CN115960749A - Leuconostoc mesenteroides producing short-chain fatty acids as well as culture method and application thereof - Google Patents
Leuconostoc mesenteroides producing short-chain fatty acids as well as culture method and application thereof Download PDFInfo
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Abstract
The invention provides leuconostoc mesenteroides producing short chain fatty acid and a culture method and application thereof. The invention relates to Leuconostoc mesenteroides I1/53 producing short chain fatty acid, which has a preservation number of GDMCC No:61720. the leuconostoc mesenteroides I1/53 provided by the invention can produce short-chain fatty acid; animal experiments prove that the bacterial powder preparation can effectively improve inflammatory bowel diseases and relieve symptoms such as bellyache, diarrhea and the like caused by inflammatory enteritis.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to leuconostoc mesenteroides producing short-chain fatty acids and a culture method and application thereof.
Background
Inflammatory Bowel Disease (IBD), which mainly includes Ulcerative Colitis (UC) and Crohn's Disease (CD), is a nonspecific, chronic, recurrent inflammatory disease that mainly affects the intestinal tract. IBD is considered to be mostly found in western europe and north america, and is closely related to lifestyles such as western high-fat, high-protein and high-sugar diets. IBD is rare in China, but in recent 20 years, the incidence rate of IBD in China has rapidly increased due to obvious changes of Chinese people's dietary habits, living rhythms, environments and the like, and the IBD has become one of the common difficult and complicated diseases of the digestive system in China at present because the IBD rapidly increases in the Zhujiang Delta region and the Yangtze Delta region. However, there is still a lack of effective drugs for treating IBD in the clinic. Recent researches show that the imbalance of intestinal flora is closely related to the occurrence and development of IBD, and the maintenance of the balance of the intestinal flora can effectively relieve the IBD and reduce the recurrence rate of the IBD. Targeting the gut flora is therefore considered a new way to treat and prevent IBD.
According to the WHO definition, probiotics (Probiotics) are a living microorganism that, when ingested in sufficient quantities, can bring benefits to the host. A large number of researches prove that the probiotics can effectively regulate the composition and the function of intestinal flora and improve the steady state and the health of the gastrointestinal tract. However, current studies on probiotic bacteria to alleviate gastrointestinal health have focused mainly on constipation and diarrhea, and there are few studies on prevention or treatment of IBD. Short Chain Fatty Acids (SCFAs) mainly include acetic acid, propionic acid, butyric acid, and the like, and are one of the main metabolites of the intestinal flora. Research proves that the SCFAs have the effects of neutralizing inflammation, inhibiting the growth of conditional pathogenic bacteria, maintaining the intestinal barrier function, enhancing immunity and the like, and therefore, the SCFAs are considered as the key material basis for the function of intestinal flora. At present, there is an increasing desire to prevent and treat IBD by amplifying the amount of short chain fatty acids in the intestinal tract, and therefore, strains that produce short chain fatty acids have been the subject of research interest.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide Leuconostoc mesenteroides producing short chain fatty acids, and a culture method and application thereof, which can be used for preventing and treating IBD by increasing the content of short chain fatty acids in intestinal tract.
The purpose of the invention is realized by the following technical scheme:
an object of the present invention is to provide Leuconostoc mesenteroides (leuconosteroids) I1/53 producing short chain fatty acids, which is deposited at the Guangdong province culture Collection of microorganisms with the deposit number GDMCC No:61720.
further, the short-chain fatty acid is one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.
Another object of the present invention is to provide a method for culturing Leuconostoc mesenteroides producing short chain fatty acids, comprising: inoculating leuconostoc mesenteroides I1/53 strain into a culture medium containing prebiotics and suitable for producing short-chain fatty acid in an inoculation amount of 1-5% by volume, controlling the temperature to be 25-45 ℃ and the pH to be 5.0-7.0, and culturing for 12-36h to finally obtain a culture solution.
Further, the prebiotics are one or more of isomaltulose, isomalt, inulin, fructo-oligosaccharide, isomaltose oligosaccharide, galacto-oligosaccharide, stachyose, dextran and dietary fiber.
Further, the culture medium comprises the following components: 2.0 to 5.0 percent of glucose, 0.5 to 2.0 percent of beef extract powder, 0.5 to 5.0 percent of peptone, 0.5 to 5.0 percent of yeast extract, 0.2 to 1.0 percent of ammonium acetate, 0.02 to 0.2 percent of magnesium sulfate, 0.01 to 0.5 percent of manganese sulfate, 0.1 to 1.0 percent of calcium chloride, 0.1 to 1.0 percent of diammonium hydrogen citrate, 0.1 to 0.5 percent of tween-80, 0.1 to 1.0 percent of dipotassium phosphate and 1 to 5 percent of prebiotics; adjusting the pH value of the culture medium to 5-7, and sterilizing at 121 ℃ for 20min.
Further, the prebiotics in the medium are sterilized separately, the other ingredients are sterilized together, and then mixed together in an aseptic process.
The invention also aims to provide application of leuconostoc mesenteroides producing short-chain fatty acid in preparing medicine for preventing and treating inflammatory bowel diseases.
Further, the dosage form of the medicament for preventing and treating the inflammatory bowel disease is oral powder, capsules or tablets.
Further, the oral powder, capsule or tablet contains leuconostoc mesenteroides I1/53 bacterial powder.
Furthermore, the addition amount of the leuconostoc mesenteroides I1/53 is 10 6 CFU/g-10 12 CFU/g。
Further, the bacteria powder of the leuconostoc mesenteroides I1/53 is live bacteria powder or dead bacteria powder, and the leuconostoc mesenteroides I1/53 is obtained by performing vacuum freeze drying or spray drying on the supernatant of a fermentation culture of the leuconostoc mesenteroides.
The invention has the outstanding effects that: the leuconostoc mesenteroides I1/53 provided by the invention can produce short-chain fatty acid; animal experiments prove that the bacterial powder preparation can effectively improve inflammatory bowel diseases and relieve symptoms such as bellyache, diarrhea and the like caused by inflammatory enteritis.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The formulation of the common reagents used in the present invention is as follows, and will not be described in detail in the following examples.
MRS medium composition: 15g/L glucose, 10g/L beef extract, 10.0g/L peptone, 5g/L yeast extract, 300mg/L magnesium sulfate, 600mg/L manganese sulfate, 2g/L diammonium hydrogen citrate, 1mg/L tween-80, 2g/L dipotassium hydrogen phosphate and pH 6.4.
Example 1 screening, isolation and identification of short chain fatty acid producing strains
The embodiment provides a method for screening and identifying a short-chain fatty acid strain, which comprises the following steps: dissolving several samples of cane juice with sterile water, diluting in gradient, placing 1mL of proper gradient in a sterile culture dish (each dilution is 5 in parallel), pouring with MRS-calcium carbonate culture medium, solidifying, and culturing in an inverted mode at 37 ℃ for 72h. A total of 50 strains with calcium-dissolving rings (expressed by the diameter of the calcium-dissolving ring and unit mm) are selected, the numbers of the strains are 1-50, the number data of the first 10 strains with larger calcium-dissolving rings are selected, and the results are shown in Table 1:
TABLE 1 Table of strains in the top 10 caldolytic ring ranking
| Strain numbering | Diameter of calcium dissolving ring (mm) |
| 5 | 2.73±0.02 ab |
| 8 | 2.91±0.04 ab |
| 12 | 3.20±0.02 ab |
| 14 | 2.84±0.01 ab |
| 19 | 3.12±0.03 ab |
| 25 | 2.78±0.01 ab |
| 28 | 2.89±0.02 ab |
| 39 | 2.98±0.04 ab |
| 42 | 2.88±0.03 ab |
| 44 | 2.79±0.01 ab |
Note: the data are marked with significant differences in the representation of different lower case letters (ab) (P < 0.05).
Example 2 screening and identification of Strain having high ability to produce short chain fatty acids
In this example, the composition and content of SCFAs in the fermentation broth of the first 10 strains with large calcium rings in example 1 were analyzed by hplc, and the specific method was to centrifuge the broth at 14000rcf for 30min at 4 ℃, filter the obtained supernatant with a nylon membrane with a pore size of 0.22 μm, and detect short-chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid) in the filtrate by hplc under the following chromatographic conditions:
the instrument comprises the following steps: waters 2965; the chromatographic column is Aminex HPX-87H,300 multiplied by 7.8mm; the mobile phase was 10mM H 2 SO 4 The flow rate is 0.6mL/min; the column temperature is 65 ℃, a 2998PDA detector is adopted, and the UV wavelength is 210nm; the amount of sample was 10. Mu.L. The results are shown in Table 2, the SCFAs producing ability of the strain No. 12 is the strongest by combining the size of the calcium dissolving ring, the strain is named as I1/53, the strain is in a circular or bean shape by observation under a microscope, the diameter of a bacterial colony is less than 1.0mm, the surface is smooth, the milk white is realized, and no pigment is produced; the cell shape is spherical, bean-shaped or short-stalk-shaped, and some cells are arranged in pairs or short chains, do not move and have no spores; gram staining was positive. The obtained strain was identified by 16s rDNA sequencingAnd determining, wherein the sequencing result is shown as SEQ ID NO.1, and the Leuconostoc mesenteroides is identified as Leuconostoc mesenteroides by performing Blast comparison with the model strain and named as Leuconostoc mesenteroides I1/53 (Leuconostoc mesenteroides I1/53), and the preservation information of the Leuconostoc mesenteroides I1/53 is as follows: the preservation unit: guangdong province microorganism strain preservation center, GDMCC for short, and the preservation address: the microbial research institute of Guangdong province, no. 59 building, no. 5 building, of the Zhonglu-Jieli, guangzhou city, with the preservation number GDMCC No:61720, preservation time 2021, 6 months and 11 days.
The leuconostoc mesenteroides I1/53 has the following biological characteristics:
the characteristics of the thallus are as follows: is gram-positive bacteria, has micro-aerobic property and good growth in anaerobic culture at pH of 5.0-7.0.
Colony characteristics: the colony on the MRS culture medium is in a round or bean shape, the diameter of the colony is less than 1.0mm, the surface is smooth, the colony is milky white, and no pigment is generated; the cells are in the shapes of spheres, beans or short stalks, and some cells are arranged in pairs or short chains, do not move and have no spores.
Biological characteristics: the strain is facultative anaerobe, but the strain grows optimally under anaerobic conditions, the growth temperature range is 2-53 ℃, and the optimal growth temperature is 30-40 ℃; has strong acid resistance, the optimal growth pH value is 5.5-6.2, the growth can be carried out in the environment with the pH value less than or equal to 5, and the growth rate is reduced under the neutral or initial alkaline condition.
TABLE 2 Leuconostoc mesenteroides producing short chain fatty acids
Example 3 Leuconostoc mesenteroides I1/53 optimal fermentation substrate determination and fermentation condition optimization
The embodiment provides a leuconostoc mesenteroides I1/53 optimal fermentation substrate determination and fermentation condition optimization, which comprises the following steps:
inoculating the screened leuconostoc mesenteroides I1/53 into a fermentation culture medium; 2.0-5.0% of glucose, 0.5-2.0% of beef extract powder, 0.5-5.0% of peptone, 0.5-5.0% of yeast extract, 0.2-1.0% of ammonium acetate, 0.02-0.2% of magnesium sulfate, 0.01-0.5% of manganese sulfate, 0.1-1.0% of calcium chloride, 0.1-1.0% of diammonium hydrogen citrate, 0.1-0.5% of tween-80, 0.1-1.0% of dipotassium hydrogen phosphate and 1-5% of prebiotics, and the sterilization conditions of the culture medium are as follows: sterilizing at 121 deg.C for 20min. Wherein the components of the culture medium of prebiotics (isomaltulose, inulin, isomalt, stachyose, etc.) are sterilized separately, the other components are sterilized together, and then added to the culture medium under aseptic conditions or not. Inoculating 1-10% of the sterilized fermentation medium at 25-45 deg.C, pH5.0-7.0, and culturing for 12-36 hr to obtain culture solution; the SCFAs were detected in the fermentation broth according to the method in example 2. As shown in Table 3, the yield of short-chain fatty acids in the supernatant of the fermentation broth of the culture medium added with the prebiotics is obviously higher for the Leuconostoc mesenteroides I1/53, and the SCFA content is the highest when the prebiotics are isomaltulose, so that the isomaltulose is selected as the optimal substrate for the Leuconostoc mesenteroides I1/53.
TABLE 3 short-chain fatty acid yield table under coculture of 12 strains and different prebiotics
Note: there was a significant difference in the representation of the data, superscripted as different lower case letters (ab) (P < 0.05).
Example 4 application of Leuconostoc mesenteroides I1/53 producing short chain fatty acid in preventing and treating IBD
The 40 male SD rats of 6-7 weeks old are divided into 4 groups on average, namely a healthy control group, a model control group and an administration group (high and low dose). The other 3 groups except the healthy control group (drinking normal water) were fed with IBD model induced by 3% Dextran Sodium Sulfate (DSS), and all animals were fed freely and under feeding conditionsThe same is true. At the same time, the healthy control group and the model control group were gavaged with 200. Mu.L of physiological saline daily, and the high and low dose groups were gavaged with Leuconostoc mesenteroides I1/53 (10) 10 CFU) and (10) 6 CFU). In the experimental process, the weight change, the excrement state, the hematochezia condition and the like of the rat are observed every day; at the end of the experiment, colon tissue was taken and analyzed for tissue ulceration index and histological score. Results are shown in table 4, with the rectal and lower colon histological score test groups being lower than the model control group, but not statistically significant; and the histologic score of the upper segment in the colon and the colonic ulcer index of the test group are lower than those of the control group of the model group, and have significant difference, which shows that the leuconostoc mesenteroides I1/53 has a certain promotion effect on the improvement of colon tissues.
TABLE 4 influence of Leuconostoc mesenteroides I1/53 on the colon
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. Leuconostoc mesenteroides (Leuconostoc mesenteroides) I1/53 producing short chain fatty acid, which has a collection number of GDMCC No:61720.
2. the Leuconostoc mesenteroides producing short chain fatty acids of claim 1, wherein: the short-chain fatty acid is one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.
3. The method for culturing Leuconostoc mesenteroides producing short chain fatty acids according to claim 1, wherein the culture medium comprises: inoculating leuconostoc mesenteroides I1/53 strain into a culture medium containing prebiotics and suitable for producing short-chain fatty acid in an inoculation amount of 1-5% by volume, controlling the temperature to be 25-45 ℃ and the pH to be 5.0-7.0, and culturing for 12-36h to finally obtain a culture solution.
4. The method for culturing Leuconostoc mesenteroides producing short chain fatty acids according to claim 3, wherein the culture medium comprises: the prebiotics are one or more of isomaltulose, isomalt, inulin, fructo-oligosaccharide, isomaltose oligosaccharide, galacto-oligosaccharide, stachyose, dextran and dietary fiber.
5. The method for culturing Leuconostoc mesenteroides producing short chain fatty acids according to claim 3, wherein the culture medium comprises: the culture medium comprises the following components: 2.0 to 5.0 percent of glucose, 0.5 to 2.0 percent of beef extract powder, 0.5 to 5.0 percent of peptone, 0.5 to 5.0 percent of yeast extract, 0.2 to 1.0 percent of ammonium acetate, 0.02 to 0.2 percent of magnesium sulfate, 0.01 to 0.5 percent of manganese sulfate, 0.1 to 1.0 percent of calcium chloride, 0.1 to 1.0 percent of diammonium hydrogen citrate, 0.1 to 0.5 percent of tween-80, 0.1 to 1.0 percent of dipotassium phosphate and 1 to 5 percent of prebiotics; adjusting the pH value of the culture medium to 5-7, and sterilizing at 121 ℃ for 20min.
6. The method for culturing Leuconostoc mesenteroides producing short chain fatty acids according to claim 3, wherein the culture medium comprises: the prebiotics in the medium are sterilized separately, the other ingredients are sterilized together, and then mixed together in an aseptic process.
7. The use of claim 1 of a Leuconostoc mesenteroides producing short chain fatty acids for the preparation of a medicament for the prevention and treatment of inflammatory bowel disease.
8. The use of claim 7, wherein: the dosage form of the medicament for preventing and treating the inflammatory bowel disease is oral powder, capsules or tablets.
9. The use of claim 8, wherein: the oral powder, capsule or tablet contains Leuconostoc mesenteroides I1/53 powder, and the addition amount of Leuconostoc mesenteroides I1/53 is 10 6 CFU/g-10 12 CFU/g。
10. The use of claim 9, wherein: the leuconostoc mesenteroides I1/53 is live bacteria powder or dead bacteria powder, and is obtained by performing vacuum freeze drying or spray drying on supernatant of a fermentation culture of the leuconostoc mesenteroides.
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