A proliferation culture medium for improving in vitro production of urolithin A by intestinal flora

Abstract

The present invention discloses a proliferation culture medium for improving in vitro production of urolithin A (Uro-A) by intestinal flora. The culture medium includes brain heart infusion broth (BHI) culture medium, amino acids and antibiotics, wherein the most preferred amino acid combination is L-arginine and L-cysteine, and the most preferred antibiotic combination is chloramphenicol and neomycin sulfate. The present invention can effectively promote the growth and metabolism of urolithin A-producing strains in intestinal flora, greatly improve the yield of urolithin A, and increase 3.02 times compared with the culture medium before optimization. This helps to produce urolithin A by in vitro fermentation of intestinal flora, and the metabolic pathways, product synthesis, enzyme activity and biocatalytic mechanism therein can also be further studied, which has great value for the application in the fields of bioengineering and biopharmaceuticals.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and in particular to a proliferation culture medium for improving in vitro production of urolithin A by intestinal flora. Background Art

[0002] Ellagitannins (ETs) are hydrolyzed in the small intestine and cecum to produce ellagic acid (EA), and the intestinal microbiota can regulate the metabolism of ellagic acid, making it a more bioavailable and absorbed urolithin. Many studies believe that the health effects of ellagic acid intake may be achieved by urolithin substances, and urolithin substances may be the key material basis for ellagic acid to play a physiological role in the body. Due to differences in individual intestinal flora, the types of urolithin ultimately metabolized after consuming ellagic acid or foods rich in ellagic acid are different, and are divided into three metabolic types: type A, type B, and type 0. Individuals who ultimately metabolize ellagic acid to produce only urolithin A are type A, individuals who metabolize ellagic acid to produce urolithin A and urolithin B are type B, and individuals who cannot produce either urolithin A or urolithin B are type 0. Urolithin A, as one of the end products of ellagic acid metabolism under the action of intestinal flora, has higher biological activities such as antioxidant, anti-inflammatory, anti-cancer, regulation of intestinal flora, and anti-estrogen effects. Therefore, obtaining urolithin A is of great significance to the study of human health. At present, urolithin A is mainly obtained by chemical synthesis, which is not only too expensive but also easy to pollute the environment. Compared with chemical synthesis, obtaining urolithin A through intestinal flora metabolic activities in vivo or in vitro is a more natural way.

[0003] Microbial culture is a technology that artificially cultivates and grows microorganisms under appropriate culture media and conditions. Through this technology, the physiological metabolism, growth dynamics, product synthesis and other characteristics of microorganisms can be studied, and microorganisms can also be used to produce antibiotics, enzymes, and fermentation products. The improved culture medium in microbial culture technology helps to improve the growth and product yield of microorganisms. In the improved fermentation medium, specific promoters, inhibitors or antibiotics are added to inhibit the growth of certain microorganisms and enhance the growth ability of target microorganisms. The use of this culture medium can significantly improve the growth and product synthesis ability of target microorganisms. Since urolithin A has good biological activity, the study of obtaining urolithin A through in vitro fermentation of intestinal flora is of great significance. At present, there are few studies on microorganisms that can convert ellagic acid into urolithin, and the corresponding fermentation medium that can increase the yield of urolithin in vitro is still lacking. Therefore, it is particularly important to formulate a corresponding fermentation medium. Summary of the invention

[0004] The technical problem to be solved by the present invention is the above-mentioned defects and shortcomings existing in the prior art, and a proliferation culture medium is provided for improving the production of urolithin A in vitro by intestinal flora.

[0005] The first object of the present invention is to provide a culture medium for use in increasing the production of urolithin A by intestinal flora in vitro.

[0006] The second object of the present invention is to provide a culture medium for increasing the production of urolithin A by intestinal flora in vitro.

[0007] In order to achieve the above object, the present invention is implemented by the following scheme:

[0008] The present invention collects feces of healthy young volunteers with urolithin metabolism type A to prepare a fecal bacterial suspension. The fecal bacterial suspension is inoculated into brain heart infusion broth (BHI) medium, GAM medium, anaerobic basal broth (ABB) medium, nutrient broth (NB) medium, Wilkins-Chalgren anaerobic broth (WCA) medium, the colony growth state is observed, and the content of urolithin metabolites is determined to obtain the optimal culture medium. Further, the optimal amino acid and antibiotic combination is obtained on the basis of the optimal culture medium to obtain a urolithin A proliferation culture medium, and the effect of the urolithin A proliferation culture medium on the in vitro fermentation of the fecal bacterial suspension to produce urolithin A is determined.

[0009] Therefore, the present invention claims the following:

[0010] A culture medium for improving in vitro urolithin A production of intestinal flora, the culture medium comprising: 0.5-1.5 g / L tryptone, 0.15-0.35 g / L disodium hydrogen phosphate, 0.4-0.6 g / L sodium chloride, 0.1-0.3 g / L glucose and 1.65-1.85 g / L brain heart extract powder;

[0011] The culture medium further contains amino acids, wherein the amino acids are L-arginine and L-cysteine;

[0012] The pH of the culture medium is 7.2-7.6.

[0013] Preferably, the culture medium contains: 0.8-1.2 g / L tryptone, 0.2-0.3 g / L disodium hydrogen phosphate, 0.45-0.55 g / L sodium chloride, 0.15-0.25 g / L glucose and 1.7-1.8 g / L brain heart extract powder; the pH of the culture medium is 7.4.

[0014] More preferably, the culture medium contains: 1.0 g / L tryptone, 0.25 g / L disodium hydrogen phosphate, 0.5 g / L sodium chloride, 0.2 g / L glucose and 1.75 g / L brain heart extract powder; and the pH of the culture medium is 7.4.

[0015] Preferably, the concentration of L-arginine is 0.05 to 0.15 g / L, and the concentration of L-cysteine ​​is 0.05 to 0.15 g / L.

[0016] More preferably, the concentration of L-arginine is 0.1 g / L, and the concentration of L-cysteine ​​is 0.1 g / L.

[0017] Preferably, the culture medium further contains antibiotics, wherein the antibiotics are chloramphenicol and / or neomycin sulfate.

[0018] More preferably, the concentration of chloramphenicol is 20-30 μg / mL, and the concentration of neomycin sulfate is 45-55 μg / mL.

[0019] More preferably, the concentration of chloramphenicol is 25 μg / mL, and the concentration of neomycin sulfate is 50 μg / mL.

[0020] Preferably, the culture medium further contains ellagic acid.

[0021] More preferably, the concentration of ellagic acid is 55-65 μM.

[0022] More preferably, the concentration of ellagic acid is 60 μM.

[0023] A culture medium for improving in vitro production of urolithin A by intestinal flora, the culture medium comprising a basal culture medium;

[0024] The basic culture medium contains: 0.5-1.5 g / L tryptone, 0.15-0.35 g / L disodium hydrogen phosphate, 0.4-0.6 g / L sodium chloride, 0.1-0.3 g / L glucose and 1.65-1.85 g / L brain heart extract powder;

[0025] The culture medium further contains amino acids, wherein the amino acids are L-arginine and L-cysteine;

[0026] The pH of the culture medium is 7.2-7.6.

[0027] Preferably, the culture medium contains: 0.8-1.2 g / L tryptone, 0.2-0.3 g / L disodium hydrogen phosphate, 0.45-0.55 g / L sodium chloride, 0.15-0.25 g / L glucose and 1.7-1.8 g / L brain heart extract powder; the pH of the culture medium is 7.4.

[0028] More preferably, the culture medium contains: 1.0 g / L tryptone, 0.25 g / L disodium hydrogen phosphate, 0.5 g / L sodium chloride, 0.2 g / L glucose and 1.75 g / L brain heart extract powder; and the pH of the culture medium is 7.4.

[0029] Preferably, the concentration of L-arginine is 0.05 to 0.15 g / L, and the concentration of L-cysteine ​​is 0.05 to 0.15 g / L.

[0030] More preferably, the concentration of L-arginine is 0.1 g / L, and the concentration of L-cysteine ​​is 0.1 g / L.

[0031] Preferably, the culture medium further contains antibiotics, wherein the antibiotics are chloramphenicol and / or neomycin sulfate.

[0032] More preferably, the concentration of chloramphenicol is 20-30 μg / mL, and the concentration of neomycin sulfate is 45-55 μg / mL.

[0033] More preferably, the concentration of chloramphenicol is 25 μg / mL, and the concentration of neomycin sulfate is 50 μg / mL.

[0034] Preferably, the culture medium further contains ellagic acid.

[0035] More preferably, the concentration of ellagic acid is 55-65 μM.

[0036] More preferably, the concentration of ellagic acid is 60 μM.

[0037] A method for culturing in vitro intestinal bacterial communities to produce urolithin A, using feces of people with urolithin metabolizer type A to prepare a fecal bacterial suspension, and inoculating the fecal bacterial suspension into the above-mentioned culture medium.

[0038] Preferably, the method for preparing the fecal bacteria suspension comprises the following steps:

[0039] S1. Collect and weigh the feces of urolithin metabolism type A;

[0040] S2. Add phosphate buffer solution to the feces at a ratio of 1:(8-10) (w:v), separate the solid and liquid, take the liquid, add glycerol to the liquid until the feces content is 9-12% (v / v).

[0041] More preferably, the method for preparing the fecal bacteria suspension comprises the following steps:

[0042] S1. Collect and weigh the feces of urolithin metabolism type A;

[0043] S2. Add phosphate buffer solution to the feces at a ratio of 1:9 (w:v), separate the solid and liquid, take the liquid, add glycerol to the liquid until the feces content is 10% (v / v).

[0044] The preparation method of the above culture medium comprises dissolving each component in water, fixing the volume to the required concentration of each component, and then sterilizing at high temperature.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention adopts a basic culture medium, amino acids and antibiotics to combine with each other to provide a proliferation culture medium for improving in vitro production of urolithin A by intestinal flora. Compared with the existing common anaerobic culture medium, the culture medium of the present invention has fewer components and a simple preparation method. The present invention can effectively promote the growth and metabolism of urolithin A-producing strains in intestinal flora, greatly improve the yield of urolithin A, and increase by 3.02 times compared with the culture medium before optimization. This helps to produce urolithin A by in vitro fermentation of intestinal flora, and the metabolic pathways, product synthesis, enzyme activity and biocatalytic mechanism therein can also be further studied, which has great value for applications in the fields of bioengineering and biopharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The results of urolithin A detection in different culture media; abc indicates significant difference, and different letters represent that the difference between two groups treated with different antibiotics is statistically significant (p < 0.05).

[0048] Figure 2 The results of urolithin A detection in the culture medium after adding different amino acids; abc refers to significant differences, different letters represent significant differences between the two groups treated with different antibiotics (p < 0.05), and the same letters represent no significant differences between the two groups treated with different antibiotics (p > 0.05).

[0049] Figure 3 Figure 2 shows the effects of different antibiotics on the production of urolithin A (K1 is salinomycin, K2 is tetracycline, K3 is lincomycin, K4 is chloramphenicol, K5 is ampicillin, K6 is vancomycin, K7 is neomycin sulfate, K8 is metronidazole, K9 is streptomycin, K10 is tylosin, K11 is ciprofloxacin, and K12 is colistin); A: The effects of different antibiotics on the production of urolithin A alone; B: The effects of combined antibiotics on the production of urolithin A and the changes in the number of viable bacteria; abcdef refer to significant differences, different letters represent significant differences between different antibiotic treatment groups (p < 0.05), and the same letters represent no significant differences between different antibiotic treatment groups (p > 0.05).

[0050] Figure 4 The effect of urolithin A proliferation medium on the production of urolithin A by in vitro fermentation of fecal microbial suspension; * represents significant difference, where * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001. DETAILED DESCRIPTION

[0051] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0052] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0053] Example 1 Preparation of Brain Heart Infusion Broth (BHI) Medium

[0054] Weigh each component according to the formula of 10.0 g / L tryptone, 2.5 g / L disodium hydrogen phosphate, 5.0 g / L sodium chloride, 2.0 g / L glucose, and 17.5 g / L brain heart extract powder, dissolve the above components with distilled water, adjust the pH to 7.4±0.2, and sterilize at 121°C in a high-temperature sterilizer for 15 min to obtain BHI medium.

[0055] Example 2 Preparation of GAM medium

[0056] Weigh the components according to the formula of 10.0 g / L of phenoxyethanol, 3.0 g / L of soytone, 5.0 g / L of yeast extract powder, 2.2 g / L of beef powder, 13.5 g / L of digested serum powder, 1.2 g / L of beef liver extract powder, 3.0 g / L of glucose, 2.5 g / L of potassium dihydrogen phosphate, 3.0 g / L of sodium chloride, 5.0 g / L of soluble starch, 0.3 g / L of L-cysteine, and 0.3 g / L of sodium thioglycolate, dissolve the above components with distilled water, adjust the pH to 7.3±0.1, and sterilize at 121°C in a high-temperature sterilizer for 15 min to obtain GAM medium.

[0057] Example 3 Preparation of Anaerobic Basic Broth (ABB) Medium

[0058] The components were weighed according to the formula of 16.0 g / L peptone, 7.0 g / L yeast extract, 5.0 g / L sodium chloride, 1.0 g / L starch, 1.0 g / L glucose, 1.0 g / L sodium pyruvate, 1.0 g / L arginine, 0.5 g / L sodium succinate, 0.5 g / L cysteine ​​hydrochloride, 0.4 g / L sodium bicarbonate, 0.5 g / L ferric pyrophosphate, 0.005 g / L hemin, 0.0005 g / L vitamin K, 0.5 g / L sodium thioglycolate, and 1.0 g / L dithiothreitol, and the above components were dissolved with distilled water, the pH was adjusted to 6.8±0.2, and the culture medium was sterilized at 121°C in a high-temperature sterilizer for 15 min to obtain ABB medium.

[0059] Example 4 Preparation of Nutrient Broth (NB) Medium

[0060] Weigh each component according to the formula of 10.0 g / L peptone, 3.0 g / L beef extract powder, and 5.0 g / L sodium chloride, dissolve the above components with distilled water, adjust the pH to 7.2±0.2, and sterilize at 121°C in a high-temperature sterilizer for 15 min to obtain NB culture medium.

[0061] Example 5 Preparation of Wilkins-Chalgren Anaerobic Broth (WCA) Medium

[0062] Weigh the components according to the formula of 10.0 g / L tryptic peptone, 5.0 g / L sodium chloride, 10.0 g / L peptone, 1.0 g / L L-arginine, 1.0 g / L glucose, 5.0 g / L yeast extract powder, 1.0 g / L sodium pyruvate, 0.005 g / L hemin chloride, and 0.0005 g / L vitamin K1, dissolve the above components with distilled water, adjust the pH to 7.1±0.1, and sterilize at 121°C in a high-temperature sterilizer for 15 min to obtain WCA medium.

[0063] Example 6 Preparation of Urolithin Metabolism Type A Fecal Bacteria Suspension

[0064] Feces of several healthy young volunteers with type A urolithin metabolism were collected and weighed, and then immediately transferred to an anaerobic workstation for processing. The feces were transferred to 50 mL centrifuge tubes, phosphate buffer solution was added at a ratio of 1:9 (w:v), and after filtering through 4 layers of gauze to remove particulate matter, glycerol was added to the feces to a content of 10%, and fecal bacterial suspension was prepared and dispensed into 15 mL centrifuge tubes.

[0065] Example 7 Determination of Urolithin A Content in Different Culture Media

[0066] 1. Experimental Methods

[0067] 100 μl of the fecal bacteria suspension prepared in Example 6 was inoculated into 5 mL of the culture medium prepared in Examples 1 to 5. 13.82 mg of ellagic acid was weighed and dissolved in 100 mL of dimethyl sulfoxide (DMSO) to obtain ellagic acid mother solution. 50 μl of ellagic acid mother solution was added to the culture medium for incubation (the final concentration of ellagic acid in the culture medium was 60 μM). After 48 h, 1 mL of the bacterial solution was diluted to a concentration gradient of 10 -5 50 μl of the culture medium was spread on the plate to observe the growth of the colonies, and the remaining 4 mL of the culture medium was used for the extraction and determination of urolithin A.

[0068] 4 mL of bacterial solution was added to an equal volume of ethyl acetate (containing 1.5% formic acid (v / v)) for extraction, vortexed for 30 seconds, and allowed to stand. After complete stratification, the upper organic phase was transferred to a clean 10 mL centrifuge tube, and the organic solvent was dried with a nitrogen stream at room temperature. The remaining precipitate after drying with a nitrogen stream was re-dissolved with 300 μL of methanol solution, vortexed, and filtered through a 0.22 μm polytetrafluoroethylene filter, and then UPLC-Q-Exactive Orbitrap / MS was used for qualitative and quantitative analysis of urolithin A.

[0069] 2. Experimental Results

[0070] The average yield of urolithin A in each fecal bacteria suspension under different culture media is as follows Figure 1 As shown, the yield of urolithin A in BHI medium was 13.06 μM, the yield of urolithin A in GAM medium was 7.42 μM, the yield of urolithin A in ABB medium was 8.34 μM, the yield of urolithin A in NB medium was 1.88 μM, and the yield of urolithin A in WCA medium was 1.30 μM. Compared with other culture media, the average yield of urolithin A in fecal bacteria suspension in BHI medium was significantly increased. Therefore, BHI medium was selected for further experiments.

[0071] Example 8 Determination of Urolithin A Content in the Culture Medium after Addition of Different Amino Acids

[0072] 1. Experimental Methods

[0073] The amino acid formula for increasing the in vitro production of urolithin A by intestinal flora is as follows: 1.0 g / L each of L-arginine (L-Arg), L-histidine (L-His), and L-cysteine ​​(L-Cys). The amino acids at a concentration of 1.0 g / L were added to BHI medium alone, in pairs, or in combination of three, inoculated with 100 μL of fecal bacteria suspension and incubated with 60 μM ellagic acid solution, and urolithin A was extracted and measured after 48 hours. The extraction and determination method of urolithin A refers to the method in Example 7.

[0074] 2. Experimental Results

[0075] The average production of urolithin A in each fecal bacteria suspension under the action of different amino acid combinations is as follows Figure 2As shown, the yield of urolithin A in BHI medium without added amino acids was 9.46 μM, the yield of urolithin A in BHI medium after adding L-Arg was 11.48 μM, the yield of urolithin A in BHI medium after adding L-His was 10.71 μM, the yield of urolithin A in BHI medium after adding L-Cys was 12.76 μM, the yield of urolithin A in BHI medium after adding L-Arg+L-His was 10.43 μM, the yield of urolithin A in BHI medium after adding L-Arg+L-Cys was 17.06 μM, the yield of urolithin A in BHI medium after adding L-His+L-Cys was 12.06 μM, and the yield of urolithin A in BHI medium after adding L-Arg+L-His+L-Cys was 14.09 μM.

[0076] Compared with the yield of urolithin A in BHI medium without the addition of amino acids, the average yield of urolithin A after the addition of amino acids was significantly increased, which is more conducive to the growth and enrichment of the target strain. At the same time, according to the comparison of the results of the average yield of urolithin A, the preferred combination is L-Arg+L-His+L-Cys, and the most preferred combination is L-Arg+L-Cys, which is more conducive to the growth of urolithin A-producing strains and increases the yield of urolithin A.

[0077] Example 9 Effects of different antibiotics on the production of urolithin A

[0078] 1. Effects of different antibiotics on the production of urolithin A

[0079] 1. Experimental Methods

[0080] The 12 antibiotics used to increase the in vitro production of urolithin A by intestinal flora are as follows: salinomycin 40 μg / mL, tetracycline 12.5 μg / mL, lincomycin 60 μg / mL, chloramphenicol 25 μg / mL, ampicillin 50 μg / mL, vancomycin 50 μg / mL, neomycin sulfate 50 μg / mL, metronidazole 50 μg / mL, streptomycin 50 μg / mL, tylosin 80 μg / mL, ciprofloxacin 100 μg / mL, and colistin 10 μg / mL. The configuration concentrations of antibiotics are shown in Table 1. The 12 antibiotics were added to BHI medium (containing 1.0 g / L of L-Arg and 1.0 g / L of L-Cys) at working concentrations, 100 μL of fecal bacterial suspension was inoculated and 60 μM of ellagic acid solution was added for incubation, and urolithin A was extracted and measured after 48 hours. The extraction and determination method of urolithin A refers to the method in Example 7.

[0081] Table 1 Storage concentration and working concentration of 12 antibiotics

[0082]

[0083] 2. Experimental results

[0084] The average production of urolithin A in each fecal bacteria suspension under the action of the blank group and each antibiotic alone is as follows Figure 3 As shown in A: blank group 17.28μM, salinomycin 0μM, tetracycline 0μM, lincomycin 0μM, chloramphenicol 28.08μM, ampicillin 19.69μM, vancomycin 1.17μM, neomycin sulfate 26.37μM, metronidazole 1.17μM, streptomycin 15.39μM, tylosin 0.26μM, ciprofloxacin 12.66μM, and colistin 22.94μM.

[0085] The results of single antibiotic treatment showed that chloramphenicol, ampicillin, neomycin sulfate and colistin could significantly promote the growth of urolithin A-producing strains, salinomycin, tetracycline, lincomycin, vancomycin, metronidazole and tylosin could almost completely kill urolithin A-producing strains, while streptomycin and ciprofloxacin could inhibit the growth and metabolism of some urolithin A-producing strains.

[0086] 2. Effects of combined antibiotics on urolithin A production and changes in viable bacterial counts

[0087] 1. Experimental Methods

[0088] Chloramphenicol, neomycin sulfate, and colistin, which have significant promoting effects, were combined in pairs or in combination of three, added to BHI medium (containing 1.0 g / L L-Arg and 1.0 g / L L-Cys) at working concentrations, inoculated with 100 μL of fecal bacteria suspension and added with 60 μM ellagic acid solution for incubation, and urolithin A was extracted and measured after 48 hours. The extraction and determination method of urolithin A refers to the method in Example 7. At the same time, the plate dilution counting method was used to count the number of viable bacteria.

[0089] 2. Experimental results

[0090] The effects of the combined action of antibiotics on the production of urolithin A and the changes in the number of viable bacteria in each fecal suspension are shown in the following table. Figure 3 As shown in B, the average production of urolithin A is as follows: sterile water blank group 19.22μM, chloramphenicol + neomycin sulfate 33μM, chloramphenicol + colistin 30.22μM, neomycin sulfate + colistin 27.52μM, chloramphenicol + neomycin sulfate + colistin 25.22μM; the number of viable bacteria is as follows: blank group 6.73×10 8 , Chloramphenicol + Neomycin Sulfate 4.1×10 8 , chloramphenicol + colistin 4.37×10 8 , Neomycin sulfate + Colistin 4.17×10 8 , chloramphenicol + neomycin sulfate + colistin 1.93×108 .

[0091] Combined with the above results, the effect of combined antibiotics is higher than that of a single antibiotic. The preferred combination is chloramphenicol, neomycin sulfate and colistin, and the most preferred combination is chloramphenicol and neomycin sulfate. The yield of urolithin A is the highest, which not only promotes the growth and metabolism of urolithin A-producing strains, but also effectively inhibits the growth and reproduction of non-target strains, greatly increasing the yield of urolithin A.

[0092] Example 10 Effect of Urolithin A Proliferation Culture Medium on Urolithin A Production by Fecal Bacteria Suspension Fermentation in Vitro

[0093] 1. Experimental Methods

[0094] The proliferation medium for increasing the in vitro production of urolithin A by intestinal flora is prepared according to the following steps: 1.0 g / L of tryptone, 0.25 g / L of disodium hydrogen phosphate, 0.5 g / L of sodium chloride, 0.2 g / L of glucose, 1.75 g / L of brain heart extract powder, 0.1 g / L of L-arginine, and 0.1 g / L of L-cysteine ​​are weighed and dissolved in 100 mL of distilled water, the pH is adjusted to 7.2, heating is used to assist dissolution, and sterilization is performed at 121°C for 15 min. 250 mg of chloramphenicol and 500 mg of neomycin sulfate are weighed and dissolved in 10 mL of distilled water, respectively, to make the storage concentrations 25 mg / mL and 50 mg / mL, respectively, and filtered for sterilization. After the sterilized BHI medium is cooled to 50°C, 100 μL of chloramphenicol and 100 μL of neomycin sulfate are added, and then mixed to obtain the urolithin A proliferation medium.

[0095] Fecal samples of multiple volunteers with urolithin metabolism type A were collected and prepared into fecal bacterial suspensions. 100 μL of fecal bacterial suspension was inoculated into urolithin A proliferation medium and 60 μM ellagic acid solution was added for incubation, with BHI medium as blank control, and samples were taken after 48 hours to detect the production of urolithin A. The extraction and determination method of urolithin A was referred to the method in Example 7.

[0096] 2. Experimental Results

[0097] The effect of increasing the production of urolithin A in the fecal suspension of each volunteer was similar. Figure 4 The effect of urolithin A proliferation medium on the in vitro fermentation of fecal bacterial suspension of four volunteers was demonstrated. The results showed that the production of urolithin A in the urolithin A proliferation medium was 41.46μM, 22.36μM, 13.69μM, and 13.25μM, respectively, which was 3.02 times, 2.89 times, 3.36 times, and 2.90 times higher than that of the control group, respectively, indicating that the urolithin A proliferation medium of the present invention can effectively improve the ability of intestinal flora to produce urolithin A in vitro.

[0098] 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 use of a culture medium for increasing the production of urolithin A by intestinal flora in vitro, characterized in that: The culture medium contains: 0.5-1.5 g / L tryptone, 0.15-0.35 g / L disodium hydrogen phosphate, 0.4-0.6 g / L sodium chloride, 0.1-0.3 g / L glucose and 1.65-1.85 g / L brain heart extract powder; The culture medium further contains amino acids, wherein the amino acids are L-arginine and L-cysteine; The concentration of the L-arginine is 0.05 to 0.15 g / L, and the concentration of the L-cysteine ​​is 0.05 to 0.15 g / L; The pH of the culture medium is 7.2 to 7.6; The medium also contained ellagic acid.

2. The use according to claim 1, characterized in that: The culture medium further contains antibiotics, wherein the antibiotics are chloramphenicol and / or neomycin sulfate.

3. The use according to claim 2, characterized in that: The concentration of chloramphenicol is 20-30 μg / mL, and the concentration of neomycin sulfate is 45-55 μg / mL.

4. A culture medium for increasing the production of urolithin A by intestinal flora in vitro, characterized in that: The culture medium comprises a basal culture medium; The basic culture medium contains: 0.5-1.5 g / L tryptone, 0.15-0.35 g / L disodium hydrogen phosphate, 0.4-0.6 g / L sodium chloride, 0.1-0.3 g / L glucose and 1.65-1.85 g / L brain heart extract powder; The culture medium further contains amino acids, wherein the amino acids are L-arginine and L-cysteine; The concentration of L-arginine is 0.05 to 0.15 g / L, and the concentration of L-cysteine ​​is 0.05 to 0.15 g / L; The pH of the culture medium is 7.2 to 7.6; The medium also contained ellagic acid.

5. The culture medium according to claim 4, characterized in that The culture medium further contains antibiotics, wherein the antibiotics are chloramphenicol and / or neomycin sulfate.

6. The culture medium according to claim 5, characterized in that The concentration of chloramphenicol is 20-30 μg / mL, and the concentration of neomycin sulfate is 45-55 μg / mL.

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