Lactobacillus rhamnosus strain with function of secreting 5-HT and application of lactobacillus rhamnosus strain

Abstract

The invention provides a lactobacillus rhamnosus strain with a function of secreting 5-HT (5-hydroxytoluene) and application of the lactobacillus rhamnosus strain, and belongs to the technical field of functional microorganisms. The lactobacillus rhamnosus strain A22004 provided by the invention has good artificial gastrointestinal fluid and cholate tolerance, and can be stably colonized in intestinal tracts. The strain A22004 not only has a function of secreting 5-HT, but also has an antibacterial effect, and the effect of the strain A22004 is superior to that of a commercial strain LGG.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and specifically relates to a Lacticaseibacillus rhamnosus strain with the function of secreting 5-HT and its application. Background Art

[0002] In recent years, the role of gut microbiota in various diseases has become increasingly prominent. A large number of studies on the "gut-brain" axis have shown that probiotics can affect the expression of neurofactors in the brain by regulating the abundance of indigenous flora in the gut and the content of its related metabolic secretions, thereby affecting central nervous system diseases. Developing probiotics with diverse functions and good adaptability is of great significance to human health.

[0003] Approximately 90% of serotonin (also known as 5-hydroxytryptamine, 5-HT) is produced by enterochromaffin (EC) cells in the gut. In EC cells, the synthesis of 5-hydroxytryptamine begins with the conversion of dietary tryptophan to 5-hydroxytryptophan (5-HTP), which is catalyzed by the rate-limiting enzyme tryptophan hydroxylase (Tph). There are two isoforms of the Tph enzyme, Tph1 is mainly present in EC cells, and Tph2 is mainly present in the brainstem and enteric neurons. It has been reported that studies on experimental models of intestinal inflammation have shown that inhibiting the production of 5-HT by Tph1 gene deletion or pharmacological manipulation can effectively improve inflammation. The patent with the publication number CN114717148A discloses a Lactobacillus paracasei strain CCFM1229, which can increase the concentrations of 5-HT and BDNF in the brains of mice and has the effect of relieving depressive mood.

[0004] Lacticaseibacillus rhamnosus belongs to Gram-positive bacteria. As a human symbiotic bacterium, it plays a role in preventing pathogen infection and gastrointestinal inflammation, and has a very broad application prospect. However, there is currently no relevant report on the function of Lacticaseibacillus rhamnosus in secreting 5-HT. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a Lacticaseibacillus rhamnosus strain A22004 with the function of secreting 5-HT.

[0006] The present invention provides a Lacticaseibacillus rhamnosus strain A22004, with the preservation number of GDMCC No: 65645.

[0007] The present invention provides a derivative product comprising the Lacticaseibacillus rhamnosus strain A22004 or the metabolite of the Lacticaseibacillus rhamnosus strain A22004.

[0008] Preferably, it includes at least one of the following: probiotic agent, drug, ferment, food, and health product.

[0009] The present invention provides the use of the Lactobacillus rhamnosus strain A22004 in the production of serotonin.

[0010] The present invention provides the use of the Lactobacillus rhamnosus strain A22004 in the preparation of a product rich in serotonin.

[0011] Preferably, the product includes at least one of the following categories: health products, foods, food additives, and pharmaceuticals.

[0012] The present invention provides the use of the Lactobacillus rhamnosus strain A22004 in the preparation of an antibacterial product.

[0013] Preferably, the bacteria in the antibacterial include Escherichia.

[0014] Preferably, the product includes drugs and / or bacteriostatic daily necessities.

[0015] Preferably, the drugs include at least one of the following dosage forms: powder, tablet, oral liquid, capsule, and drop;

[0016] The bacteriostatic daily necessities include at least one of the following: disinfectants, washing products, oral care products, cosmetics, and washing and care products.

[0017] The present invention provides a Lactobacillus rhamnosus strain A22004 with a preservation number of GDMCC No: 65645. The strain A22004 was isolated from the feces of healthy centenarians in Guangxi, has good tolerance to artificial gastrointestinal fluid and bile salts, and can stably colonize in the intestine to exert biological functions. The strain A22004 has good inhibitory ability against Escherichia coli, and the effect is better than that of the commercial strain LGG. The strain A22004 also has the function of autocrine 5-HT, and the effect is better than that of the commercial strain LGG. It can be seen that the Lactobacillus rhamnosus strain A22004 provided by the present invention has the characteristic of stably colonizing in the intestinal tract of the body, and has good antibacterial function, providing a new idea for the development of antibacterial drugs. At the same time, the strain A22004 has the characteristic of secreting 5-HT, providing a new way for the development of related products rich in 5-HT. Description of the Drawings

[0018] Figure 1 It is a Gram staining result diagram of the Lactobacillus rhamnosus strain A22004;

[0019] Figure 2 It is the detection result of the tolerance of the Lactobacillus rhamnosus strain A22004 to artificial gastrointestinal fluid;

[0020] Figure 3 It is the detection result of the tolerance of the Lactobacillus rhamnosus strain A22004 to bovine bile salts;

[0021] Figure 4 is the 5-HT standard working curve;

[0022] Figure 5 is the detection result of 5-HT secreted by Lactobacillus rhamnosus strain A22004 itself;

[0023] Figure 6 is the detection result of 5-HT secreted by the control strain LGG.

[0024] Biological material preservation information

[0025] Lacticaseibacillus rhamnosus strain A22004 was deposited in the Guangdong Provincial Microbial Culture Collection Center, located on the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, abbreviated as GDMCC, with the deposit number GDMCC No: 65645, and the deposit date was December 16, 2024. Detailed implementation manners

[0026] The present invention provides a Lactobacillus rhamnosus strain A22004 with the deposit number GDMCC No: 65645.

[0027] In the present invention, the strain A22004 was isolated and purified from the feces of healthy centenarians in Guangxi. The morphological characteristics of the strain A22004 on the MRS plate are as follows: the colonies are round, white, convex, with a smooth and moist surface and neat edges. The strain A22004 was identified by 16S rDNA, and the obtained 16S rDNA (SEQ ID NO: 1) was confirmed to be Lactobacillus rhamnosus by sequence alignment.

[0028] In the present invention, for the culturing method of the Lactobacillus rhamnosus strain A22004, it is preferred to inoculate the activated Lactobacillus rhamnosus strain A22004 into an MRS liquid medium for anaerobic culture to obtain a bacterial liquid of the Lactobacillus rhamnosus strain A22004. The activation method of the Lactobacillus rhamnosus strain A22004 preferably includes the following steps: The Lactobacillus rhamnosus strain A22004 preserved in glycerol is streaked on an MRS plate for culturing to obtain single colonies. The temperature of the streaking culture is preferably 36 - 38°C, and can be 37°C. The time of the streaking culture is preferably 22 - 26 h, and can be 24 h. The streaking culture is preferably carried out under anaerobic conditions, preferably in an anaerobic workstation. The temperature of the anaerobic culture is preferably 36 - 38°C, and can be 37°C. The time of the anaerobic culture is preferably 7 - 9 h, and can be 8 h. During the anaerobic culture, the inoculation amount of the activated Lactobacillus rhamnosus strain A22004 is preferably 0.5% - 1%, and can be 0.8%. The present invention does not impose special restrictions on the formulations of the MRS liquid medium and the MRS plate, and the well-known MRS liquid medium and MRS plate formulations in the art can be used. For example, the MRS liquid medium formulation is 20 g / L glucose, 10 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 0.1% Tween - 80, and the pH value is 6.6 - 6.8. The difference between the MRS plate and the MRS liquid medium is only that agar with a mass percentage of 1.5% - 2% is additionally added.

[0029] The present invention provides a derivative product comprising the Lactobacillus rhamnosus strain A22004 or the metabolite of the Lactobacillus rhamnosus strain A22004.

[0030] In the present invention, the derivative product preferably includes at least one of the following: probiotic agents, drugs, fermenting agents, foods, and health products. The morphology of the Lactobacillus rhamnosus strain A22004 preferably includes the bacterial cells, spores, culture broth, fermentation broth, or bacterial suspension of the Lactobacillus rhamnosus strain A22004.

[0031] The present invention provides the application of the Lactobacillus rhamnosus strain A22004 in the production of 5 - hydroxytryptamine.

[0032] In the present invention, the method for the Lactobacillus rhamnosus strain A22004 to produce 5 - hydroxytryptamine preferably includes the following steps:

[0033] The bacterial liquid of the Lactobacillus rhamnosus strain A22004 prepared in the above technical solution is subjected to solid - liquid separation, the solid - phase bacterial cells are collected, the bacterial cells are washed and then resuspended, cultured statically, the liquid phase is collected, and 5 - hydroxytryptamine is separated and purified.

[0034] In the present invention, the solution for washing and resuspension is preferably HBSS solution. The temperature for static culture is preferably 36 - 38 °C, and can be 37 °C. The time for static culture is preferably 1.5 - 2.5 h, and can be 2 h. The method for solid-liquid separation is preferably centrifugation. The present invention has no special limitation on the method for separating and purifying 5-hydroxytryptamine, and any method well-known in the art for separating and purifying 5-hydroxytryptamine can be used.

[0035] In the present invention, the liquid phase in the above technical solution was determined by 5-hydroxytryptamine HPLC method. The results showed that the 5-HT secreted by Lactobacillus rhamnosus A22004 itself was completely separated within 13.487 min. 9 For the strain A22004 with 10 CFU / mL, the yield of self-secreted 5-HT was 6.419 μg / mL, while the yield of 5-HT of the commercial strain LGG at the same concentration was 6.073 μg / mL. It can be seen that the Lactobacillus rhamnosus A22004 provided by the present invention has the function of self-secreting 5-HT and is slightly stronger than the commercial strain LGG.

[0036] In view of the characteristic that the strain A22004 can self-secrete 5-hydroxytryptamine, the present invention provides the application of the Lactobacillus rhamnosus strain A22004 in the preparation of products rich in 5-hydroxytryptamine.

[0037] In the present invention, the method for preparing a product rich in 5-hydroxytryptamine using the Lactobacillus rhamnosus strain A22004 preferably involves fermenting with the Lactobacillus rhamnosus strain A22004, and the prepared fermented product is rich in 5-hydroxytryptamine. The products include health products, foods, food additives, drugs, etc.

[0038] The present invention provides the application of the Lactobacillus rhamnosus strain A22004 in the preparation of antibacterial products.

[0039] In the present invention, the bacteria in the antibacterial preferably include Escherichia. In the examples of the present invention, Escherichia coli O157:H7 and Escherichia coli were used as representatives of pathogenic bacteria to illustrate the antibacterial characteristics of the strain A22004. The results showed that the Lactobacillus rhamnosus A22004 could significantly inhibit the growth of pathogenic bacteria, and its inhibitory effect was better than that of the commercial strain LGG.

[0040] In the present invention, the product preferably includes drugs and / or antibacterial daily necessities. The dosage form of the drug preferably includes at least one of the following: tablets, powders, oral liquids, capsules, drops, etc. The antibacterial daily necessities include disinfectants, washing products, oral care products, cosmetics, hair and skin care products, etc. The viable bacteria concentration of Lactobacillus rhamnosus A22004 in the product is not less than 2.5×108 CFU / mL, which can be 5×10 8 CFU / mL, 8×10 8 CFU / mL, 10×10 8 CFU / mL, 20×10 8 CFU / mL, 50×10 8 CFU / mL, 1×10 10 CFU / mL, 1×1011CFU / mL.

[0041] The following is a detailed description of a Lactobacillus rhamnosus strain with the function of secreting 5-HT and its application provided by the present invention in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] Isolation and identification of Lactobacillus rhamnosus strain A22004

[0044] The intestinal microbial flora samples of centenarians were diluted by concentration gradient and spread on MRS plates, and cultured in a biochemical incubator at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium, and cultured on a shaker at 37°C for 24 h. The bacterial liquid was picked and streaked on MRS plates, and cultured to obtain single colonies. Then single colonies were picked and streaked again, and cultured to obtain single colonies, which were the pure isolated lactobacilli obtained. It was determined to be a Gram-positive bacterium by Gram staining, as Figure 1 shown. Single colonies of the pure isolated lactobacilli were picked for Gram staining, specific detection and 16S rDNA sequencing, and the similarity with Lactobacillus rhamnosus 6551 was 99.73%. Therefore, the isolated strain was Lactobacillus rhamnosus, named A22004. The 16S rDNA sequence of the strain was:

[0045] CGACGGGGGGCCTATACTGCAGTCGAACGAGTTCTGATTATTGAAAGGTGC

[0046] TTGCATCTTGATTTAATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTG

[0047] GGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACC

[0048] GCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTAT

[0049] CGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTC

[0050] ACCAAGGCAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGG

[0051] GACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTC

[0052] CACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGC

[0053] TTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAAC

[0054] TGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGC

[0055] CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGC

[0056] GTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTT

[0057] AACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGAC

[0058] AGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACC

[0059] AGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGC

[0060] ATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATG

[0061] AATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATT

[0062] AAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATT

[0063] GACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG

[0064] CGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGT

[0065] TTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGT

[0066] GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAG

[0067] TTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGG

[0068] AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC

[0069] ACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAG

[0070] CTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTA

[0071] CACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATA

[0072] CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACA

[0073] CCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGGGACCAAATT(SEQ ID NO:1)。

[0074] Example 2

[0075] Detection experiment on the characteristics of Lactobacillus rhamnosus strain A22004 against artificial gastrointestinal fluids

[0076] Pick the monoclonal colonies of Lactobacillus rhamnosus A22004 and commercial Lactobacillus rhamnosus GG, and inoculate them into MRS liquid medium respectively, and culture overnight in a shaker at 37°C. According to an inoculation amount of 10%, inoculate the bacterial liquid into artificial gastric juice with a pH of 2.5 (formula: 10 g of pepsin, 16.4 ml of hydrochloric acid, and make up to 100 ml with distilled water), set 3 time points (0 h, 1 h, 3 h), take 100 μl each and spread it on an MRS petri dish, and culture in a biochemical incubator at 37°C for 48 h, then count the number of single colonies; take the bacterial liquid after being treated with artificial gastric juice for 3 h, according to an inoculation amount of 10%, inoculate it into artificial intestinal juice with a pH of 8 (formula: 10 g of pancreatin, 6.8 g of potassium dihydrogen phosphate, make up to 1000 ml with distilled water, and adjust the pH value with NaOH), set 4 time points (0 h, 2 h, 4 h, 7 h), take 100 μl each and spread it on an MRS petri dish, and culture in a biochemical incubator at 37°C for 48 h, then count the number of single colonies, and calculate the survival rate according to formula I:

[0077] Survival rate (%) = (number of single colonies at different time points / number of single colonies at 0 h) × 100% Formula I.

[0078] The results are as Figure 2 shown that the survival rates of the strain A22004 at 1 h and 3 h in artificial gastric juice are similar to those of the commercial strain GG. The survival rate of the strain A22004 at 4 h in artificial intestinal juice with a pH of 8 is significantly better than that of the commercial strain GG, indicating that the strain A22004 can successfully pass through the stimulation of gastrointestinal fluids and reach the intestine to play a role.

[0079] Example 3

[0080] Detection experiment on the tolerance of Lactobacillus rhamnosus strain A22004 to bile salts

[0081] Prepare a bacterial suspension of the strain A22004 according to the method of Example 2, take 1 mL each and add it to MRS medium containing 0.3 g / L and 0.5 g / L bile salts, and culture anaerobically at 37°C for 18 h. Take 100 μL of the culture broth at 0 h, 2 h, and 24 h respectively for gradient dilution and spreading, and culture under anaerobic conditions at 37°C for 1 - 2 days, and count the viable bacteria. Calculate the bile salt tolerance survival rate of the strain according to formula II:

[0082] Bile salt tolerance survival rate = (number of viable bacteria at N h / number of viable bacteria at 0 h) × 100% Formula II.

[0083] Among them, N represents 2 h or 24 h.

[0084] The results are as Figure 3As shown, the survival rate of the strain A22004 still exceeded 100% after 24 hours in the presence of 0.5 g / L of bile salts, and its tolerance ability was comparable to that of the commercial strain LGG. This indicates that the strain A22004 has excellent bile salt tolerance ability.

[0085] Example 4

[0086] Function of Lactobacillus rhamnosus strain A22004 in inhibiting pathogenic bacteria

[0087] The pathogenic bacteria Escherichia coli O157:H7 (ATCC 35150) and Escherichia coli (ATCC 25922) in the logarithmic growth phase were spread on an LB plate, and Oxford cups were placed. The activated strain A22004 was prepared into a bacterial suspension, and the OD 600 was adjusted to 0.5. 200 μl was taken and inoculated into the Oxford cup, and incubated overnight to observe its effect on the pathogenic bacteria.

[0088] The results are shown in Table 1 below.

[0089] Table 1 Bacteriostatic effect of Lactobacillus rhamnosus strain A22004

[0090]

[0091] The results showed that the Lactobacillus rhamnosus strain A22004 could significantly inhibit the growth of pathogenic bacteria, and its inhibitory effect was better than that of the commercial strain LGG.

[0092] Example 5

[0093] Detection of 5-HT secreted by Lactobacillus rhamnosus strain A22004

[0094] Single colonies of the purified strain A22004 and the commercial strain LGG were picked and inoculated into MRS liquid medium, and cultured overnight in a shaker at 37°C. The cells were collected, centrifuged, and washed 3 times with HBSS (Hanks balanced salt solution); then the cells were resuspended with HBSS solution (formula shown in Table 2) and left standing in a biochemical incubator at 37°C for 2 h. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter head into a liquid phase bottle for standby.

[0095] Table 2 Formula of HBSS solution

[0096]

[0097]

[0098] Preparation of 5-HT standard solution: Dissolve 5-HT standard product in the dark with HBSS to concentrations of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, 6.0 μg / mL, and 7.5 μg / mL. Filter through a 0.22 μm filter head into a liquid phase bottle for standby.

[0099] Quantitatively detect 5-HT by high performance liquid chromatography (HPLC). The model of the HPLC chromatograph is Chromai Leaps. The chromatographic conditions are as follows: C18 reverse phase chromatographic column (200 mm × 4.6 mm, 5 μm), the mobile phase is a 0.02 mol / L potassium dihydrogen phosphate - methanol (volume ratio 25:2) solution. Perform detection according to the initial gradient program, the flow rate is 0.5 ml / min, ultraviolet detector, the detection wavelength is 275 nm, and quantitatively evaluate the content of 5-HT autocrine by strain A22004. Standard working curve: In the concentration range of 1.0 - 7.5 μg / mL, there is a good linear relationship between the peak area and the 5-HT concentration. The regression equation is shown in Formula III (see Figure 4 ).

[0100] The regression equation is y = 4.7793x - 0.3927, Formula III.

[0101] Among them, the correlation coefficient R 2 = 0.9959, y represents the peak area, and x represents the 5-HT concentration.

[0102] Figure 5 It shows that the 5-HT autocrine by the Lactobacillus rhamnosus strain A22004 of the present invention is completely separated within 13.487 min. The yield of 5-HT autocrine by the strain A22004 at 10 9 CFU / mL is 6.419 μg / mL. Figure 6 It shows that the commercial strain LGG at the same concentration is completely separated within 13.497 min, and the yield of 5-HT autocrine is 6.073 μg / mL. It can be seen that the Lactobacillus rhamnosus A22004 provided by the present invention has the function of autocrine 5-HT and the ability to secrete 5-HT is stronger than that of the commercial strain LGG.

[0103] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Lacticaseibacillus rhamnosus strain A22004, characterized in that: The deposit number is GDMCC No:65645.

2. A derivative product comprising the Lactobacillus rhamnosus strain A22004 of claim 1 or a metabolite of the Lactobacillus rhamnosus strain A22004.

3. The derivative product according to claim 2, characterized in that: Includes at least one of the following: probiotics, drugs, fermentation cultures, foods and health products.

4. Use of the Lactobacillus rhamnosus strain A22004 according to claim 1 in producing 5-hydroxytryptamine.

5. Use of the Lactobacillus rhamnosus strain A22004 according to claim 1 in preparing a product rich in 5-hydroxytryptamine.

6. The use according to claim 5, characterized in that: The products include at least one of the following categories: health products, food, food additives and medicines.

7. Use of the Lactobacillus rhamnosus strain A22004 according to claim 1 in the preparation of antibacterial products.

8. The use according to claim 7, characterized in that: The antibacterial agents include Escherichia.

9. The use according to claim 7 or 8, characterized in that: The products include medicines and / or antibacterial daily necessities.

10. The use according to claim 9, characterized in that: The drug comprises at least one of the following dosage forms: powder, tablet, oral solution, capsule and drops; The antibacterial daily necessities include at least one of the following: disinfectants, detergents, oral care products, cosmetics, and toiletries.

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