CN115747098A - Streptococcus thermophilus FUA329 and method for producing urolithin A by fermenting same - Google Patents
Streptococcus thermophilus FUA329 and method for producing urolithin A by fermenting same Download PDFInfo
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- CN115747098A CN115747098A CN202211224627.6A CN202211224627A CN115747098A CN 115747098 A CN115747098 A CN 115747098A CN 202211224627 A CN202211224627 A CN 202211224627A CN 115747098 A CN115747098 A CN 115747098A
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- urolithin
- fua329
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- abb
- fermentation
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- RIUPLDUFZCXCHM-UHFFFAOYSA-N Urolithin A Chemical compound OC1=CC=C2C3=CC=C(O)C=C3OC(=O)C2=C1 RIUPLDUFZCXCHM-UHFFFAOYSA-N 0.000 title claims abstract description 93
- 241000194020 Streptococcus thermophilus Species 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 claims abstract description 31
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 claims abstract description 31
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- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 claims abstract description 31
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Abstract
The invention provides a streptococcus thermophilus FUA329 from a human breast source and capable of converting ellagic acid into urolithin A, and provides a new way for preparing the urolithin A. The technology has the advantages of convenient operation and high yield (the conversion rate is 82 percent), and is suitable for mass production of the urolithin A. In addition, the streptococcus thermophilus FUA329 provided by the invention has high safety and good probiotics, and has the potential to become a novel probiotic.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and provides streptococcus thermophilus FUA329 from human breast milk sources and a method for producing urolithin A by fermenting ellagic acid with the streptococcus thermophilus FUA329.
Background
In 1980, urolithin A was first discovered in the metabolite of rats fed ellagic acid and has the molecular formula C 13 H 8 O 4 The relative molecular mass was 228.2. Subsequently, urolithin a was found to have a variety of biological activities. Such as modulation of mitochondrial biosynthesis and clearance of autophagy-induced mitochondrial damage to enhance muscle function; modulating the probability of estrogen receptors inhibiting high fat diet-mediated uterine tumorigenesis; preventing and treating prostate cancer by modulating androgen receptor; can remarkably inhibit typical inflammatory factors, such as enteritis, osteoarthritis, neuritis, nephritis, intervertebral disc pathological changes and the like; the risk of heart metabolism can be effectively reduced; can prevent D-galactose induced brain aging, and improve cognitive disorder; can be used for preventing and reversing obesity caused by high fat diet, and improving metabolism function, and has no side effects.
A large number of studies indicate that various intestinal microorganisms and enzymes are involved in the process of metabolizing ellagic acid into urolithin. Depending on whether or not intestinal microorganisms metabolize ellagic acid to produce urolithins and the kind of urolithins produced, humans can be classified into three types: urolithin a type, the final product being urolithin a; urolithin B type, the final products being urolithin B and isourolithin A; urolithin type 0, no production of urolithin. It has been found that the proportion of urolithin B is significantly increased in people with chronic diseases such as metabolic syndrome and colorectal cancer, and only about 50% of people over 40 years old are urolithin A. Since urolithin a has multiple probiotic functions and the proportion of urolithin a type population is low in the elderly and in people with chronic diseases, in 2018, the U.S. FDA approved urolithin a for use in dietary supplements. Amazentis corporation developed a nutritional supplement product Mitopure with high purity urolithin a as a major component for combating cell and muscle decline.
At present, the industrial production of urolithin mainly uses a chemical method, but the method has long reaction time, high cost, large energy consumption and high product price. If a new generation of probiotics capable of converting the ellagic acid into the urolithin A is developed, the urolithin A can be prepared by a microbial fermentation method, and tea and food rich in the ellagic acid can be fermented to prepare a probiotic fermented drink rich in the urolithin A. However, it has been reported that only Gordonibacter urolithinacinensis and Gordonibacter pamecene can convert ellagic acid in vitro to produce isourolithin A, and that only Birdobacterium pseudoatellum can convert ellagic acid to produce urolithin A and urolithin B, which have been isolated from human intestinal tracts. The obtained cellulosate can be converted into ellagic acid by mixed fermentation of strains such as Eggerthella sp. Therefore, the method can be used for exploring a plurality of strains capable of converting ellagic acid into urolithin A, screening a strain capable of producing urolithin A in vivo and in vitro, can make up the defects of the current preparation technology of urolithin A, and has important significance for industrial production.
Disclosure of Invention
Aiming at the defects existing in the technology, the invention provides a Streptococcus thermophilus FUA329 (Streptococcus thermophilus) derived from human breast milk.
Streptococcus thermophilus FUA329 has been preserved in China general microbiological culture Collection center in 2022, 5-23.D., with the preservation number of CGMCC NO.24963.
The streptococcus thermophilus FUA329 provided by the invention is used for producing urolithin A.
The invention provides application of a composition containing streptococcus thermophilus FUA329 in preparing a medicine for improving intestinal flora.
In one aspect, the present invention provides a screening method of streptococcus thermophilus FUA329, comprising the following steps:
i) Verifying the human milk sample producing urolithin a: inoculating a sample of breast milk containing ellagic acid under aseptic conditionsCulturing in ABB liquid culture medium under anaerobic condition for 4 days, comparing with culture medium containing ellagic acid and no breast milk, collecting sample once per day, and adding organic solvent C 2 H 3 N:H 2 HCOOH (80, 19.9, V;
ii) screening for urolithin A producing strains: the method comprises the steps of diluting a mother milk sample fermentation liquor which is verified to produce urolithin A by adding normal saline, coating 0.1mL of the diluted fermentation liquor on an ABB solid plate, culturing under anaerobic conditions to obtain a plurality of microbial colonies, respectively inoculating each microbial colony into an ABB liquid culture medium containing ellagic acid, performing fermentation culture under anaerobic conditions, performing HPLC (high performance liquid chromatography) and HPLC-MS (high performance liquid chromatography) -MS/MS (mass spectrometry) analysis on the fermentation liquor, and screening a strain which produces the urolithin A, wherein the strain is named as FUA329.
In another aspect, the present invention provides a method for producing urolithin a by using streptococcus thermophilus FUA329, comprising the following steps:
1) Activating strains: taking FUA329 stored in glycerin pipe in a refrigerator at-80 deg.C, and performing three-zone streaking on ABB solid culture medium to obtain single colony;
2) Seed culture: inoculating the activated single colony of the strain FUA329 into a seed culture medium to culture to obtain a seed solution;
3) Fermentation culture: inoculating the seed solution into a fermentation culture medium added with ellagic acid for fermentation culture;
4) Separation and extraction: and centrifuging the fermentation liquor to obtain supernatant, namely the crude urolithin A extraction liquid.
The ABB solid culture medium formula in the step 1) is an ABB anaerobic basal broth culture medium added with 2% agar.
The seed culture medium in the step 2) is ABB anaerobic basic broth culture medium, the pH value is 6.8 +/-0.2, and the formula comprises: 16.0g/l of peptone, 7.0g/l of yeast extract powder, 5.0g/l of sodium chloride, 1.0g/l of starch, 1.0g/l of glucose, 1.0g/l of sodium pyruvate, 1.0g/l of arginine, 0.5g/l of sodium succinate, 0.5g/l of cysteine hydrochloride, 0.4g/l of sodium bicarbonate, 0.5g/l of ferric pyrophosphate, 0.005g/l of hemin, 0.0005g/l of vitamin K, 0.5g/l of sodium thioglycolate and 1.0g/l of dithiothreitol.
The culture condition in the step 2) is to culture for 24 hours under anaerobic condition at 37 ℃.
The fermentation medium in step 3) is ABB anaerobic basal broth containing 20 μ M ellagic acid.
The inoculation amount of the seed solution in the step 3) is 2%.
The fermentation culture condition in the step 3) is to culture for 48 hours at 37 ℃ under anaerobic condition.
The centrifugation condition in the step 4) is that the centrifugation is carried out for 10min at 12000 Xg and 4 ℃.
The invention has the beneficial effects that: the invention provides a streptococcus thermophilus FUA329 from a human breast source and capable of converting ellagic acid into urolithin A, and provides a new way for preparing the urolithin A. The technology has the advantages of convenient operation and high yield (the conversion rate is 82 percent), and is suitable for mass production of the urolithin A. In addition, the streptococcus thermophilus FUA329 provided by the invention has high safety and good probiotics, and has the potential to become novel probiotics.
Drawings
FIG. 1 is a gram stain (× 1000) of strain FUA 329;
FIG. 2 is a colony morphology of the strain FUA 329;
FIG. 3 is a phylogenetic tree of the strain FUA 329;
FIG. 4 is a liquid phase diagram (A) of a urolithin standard sample, wherein the number of the peaks in the A diagram is 1 for urolithin D,2 for ellagic acid, 3 for urolithin M6,4 for urolithin C,5 for isourolithin A,6 for urolithin A, and 7 for urolithin B; a urolithin A standard sample mass spectrogram (a) a liquid phase diagram (B) of a strain FUA329 fermentation liquor in a figure 5; the strain FUA329 converts ellagic acid to produce a urolithin A fermentation liquid mass spectrogram (b);
FIG. 6 is a graph showing the results of the hemolytic plate experiment with the strain FUA 329;
FIG. 7 shows the conversion process of strain FUA329 for fermentation and conversion of ellagic acid into urolithin A;
FIG. 8 shows the results of the acid tolerance and bile salt tolerance experiments of the strain FUA329 after in vitro treatment at different pH and bile salt concentrations.
Detailed Description
The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.
EXAMPLE 1 screening of strains
Example 1: isolation and characterization of strains
The strain FUA329 related to the invention is Streptococcus thermophilus FUA329 separated from human breast milk source, and the strain is preserved in China general microbiological culture Collection center at 23.5.2022, and the preservation unit address is as follows: west road No. 1, north west of the republic of kyo, 3, the collection of strains of the institute of microbiology, academy of sciences of china. The contact telephone is 010-64807355, and the preservation number is CGMCC NO.24963. GenBank accession number of the 16S rRNA gene sequence of the strain is OM892001.
1. Screening of strains:
1) Verifying the human milk sample producing urolithin a: inoculating 1ml of breast milk sample into ABB liquid culture medium containing 20 μ M ellagic acid under aseptic condition, culturing under anaerobic condition for 4 days, collecting 10ml of sample once per day by using ellagic acid-containing breast milk-free culture medium and breast milk-containing ellagic acid-free culture medium as control, and adding organic solvent C 2 H 3 N:H 2 O:CH 2 O 2 (80.9, 0.1, V;
2) Screening a strain producing urolithin A: diluting with fermentation broth of breast milk sample with verified production of urolithin A to 10% with physiological saline -3 -10 -7 Then, 0.1mL of the suspension was spread on an ABB solid plate, and after culturing for 24-48 hours at 37 ℃ under anaerobic conditions, 93 microbial colonies were obtained, each colony was inoculated into an ABB liquid medium containing 20. Mu.M ellagic acid, and fermentation-cultured for 48 hours at 37 ℃ under anaerobic conditions, and HPLC-MS/MS analyses were performed to screen out a urolithin A-producing strain, which was designated FUA329.
2. Morphological identification:
the strain FUA329 is gram-positive coccus, and can grow in ABB anaerobic basic broth culture medium at 37 deg.C for 48h, and the bacterial colony is round, milky semitransparent, wet in surface, regular in edge, free of halo, and has a central bulge with a diameter of 0.5-1.0mm, and is easy to pick up, as shown in FIG. 1 and FIG. 2.
3. Physiological and biochemical identification:
the strain was subjected to salicylic acid test with reference to a manual of identification of common bacteria systems (Dongxiu bead, chuia Miaoying. Manual of identification of common bacteria systems. Beijing, scientific Press, 2001), H 2 O 2 The strain species is preliminarily determined by tests such as a gelatin liquefaction test, an indole test, a nitrate reduction test, mannitol, sorbitol, maltose, sucrose, fructose utilization and the like. The experimental result shows that the strain is positive in a mannitol test and a salicylic acid test, H 2 O 2 The test results of the test, the gelatin liquefaction test, the indole test, the bile lysis test and the test of sorbitol, maltose, sucrose and fructose are all negative. Some physiological and biochemical results are shown in table 1.
TABLE 1 results of physiological and biochemical tests of the strain FUA329
Note: +: positive; -: negative;
4. amplification and analysis of FUA 329S rRNA sequence of Strain
The genome of FUA329 is extracted by an Axygen kit, and a universal primer for amplifying the 16S rRNA sequence of the prokaryotic microorganism is selected to react in a PCR mix system.
The universal primers for PCR reaction: 27F:5 'AGAGAGTTTGATCCTGGCTCAG-3'; 1492R:5'-GGTTACCTTGTTACGACTT-3'.
The reaction system (25. Mu.l) was: 2 Xmax Premix (12.5. Mu.l), upstream and downstream primers (0.75. Mu.l each), DNA template (0.5. Mu.l), ddH2O (10.5. Mu.l). Reaction procedure: denaturation at 98 deg.C for 2min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 10s, and 30-35 cycles; final extension at 72 ℃ for 2min. The sequence was sequenced and submitted to GenBank (accession number: OM 892001) and the sequence was compared to sequences in the GenBank database for homologyOn the other hand, it was found that the strain Streptococcus thermophilus DSM20617 was used T (accession number: NR 118998) the similarity of the 16S rRNA gene sequence reaches 99.57 percent. The phylogenetic tree showed that the strain FUA329 has a closest relationship to Streptococcus thermophilus, as shown in FIG. 3.
The GenBank accession number of the gene sequence of the Streptococcus thermophilus FUA329 16S rRNA is OM892001, and the specific sequence is as follows:
TGCGGCAGCTATAATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACCCATGTCATTTATTTGAAAGGGGCAAATGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAACGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGAGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGTCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGAATGCAG。
EXAMPLE 2 Process for the fermentative conversion of ellagic acid to urolithin A by the Strain FUA329 and identification thereof
The seed culture medium is ABB anaerobic basic broth culture medium, and the formula comprises 16.0g/l of peptone, 7.0g/l of yeast extract powder, 5.0g/l of sodium chloride, 1.0g/l of starch, 1.0g/l of glucose, 1.0g/l of sodium pyruvate, 1.0g/l of arginine, 0.5g/l of sodium succinate, 0.5g/l of cysteine hydrochloride, 0.4g/l of sodium bicarbonate, 0.5g/l of ferric pyrophosphate, 0.005g/l of hemin chloride, 0.0005g/l of vitamin, 0.5g/l of sodium thioglycolate, 1.0g/l of dithiothreitol and pH6.8 +/-0.2.
The fermentation medium was ABB anaerobic basal broth containing 20. Mu.M ellagic acid.
1. Activation of the strain: the streptococcus thermophilus FUA329 inoculated in a refrigerator at the temperature of-80 ℃ is streaked and activated in three areas on an ABB solid culture medium;
2. preparing a seed solution: inoculating a single colony of the strain FUA329 into a seed culture medium, and culturing for 24h at 37 ℃ under an anaerobic condition to obtain a seed solution;
3. preparing fermentation liquor: inoculating the seed liquid to a fermentation culture medium by 1% of inoculation amount, culturing at 37 ℃ under anaerobic condition for 48h, centrifuging at 12000 Xg and 4 ℃ for 10min, and taking supernatant as urolithin A fermentation liquid;
HPLC and UPLC-MS identification and analysis: taking 1.0ml of fermentation liquor and C 2 H 3 N:H 2 O:CH 2 O 2 (80.19.9, V.
1) HPLC analysis: the chromatographic analysis of the samples was carried out on a ZORBAX SB-C18 column (Agilent, USA) of 250X 4.6mm and a particle size of 5 μm. Acetonitrile and 1% methanol are used as mobile phases, the sample amount is 5 mul, and the flow rate is 1ml/min. Recording was carried out at 305 nm. The gradient of elution was: 0-15min, 0-20% acetonitrile; 15-20min, 20-70% acetonitrile; 20-21min, 70-95% acetonitrile; 21-24min, 95-100% acetonitrile; 24-25min, 100-20% acetonitrile; preparing a urolithin standard sample: respectively weighing 0.005g of urolithin A, urolithin C, urolithin D and isourolithin A,0.004g of urolithin B and 0.006g of urolithin M6, adding 1ml of water and 1ml of C 2 H 3 N:H 2 O:CH 2 O 2 (80.19.9, vThe bottle was loaded to obtain a 20. Mu.M concentration of the urolithin standard.
2) HPLC-MS/MS analysis: samples were analyzed 150-1000 Da using an ACQUITY QDa ESIMS using a C18 column (ACQUITY UPLC BEH C18, 2.1X 50mm,1.7 μm) with 0.2% formic acid and acetonitrile as mobile phases, a flow rate of 0.5ml/min, and a sample loading of 50 μ l. The gradient of elution was: 0-6 min, 10-100% acetonitrile; 6-7min, 100% acetonitrile; 7-8min, 100-10% acetonitrile; 8-9min and 10% acetonitrile.
Compared with the standard samples of urolithin A, urolithin B, urolithin C, urolithin D, urolithin M6, isourolithin A and ellagic acid, FUA329 was confirmed to produce urolithin A, and the analysis results are shown in FIG. 4 and FIG. 5.
Example 3: safety and probiotic analysis of the strain FUA329
1. Safety analysis of strain FUA329
Hemolysis experiment: 1ml of activated single bacterial colony is inoculated in an ABB liquid culture medium, anaerobic culture is carried out for 12h at 37 ℃, bacterial liquid is taken for centrifugation (8000 rpm is multiplied by 4 ℃,5 min), bacterial precipitation is re-suspended by sterile physiological saline, 100 mul of re-suspended bacterial liquid is sucked and placed on a sheep blood agar plate containing 5 percent for three-zone streaking, staphylococcus aureus is taken as a positive control, and after culture is carried out for 48h at 37 ℃, the hemolysis condition of bacterial colony growing on the plate is observed (figure 6).
Drug sensitivity test: 27 antibiotics (amikacin, norfloxacin, ofloxacin, ciprofloxacin, levofloxacin, erythromycin, tetracycline, cefuroxime, cefazolin, cephalothin, cefotaxime, ceftriaxone, ceftazidime, piperacillin, ampicillin, oxacillin, penicillin G, aztreonam, sulfamethoxazole, nitrofurantoin, chloramphenicol, polymyxin B, clindamycin, kanamycin, gentamicin, streptomycin, vancomycin) were selected and tested according to a drug sensitive kit (Hangzhou river microbiology reagent, inc.). Activating and culturing the strain to logarithmic phase, and taking 100 mul 10 -3 The bacterial liquid with the concentration is coated on an ABB solid culture medium. Placing drug sensitive paper sheets containing different concentrations on ABB solid flat plate, anaerobically culturing at 37 deg.C for 48 hr, measuring the size of antibacterial zone, repeating each test for 3 times, averaging, and making referenceThe sensitivity of the strain to different drugs is judged according to the performance standard of the sensitivity test of the antibacterial drug paper method.
The safety of the strain FUA329 is studied by hemolytic activity experiment and drug sensitive test paper experiment. The strain FUA329 was found to produce no haemolysis by comparison with β -haemolytically active staphylococcus aureus (s. Aureus), as shown in figure 3. The strain FUA329 was found to be resistant to 9 antibiotics in 27 drug susceptibility tests, see Table 2 below.
TABLE 2 results of the drug susceptibility test of the strain FUA329
Note: r: resistance; i: an intermediary; s: sensitivity.
2. Probiotic analysis of strain FUA329
Acid resistance experiment: preparing ABB liquid culture media with pH values of 2.0, 3.0, 4.0, 5.0 and 6.0 respectively, inoculating the activated strain FUA329 into the ABB liquid culture media with different pH values by 1 percent of inoculation amount, performing anaerobic culture at 37 ℃, and sampling at 0h, 1h, 2h and 3h respectively. Viable count of each test was calculated by dilution spread plate method, and log CFU/ml was used as a control with the original ABB liquid medium results at pH6.8 + -0.2, and each test was repeated three times. The tolerance to acid environment of the strain was calculated by the following formula.
Survival rate (%) = a 1 /A 0 ×100％
In the formula: a. The 0 、A 1 Viable counts of the control group and the experimental group were obtained.
After being ingested, the probiotics reach the stomach through the digestive tract, so the tolerance to gastric juice is an important index for developing and utilizing probiotic resources, the pH of human gastric juice is generally 2.0-3.0, the gastric emptying time is about 2-3 h, the probiotic product is generally recommended to be eaten 0.5h after meal, so the survival rate of acid tolerance of the screened strain is evaluated by adopting the pH of 2.0, 3.0, 4.0, 5.0 and 6.0 and the retention time of 1, 2 and 3h, and the tolerance of the strain to extreme acid environment in gastric juice is tested. The results show that the survival rate of the strain is reduced to different degrees along with the reduction of the pH value and the prolonging of the culture time, but the survival rate of the FUA329 treated for 3 hours under different pH conditions is more than 58 percent, and the figure 8 shows that the survival rate is higher than that of the strain.
Bile salt resistance test: preparing ABB culture medium containing 0.1%, 0.2%, 0.3%, 0.4%, 0.5% bile salt and no bile salt, inoculating activated strain FUA329 into ABB liquid culture medium with 1% inoculum size, anaerobically culturing at 37 deg.C, and sampling at 0h, 1h, 2h, and 3 h. And calculating the number of viable bacteria in each group of test by adopting a dilution coating plate method, recording the number of the viable bacteria in each group by log CFU/ml, taking the result of the original ABB liquid culture medium without bile salt as a control group, repeating the test in each group for three times, and calculating the tolerance rate of the strain to the bile salt environment according to the following formula.
Survival rate (%) = B 1 /B 0 ×100％
In the formula: b is 0 、B 1 The viable count of the control group and the experimental group are respectively.
In order to exert the probiotic effect in human intestinal tracts, the probiotic strains must be capable of resisting the bacteriostatic effect of bile salts widely distributed in intestinal cavities, so the tolerance capability of the strains to the bile salts is one of important standards for screening probiotics. Thus, in this study comparing the ability of the selected strains to tolerate bile from 0.1% to 0.5%, the survival rates of FUA329 were less than 60% after 3h in 0.4% and 0.5% bile salt medium, and 61%, 70.9% and 77.7% after 3h in 0.3%, 0.2% and 0.1% bile salt medium, respectively. The research proves that the strain FUA329 has good probiotic activity and potential to become novel probiotics through in-vitro acid and bile salt resistance experiments, and is shown in figure 8.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and shall cover the scope of the present invention.
Claims (10)
1. A streptococcus thermophilus FUA329, which has been preserved in China general microbiological culture collection center in 2022, 5 months and 23 days, with the preservation number of CGMCC NO.24963.
2. Streptococcus thermophilus FUA329 according to claim 1 for the production of urolithin A.
3. Use of a composition comprising streptococcus thermophilus FUA329 according to claim 1 for the preparation of a medicament for improving the intestinal flora.
4. The method of screening streptococcus thermophilus FUA329 according to claim 1, comprising the steps of:
i) Breast milk samples for verification of urolithin a production: inoculating breast milk sample into ABB liquid culture medium containing ellagic acid under aseptic condition, culturing under anaerobic condition for 4 days, comparing with ellagic acid-containing and breast milk-free culture medium and breast milk-containing and ellagic acid-free culture medium, collecting sample once per day, and adding organic solvent C 2 H 3 N:H 2 HCOOH (80, 19.9, V;
ii) screening for urolithin A producing strains: diluting the mother milk sample fermentation liquor which is verified to produce the urolithin A by adding normal saline, coating 0.1mL of the diluted fermentation liquor on an ABB solid plate, culturing under anaerobic conditions to obtain a plurality of microbial colonies, respectively inoculating each microbial colony into an ABB liquid culture medium containing ellagic acid, performing fermentation culture under anaerobic conditions, performing HPLC and HPLC-MS/MS analysis on the fermentation liquor, and screening a strain producing the urolithin A, wherein the strain is named FUA329.
5. The method of streptococcus thermophilus FUA329 for production of urolithin a according to claim 1, comprising the steps of:
1) Activating strains: taking FUA329 stored in glycerin pipe in a refrigerator at-80 deg.C, and performing three-zone streaking on ABB solid culture medium to obtain single colony;
2) Seed culture: inoculating the activated single colony of the strain FUA329 into a seed culture medium to culture to obtain a seed solution;
3) Fermentation culture: inoculating the seed solution into a fermentation culture medium added with ellagic acid for fermentation culture;
4) Separation and extraction: and centrifuging the fermentation liquor to obtain supernatant, namely the crude urolithin A extraction liquid.
6. The method for producing urolithin a according to claim 5, wherein said ABB solid medium formulation in step 1) is ABB anaerobic base broth supplemented with 2% agar.
7. The method for producing urolithin a according to claim 5, wherein said seed culture medium in step 2) is ABB anaerobic basal broth, ph6.8 ± 0.2, and the formulation comprises: 16.0g/l of peptone, 7.0g/l of yeast extract powder, 5.0g/l of sodium chloride, 1.0g/l of starch, 1.0g/l of glucose, 1.0g/l of sodium pyruvate, 1.0g/l of arginine, 0.5g/l of sodium succinate, 0.5g/l of cysteine hydrochloride, 0.4g/l of sodium bicarbonate, 0.5g/l of ferric pyrophosphate, 0.005g/l of hemin, 0.0005g/l of vitamin, 0.5g/l of sodium thioglycolate and 1.0g/l of dithiothreitol; the culture condition in the step 2) is to culture for 24 hours under anaerobic condition at 37 ℃.
8. The method for producing urolithin A according to claim 5, wherein the fermentation medium in step 3) is ABB anaerobic basal broth medium containing 20 μ M ellagic acid.
9. The method for producing urolithin A according to claim 5, wherein the amount of inoculation of the seed solution in step 3) is 2%, and the fermentation culture conditions are anaerobic culture at 37 ℃ for 48h.
10. The method for producing urolithin A according to claim 5, wherein the centrifugation conditions in step 4) are 12000 Xg at 4 ℃ for 10min.
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| CN110693880A (en) * | 2019-10-24 | 2020-01-17 | 广州中医药大学第一附属医院 | Urolithin preparation and application thereof |
| CN111978282A (en) * | 2020-09-10 | 2020-11-24 | 四川轻化工大学 | Synthesis method of urolithin A |
| CN112074510A (en) * | 2018-02-27 | 2020-12-11 | 阿马曾提斯公司 | Industrial-Scale Synthesis of urolithin A |
| CN113813255A (en) * | 2021-10-20 | 2021-12-21 | 山东大学 | Application of urolithin A and derivatives thereof in tumor immunotherapy |
| CN114404407A (en) * | 2022-03-03 | 2022-04-29 | 苏州大学附属第一医院 | Application of urolithin A in preparation of medicine for preventing or treating senile osteoporosis |
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| CN112074510A (en) * | 2018-02-27 | 2020-12-11 | 阿马曾提斯公司 | Industrial-Scale Synthesis of urolithin A |
| CN110693880A (en) * | 2019-10-24 | 2020-01-17 | 广州中医药大学第一附属医院 | Urolithin preparation and application thereof |
| CN111978282A (en) * | 2020-09-10 | 2020-11-24 | 四川轻化工大学 | Synthesis method of urolithin A |
| CN113813255A (en) * | 2021-10-20 | 2021-12-21 | 山东大学 | Application of urolithin A and derivatives thereof in tumor immunotherapy |
| CN114404407A (en) * | 2022-03-03 | 2022-04-29 | 苏州大学附属第一医院 | Application of urolithin A in preparation of medicine for preventing or treating senile osteoporosis |
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