A strain of Weissella mesenteroides MbWp-25 and its products and applications

Abstract

The present invention discloses a strain of Enterobacteria mesenteroides MbWp‑25 and its products and applications. The Enterobacteria mesenteroides was deposited in the Guangdong Provincial Microbial Culture Collection Center on February 11, 2022, with a deposit number of GDMCC No: 62252. The Enterobacteria mesenteroides has good acid production ability and anti-bile salt effect, and thus also has good antibacterial properties, can inhibit pathogenic bacteria, promote host growth and development, and has extremely wide application value. Moreover, the bacteria also has an inflammatory treatment effect, and also has a good application effect in food fermentation. The freeze-dried powder product prepared by the bacteria is low in cost, high in storage survival rate, and simple in preparation process, and also has extremely high application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Weissella mesenteroides MbWp-25 and a product and application thereof. Background Art

[0002] Lactic acid bacteria (LAB) is a general term for bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. Lactic acid bacteria have a wide range of applications. According to the actual situation of the strain, it can be used as a fungus with auxiliary treatment of intestinal diseases, antibiotic substitution or food fermentation functions, and has been maturely applied to all walks of life. Related studies have shown that lactic acid bacteria can promote animal growth, regulate intestinal pH, maintain intestinal microecological balance, thereby improving gastrointestinal function, improving digestibility and biological efficacy, inhibiting the growth of putrefactive bacteria in the intestine, and improving the body's immunity. However, most of the lactic acid bacteria currently available on the market grow anaerobic, have certain requirements for the use environment, and the applicable species are single, which cannot meet the actual market demand. Therefore, the development of a new lactic acid bacteria strain with low requirements for use conditions is of great significance for fermentation and microbial application fields. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a strain of Weissella mesenteroides MbWp-25 and its products and applications. The Weissella mesenteroides MbWp-25 in the present invention has a good bile salt resistance effect and a strong acid production ability, can be widely used in various fermentation products, and has an extremely wide range of applications and practical value.

[0004] The first aspect of the present invention provides a strain of Weissella paramesenteroides, which is Weissella paramesenteroides MbWp-25. The Weissella paramesenteroides MbWp-25 was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on February 11, 2022, with a collection number of GDMCC No: 62252.

[0005] In the embodiment of the present invention, the Weissella paramesenteroides MbWp-25 is isolated and obtained by the inventors from mulberry wine naturally fermented for 6 to 18 months in a mulberry orchard in Lingnan, and has good acid and bile acid resistance.

[0006] In some embodiments of the present invention, the specific breeding method of the Weissella mesenteroides MbWp-25 is:

[0007] (1) Mulberry wine collected aseptically from Lingnan mulberry orchards and fermented for 6 to 18 months was diluted 10 times in a continuous gradient under anaerobic conditions. MRS medium (Lactobacillus selective medium pour plate) was used in an anaerobic incubator with an anaerobic gas production bag at 36.5-37.5°C for 24-72 h to isolate potential Lactobacillus bacteria.

[0008] (2) Pick up small transparent colonies with slightly raised circular outlines from the plate, and after Gram staining and microscopic examination, select Gram-positive bacteria (the bacteria are irregular short rods with round or slightly thin ends, arranged in pairs or short chains), and continue to separate and purify by streaking 2 to 3 times until a pure culture of Weissella mesenteroides is isolated.

[0009] (3) The pure culture of Weissella mesenolytica was cultured for 4-6 hours on an MRS plate adjusted to pH 2.0 and an MRS plate containing 0.9% bile salt to obtain the target Weissella mesenolytica.

[0010] (4) The target mesenteroides-like Weissella was identified and the mesenteroides-like Weissella MbWp-25 was obtained.

[0011] Weissella mesenteroides mainly comes from plants such as fruit wine and pickles, and is mainly used for the fermentation of plant products. In the embodiments of the present invention, the isolated Weissella mesenteroides has good acid production ability and thus has good antibacterial properties, can inhibit pathogens, and promote host growth and development, thus having certain potential application value in terms of antibiotic substitutes.

[0012] In the embodiment of the invention, the Weissella mesenteroides MbWp-25 isolated by the inventor has the following biological characteristics: Gram-positive bacteria, no flagella, no movement, no spore formation, facultative anaerobic; the bacterial body is irregular short rod-shaped, with round or slightly small ends, arranged in pairs or short chains, and its colony is smooth with complete edges and milky white. The optimal growth temperature is 36.5-37.5°C; the optimal pH value is 5.7-7.0.

[0013] According to the first aspect of the present invention, in some embodiments of the present invention, the nucleotide sequence of the 16S rRNA gene of Weissella mesenteroides is shown as SEQ ID NO:1.

[0014] In the examples of the invention, the inventors constructed a phylogenetic tree for Weissella mesenteroides MbWp-25 and found that it had certain homology differences with Weissella mesenteroides found in the prior art.

[0015] The second aspect of the present invention provides a product containing the Weissella mesenteroides described in the first aspect of the present invention, wherein the dosage form of the product includes solid powder, liquid bacterial agent, granular inoculated bacterial agent, frozen bacterial slurry, and agar carrier bacterial agent.

[0016] Of course, those skilled in the art can also reasonably adjust the dosage form selection according to actual use requirements to achieve a higher storage survival rate or use effect.

[0017] According to the second aspect of the present invention, in some embodiments of the present invention, the product is Weissella mesenteroides-encapsulated freeze-dried powder.

[0018] In the embodiment of the present invention, the inventors have effectively improved the storage survival rate by adopting the freeze-dried powder method, and the effective live bacteria count has increased by nearly 10,000 times compared with normal storage. In the embodiment of the present invention, the live bacteria count of the embedded freeze-dried powder of Enterobacteriaceae is greater than 100 billion CFU / g, and the live bacteria survival rate is more than 60% after storage at 25°C for 6 months. Moreover, the preparation method in the embodiment of the present invention is to carry out embedding freeze-drying treatment under a non-strict anaerobic environment, and to microencapsulate and embed the Enterobacteriaceae in steps using a variety of inexpensive and easily available antioxidant ingredients before freeze-drying treatment, so as to effectively prevent the interaction between oxygen and the cell membrane system of Enterobacteriaceae, thereby preventing the behavior of damaging DNA synthesis, and being able to remove free radicals generated before the strain is freeze-dried, thereby preventing oxidative damage during the drying process, and making the Enterobacteriaceae less likely to be inactivated under normal temperature storage conditions.

[0019] The above method is not limited to freeze-drying the Weissella mesenteroides MbWp-25 of the present invention, but is also applicable to freeze-drying other facultative anaerobic bacteria to obtain the same or similar effects.

[0020] In some embodiments of the present invention, the raw materials for preparing the Weissella mesenteroides embedded freeze-dried powder include the Weissella mesenteroides described in the first aspect of the present invention, sodium alginate, calcium carbonate, milk powder, soybean oil, acetate and adjuvants.

[0021] Of course, those skilled in the art can reasonably add other materials for embedding or improving the embedding effect according to actual use requirements, including but not limited to sodium alginate, calcium carbonate, milk powder, and soybean oil.

[0022] In some embodiments of the present invention, glacial acetic acid or Tween is added to the soybean oil.

[0023] In some embodiments of the present invention, the soybean oil is soybean oil containing 0.3-0.7% (v / v) glacial acetic acid.

[0024] In some embodiments of the present invention, the soybean oil is soybean oil containing 0.5% (v / v) glacial acetic acid.

[0025] In some embodiments of the present invention, the soybean oil is soybean oil containing 1-2% (v / v) Tween.

[0026] In some embodiments of the present invention, the soybean oil is soybean oil containing 1.5% (v / v) Tween.

[0027] In some embodiments of the present invention, the sodium alginate, calcium carbonate and milk powder are all aqueous solutions prepared by mixing them with water.

[0028] In some embodiments of the present invention, the mass ratio of sodium alginate aqueous solution, calcium carbonate aqueous solution, Weissella mesenteroides bacterial solution, acetate aqueous solution, milk powder aqueous solution, and soybean oil containing glacial acetic acid is 10-15: 3-6: 1-4: 200-230: 350-370: 390-420.

[0029] In some embodiments of the present invention, the mass ratio of the sodium alginate aqueous solution, the calcium carbonate aqueous solution, the Weissella mesenteroides bacterial solution, the acetate aqueous solution, the milk powder aqueous solution, and the soybean oil containing glacial acetic acid is 13:5:3:214:366:400.

[0030] In some embodiments of the present invention, the number of viable bacteria in the Weissella mesenteroides bacterial solution is 200 to 300 billion CFU / g.

[0031] In some embodiments of the present invention, the number of viable bacteria in the Weissella mesenteroides bacterial solution is 250 billion CFU / g.

[0032] In some embodiments of the present invention, the mass concentration of the sodium alginate aqueous solution is 3-8 wt %.

[0033] In some embodiments of the present invention, the mass concentration of the sodium alginate aqueous solution is 5 wt %.

[0034] In some embodiments of the present invention, the mass concentration of the calcium carbonate aqueous solution is (45-55) wt%.

[0035] In some embodiments of the present invention, the mass concentration of the calcium carbonate aqueous solution is 50 wt %.

[0036] In some embodiments of the present invention, the mass concentration of the acetate aqueous solution is (40-60) wt%.

[0037] In some embodiments of the present invention, the mass concentration of the acetate aqueous solution is 50 wt %.

[0038] In some embodiments of the present invention, the acetate is sodium acetate.

[0039] In some embodiments of the present invention, the milk powder is skimmed milk powder, and the mass concentration of the skimmed milk powder aqueous solution is (10-20) wt%.

[0040] In some embodiments of the present invention, the mass concentration of the skimmed milk powder aqueous solution is 15 wt %.

[0041] In some embodiments of the present invention, the adjuvant includes an emulsifier, a pH adjuster, a lyophilization protectant, a carrier, and a solvent.

[0042] Of course, those skilled in the art can reasonably add other adjuvants according to actual use requirements, including but not limited to emulsifiers, pH adjusters, lyophilization protectants, carriers, and solvents.

[0043] In some embodiments of the present invention, the activation method of the Enterobacteriaceae-encapsulated freeze-dried powder is: adding 0.3-0.5 mL of a resuscitation solution to the Enterobacteriaceae-encapsulated freeze-dried powder, and then transferring the solution to 1-2 MRS agar plates and MRS liquid culture medium, and resuscitating and culturing at 36.5-37.5°C for 24-72 hours. After the identification is correct, confirming its purity and continuing the subculture.

[0044] In some embodiments of the present invention, the resuscitation solution is MRS liquid culture medium.

[0045] In some embodiments of the present invention, the components of the MRS liquid culture medium are: by mass, 10 parts of casein digest, 10 parts of beef extract, 4 parts of yeast extract powder, 2 parts of dipotassium hydrogen phosphate, 2 parts of triammonium citrate, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate (MgSO4·7H2O), 0.05 parts of manganese sulfate (MnSO4·4H2O), 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose, 1 part of Tween 80, 1000 parts of water, and the pH value is 7.0±0.2.

[0046] When using the Weissella mesenteroides-like embedded freeze-dried powder in the embodiment of the present invention in the process of preparing feed and medical and health products, it is not easy to be inactivated during storage at room temperature, and the number of live bacteria can be effectively guaranteed during the shelf life, thereby ensuring the efficacy of products such as feed and medical and health products.

[0047] The third aspect of the present invention provides a method for preparing a freeze-dried powder of Weissella mesenteroides, comprising the following steps:

[0048] Take the Weissella mesenteroides described in the first aspect of the present invention, add sodium alginate, calcium carbonate and part of soybean oil, add milk powder, remaining soybean oil and acetate after sufficient emulsification, centrifuge, remove the supernatant and freeze-dry to obtain the product.

[0049] In some embodiments of the present invention, the preparation method is specifically:

[0050] In an anaerobic environment, take the Weissella mesenteroides described in the first aspect of the present invention, add the pre-sterilized sodium alginate solution and calcium carbonate solution, and then add a portion of soybean oil (the soybean oil is added with an emulsifier), and stir and emulsify thoroughly. In a non-strict anaerobic environment, add skim milk powder and the remaining soybean oil (the soybean oil is added with a pH regulator) to the emulsified solution, and stir and mix thoroughly. Then add acetate, let stand for 2 to 2.5 hours, centrifuge, remove the supernatant to obtain the Weissella mesenteroides embedded microcapsules, and freeze-dry to obtain freeze-dried powder.

[0051] In some preferred embodiments of the present invention, the emulsifier is Tween.

[0052] In some embodiments of the present invention, the soybean oil added for the first time is soybean oil containing 1-2% (v / v) Tween.

[0053] In some embodiments of the present invention, the soybean oil added for the first time is soybean oil containing 1.5% (v / v) Tween.

[0054] In some preferred embodiments of the present invention, the pH adjuster is glacial acetic acid.

[0055] In some embodiments of the present invention, the soybean oil added for the second time is soybean oil containing 0.3-0.7% (v / v) glacial acetic acid.

[0056] In some embodiments of the present invention, the soybean oil added for the second time is soybean oil containing 0.5% (v / v) glacial acetic acid.

[0057] Of course, those skilled in the art can also reasonably select other emulsifiers and pH adjusters according to actual use requirements, including but not limited to Tween 80 and glacial acetic acid.

[0058] In some preferred embodiments of the present invention, the preparation method further comprises washing the Weissella mesenteroides embedded microcapsules, the washing solution is physiological saline, and the washing times are 2 to 3 times.

[0059] In some embodiments of the present invention, the mass ratio of sodium alginate aqueous solution, calcium carbonate aqueous solution, Weissella mesenteroides bacterial solution, acetate aqueous solution, milk powder aqueous solution, and soybean oil containing glacial acetic acid is 10-15: 3-6: 1-4: 200-230: 350-370: 390-420.

[0060] In some embodiments of the present invention, the mass ratio of the sodium alginate aqueous solution, the calcium carbonate aqueous solution, the Weissella mesenteroides bacterial solution, the acetate aqueous solution, the milk powder aqueous solution, and the soybean oil containing glacial acetic acid is 13:5:3:214:366:400.

[0061] In some embodiments of the present invention, the number of viable bacteria in the Weissella mesenteroides bacterial solution is 200 to 300 billion CFU / g.

[0062] In some embodiments of the present invention, the number of viable bacteria in the Weissella mesenteroides bacterial solution is 250 billion CFU / g.

[0063] In some embodiments of the present invention, the mass concentration of the sodium alginate aqueous solution is 3-8 wt %.

[0064] In some embodiments of the present invention, the mass concentration of the sodium alginate aqueous solution is 5 wt %.

[0065] In some embodiments of the present invention, the mass concentration of the calcium carbonate aqueous solution is (45-55) wt%.

[0066] In some embodiments of the present invention, the mass concentration of the calcium carbonate aqueous solution is 50 wt %.

[0067] In some embodiments of the present invention, the mass concentration of the acetate aqueous solution is (40-60) wt%.

[0068] In some embodiments of the present invention, the mass concentration of the acetate aqueous solution is 50 wt %.

[0069] In some embodiments of the present invention, the acetate is sodium acetate.

[0070] In some embodiments of the present invention, the milk powder is skimmed milk powder, and the mass concentration of the skimmed milk powder aqueous solution is (10-20) wt%.

[0071] In some embodiments of the present invention, the mass concentration of the skimmed milk powder aqueous solution is 15 wt %.

[0072] The fourth aspect of the present invention provides use of the Weissella mesenteroides described in the first aspect of the present invention in the preparation of a medicament for improving or treating inflammation.

[0073] In some embodiments of the invention, the inflammation comprises colitis.

[0074] In the examples of the present invention, the inventors verified through a colitis mouse model that the Weissella mesenteroides described in the first aspect of the present invention can significantly improve or treat colitis, thereby indicating that the Weissella mesenteroides MbWp-25 has the function of improving or treating inflammation.

[0075] The fifth aspect of the present invention provides use of the Weissella mesenteroides described in the first aspect of the present invention in preparing a fermentation product.

[0076] According to the fifth aspect of the present invention, in some embodiments of the present invention, the product includes medicine, feed, feed additives and cosmetics.

[0077] The beneficial effects of the present invention are:

[0078] 1. The present invention provides a type of Weissella mesenteroides, which is named as Weissella mesenteroides MbWp-25. The type of Weissella mesenteroides has good acid production ability and bile salt resistance, and thus has good antibacterial properties, can inhibit pathogens, promote host growth and development, and has extremely wide application value. Moreover, through experimental verification, it is also found that the type of Weissella mesenteroides has an inflammatory treatment effect and also has a good application effect in fermentation.

[0079] 2. The present invention also provides a freeze-dried powder product containing the above-mentioned Weissella mesenolyticus MbWp-25 and a preparation method thereof. The method has a simple process and low raw material cost, and can greatly improve the storage survival rate of Weissella mesenolyticus, thereby providing effective technical support for the redevelopment and utilization of Weissella mesenolyticus products. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is a Gram-stained microscope observation of Weissella mesenteroides MbWp-25.

[0081] Figure 2 This is the phylogenetic tree of Weissella mesenteroides MbWp-25.

[0082] Figure 3 This is the growth curve of Weissella mesenteroides MbWp-25, and the abscissa is time (h).

[0083] Figure 4 This is a graph showing the storage stability results of the freeze-dried powder of Weissella mesenteroides MbWp-25 in the examples of the present invention.

[0084] Figure 5 This is a disease activity index (DAI) score diagram for each group of mice in the examples of the present invention.

[0085] Figure 6These are the test results of IFN, IL-6, IL-10, IL-1β, and LPS levels in the serum of each group of mice in the examples of the present invention.

[0086] Figure 7 These are the test results of NO, MPO and GSH-Px levels in the serum of each group of mice in the examples of the present invention.

[0087] Figure 8 Pathological sections of the small intestine of each group of mice in the examples of the present invention.

[0088] Fig. 9 4 is a comparison chart of the colon lengths of mice in each group in the examples of the present invention.

[0089] Fig.10 It is a liquid chromatogram of DSS+MbWp-25 live bacteria (L25) in the example of the present invention.

[0090] Fig.11 This is a graph showing the changes in rheological properties of the fermentation of Weissella mesenteroides MbWp-25.

[0091] Fig.12 This is a comparison of Weissella mesenteroides MbWp-25 under different temperature conditions.

[0092] Fig.13 This is a graph showing the acid production changes of Weissella mesenteroides MbWp-25 over fermentation time, with the horizontal axis representing time (min). DETAILED DESCRIPTION

[0093] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0094] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0095] Obtaining Weissella mesenteroides MbWp-25

[0096] Weissella paramesenteroides MbWp-25 in the embodiment of the present invention is a probiotic with acid and bile acid resistance isolated from mulberry wine (purchased from Chuangxian Fruit Mulberry Picking Garden, Yonghe Town, Guangzhou) that was naturally fermented for 6 to 18 months in Lingnan mulberry orchards. The strain was deposited at the Guangdong Provincial Microbiological Culture Collection Center (deposit address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou) on February 11, 2022, with a deposit number of GDMCC No: 62252. Its classification name is Weissella paramesenteroides.

[0097] In the present embodiment, the Weissella mesenteroides MbWp-25 isolated by the inventors has the following biological characteristics: Gram-positive bacteria (Gram staining results are as follows Figure 1 As shown in the figure, it has no flagella, does not move, does not form spores, and is facultatively anaerobic; the bacterial body is irregular short rod-shaped, with round or slightly smaller ends, arranged in pairs or short chains, and its colonies are smooth with complete edges and are milky white. The optimal growth temperature is 36.5-37.5℃; the optimal pH value is 5.7-7.0.

[0098] The above-mentioned Weissella mesenteroides MbWp-25 was identified by 16S rRNA Sanger dideoxy sequencing (completed by Guangzhou Blue Light Biotechnology Co., Ltd.), and its sequence is:

[0099] 5’-TACATGCAAGTCGAACGCTTTGTCTTTAATTGATCTGACGAGCTTGCTCTGATGTGATTTTATCTGACAAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTACCTCTTAGCAGGGGATAACATTTGGAAACAAGTGCTAATACCGTATAATACCAACAACCGCATGGTTGTTGGTTGAAAGATGGTTCTGCTATCACTAAGAGATGGACCCGCGGTGCATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGGTTTCGGCTCGTAAAACACTGTTATAAGAGAAGAACGGCACTGAGAGTAACTGTTCAGTGTGTGACGGTATCTTACCAGAAAGGAACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTTCCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTATTTAAGTCTGAAGTGAAAGCCCTCAGCTCAACTGAGGAATGGCTTTGGAAACTGGATGACTTGAGTGCAGTAGAGGAAAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGTTGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAGTGCTAGATGTTCGAGGGTTTCCGCCCTTGAGTGTCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTTGCTAATCCTAGAAATAGGACGTTCCCTTCGGGGACAAGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA AATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGCATATACAACGAGTCGCCAACCCGCGAGGGTGCGCTAATCTCTTAAAGTATGTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGG-3'

[0100] (SEQ ID NO: 1).

[0101] The phylogenetic tree of Weissella mesenteroides MbWp-25 was constructed. Figure 2 shown.

[0102] Growth curve and related physicochemical properties of Weissella mesenteroides MbWp-25 Figure 3 And as shown in Table 1.

[0103] Among them, the physical and chemical properties tests include sugar fermentation test, milk peptone effect test, optimal carbon source test, lactic acid production test, bile salt resistance test (using MRS culture medium containing different amounts of bile salts for cultivation), and catalase test.

[0104] Among them, the sugar fermentation test, milk peptone effect test and catalase test methods refer to "Probiotics" (Editorial Committee of "Dairy Science and Technology Series", National Key Laboratory of Dairy Biotechnology. Dairy Science and Technology Series Probiotics. Beijing: Chemical Industry Press, 2016.01.). The most suitable carbon source test method reference (Zhao Guosheng, Han Hao. Research on suitable carbon and nitrogen sources in Cordyceps militaris mycelium liquid culture medium [J]. Hebei Agricultural Science, 2011, 15(08): 39-41.); lactic acid production detection method reference (Wu Jinlan, Fu Yulin, Zhou Xiaoling, Chen Zhengpei, Xiong Jianwen, Cui Na, Gong Xi. Screening, identification and fermentation condition optimization of high-yield lactic acid lactic acid bacteria in sour bamboo shoots [J]. China Brewing, 2021, 40(01): 65-69.).

[0105] Table 1 Physicochemical properties of Weissella mesenteroides MbWp-25

[0106]

[0107] Example 1 Weissella mesenteroides MbWp-25 lyophilized powder

[0108] (1) Preparation of Weissella mesenteroides MbWp-25 bacterial sludge:

[0109] Take the mesangial-like Weissella MbWp-25 isolated and obtained in the above embodiment, use MRS liquid culture medium (purchased from Guangdong Huankai Microorganism) to culture anaerobically at 37°C for 24 hours, and subculture twice to obtain seed liquid. Take an appropriate amount of seed liquid and inoculate it into the fermentation medium for constant temperature anaerobic fermentation at 37°C to obtain a high-density culture solution of mesangial-like Weissella MbWp-25 (the number of viable bacteria of mesangial-like Weissella MbWp-25 in the culture solution is measured to be 1 billion CFU / mL). Centrifuge at 5000rpm for 15min, remove the supernatant, and obtain a bacterial mud of mesangial-like Weissella MbWp-25 (the number of viable bacteria of mesangial-like Weissella MbWp-25 in the bacterial mud is measured to be 250 billion CFU / g).

[0110] Among them, the components of the above-mentioned fermentation medium are: by mass, 10 parts of casein digest (purchased from Guangdong Huankai Microorganism), 10 parts of beef extract, 4 parts of yeast extract powder, 2 parts of dipotassium hydrogen phosphate, 2 parts of triammonium citrate, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate (MgSO4·7H2O), 0.05 parts of manganese sulfate (MnSO4·4H2O), 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose, 1 part of Tween 80, 1000 parts of water, and the pH value is 7.0.

[0111] (2) Microencapsulation:

[0112] In this embodiment, the embedding preformed liquid is first prepared according to the following method, which specifically includes:

[0113] Embedding preformed solution 1: Add 0.02 kg of sodium alginate into water to prepare a 5% aqueous solution, and sterilize at 115°C for 15 minutes.

[0114] Embedding preformed solution 2: Add 0.07 kg of calcium carbonate into water to prepare a 50% solution, and sterilize at 115°C for 15 minutes;

[0115] Embedding preformed solution 3: add 0.05 kg of acetate (in this embodiment, acetate is sodium acetate) into water to prepare a 50% solution, and sterilize at 115° C. for 15 min to obtain;

[0116] Embedding preformed solution 4: Add 1 kg of skim milk powder to water to prepare a 15% solution, sterilize at 115°C for 7 minutes;

[0117] Embedding preformed solution 5: Soybean oil containing 0.5% (v / v) glacial acetic acid is sterilized at 115° C. for 7 min.

[0118] In an anaerobic environment, add the embedding preformed liquid 1 and 2 (i.e., 5wt% sodium alginate solution and 50wt% calcium carbonate solution) to the bacterial sludge of the enterobacteria MbWp-25 obtained in step (1), mix well, then add 40mL of soybean oil containing 600μL Tween 80, stir at 400r / min for 15min at 4°C, and fully emulsify and mix. In a non-strict anaerobic environment, continue to add 10mL of embedding preformed liquid 4 (i.e., 15wt% skim milk powder solution) and 10mL of embedding preformed liquid 5 (i.e., soybean oil containing 0.5% glacial acetic acid) to the mixed solution, and stir at 400r / min for 30min at 4°C. After stirring, add 60mL of embedding preformed liquid 3 (i.e., 50wt% acetate solution) with a pH of 5.5 and let stand for 2h. Centrifuge at 4000rpm for 10min to obtain microcapsules of enterobacteria MbWp-25.

[0119] (3) Preparation of Weissella mesenteroides MbWp-25 lyophilized powder:

[0120] The Weissella mesenteroides MbWp-25 microcapsules were washed three times with physiological saline, quickly frozen to -50°C with liquid nitrogen, and vacuum dried (vacuum degree ≤ 30 Pa) for 35 hours to obtain the Weissella mesenteroides MbWp-25 lyophilized powder.

[0121] Example 2 Weissella mesenteroides MbWp-25 lyophilized powder

[0122] The steps are the same as those of Example 1, except that in step (2), the amount of 15 wt % skim milk powder solution added is 20 mL, and the amount of soybean oil containing 0.5 wt % glacial acetic acid added is 20 mL.

[0123] Effect test of Weissella mesenteroides MbWp-25 freeze-dried powder

[0124] The Weissella mesenteroides MbWp-25 lyophilized powder in Example 1 was taken, and the number of live bacteria after storage at 25° C. for 6 months (180 days) was measured using a conventional viable bacteria detection method in the art, and the viable bacteria survival rate was calculated. The Weissella mesenteroides MbWp-25 lyophilized powder without embedding was used as a control.

[0125] The results are as follows Figure 4 shown.

[0126] It can be found that the viable bacteria count of the uncoated Weissella mesenteroides MbWp-25 freeze-dried powder is about 1 billion CFU / g after being stored at 25°C for 6 months, and the survival rate is only 0.06% compared with before storage. The viable bacteria count of the Weissella mesenteroides MbWp-25 freeze-dried powder in Example 1 is about 100 billion CFU / g after being stored at 25°C for 6 months, and the survival rate is 60% compared with before storage. From the above results, it can be seen that the Weissella mesenteroides MbWp-25 freeze-dried powder prepared by the above method effectively reduces the stress of the bacteria to the external adverse environment, reduces cell damage, improves the storage stability of Weissella mesenteroides MbWp-25, avoids its easy inactivation during storage at room temperature, and cannot effectively guarantee the viable bacteria count during the shelf life, which affects its efficacy. The disadvantages can be widely used in feed and pharmaceutical products to stabilize product quality and exert its health effects.

[0127] The test results of the Weissella mesenteroides MbWp-25 freeze-dried powder in Example 2 are the same as those in Example 1.

[0128] Improvement and treatment effects of Weissella mesenteroides MbWp-25 on acute colitis

[0129] In the examples of the present invention, the inventors used animal experiments to verify the effect of the above-mentioned Weissella mesenteroides MbWp-25 in improving and treating acute colitis.

[0130] In this embodiment, the test animals used were 48 SPF-grade C57BL / 6J mice (weighing 19-21 g), which were purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd. All experimental mice were placed in a sterile SPF laboratory of the Experimental Animal Center of the School of Food Science and Engineering of Foshan University of Science and Technology for a 3-day safety inspection. After 7 days of adaptation to the environment through free drinking water and food, the 48 C57BL / 6J mice were divided into a blank group, a DSS modeling group, a positive drug group, a MbWp-25 live bacteria group, a MbWp-25 dead bacteria group, and a MbWp-25 prebiotic group according to their weight. There were eight mice in each group, and each group was divided into two cages, with 4 mice in each cage. At the same time, the mice were marked with ear tags when they were grouped, and the cages were marked. During this period, the mice were given sufficient water and feed, the temperature of the SPF laboratory was controlled at a constant 23-25°C, the relative humidity was 40-50%, the lighting cycle of 12 h light and 12 h dark was used to control the automatic switching system, and the indoor light intensity was controlled at 15-20 lux.

[0131] The specific test steps are:

[0132] The experimental mice were familiarized with a normal diet at 23-25°C and a 12h light / dark cycle for 7 days of adaptation. Then 40 C57BL / 6J mice in five groups, namely, DSS modeling group, positive drug group, live bacteria group, dead bacteria group, and prebiotic group, were allowed to drink 4% DSS solution freely and were treated as follows to construct an acute colitis (UC) model. The water was changed three times on the 1st, 3rd, and 5th days, respectively. There was no restriction on the feed of all groups and they could eat freely. During the 7-day experiment, mice in all groups were weighed, gavaged, and observed every day, and the mouse feces were collected and observed. The mice were killed on the 8th day and all mice were euthanized.

[0133] The specific treatment of each group is as follows:

[0134] (1) Blank group (ck): no drinking of 4% DSS solution, free drinking of distilled water, and daily intragastric administration of normal saline;

[0135] (2) DSS model group (dss): free drinking of 4% DSS solution and daily intragastric administration of normal saline;

[0136] (3) Positive drug group (sasp): The mice were allowed to drink 4% DSS solution freely and were intragastrically administered with 0.25 g / L sulfasalazine aqueous solution every day, with the intragastrically administered volume being 0.2 mL / 10 g of mouse body weight.

[0137] (4) MbWp-25 live bacteria group (L25): free drinking of 4% DSS solution, intragastric administration of 1×10 8 cfu / mL MbWp-25 live bacterial solution, the intragastric volume is 0.2mL / 10g mouse body weight. ;

[0138] (5) MbWp-25 dead bacteria group (D25): free drinking of 4% DSS solution, intragastric administration of 1×10 8 cfu / mL MbWp-25 dead bacteria solution (rehydrated and kept at 65℃ for 10min), the intragastric volume was 0.2mL / 10g mouse body weight.

[0139] (6) MbWp-25 prebiotic group (P25): The mice were allowed to drink 4% DSS solution freely and were gavaged with 0.2 mL / 10 g of MbWp-25 culture medium metabolite solution every day.

[0140] The bacterial solution of Weissella mesenteroides MbWp-25 was prepared by mixing the freeze-dried powder of Weissella mesenteroides MbWp-25 prepared in Example 1 with sterile saline to prepare 1×10 8 The preparation method of the MbWp-25 culture broth metabolite solution is as follows: centrifuge the MbWp-25 live bacterial suspension, and filter to remove the bacterial sludge to obtain the filtrate.

[0141] During the period, the physiological state of each group of experimental mice was observed daily, including the glossiness and roughness of their hair, stool viscosity, diarrhea and bloody stools, response to stimulation, tremors, convulsions, and sleeping and lying down. The mouse feces were collected regularly every day and the body weight was measured.

[0142] Among them, the fecal occult blood kit was used to record the bloody stool, stool consistency and weight loss of the mice. The disease activity index (DAI) score is the sum of the three indicators of weight loss percentage, stool viscosity representing diarrhea, and fecal occult blood. The details of the scoring criteria are shown in Table 2 below.

[0143] Table 2 DAI scoring criteria

[0144] score Percentage of weight loss Stool consistency Fecal occult blood 0 0 normal Negative 1 1～5％ Soft stools Light blue 2 5～10％ Mucoid stool blue 3 10～20％ Thin liquid stool Dark Blue 4 >20％ Naked bloody stool

[0145] The disease activity score was calculated by combining the percentage of weight loss, fecal viscosity, and fecal occult blood of each group of mice. Figure 5 shown.

[0146] The disease activity score was calculated by combining the scores of weight loss percentage, stool viscosity, and fecal occult blood. Figure 5 The results showed that the dss group had significant weight loss, liquefied feces and visible blood in the stool, indicating that the dss colitis model was successful. Compared with the dss model group, MbWp-25 had no significant effect on normal mice, and could significantly alleviate the symptoms of weight loss, liquefied feces and blood in the stool of dss mice, while lowering the DAI score.

[0147] After anesthetizing the experimental mice, blood was collected from the eyeballs, and the obtained plasma was centrifuged at 3 000 rpm for 5 min at 4°C. The supernatant was aspirated and stored at -20°C for later use. ELISA kits were used to measure the levels of IFN, IL-6, IL-10, IL-1β, LPS, NO, MPO, and GSH-Px in serum.

[0148] The results are as follows Figure 6 and Figure 7 shown.

[0149] Compared with the dss group, the mouse sera of the oral administration of L25, D25, P25 and sulfasalazine groups all had the effect of reducing the levels of pro-inflammatory cytokines and increasing the levels of anti-inflammatory cytokines to varying degrees. The overall effect of oral administration of L25 was better than that of D25, and D25 was better than that of P25, while the effect of sulfasalazine was poor. The effects of L25, D25 and P25 were more significant than those of the sulfasalazine group.

[0150] IFN-γ is considered a proinflammatory cytokine because it can enhance the activity of TNF and induce nitric oxide (NO); IL-1β is a potent proinflammatory cytokine, mainly secreted by lymphocytes, macrophages, and monocytes. When there is a viral infection or inflammation, the increased expression of pattern recognition receptors (PRRs) and Toll-like receptors (TLRs) leads to enhanced expression of IL-1β. IL-6 is a pleiotropic proinflammatory cytokine that not only affects the immune system, but also plays a role in other systems and physiological mechanisms, such as regulating cell growth, as well as cell activation, proliferation, survival, and differentiation; interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine; lipopolysaccharide (LPS) is a strong activator of immune cells (including B cells, monocytes, macrophages, and other LPS-responsive cells), which require immunogenic stimuli such as LPS to produce cytokines.

[0151] Compared with the dss group, oral administration of L25, D25, P25 and sulfasalazine had significant effects on reducing the level of IFN-γ, but the effects on reducing the levels of IL-1β and IL-6 were less significant. The intake of L25 played an extremely significant role in promoting the regulation of two cytokines and immune response markers, IL-1β, IL-6 and LPS; the intake of D25 played a significant role in promoting the regulation of four cytokines, IFN-γ, IL-1β, IL-6 and IL-10; the intake of P25 played a significant role in promoting the regulation of three cytokines and immune response markers, IFN-γ, IL-1β and IL-10; sulfasalazine only played a relatively significant role in the regulation of two cytokines and immune response markers, IL-6 and IL-10.

[0152] The colon was separated from the mouse organs, measured with a steel ruler, and the length of the colon was recorded and photographed. After the mouse colon was cleaned, about 1 cm of the colon at 1-2 cm from the anus was cut and collected into a 1.5 mL ep tube, and 1 mL of paraformaldehyde solution was injected for fixation, marked, and set aside. The small intestinal tissue of the experimental mouse was taken to make small intestinal tissue sections and stained with H&E.

[0153] Nitric oxide (NO) is a reactive free radical that plays an important role in important physiological functions such as neurotransmission, immune response, and cell apoptosis. NO levels and signal disorders in the body often occur in certain disease states. NO with appropriate concentration levels is important for protecting organs such as the liver from ischemic damage. Long-term high concentrations of NO may cause a variety of cancers and inflammations, including juvenile diabetes, multiple sclerosis, arthritis, and ulcerative colitis. MPO is a functional marker and activation marker of neutrophils, and its level and activity changes represent the function and activity state of neutrophil polymorphonuclear leukocytes (PMN). Glutathione peroxidase (GSH-Px) is an important peroxide decomposition enzyme widely present in the body. The active center of GSH-Px is selenocysteine, and its activity can reflect the body's selenium level. Compared with the dss group, oral administration of L25, D25, and P25 had significant effects on regulating the levels of NO, MPO, and GSH-Px, while sulfasalazine was not significant.

[0154] The results are as follows Figure 8 and Fig. 9 shown.

[0155] Based on the histological analysis and scoring of each group of mice, combined with the results of mouse small intestinal pathological sections ( Figure 8 ) and the comparison of colon length in each group ( Fig. 9 ), it can be found that compared with the ck group, the dss group showed significant pathological characteristics of colitis, indicating that the colitis model was correct. Compared with the ck group, the L25 group did not show significant changes, its tissue morphology was good, and there was no inflammatory infiltration and other phenomena, indicating that the mesenteroides MbWp-25 does not cause inflammation and other related problems. Compared with the dss group, the inflammatory infiltration of the L25 group was significantly alleviated, indicating that the mesenteroides MbWp-25 significantly alleviated the intestinal damage caused by DSS. And the mice fed with mesenteroides MbWp-25 and its metabolites had the best colon length, which proved that the mesenteroides MbWp-25 can alleviate the intestinal damage caused by DSS and has the effect of improving or treating colitis.

[0156] Liquid chromatography (LC) was used to determine the concentrations of short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, n-butyric acid, and isovaleric acid) in feces.

[0157] The results are as follows Fig.10 (Liquid chromatogram of short-chain fatty acids in feces of mice in group L25).

[0158] Compared with the dss model group, MbWp-25 can significantly increase the level of fecal short-chain fatty acids, which is beneficial to intestinal health.

[0159] Test on the fermentation effect of Weissella mesenteroides MbWp-25

[0160] In order to further explore its feasibility in the fermentation field, the inventors took the lyophilized powder of Weissella mesenteroides MbWp-25 prepared in Example 1 and prepared it into 1×10 8 CFU / mL of bacterial suspension, then add 10wt% skim milk powder and 8wt% white sugar for mixed fermentation at a fermentation temperature of 37°C.

[0161] (1) Rheological properties testing:

[0162] A viscometer was used to analyze the changes in rheological properties during the fermentation process.

[0163] The results are as follows Fig.11 shown.

[0164] (2) Fermentation temperature optimization:

[0165] According to the above-mentioned component formula, the live bacteria (characterized by OD600) in the fermentation products of Weissella mesenteroides MbWp-25 at different fermentation temperatures (25, 28, 31, 34, 37 and 42°C) were detected respectively.

[0166] The results are shown in Table 3 and Fig.12 shown.

[0167] Table 3 The viable bacteria of Weissella mesenteroides MbWp-25 at different fermentation temperatures

[0168]

[0169]

[0170] The results showed that the fermentation effect of Weissella mesenteroides MbWp-25 was best at 28-42℃, when the number of live bacteria and bacterial activity were the best.

[0171] (3) Acid production effect:

[0172] According to the above component formulation, a pH meter was used to monitor the acidity changes in the fermentation product 3 hours after the start of fermentation.

[0173] The results are as follows Fig.13 shown.

[0174] It can be found that the acid production effect of Weissella mesenteroides MbWp-25 is stable. It enters the stable period after 1410 minutes of fermentation. The pH value of the obtained product is about 4.2, which has good acid production performance.

[0175] The above results indicate that Weissella mesenteroides MbWp-25 can be effectively used in the fermentation field and has excellent fermentation effect.

[0176] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Weissella mesenteroides, characterized in that The Weissella mesenteroides is Weissella mesenteroides ( Weissella paramesenteroides ) MbWp-25, the Weissella mesenteroides ( Weissella paramesenteroides )MbWp-25 was deposited in Guangdong Provincial Microbiological Culture Collection Center on February 11, 2022, with the deposit number GDMCC No: 62252.

2. The Weissella mesenteroides according to claim 1, characterized in that The nucleotide sequence of the 16SrRNA gene of Weissella mesenteroides is shown in SEQ ID NO:

1.

3. A product containing Weissella mesenteroides according to claim 1 or 2, characterized in that: The dosage forms of the product include solid powder, liquid bacterial agent, granular inoculated bacterial agent, frozen bacterial slurry, and agar carrier bacterial agent.

4. The product according to claim 3, characterized in that The product is a freeze-dried powder encapsulated with Weissella mesenteroides.

5. The product according to claim 4, characterized in that The raw materials for preparing the Weissella mesenteroides embedded freeze-dried powder include the Weissella mesenteroides described in claim 1 or 2, sodium alginate, calcium carbonate, milk powder, soybean oil, acetate and adjuvants.

6. The product according to claim 5, characterized in that The auxiliary agents include emulsifiers, pH regulators, freeze-drying protective agents, carriers, and solvents.

7. A method for preparing a freeze-dried powder of Weissella mesenteroides, comprising the following steps: Take the Weissella mesenteroides described in claim 1 or 2, add sodium alginate, calcium carbonate and part of soybean oil, add milk powder, remaining soybean oil and acetate after sufficient emulsification, centrifuge and freeze-dry to obtain the product.

8. Use of the Weissella mesenteroides according to claim 1 or 2 in the preparation of a medicament for improving or treating inflammation, wherein the inflammation is colitis.

9. Use of the Weissella mesenteroides according to claim 1 or 2 in the preparation of fermentation products; the products are medicines, feeds and feed additives.

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