seen that the pH after fermentation in Example 2 is the lowest. The lower the pH, the more complete the fermentation. Therefore, the fermentation degree of Example 2 is the highest.

[0074] The total number of colonies was 10 5 CFU, 10 6 CFU and 10 7 CFU of Propionibacterium acnes were evenly spread on a fortified Clostridium culture medium plate, and then a drug-sensitive paper disc was placed on the coated Propionibacterium acnes. The fermentation product powders prepared in Examples 2, 3, and 4 were dissolved in 10 mL of sterile water, and 20 μL of each was dripped onto the drug-sensitive paper disc. The results are shown in Table 4:

[0075] Table 4

[0076]

[0077] Combining Examples 2 to 4 and Table 4, it can be seen that Example 2 has the largest inhibition zone, and therefore, Example 2 has the best effect in inhibiting Propionibacterium acnes.

[0078] 100 mg of the fermentation product powder prepared in Example 2 was dissolved in 500 μL of a solvent having an acetonitrile / water volume ratio of 1:1. Liquid chromatography and mass spectrometry analysis were performed using a mass spectrometer. Mobile phase A and mobile phase B were 25 mM ammonium formate and 0.1% acetonitrile formic acid solution, respectively. At the same time, multiple reaction monitoring mode was used to selectively and quantitatively monitor tryptophan metabolites in the fermentation product. Peak areas and retention times were extracted using Multiquant 3.0.2 software. Standard curves of 13 tryptophan metabolites at different concentrations were established for quantification. The results are shown in FIG. Figure 3 , combined with Example 2 and Figure 3 Compared with whey protein, the fermentation product powder prepared in Example 2 showed significant differences in the expression levels of tryptophan metabolites produced in Example 2, such as a significant increase in the content of indole-3-lactic acid (ILA) and indole-3-acetic acid (IAA) that can reduce inflammation.

[0079] The zebrafish skin reacts similarly to the human skin in response to inflammatory stimulation. After the skin is subjected to inflammatory stimulation, neutrophils can be induced to gather on the skin surface. A zebrafish inflammation induction model was constructed using copper sulfate. After treatment with the test substance, the number of neutrophils on the skin surface was measured to evaluate whether the cosmetics had soothing and anti-inflammatory effects. Model group: copper sulfate (modeling drug), positive drug group: dexamethasone, sample group: fermentation product powder prepared in Example 2, the results are shown in Figure 4 , combined with Example 2 and Figure 4 Compared with the model group, the inhibition rate of neutrophil aggregation in the specific area of ​​zebrafish in Example 2 was 84.35%, which was significantly different (p<0.05).

Claims

1. A strain of Leuconostoc mesenteroides, characterized by: The Leuconostoc mesenteroides is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2024452.

2. Use of the Leuconostoc mesenteroides fermentation product according to claim 1 in inhibiting Propionibacterium acnes.

3. The use according to claim 2, characterized in that: The application method comprises the following steps: contacting the fermentation product of Leuconostoc mesenteroides with Propionibacterium acnes.

4. The use according to claim 2, characterized in that: The preparation method of the Leuconostoc mesenteroides fermentation product comprises the following steps: inoculating Leuconostoc mesenteroides into a fermentation medium containing protein powder and fermenting the culture medium.

5. The use according to claim 4, characterized in that: The protein powder is skimmed milk powder, whey protein or milk protein.

6. The use according to claim 4, characterized in that: The fermentation medium also includes isomaltooligosaccharide, glucose, medium chain triglyceride and water.

7. The use according to claim 4, characterized in that: The fermentation medium comprises, by weight, 1-5 parts of protein powder, 1-3 parts of isomaltooligosaccharide, 1-3 parts of glucose, 1-3 parts of medium-chain triglycerides and 100 parts of water.

8. The use according to claim 2, characterized in that: The preparation method of the fermentation product of Leuconostoc mesenteroides is as follows: inoculating Leuconostoc mesenteroides into a fermentation medium, and adding Leuconostoc mesenteroides in an amount of 10 7 -10 9 CFU / mL, ferment at 25-37℃ for 24-48h, and vacuum freeze-dry after fermentation.

Images