An isolation and culture method of Megasphaera elsdenii in the intestine and its application

Abstract

The invention discloses a method for isolating and culturing Megasphaera elsdenii in the intestine and its application, belonging to the technical field of microbiology. The Megasphaera elsdenii (Megasphaera elsdenii) pSML provided by the invention was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 19, 2022, and the deposit number is: GDMCC No: 62902. The method for isolating and culturing the Megasphaera elsdenii includes the following steps: S1 preparing a fecal suspension; S2 enrichment culture; S3 preparing a sample dilution; S4 strain screening; S5 isolation and purification culture; S6 in vitro fermentation. The invention uses human feces and performs enrichment culture before isolation and purification, which increases the proportion of the target bacteria in the screening process, reduces the isolation difficulty and improves the isolation efficiency. By using this bacterium for in vitro fermentation, it is found that it can utilize prebiotics to produce glutathione and has the potential to improve in vivo oxidative stress, providing new ideas for the future development and utilization of this bacterium.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a method for isolating and culturing Megasphaera elsdenii in the intestine and its application. Background Art

[0002] Intestinal microorganisms refer to a large number of microorganisms existing in the animal intestine. This group of microorganisms lives on the animal intestine and at the same time helps the host to complete a variety of physiological and biochemical functions. The human intestinal flora is huge in number, with about 10 trillion bacteria inhabiting the intestine, more than 1,000 species of bacteria, accounting for 90% of the total human bacteria. The vast majority of the intestinal flora of adults is Firmicutes and Bacteroidetes, followed by Actinobacteria, Proteobacteria, Fusobacteria, etc., which can be divided into three major categories: beneficial bacteria (such as Bifidobacterium, Lactobacillus, etc.), conditional pathogenic bacteria (such as Enterococcus, Enterobacter, etc.), and harmful bacteria (such as Proteus, Pseudomonas, Staphylococcus aureus, etc.), and also includes some archaea such as methanogens, eukaryotes and viruses. The intestinal flora not only coexists and evolves with the human body, but also affects multiple aspects of the host's digestion, absorption, metabolism, immunity, etc. Its importance and key role are equivalent to an important "organ" of the human body. Now more and more relevant studies show that a healthy gut microbiome is considered to improve metabolic capacity and can effectively resist infections and prevent the occurrence of autoimmune diseases and tumors. On the contrary, the imbalance of the intestinal flora can cause problems such as regulating the host's fat metabolism genes and causing low-level chronic inflammation throughout the body.

[0003] Megasphaera elsdenii ( Megasphaera elsdenii ) formerly known as Peptostreptococcus elsdenii belongs to Gram-negative anaerobic bacteria, is a lactic acid-decomposing bacterium that produces propionic acid in the rumen of cattle and sheep, can decompose lactic acid to effectively prevent the occurrence of lactic acidosis, and there are already plant materials for producing silage using it. In addition, it can also ferment a variety of carbohydrates to produce bioactive factors such as short-chain fatty acids to promote intestinal health. Therefore, this bacterium has broad application prospects in the medical, food, livestock and other industries. Before conducting experimental analysis on Megasphaera elsdenii, it is necessary to isolate and purify Megasphaera elsdenii in order to carry out research on its high-density fermentation. At present, most of the Megasphaera elsdenii used in research are screened from the rumen fluid of cattle and sheep, and it is difficult to obtain raw material samples. When screening from human feces, due to the low abundance of this bacterium, it is easily contaminated by other bacterial species in the intestine, and the separation is difficult. Therefore, it is necessary to provide a new and efficient method for isolating and extracting Megasphaera elsdenii to achieve its isolation and extraction.

[0004] Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is a bioactive tripeptide compound composed of L-glutamic acid, L-cysteine, and glycine. It is an important antioxidant and the main non-protein thiol compound ( > 90%) in cells, and is widely used in industries such as medicine and health care, skin care and beauty, and food additives. Currently, the main production methods of GSH include solvent extraction, chemical synthesis, enzymatic catalysis, and biological fermentation. In comparison, biological fermentation has advantages such as mild reaction conditions, simple reaction steps, low cost, high conversion efficiency, and fast production rate, and is the main trend for future GSH production. However, most current research remains at the laboratory stage. In biological fermentation, recombinant strains are often used for GSH production. There are few natural strains that can produce GSH. Currently, a patent with publication number CN101880702A discloses the production of GSH by Candida utilis fermentation using glucose as the carbon source. A patent with publication number CN101633944A discloses a Trichosporon sp. Trichosporonmontevideense CGMCC No.3144, which can produce GSH while degrading phenol. A patent with publication number CN106119139A discloses a Saccharomyces cerevisiae NAU-LYH-SGS strain that produces GSH and ferments with Lactobacillus acidophilus in a medium with glucose as the carbon source. It can be seen that none of the existing technologies have a natural strain that can produce GSH using prebiotics. Summary of the Invention

[0005] The researchers of the present invention screened a medium that can effectively isolate Megasphaera elsdenii through a large number of experiments. Furthermore, in vitro fermentation experiments with this bacterium showed that Megasphaera elsdenii can produce a large amount of glutathione by fermenting prebiotics, which provides new solutions and ideas for the development of biological products with functions such as improving oxidative stress in the body and assisting in the treatment of corresponding diseases, and for enriching this bacterial community in the body through diet therapy to assist in treatment.

[0006] The purpose of the present invention is to provide a Megasphaera elsdenii ( Megasphaera elsdenii ) pSML, as well as an optimized isolation method for this bacterium, to achieve the efficient isolation of this bacterium from human feces. In addition, the present invention uses this bacterium to ferment prebiotics and discovers that it can produce glutathione using prebiotics and has the potential to improve oxidative stress in the body, providing new ideas for the future development and utilization of this bacterium.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions:

[0008] The present invention provides a Megasphaera elsdenii ( Megasphaera elsdenii)pSML was deposited at the Guangdong Provincial Culture Collection of Microorganisms on October 19, 2022, with the deposit number: GDMCC No: 62902.

[0009] The nucleotide sequence of the 16S rRNA of the Megasphaera elsdenii pSML provided by the present invention is shown in SEQ ID NO. 1.

[0010] The present invention provides a bacterial agent, which contains the above-mentioned Megasphaera elsdenii pSML.

[0011] In one embodiment of the present invention, the bacterial agent contains wet cells or freeze-dried cells of Megasphaera elsdenii pSML.

[0012] In one embodiment of the present invention, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0013] The present invention provides a method for producing glutathione, which is to inoculate the above-mentioned Megasphaera elsdenii pSML or the above-mentioned bacterial agent into a medium containing xylooligosaccharide and ferment to produce glutathione.

[0014] In one embodiment of the present invention, the fermentation conditions are an anaerobic environment at 35-38 °C.

[0015] In one embodiment of the present invention, the components of the medium include 10-12 g / L of peptone, 10-12 g / L of yeast extract, 5-6 g / L of beef extract, 5-10 g / L of prebiotic, 1.5-2.5 g / L of dipotassium hydrogen phosphate, 1-2 g / L of Tween 80, 40-50 ml / L of salt solution, 50-100 μg / L of vitamin K1, 5-10 mg / L of hemin, 0.5-1 g / L of cysteine hydrochloride, and the pH is adjusted to 6.8-7.2.

[0016] In one embodiment of the present invention, the salt solution is 0.2-0.3 g / L of calcium chloride, 0.4-0.6 g / L of magnesium sulfate, 1-1.5 g / L of dipotassium hydrogen phosphate, 1-1.5 g / L of potassium dihydrogen phosphate, 10-12 g / L of sodium bicarbonate, and 2-2.5 g / L of sodium chloride.

[0017] In one embodiment of the present invention, the prebiotic is xylooligosaccharide.

[0018] The present invention also provides the application of the above-mentioned Megasphaera elsdenii pSML or the above-mentioned bacterial agent or the above-mentioned method in the preparation of products containing glutathione.

[0019] In one embodiment of the present invention, the products include food, medicine or feed.

[0020] The present invention also provides a feed additive, which comprises the above-mentioned Megasphaera elsdenii pSML or the above-mentioned bacterial agent.

[0021] The present invention also provides an optimized isolation method and application of Megasphaera elsdenii, and the specific steps are as follows:

[0022] S1: Prepare a fecal suspension. Weigh adult feces into a sterile buffer, shake and mix well, transfer the supernatant to a sterile centrifuge tube and add an equal volume of sterile physiological saline containing 20% glycerol.

[0023] S2: Enrichment culture. Take a certain amount of the fecal suspension and add it to an enrichment medium, and culture it under anaerobic conditions for 24 - 48 h.

[0024] S3: Prepare a sample dilution. Pipette the bacterial liquid after enrichment culture in S2 and mix it with sterile water, and perform gradient dilution.

[0025] S4: Strain screening. Take a certain amount of the diluted samples at different dilution gradients in S3 and evenly spread them on the screening medium plates, culture them under anaerobic conditions, observe the colony morphology after the culture is completed, and select the brown, smooth, moist single colonies for microscopic examination and Gram staining.

[0026] S5: Isolation and purification culture. The bacteria that are spherical under microscopic examination and stained red by Gram staining are subjected to purification culture and preservation.

[0027] In an embodiment of the present invention, in S2 and S4, the culture is carried out at 35 - 38 °C.

[0028] In an embodiment of the present invention, in S3, the bacterial liquid and sterile water are mixed at a volume ratio of 1:10.

[0029] In an embodiment of the present invention, the enrichment medium comprises 10 - 12 g / L of peptone, 2 - 5 g / L of yeast extract, 2 - 4 g / L of sodium bicarbonate, 2 - 3 g / L of D-lactic acid, 0.5 - 1 g / L of cysteine hydrochloride, 0.4 - 0.5 g / L of dipotassium hydrogen phosphate, 0.4 - 0.5 g / L of potassium dihydrogen phosphate, 0.05 - 0.1 g / L of magnesium sulfate, 0.05 - 0.1 g / L of calcium chloride, 10 - 20 mg / L of heme, 200 - 300 μg / L of growth factor, and the pH is adjusted to 5.0 - 5.5.

[0030] In one embodiment of the present invention, the screening culture medium includes 10-12 g / L of peptone, 10-12 g / L of yeast extract, 5-6 g / L of beef extract, 5-10 g / L of glucose, 1.5-2.5 g / L of dipotassium hydrogen phosphate, 1-2 g / L of Tween 80, 40-50 ml / L of saline solution, 150-100 μg / L of vitamin K, 5-10 mg / L of hemin, 0.5-1 g / L of cysteine hydrochloride, and 15-25 g / L of agar, and the pH is adjusted to 6.8-7.2.

[0031] In one embodiment of the present invention, the growth factors are biotin 10-15 mg / L, cobalamin 10-15 mg / L, pyridoxamine 150-200 mg / L, thiamine 0.5-1 mg / L, and folic acid 50-60 mg / L.

[0032] The beneficial effects of the present invention are:

[0033] 1. The present invention provides a method for screening and isolating Megasphaera elsdenii from human feces. Compared with the traditional method of isolating the bacteria from the rumen fluid of cattle and sheep, the raw materials required for the method provided by the present invention are more convenient to obtain; if it is screened from human feces, the bacteria are easy to be contaminated by other bacteria in the intestines due to their low abundance, making separation difficult. By using this method to first enrich and then separate, the quality of separation can be guaranteed, providing support for subsequent research on the bacteria.

[0034] 2. The Megasphaera elsdenii pSML provided by the present invention can ferment prebiotics to produce a large amount of glutathione. After 12 hours of fermentation culture, the glutathione production reaches a maximum of 60.45 μmol / L, which provides new solutions and ideas for developing biological products that can improve oxidative stress in organisms and assist in the treatment of corresponding diseases, as well as enriching the flora in the body through diet therapy to assist in treatment.

[0035] Biological Deposit

[0036] Giant coccus elsdenii is ( Megasphaera elsdenii ) pSML, which was deposited in Guangdong Microbial Culture Collection Center on October 19, 2022, and its taxonomic name is Megasphaera elsdenii , the deposit number is: GDMCCNo: 62902, and the deposit address is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the morphological diagram of Megasphaera elsdenii pSML;

[0038] Figure 2 This is the PCR electrophoresis detection diagram of Megasphaera elsdenii pSML;

[0039] Figure 3 Growth curve of Megasphaera elsdenii pSML;

[0040] Figure 4 Determination chart of glutathione production by Megasphaera elsdenii pSML fermentation;

[0041] Figure 5 Detect glutathione production by Megasphaera elsdenii pSML in the fermentation broth at different time points. Specific implementation mode

[0042] The present invention will be further described in detail below in conjunction with the figures and specific implementation modes. The embodiments of the present invention are given for the convenience of illustration and description, and do not show all attempts without omission or limit the present invention to the disclosed form. Many modifications and variations will be obvious to those skilled in the art. The embodiments are selected and described to better illustrate the principles and practical applications of the present invention, and enable those of ordinary skill in the art to understand the present invention and thus design implementation modes with various modifications suitable for specific purposes.

[0043] Detection method of glutathione: Measured by the DTNB method. The specific steps are as follows: Take 10 mg of glutathione standard product and make up the volume to 100 ml to obtain a 0.1 g / L glutathione standard solution. Take 0.0, 0.2, 0.4, 0.5, 0.8, 1.0 mL of the standard solution, add water to 1.0 mL, add 3 mL of Tris-HCl buffer solution, mix well, take 1.0 mL and add it to 5 mL of DTNB solution, mix well and react for 5 min, measure the absorbance at a wavelength of 412 nm, and draw a standard curve. The in vitro fermentation broth is centrifuged at 8000 r / min for 5 min, and 1.0 mL of the supernatant is taken. The absorbance is measured according to the above method, and the content of glutathione in the fermentation broth is obtained by substituting it into the standard curve.

[0044] Example 1

[0045] The technical solution of the present invention provides a screening method for Megasphaera elsdenii, including the following steps:

[0046] S1: Prepare fecal suspension. Weigh 1.0 g of adult feces, place it in 20 mL of sterile buffer, vortex for 1 min, take the supernatant after standing, transfer the supernatant to a sterile centrifuge tube and add an equal volume of sterile physiological saline containing 20% (v / v) glycerol.

[0047] S2: Enrichment culture. Take a certain amount of the fecal suspension from S1 and add it to the enrichment medium, and culture it under anaerobic conditions at 36 °C for 36 h.

[0048] S3: Prepare a sample dilution solution. Pipette the bacterial solution after enrichment culture in S2 and mix it with sterile water at a volume ratio of 1:10, and continue to perform ten-fold serial dilution with a dilution gradient of 10 -1 -10 -9 .

[0049] S4: Strain screening. Take a certain amount of the dilution samples with dilution gradients from 10 -4 to 10 -9 in S3 and evenly spread them on the screening medium plates. Make 2 - 3 parallels for the dilution samples of each dilution gradient and culture them under anaerobic conditions at 36°C for 36 h. After the culture ends, observe the colony morphology, and select the brown, smooth, and moist single colonies for microscopic examination and Gram staining.

[0050] S5: Isolation and purification culture. Purify and preserve the bacteria that are spherical under microscopic examination and stained red by Gram staining.

[0051] Enrichment medium: Peptone 12 g / L, yeast extract 5 g / L, sodium bicarbonate 4 g / L, D-lactic acid 2 g / L, cysteine hydrochloride 0.5 g / L, dipotassium hydrogen phosphate 0.4 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate 0.05 g / L, calcium chloride 0.05 g / L, hemin 20 mg / L, growth factor 200 μg / L, pH adjusted to 5.5; Growth factor: Biotin 10 mg / L, cobalamin 10 mg / L, pyridoxamine 150 mg / L, thiamine 0.5 mg / L, folic acid 50 mg / L.

[0052] Screening medium: Peptone 12 g / L, yeast extract 10 g / L, beef extract 5 g / L, glucose 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, Tween 80 2 g / L, salt solution 40 ml / L, vitamin K1 50 μg / L, hemin chloride 10 mg / L, cysteine hydrochloride 1 g / L, agar 15 g / L, pH adjusted to 7.0; Salt solution: Calcium chloride 0.2 g / L, magnesium sulfate 0.4 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, sodium bicarbonate 10 g / L, sodium chloride 2 g / L.

[0053] Example 2

[0054] The technical solution of the present invention provides a method for screening Megasphaera elsdenii, including the following steps:

[0055] S1: Prepare a fecal suspension. Weigh 0.5 g of adult feces, vortex in 20 mL of sterile buffer for 1 min, transfer the supernatant to a sterile centrifuge tube and add an equal volume of sterile physiological saline containing 20% glycerol.

[0056] S2: Enrichment culture. Take a certain amount of fecal suspension from S1 and add it to the enrichment medium, and culture it under anaerobic conditions at 37°C for 48 h.

[0057] S3: Prepare sample diluent. Pipette the bacterial liquid after enrichment culture in S2 and mix it with sterile water at a ratio of 1:10, and continue to perform ten-fold serial dilutions. The dilution gradient is 10 -1 -10 -9 。

[0058] S4: Strain screening. Take a certain amount of the dilution samples with dilution gradients from 10 -4 to 10 -9 in S3 and evenly coat them on the screening medium plates. Make 2 - 3 parallels for the dilution samples of each dilution gradient, and culture them under anaerobic conditions at 37°C for 48 h. After the culture, observe the colony morphology, and select the brown, smooth, and moist single colonies for microscopic examination and Gram staining.

[0059] S5: Isolation and purification culture. The bacteria that are spherical under microscopic examination and stained red by Gram staining are subjected to purification culture and preservation.

[0060] Enrichment medium: Peptone 10 g / L, yeast extract 2.5 g / L, sodium bicarbonate 4 g / L, D-lactic acid 2.5 g / L, cysteine hydrochloride 1 g / L, dipotassium hydrogen phosphate 0.45 g / L, potassium dihydrogen phosphate 0.45 g / L, magnesium sulfate 0.1 g / L, calcium chloride 0.1 g / L, hemin 10 mg / L, growth factor 200 μg / L, adjust the pH to 5.0; The growth factor is: biotin 15 mg / L, cobalamin 15 mg / L, pyridoxamine 200 mg / L, thiamine 1 mg / L, folic acid 60 mg / L.

[0061] Screening medium: Peptone 10 g / L, yeast extract 10 g / L, beef extract 5 g / L, glucose 5 g / L, dipotassium hydrogen phosphate 2 g / L, Tween 80 1 g / L, salt solution 40 ml / L, vitamin K1 50 μg / L, hemin chloride 10 mg / L, cysteine hydrochloride 1 g / L, agar 20 g / L, adjust the pH to 7.2; The salt solution is: calcium chloride 0.2 g / L, magnesium sulfate 0.4 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, sodium bicarbonate 10 g / L, sodium chloride 2 g / L.

[0062] Example 3

[0063] 1. Screening of strains

[0064] Refer to the method of Example 1 or Example 2 for strain screening. After screening on the plate, select the brown, smooth, and moist single colonies, and the colony morphology results of the Megasphaera elsdenii obtained after subsequent multiple purification cultures on the plate are as Figure 1as shown

[0065] 2. Identification of the Strain

[0066] Design specific primers for Megasphaera elsdenii:

[0067] MF: 5’-CTCCGAAGAGCCTCCCTTTC-3’;

[0068] MR: 5’-AGATTCGAGTGGCAAACGGG-3’,

[0069] The fragment of this strain obtained after PCR is about 350 bp. Observe the electrophoresis result of the strain screened above after PCR as Figure 2 as shown

[0070] 3. Detection of Glutathione Content by In Vitro Fermentation

[0071] Carry out in vitro fermentation on the 8 strains of Megasphaera elsdenii screened in step 1 respectively. Inoculate the strains into the fermentation medium and culture them under anaerobic conditions at 37°C, and detect their growth curves ( Figure 3 ). Then, when the bacteria grow to the logarithmic phase, inoculate the obtained bacterial liquid into 50 mL of fermentation medium at an inoculation amount of 2% (v / v), and continue to culture it under anaerobic conditions at 37°C for 12 h. Measure the glutathione content in the fermentation broth. The results are as Figure 4 shown, among which the 5th strain (i.e., the strain provided by the present invention Megasphaera elsdenii ) has the strongest ability to produce glutathione by pSML. The change result of the glutathione content in its fermentation broth over time is as Figure 5 shown. When fermented and cultured for 12 h, the yield of glutathione reaches the highest, which is 60.45 μmol / L.

[0072] Fermentation medium: peptone 10 g / L, yeast extract 10 g / L, beef extract 5 g / L, xylooligosaccharide 5 g / L, dipotassium hydrogen phosphate 2 g / L, Tween 80 1 g / L, salt solution 40 ml / L, vitamin K1 50 μg / L, hemin 10 mg / L, cysteine hydrochloride 1 g / L, pH adjusted to 7.2; Salt solution: calcium chloride 0.2 - 0.3 g / L, magnesium sulfate 0.4 - 0.6 g / L, dipotassium hydrogen phosphate 1 - 1.5 g / L, potassium dihydrogen phosphate 1 - 1.5 g / L, sodium bicarbonate 10 - 12 g / L, sodium chloride 2 - 2.5 g / L.

[0073] The present invention provides a Megasphaera elsdenii pSML strain isolated from human feces and a method for screening and isolating the same. Most of the Megasphaera elsdenii used in previous studies were screened from the rumen fluid of cattle and sheep, and it was difficult to obtain raw material samples. When screening from human feces, due to the low abundance of this bacterium and easy contamination by other bacterial species in the intestine, the isolation is difficult. Therefore, a new and efficient method for isolating and extracting Megasphaera elsdenii is needed to achieve its isolation and extraction.

[0074] The Megasphaera elsdenii provided by the present invention can ferment prebiotics to produce a large amount of glutathione, which provides new solutions and ideas for the development of biological products with functions such as improving oxidative stress in organisms and assisting in the treatment of corresponding diseases, and for enriching this bacterial community in the body through diet therapy to assist in treatment.

[0075] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An Escherichia megaterium ( Megasphaera elsdenii ) pSML was deposited at the Guangdong Microbial Culture Collection Center on October 19, 2022, with the deposit number: GDMCC No: 62902.

2. A bacterial agent, characterized in that, The microbial agent contains Megasphaera elsdenii pSML described in claim 1.

3. A method for producing glutathione, characterized in that, The method is to inoculate Megasphaera elsdenii pSML described in claim 1 or the microbial agent described in claim 2 into a culture medium containing prebiotics for fermentative production of glutathione; the prebiotic is xylooligosaccharide.

4. The method according to claim 3, characterized in that The conditions for the fermentation are an anaerobic environment at 35 - 38 °C.

5. Use of Megasphaera elsdenii pSML described in claim 1 or the microbial agent described in claim 2 or the method described in claim 3 or 4 in the preparation of a product containing glutathione.

6. A feed additive, characterized in that, The feed additive includes Megasphaera elsdenii pSML described in claim 1 or the microbial agent described in claim 2.

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