CN117106590B - A proliferation culture medium for improving in vitro production of urolithin A by intestinal flora - Google Patents
A proliferation culture medium for improving in vitro production of urolithin A by intestinal flora Download PDFInfo
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Abstract
The invention discloses a proliferation culture medium for improving the exogenesis of urolithin A (Uro-A) in intestinal bacteria. The culture medium comprises brain-heart extract 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 invention can effectively promote the growth and metabolism of the urolithin A-producing strain in intestinal flora, greatly improve the output of urolithin A, and improve 3.02 times compared with the culture medium before optimization. The method is favorable for producing the urolithin A by the external fermentation of intestinal bacteria, can further research metabolic pathways, product synthesis, enzyme activity and biocatalysis mechanisms in the urolithin A, and has great value for application in the fields of bioengineering, biopharmaceuticals and the like.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a proliferation culture medium for improving the exogenesis of urolithin A in intestinal bacteria.
Background
Ellagitannins (ETs) hydrolyze in the small intestine and cecum to produce Ellagic Acid (EA), and intestinal microbiota can regulate the metabolism of ellagic acid, making it a more bioavailable, more absorbing urolithin. Many studies suggest that the health effects exhibited upon intake of ellagic acid may be achieved by uroliths, which may be the key material basis for physiological effects of ellagic acid in vivo. Due to the difference of intestinal flora of individuals, the types of urolithin finally metabolized and generated after ellagic acid or ellagic acid-rich food is eaten are different, and are divided into three metabolic types, namely A type, B type and 0 type. The final metabolic ellagic acid produced only urolithin A in the A-type and urolithin A and B in the B-type, while none of them produced urolithin A and B in the 0-type. Urolithin A is taken as one of final products of the metabolism of ellagic acid under the action of intestinal flora, has higher biological activities of resisting oxidation, resisting inflammation, resisting cancer, regulating intestinal flora, resisting estrogen and the like, so that the acquisition of the urolithin A has important significance for the research of human health. At present, the urolithin A is mainly synthesized by a chemical process, so that the cost is too high and the environment is easy to be polluted. The acquisition of urolithin a by metabolic activity of the intestinal flora in vivo or in vitro is a more natural route than chemical synthesis.
Microorganism culture is a technique of artificially culturing and growing microorganisms under proper culture medium and conditions, by which the characteristics of the microorganisms in terms of physiological metabolism, growth kinetics, product synthesis and the like can be studied, and antibiotics, enzymes and fermentation products can be produced by using the microorganisms. The improved culture medium in the microorganism culture technology helps to increase the growth of microorganisms and the yield of products. In the improved fermentation medium, specific promoter, inhibitor or antibiotic is added to inhibit the growth of some microbe and raise the growth capacity of target microbe, so that the growth and product synthesizing capacity of target microbe may be improved obviously. Because urolithin A has good biological activity, the research of obtaining urolithin A through the external fermentation of intestinal bacteria colony has important significance. At present, few studies on microorganisms capable of converting ellagic acid into urolithin are carried out, and a corresponding fermentation medium capable of improving the yield of urolithin in vitro is still lacking. Therefore, the establishment of a corresponding fermentation medium is of particular importance.
Disclosure of Invention
The invention aims to solve the technical problems and defects in the prior art and provide a proliferation culture medium for improving the exogenesis of urolithin A in intestinal bacteria groups.
The first object of the invention is to provide an application of a culture medium in improving the exogenesis of urolithin A in intestinal bacteria.
The second object of the invention is to provide a culture medium for improving the exogenesis of urolithin A in intestinal bacteria.
In order to achieve the above object, the present invention is realized by the following means:
The invention collects the feces of healthy young volunteers with urolithin metabolism A and prepares the fecal bacterial suspension. The culture medium was optimized by inoculating fecal suspension into brain-heart extract Broth (BHI) medium, GAM medium, anaerobic Base Broth (ABB) medium, nutrient Broth (NB) medium, wilkins-Chalgren anaerobic bacteria broth (WCA) medium, observing colony growth state, and measuring the content of urolithin-like metabolites. Furthermore, the optimal combination of amino acid and antibiotics is obtained on the basis of the optimal culture medium, so that the urolithin A proliferation culture medium is obtained, and the influence of the urolithin A proliferation culture medium on the in-vitro fermentation of fecal bacterial suspension to produce urolithin A is measured.
Accordingly, the present invention claims the following:
The application of a culture medium in improving the exogenesis of urolithin A in an intestinal flora comprises 0.5-1.5 g/L of tryptone, 0.15-0.35 g/L of disodium hydrogen phosphate, 0.4-0.6 g/L of sodium chloride, 0.1-0.3 g/L of glucose and 1.65-1.85 g/L of brain heart leaching powder;
the culture medium also contains amino acids, wherein the amino acids are L-arginine and L-cysteine;
The pH of the culture medium is 7.2-7.6.
Preferably, the culture medium comprises 0.8-1.2 g/L of tryptone, 0.2-0.3 g/L of disodium hydrogen phosphate, 0.45-0.55 g/L of sodium chloride, 0.15-0.25 g/L of glucose and 1.7-1.8 g/L of brain heart leaching powder, and the pH of the culture medium is 7.4.
More preferably, the culture medium contains 1.0g/L tryptone, 0.25g/L disodium hydrogen phosphate, 0.5g/L sodium chloride, 0.2g/L glucose and 1.75g/L brain heart extract powder, and the pH of the culture medium is 7.4..
Preferably, the concentration of the L-arginine is 0.05-0.15 g/L, and the concentration of the L-cysteine is 0.05-0.15 g/L.
More preferably, the concentration of L-arginine is 0.1g/L and the concentration of L-cysteine is 0.1g/L.
Preferably, the medium further comprises an antibiotic, wherein the antibiotic is chloramphenicol and/or neomycin sulfate.
More preferably, the chloramphenicol concentration is 20-30 mug/mL, and the neomycin sulfate concentration is 45-55 mug/mL.
More preferably, the chloramphenicol concentration is 25 μg/mL and the neomycin sulfate concentration is 50 μg/mL.
Preferably, the medium further contains ellagic acid.
More preferably, the concentration of ellagic acid is 55-65 mu M.
More preferably, the concentration of ellagic acid is 60 μm.
A culture medium for increasing exogenesis of an intestinal flora to urolithin a, the culture medium comprising a basal medium;
the basic culture medium comprises 0.5-1.5 g/L of tryptone, 0.15-0.35 g/L of disodium hydrogen phosphate, 0.4-0.6 g/L of sodium chloride, 0.1-0.3 g/L of glucose and 1.65-1.85 g/L of brain heart leaching powder;
the culture medium also contains amino acids, wherein the amino acids are L-arginine and L-cysteine;
The pH of the culture medium is 7.2-7.6.
Preferably, the culture medium comprises 0.8-1.2 g/L of tryptone, 0.2-0.3 g/L of disodium hydrogen phosphate, 0.45-0.55 g/L of sodium chloride, 0.15-0.25 g/L of glucose and 1.7-1.8 g/L of brain heart leaching powder, and the pH of the culture medium is 7.4.
More preferably, the culture medium contains 1.0g/L tryptone, 0.25g/L disodium hydrogen phosphate, 0.5g/L sodium chloride, 0.2g/L glucose and 1.75g/L brain heart extract powder, and the pH of the culture medium is 7.4.
Preferably, the concentration of the L-arginine is 0.05-0.15 g/L, and the concentration of the L-cysteine is 0.05-0.15 g/L.
More preferably, the concentration of L-arginine is 0.1g/L and the concentration of L-cysteine is 0.1g/L.
Preferably, the medium further comprises an antibiotic, wherein the antibiotic is chloramphenicol and/or neomycin sulfate.
More preferably, the chloramphenicol concentration is 20-30 mug/mL, and the neomycin sulfate concentration is 45-55 mug/mL.
More preferably, the chloramphenicol concentration is 25 μg/mL and the neomycin sulfate concentration is 50 μg/mL.
Preferably, the medium further contains ellagic acid.
More preferably, the concentration of ellagic acid is 55-65 mu M.
More preferably, the concentration of ellagic acid is 60 μm.
A method for producing urolithin A by in vitro culturing intestinal bacteria population comprises preparing fecal suspension from human feces of urolithin metabolism A type, and inoculating the fecal suspension into the culture medium.
Preferably, the preparation method of the fecal suspension comprises the following steps:
s1, respectively collecting feces of urolithin metabolism A type, and weighing;
s2, adding phosphate buffer solution into the excrement according to the proportion of 1 (8-10) (w: v), carrying out solid-liquid separation, taking liquid, and adding glycerol into the liquid until the excrement content is 9-12% (v/v), thus obtaining the excrement.
More preferably, the preparation method of the fecal suspension comprises the following steps:
s1, respectively collecting feces of urolithin metabolism A type, and weighing;
S2, adding phosphate buffer solution into the excrement according to the proportion of 1:9 (w: v), carrying out solid-liquid separation, taking liquid, and adding glycerol into the liquid until the excrement content is 10% (v/v), thus obtaining the novel excrement.
The preparation method of the culture medium comprises the steps of dissolving each component in water, then fixing the volume to the concentration required by each component, and then sterilizing at high temperature.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a proliferation culture medium for improving the exogenesis of urolithin A of intestinal bacteria by combining a basic culture medium, amino acid and antibiotics. Compared with the existing common anaerobic culture medium, the culture medium has fewer components and simple preparation method. The invention can effectively promote the growth and metabolism of the urolithin A-producing strain in intestinal flora, greatly improve the output of urolithin A, and improve 3.02 times compared with the culture medium before optimization. The method is favorable for producing the urolithin A by the external fermentation of intestinal bacteria, can further research metabolic pathways, product synthesis, enzyme activity and biocatalysis mechanisms in the urolithin A, and has great value for application in the fields of bioengineering, biopharmaceuticals and the like.
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FIG. 1 shows the results of the detection of urolithin A in different media, abc refers to the significant differences, and different letters represent the differences between the different antibiotic treatment groups in a statistical sense (p < 0.05).
FIG. 2 shows the results of detection of urolithin A in the medium after addition of different amino acids, abc indicates a significant difference, different letters indicate a significant difference between the different antibiotic treatment groups (p < 0.05), and the same letters indicate no significant difference between the different antibiotic treatment groups (p > 0.05).
FIG. 3 shows the effect 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, K12 is colistin), A is the effect of different antibiotics alone on the production of urolithin A, B is the effect of the combined effect of antibiotics on the production of urolithin A and the result of the change in the number of viable bacteria, abcdef is a difference in significance, different letters represent a difference in significance between the different antibiotic treatment groups (p < 0.05), and the same letters represent no difference in significance between the different antibiotic treatment groups (p > 0.05).
Fig. 4 shows the effect of urolithin a proliferation medium on urolithin a production by in vitro fermentation of a suspension of fecal bacteria, wherein p <0.05, p <0.01, p <0.001, and p <0.0001.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of brain-heart extract Broth (BHI) Medium
Weighing each component according to a formula of 10.0g/L tryptone, 2.5g/L disodium hydrogen phosphate, 5.0g/L sodium chloride, 2.0g/L glucose and 17.5g/L brain heart leaching powder, dissolving each component by distilled water, adjusting the pH value to 7.4+/-0.2, and sterilizing for 15min at 121 ℃ in a high-temperature sterilizing pot to obtain the BHI culture medium.
EXAMPLE 2 preparation of GAM Medium
Weighing each component according to a formula of 10.0g/L of lunar peptone, 3.0g/L of soybean peptone, 5.0g/L of yeast extract powder, 2.2g/L of beef powder, 13.5g/L of digested serum powder, 1.2g/L of beef liver extract powder, 3.0g/L of glucose, 2.5g/L of monopotassium phosphate, 3.0g/L of sodium chloride, 5.0g/L, L-cysteine, and 0.3g/L of sodium thioglycolate, dissolving the components by distilled water, adjusting the pH value to 7.3+/-0.1, and sterilizing in a high-temperature sterilizing pot at 121 ℃ for 15min to obtain the GAM culture medium.
Example 3 preparation of Anaerobic Base Broth (ABB) Medium
Weighing each component according to a formula of 16.0g/L peptone, 7.0g/L yeast extract powder, 5.0g/L sodium chloride, 1.0g/L starch, 1.0g/L glucose, 1.0g/L sodium pyruvate, 1.0g/L arginine, 0.5g/L sodium succinate, 0.5g/L cysteine hydrochloride, 0.4g/L sodium bicarbonate, 0.5g/L ferric pyrophosphate, 0.005g/L chlorhydrin, 0.0005g/L vitamin K, 0.5g/L sodium thioglycolate and 1.0g/L dithiothreitol, dissolving each component by distilled water, adjusting the pH value to 6.8+/-0.2, and sterilizing for 15min at 121 ℃ in a high-temperature sterilizing pot to obtain the ABB culture medium.
EXAMPLE 4 preparation of Nutrient Broth (NB) Medium
Weighing the components according to a formula of 10.0g/L peptone, 3.0g/L beef extract powder and 5.0g/L sodium chloride, dissolving the components by distilled water, adjusting the pH value to 7.2+/-0.2, and sterilizing in a high-temperature sterilizing pot at 121 ℃ for 15min to obtain the NB culture medium.
EXAMPLE 5 preparation of Wilkins-Chalgren anaerobic broth (WCA) Medium
Weighing each component according to a formula of 10.0g/L of tryptone, 5.0g/L of sodium chloride, 10.0g/L, L-arginine 1.0g/L of peptone, 1.0g/L of glucose, 5.0g/L of yeast extract powder, 1.0g/L of sodium pyruvate, 0.005g/L of hemin and 0.0005g/L of vitamin K, dissolving the components by distilled water, regulating the pH value to 7.1+/-0.1, and sterilizing in a high-temperature sterilizing pot at 121 ℃ for 15min to obtain the WCA culture medium.
EXAMPLE 6 preparation of urolithin metabolizing A-type fecal suspension
And respectively collecting the feces of a plurality of healthy young volunteers with urolithin metabolism A, and immediately transferring the feces to an anaerobic workstation for treatment after collecting and weighing the feces. Transferring the feces into 50mL centrifuge tubes respectively, adding phosphate buffer solution according to a ratio of 1:9 (w: v), filtering with 4 layers of gauze to remove particles, adding glycerol until the feces content is 10%, preparing fecal bacterial suspension, and subpackaging into 15mL centrifuge tubes.
EXAMPLE 7 determination of the content of urolithin A in different media
1. Experimental method
100 Μl of the fecal suspension prepared in example 6 was inoculated into 5mL of the culture medium prepared in examples 1 to 5, respectively. 13.82mg of ellagic acid was weighed and dissolved in 100mL of dimethyl sulfoxide (DMSO) to obtain a mother liquor of ellagic acid. 50 μl of ellagic acid mother liquor is added into the culture medium for incubation (the final concentration of ellagic acid in the culture medium is 60 μM), 1mL of bacterial liquid is taken after 48 hours for concentration gradient dilution to 10 -5, 50 μl of bacterial liquid is respectively taken and coated on a flat plate for observing the growth state of bacterial colonies, and the remaining 4mL of bacterial liquid is used for extraction and measurement of urolithin A.
4ML of the bacterial liquid was extracted with an equal volume of ethyl acetate (containing 1.5% formic acid (v/v)), and after vortexing and mixing for 30s, the bacterial liquid was allowed to stand. After complete delamination, the upper organic phase was transferred to a clean 10mL centrifuge tube and the organic solvent was dried with a stream of nitrogen at room temperature. The residual precipitate after blow-drying with nitrogen stream was redissolved with 300. Mu.L of methanol solution, and after vortex mixing, filtered through a 0.22 μm polytetrafluoroethylene filter, and then qualitative and quantitative analysis of urolithin A was performed by UPLC-Q-Exactive Orbitrap/MS.
2. Experimental results
The average urolithin A yields of the various fecal suspensions under the action of the different media are shown in FIG. 1, wherein the BHI media has 13.06. Mu.M urolithin A yield, the GAM media has 7.42. Mu.M urolithin A yield, the ABB media has 8.34. Mu.M urolithin A yield, the NB media has 1.88. Mu.M urolithin A yield and the WCA media has 1.30. Mu.M urolithin A yield. Compared with other culture mediums, the average yield of urolithin A in the BHI culture medium is obviously improved. Thus, BHI medium was subsequently selected for further experiments.
Example 8 determination of the content of urolithin A in the Medium after addition of different amino acids
1. Experimental method
The amino acid formula for improving the exogenesis of urolithin A of the intestinal flora is as follows, L-arginine (L-Arg), L-histidine (L-His) and L-cysteine (L-Cys) are respectively 1.0g/L. Amino acids with the concentration of 1.0g/L are singly, pairwise or three are combined together and added into a BHI culture medium, 100 mu L of fecal bacterial suspension is inoculated, 60 mu M of ellagic acid solution is added for incubation, and urolithin A is extracted after 48 hours for measurement. The method of measuring the extraction of urolithin A was as described in example 7.
2. Experimental results
The average yield of urolithin A in the BHI culture medium without adding amino acid is shown in figure 2, wherein the yield of urolithin A in the BHI culture medium after adding L-Arg is 9.46 mu M, the yield of urolithin A in the BHI culture medium after adding L-His is 11.48 mu M, the yield of urolithin A in the BHI culture medium after adding L-His is 10.71 mu M, the yield of urolithin A in the BHI culture medium after adding L-Cys is 12.76 mu M, the yield of urolithin A in the BHI culture medium after adding L-Arg+L-His is 10.43 mu M, the yield of urolithin A in the BHI culture medium after adding L-Arg+L-Cys is 17.06 mu M, the yield of urolithin A in the BHI culture medium after adding L-His+L-Cys is 12.06 mu M, and the yield of urolithin A in the BHI culture medium after adding L-Arg+L-Cys is 14.09 mu M.
Compared with the yield of the urolithin A in the BHI culture medium without the added amino acid, the average yield of the urolithin A after the amino acid is added is obviously improved, and the growth and the enrichment of the target strain are facilitated. Meanwhile, according to the comparison of the average yield of the urolithin A, the preferable combination is L-Arg+L-His+L-Cys, and the most preferable combination is L-Arg+L-Cys, thereby being more beneficial to the growth of the urolithin A-producing strain and improving the yield of the urolithin A.
Example 9 Effect of different antibiotics on urolithin A production
1. Effect of individual antibiotics on urolithin A production
1. Experimental method
The 12 antibiotics used for improving the exourolithin A production of the intestinal flora are salinomycin 40 mug/mL, tetracycline 12.5 mug/mL, lincomycin 60 mug/mL, chloramphenicol 25 mug/mL, ampicillin 50 mug/mL, vancomycin 50 mug/mL, neomycin sulfate 50 mug/mL, metronidazole 50 mug/mL, streptomycin 50 mug/mL, tylosin 80 mug/mL, ciprofloxacin 100 mug/mL, colistin 10 mug/mL. The antibiotic concentrations are set forth in Table 1. 12 antibiotics were added to BHI medium (containing 1.0 g/L-Arg and 1.0 g/L-Cys) at working concentration, inoculated with 100. Mu.L of fecal suspension and incubated with 60. Mu.M ellagic acid solution, after 48h urolithin A was extracted for measurement. The method of measuring the extraction of urolithin A was as described in example 7.
TABLE 1 storage and working concentrations of 12 antibiotics
2. Experimental results
The average urolithin A yields of each fecal suspension in the blank and each antibiotic alone are shown in FIG. 3A as 17.28. Mu.M, salinomycin 0. Mu.M, tetracycline 0. Mu.M, lincomycin 0. Mu.M, chloramphenicol 28.08. Mu.M, ampicillin 19.69. Mu.M, vancomycin 1.17. Mu.M, neomycin sulfate 26.37. Mu.M, metronidazole 1.17. Mu.M, streptomycin 15.39. Mu.M, tylosin 0.26. Mu.M, ciprofloxacin 12.66. Mu.M, colistin 22.94. Mu.M.
The results of single antibiotic treatment show that chloramphenicol, ampicillin, neomycin sulfate and colistin significantly promote the growth of urolithin A-producing strains, salinomycin, tetracycline, lincomycin, vancomycin, metronidazole and tylosin substantially completely kill urolithin A-producing strains, while streptomycin and ciprofloxacin inhibit the growth metabolism of a portion of urolithin A-producing strains.
2. Influence of antibiotic combination on urolithin A production and variation of viable count
1. Experimental method
Chloramphenicol, neomycin sulfate, colistin with remarkable promoting effect were combined two by two or three together, added to BHI medium (containing 1.0 g/L-Arg and 1.0 g/L-Cys) at working concentration, inoculated with 100. Mu.L of fecal suspension and incubated with 60. Mu.M ellagic acid solution, and after 48 hours, urolithin A was extracted for measurement. The method of measuring the extraction of urolithin A was as described in example 7. And simultaneously counting the viable count by adopting a plate dilution counting method.
2. Experimental results
The effect of each fecal suspension on the production of urolithin A by antibiotic combination and the results of the change in viable count are shown in FIG. 3B, wherein the average production of urolithin A is as follows, sterile water blank 19.22. Mu.M, chloramphenicol+neomycin sulfate 33. Mu.M, chloramphenicol+colistin 30.22. Mu.M, neomycin sulfate+colistin 27.52. Mu.M, chloramphenicol+neomycin sulfate+colistin 25.22. Mu.M, viable count is as follows, blank 6.73X10 8, chloramphenicol+neomycin sulfate 4.1X10 8, chloramphenicol+colistin 4.37X10 8, neomycin sulfate+colistin 4.17X10 8, chloramphenicol+neomycin sulfate+colistin 1.93X10 8.
By combining the analysis of the results, the effect of the combined action of antibiotics is higher than that of single antibiotics, chloramphenicol, neomycin sulfate and colistin are preferably combined, chloramphenicol and neomycin sulfate are most preferably combined, the yield of urolithin A is highest, the growth metabolism of urolithin A-producing strains is promoted, the growth and propagation of non-target strains are effectively inhibited, and the yield of urolithin A is greatly improved.
EXAMPLE 10 Effect of urolithin A proliferation Medium on urolithin A production by in vitro fermentation of fecal suspension
1. Experimental method
The proliferation culture medium for improving the exogenesis of the urolithin A of the intestinal flora is prepared according to the following steps of weighing 1.0g/L of tryptone, 0.25g/L of disodium hydrogen phosphate, 0.5g/L of sodium chloride, 0.2g/L of glucose, 1.75g/L, L-0.1 g/L, L-0.1 g/L of cysteine of brain heart leaching powder, dissolving in 100mL of distilled water, adjusting pH to 7.2, heating to assist dissolution, and sterilizing at 121 ℃ for 15min. 250mg of chloramphenicol and 500mg of neomycin sulfate were weighed and dissolved in 10mL of distilled water, respectively, to give storage concentrations of 25mg/mL and 50mg/mL, respectively, and filtered for sterilization. After the sterilized BHI culture medium is cooled to 50 ℃, 100 mu L of chloramphenicol and 100 mu L of neomycin sulfate are added, and then the mixture is uniformly mixed to prepare the urolithin A proliferation culture medium.
And respectively collecting a plurality of urine samples of volunteers with urolithin metabolism A type to prepare fecal bacterial suspension. 100 mu L of fecal suspension is inoculated into urolithin A proliferation culture medium, and 60 mu M of ellagic acid solution is added for incubation, BHI culture medium is used as blank control, and sampling is carried out after 48 hours to detect the yield of urolithin A. The method of measuring the extraction of urolithin A was as described in example 7.
2. Experimental results
The effect of increasing the production of urolithin a in fecal suspension of each volunteer was similar and figure 4 shows the effect of urolithin a proliferation medium on urolithin a production by fermentation of fecal suspension of four of these volunteers in vitro. The results show that the yields of the urolithin A in the urolithin A proliferation culture medium are respectively 41.46 mu M, 22.36 mu M, 13.69 mu M and 13.25 mu M, and are respectively improved by 3.02 times, 2.89 times, 3.36 times and 2.90 times compared with the control group, so that the urolithin A proliferation culture medium can effectively improve the capability of intestinal flora in producing the urolithin A in vitro.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
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