CN115747098B - Streptococcus thermophilus FUA329 and method for producing urolithin A by fermentation of streptococcus thermophilus FUA329 - Google Patents

Streptococcus thermophilus FUA329 and method for producing urolithin A by fermentation of streptococcus thermophilus FUA329 Download PDF

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CN115747098B
CN115747098B CN202211224627.6A CN202211224627A CN115747098B CN 115747098 B CN115747098 B CN 115747098B CN 202211224627 A CN202211224627 A CN 202211224627A CN 115747098 B CN115747098 B CN 115747098B
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urolithin
fua329
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streptococcus thermophilus
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房耀维
刘姝
杨光
候晓月
刘琪彤
叶沁文
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Jiangsu Ocean University
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Abstract

The invention provides a streptococcus thermophilus FUA329 which is derived from human breast milk and can convert ellagic acid into urolithin A, and provides a new way for preparing the urolithin A. The technology has convenient operation and high yield (the conversion rate is 82%), and is suitable for mass production of urolithin A. In addition, the streptococcus thermophilus FUA329 provided by the invention has the advantages of high safety, good probiotics and potential of becoming novel probiotics.

Description

Streptococcus thermophilus FUA329 and method for producing urolithin A by fermentation of streptococcus thermophilus FUA329
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 by using the streptococcus thermophilus FUA329.
Background
In 1980, urolithin A was first found in ellagic acid fed rat metabolites of formula C 13 H 8 O 4 The relative molecular mass was 228.2. Subsequently, urolithin A was found to have various biological activities. Such as modulating mitochondrial biosynthesis and autophagy-induced mitochondrial lesion clearance to enhance muscle function; modulating estrogen receptors inhibits the probability of high-fat diet-mediated uterine tumorigenesis; prevention and treatment of prostate cancer by modulation of androgen receptor; can significantly inhibit typical inflammatory factors, such as enteritis and osteoarticular diseasesHas inhibiting effects on inflammation, neuritis, kidney inflammation, intervertebral disc lesion, etc.; the risk of cardiac metabolism can be effectively reduced; can prevent D-galactose-induced brain aging and improve cognitive dysfunction; can prevent and reverse obesity caused by high fat diet, improve metabolism, and has no side effects.
A great deal of research shows that a plurality of intestinal microorganisms and enzymes participate in the process of metabolizing ellagic acid into urolithin. Humans can be classified into three types according to whether ellagic acid is metabolized by intestinal microorganisms to produce urolithin and the kinds of urolithin produced thereby: urolithin A type, wherein the final product is urolithin A; urolithin B type, wherein the final products are urolithin B and isourolithin A; urolithin type 0, no urolithin is produced. It has been found that the proportion of urolithin type B increases significantly in people suffering from chronic diseases such as metabolic syndrome and colorectal cancer, and only about 50% of people over 40 years old have artificial urolithin type a. Since urolithin a has various probiotic functions and a low proportion of the population of urolithin a is in the elderly and those suffering from chronic diseases, the us FDA approved urolithin a for use in dietary supplements in 2018. Amazentis company developed a nutritional supplement commercial product, mitopure, with high purity urolithin a as the major ingredient, for combating cell and muscle decline.
At present, the industrial production of the urolithin mainly utilizes a chemical method, but the method has the advantages of long reaction time, high cost, high energy consumption and high product price. If a new generation of probiotics which can convert ellagic acid into urolithin A is developed and obtained, the method can be used for preparing the urolithin A by utilizing a microbial fermentation method, fermenting tea and food rich in ellagic acid and preparing a probiotic fermented drink rich in the urolithin A. However, only Gordonibacter urolithinfaciens and Gordonibacter pamelaece are reported to convert ellagic acid in vitro to produce isourolithin A, bifidobacterium pseudocatenulatum is reported to convert ellagic acid to produce urolithin A and urolithin B, and the strains are isolated from human intestinal tracts. Liriol A was prepared from K.K. cellophane by mixed fermentation of Eggerthella sp., clostridium boltec, clostridium citroniae, clostridium asparagiforme, etc. strains to convert ellagic acid. Therefore, the strain capable of converting ellagic acid into urolithin A in multiple channels is explored, and the strain capable of generating urolithin A in vivo and in vitro is screened, so that the defect of the current urolithin A preparation technology can be overcome, and the method has important significance for industrial production.
Disclosure of Invention
The invention provides a streptococcus thermophilus FUA329 (Streptococcus thermophilus) which is derived from human breast milk and aims at overcoming the defects of the prior art.
The streptococcus thermophilus FUA329 has been preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 24963 in 2022, 5 and 23.
The streptococcus thermophilus FUA329 provided by the invention is used for producing urolithin A.
The invention provides a use of a composition for preparing a medicament for improving intestinal flora, wherein the composition contains streptococcus thermophilus FUA329.
In one aspect, the invention provides a screening method of streptococcus thermophilus FUA329, comprising the following steps:
i) Verification of a urolithin a-producing breast milk sample: inoculating breast milk sample into ABB liquid culture medium containing ellagic acid under aseptic condition, culturing under anaerobic condition for 4 days, collecting sample once daily with organic solvent C 2 H 3 N:H 2 O is HCOOH (80:19.9:0.1, V: V) is extracted in an equal volume, HPLC and HPLC-MS/MS analysis are carried out, and breast milk samples for producing urolithin A are screened;
ii) screening for urolithin A-producing strains: after diluting with normal saline, 0.1mL of the mother milk sample fermentation broth which is verified to produce urolithin A is coated on an ABB solid plate, a plurality of microbial colonies are obtained after culturing under anaerobic conditions, each colony is respectively inoculated into an ABB liquid culture medium containing ellagic acid, fermentation culture is carried out under anaerobic conditions, HPLC and HPLC-MS/MS analysis are carried out on the fermentation broth, and a strain which produces urolithin A is screened out and named FUA329.
In another aspect, the invention provides a method for producing urolithin A by using the streptococcus thermophilus FUA329, which comprises the following steps:
1) Activating strains: taking FUA329 preserved in a glycerol tube in a refrigerator at the temperature of minus 80 ℃ to carry out three-area lineation on an ABB solid culture medium to obtain a single colony;
2) Seed culture: inoculating single colony of activated strain FUA329 into a seed culture medium for culturing to obtain seed solution;
3) Fermentation culture: inoculating the seed solution into a fermentation medium added with ellagic acid for fermentation culture;
4) Separating and extracting: and centrifuging the fermentation liquor, and obtaining supernatant fluid which is the urolithin A crude extract.
The ABB solid medium formulation in step 1) was ABB anaerobic base broth medium supplemented with 2% agar.
The seed culture medium in the step 2) is an ABB anaerobic basic broth culture medium with the pH value of 6.8+/-0.2, and the formula comprises: 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 hemin, 0.0005g/l vitamin K, 0.5g/l sodium thioglycolate, and 1.0g/l dithiothreitol.
The culture condition in the step 2) is that the culture is performed under anaerobic condition at 37 ℃ for 24 hours.
The fermentation medium in step 3) is an ABB anaerobic basal broth medium containing 20 μm ellagic acid.
The seed liquid inoculation amount in the step 3) is 2%.
The fermentation culture condition in the step 3) is that the fermentation culture is performed under anaerobic condition at 37 ℃ for 48 hours.
The centrifugation conditions in step 4) were centrifugation at 12000 Xg at 4℃for 10min.
The invention has the beneficial effects that: the invention provides a streptococcus thermophilus FUA329 which is derived from human breast milk and can convert ellagic acid into urolithin A, and provides a new way for preparing the urolithin A. The technology has convenient operation and high yield (the conversion rate is 82%), and is suitable for mass production of urolithin A. In addition, the streptococcus thermophilus FUA329 provided by the invention has high safety and good probiotics and has potential to become novel probiotics.
Drawings
FIG. 1 is a gram of strain FUA329 (x 1000);
FIG. 2 is a colony morphology of strain FUA 329;
FIG. 3 is a phylogenetic tree of strain FUA 329;
FIG. 4 is a liquid phase diagram (A) of a urolithin standard sample, wherein the peak number 1 in the A diagram is urolithin D,2 is ellagic acid, 3 is urolithin M6,4 is urolithin C,5 is isourolithin A,6 is urolithin A, and 7 is urolithin B; urolithin A standard mass spectrum (a) the liquid phase diagram (B) of the fermentation broth of the strain FUA329 shown in fig. 5; the strain FUA329 converts ellagic acid to produce a urolithin A fermentation broth mass spectrum (b);
FIG. 6 is a graph showing the results of a hemolysis plate experiment of strain FUA 329;
FIG. 7 shows the transformation process of ellagic acid to urolithin A by fermentation of strain FUA 329;
FIG. 8 shows the results of acid tolerance and bile salt tolerance experiments of strain FUA329 after treatment at different pH and bile salt concentrations in vitro.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
Example 1 screening of strains
Example 1: isolation and identification 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 (China general microbiological culture Collection center) at the date of 5 and 23 of 2022, and the preservation unit address: the national institute of sciences of China, the collection of bacterial species, north Chen Xielu No. 1, 3, the Korean region of Beijing. The contact telephone is 010-64807355 with the preservation number of CGMCC NO.24963. The GenBank accession number of the 16S rRNA gene sequence of the strain is OM892001.
1. Screening of strains:
1) Verification of urolithin A-producing Breast milk sampleThe method comprises the following steps: inoculating 1ml of breast milk sample into ABB liquid culture medium containing 20 μm ellagic acid under aseptic condition, culturing under anaerobic condition for 4 days, taking culture medium containing ellagic acid and no breast milk and culture medium containing breast milk and no ellagic acid as control, collecting 10ml sample once daily, and using organic solvent C 2 H 3 N:H 2 O:CH 2 O 2 (80:19.9:0.1, v:v: equal volume extraction, HPLC and HPLC-MS/MS analysis, screening out breast milk sample for producing urolithin A;
2) Screening for urolithin A-producing strains: diluting the fermentation broth with normal saline to 10 -3 -10 -7 Then, 0.1mL of the culture medium was applied to an ABB solid plate, cultured at 37℃for 24 to 48 hours 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 culture was performed at 37℃for 48 hours 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 grows for 48 hours at 37 ℃ in an ABB anaerobic basic broth culture medium, and the colony is round, milk white and semitransparent, has a moist surface, regular edges, no halo and a central protuberance, has a diameter of 0.5-1.0mm and is easy to pick, and is shown in figures 1 and 2.
3. And (3) physiological and biochemical identification:
the strain was subjected to salicylic acid test, H, by referring to the common bacteria System identification Manual (Dongxiu bead, cai Miaoying. Common bacteria System identification Manual Beijing, scientific Press, 2001) 2 O 2 The strain species are preliminarily determined by tests such as gelatin liquefaction tests, indole tests, nitrate reduction tests, mannitol, sorbitol, maltose, sucrose, fructose utilization and the like. Experimental results show that the strain is positive in mannitol test and salicylic acid test, H 2 O 2 The test, gelatin liquefaction test, indole test, bile lysis test, sorbitol, maltose, sucrose and fructose test results are all negative. The results of some physiological and biochemical processes are shown in Table 1.
TABLE 1 physiological and biochemical test results of strain FUA329
Note that: +: positive; -: negative;
4. amplification and analysis of the FUA 329S rRNA sequence of the Strain
The genome of FUA329 is obtained by extracting with 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 reactions: 27F:5'-AGAGTTTGATCCTGGCTCAG-3';1492R:5'-GGTTACCTTGTTACGACTT-3'.
The reaction system (25 μl) 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). The reaction procedure: denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 10s,30-35 cycles; final extension at 72℃for 2min. After sequencing, submitted to GenBank (accession number: OM 892001), the sequence was aligned with sequences in GenBank database to find strain Streptococcus thermophilus DSM20617 T (accession number: NR 118998) 16S rRNA gene sequence similarity was 99.57%. Phylogenetic tree showed that strain FUA329 was closest to Streptococcus thermophilus, see figure 3.
The GenBank accession number of the streptococcus thermophilus FUA329 16S rRNA gene sequence is OM892001, and the specific sequence is as follows:
TGCGGCAGCTATAATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACCCATGTCATTTATTTGAAAGGGGCAAATGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAACGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGAGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGTCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGAATGCAG。
example 2 fermentation of Strain FUA329 to convert ellagic acid to produce urolithin A and identification thereof
The seed culture medium is an ABB anaerobic basic broth culture medium, and the formula of the seed culture medium 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, 1.0g/l of dithiothreitol and pH6.8+/-0.2.
The fermentation medium was ABB anaerobic basal broth medium containing 20 μm ellagic acid.
1. Activation of the strain: activating three-area lines of streptococcus thermophilus FUA329 kept in a refrigerator at-80 ℃ on an ABB solid culture medium;
2. preparing seed liquid: inoculating single colony of strain FUA329 into a seed culture medium, and culturing for 24 hours at 37 ℃ under anaerobic condition to obtain seed solution;
3. preparation of fermentation liquor: inoculating 1% seed solution to fermentation medium, culturing at 37deg.C under anaerobic condition for 48h, centrifuging at 12000 Xg at 4deg.C for 10min, and collecting supernatant to obtain urolithin A fermentation broth;
hplc and UPLC-MS identification analysis: taking 1.0ml of fermentation liquor and C 2 H 3 N:H 2 O:CH 2 O 2 The (80:19.9:0.1, V: V) solution was mixed in equal volumes, and after thorough mixing, 1.0ml was aspirated by syringe, and after passing through 0.22 μm organic filter, added to HPLC sample bottles and labeled for detection analysis.
1) HPLC analysis: chromatographic analysis of the samples was performed on a ZORBAX SB-C18 column (Agilent, USA) of 250X 4.6mm, particle size 5 μm. Acetonitrile and 1% methanol are used as mobile phases, the sample injection amount is 5 μl, and the flow rate is 1ml/min. Recording was performed at 305 nm. The gradient of elution is: 0-15 min, 0-20% acetonitrile; 15-20 min, 20-70% acetonitrile; 20-21 min, 70-95% acetonitrile; 21-24 min, 95-100% acetonitrile; 24-25 min, 100-20% acetonitrile; urolithin standard sample configuration: 0.005g of urolithin A, urolithin C, urolithin D and isourolithin A,0.004g of urolithin B and 0.006g of urolithin M6 are weighed out respectively, 1ml of water and 1ml of C are added 2 H 3 N:H 2 O:CH 2 O 2 (80:19.9:0.1, V: V) solution, and after mixing, passing through an organic filter membrane of 0.22 μm into a sample bottle to obtain a urolithin standard sample with the concentration of 20 μm.
2) HPLC-MS/MS analysis: the sample was analysed using a C18 column (ACQUITY UPLC BEH C, 2.1X10 mm,1.7 μm) using ACQUITY QDa ESIMS for 150-1000 Da with 0.2% formic acid and acetonitrile as mobile phase, 0.5ml/min flow rate and 50. Mu.l sample volume. The gradient of elution is: 0-6 min, 10-100% acetonitrile; 6-7 min,100% acetonitrile; 7-8 min, 100-10% acetonitrile; 8-9 min,10% acetonitrile.
Comparison with standard samples of urolithin A, urolithin B, urolithin C, urolithin D, urolithin M6, isourolithin A and ellagic acid proves that FUA329 can produce urolithin A, and the analysis results are shown in FIG. 4 and FIG. 5.
Example 3: safety and probiotic analysis of strain FUA329
1. Strain FUA329 safety analysis
Hemolysis experiment: 1ml of the activated single colony was inoculated into ABB liquid medium, anaerobic culture was performed at 37℃for 12 hours, bacterial solution was centrifuged (8000 rpm. Times.4℃,5 minutes), bacterial cell pellet was resuspended in sterile normal saline, 100. Mu.l of the resuspended bacterial solution was pipetted onto a sheep blood agar plate containing 5% to perform three-region streaking, and after culturing at 37℃for 48 hours with staphylococcus aureus as a positive control, hemolysis of the colony grown on the plate was observed (FIG. 6).
Drug sensitivity test: the test was performed using 27 antibiotics (amikacin, norfloxacin, ofloxacin, ciprofloxacin, levofloxacin, erythromycin, tetracycline, cefuroxime, cefazolin, cefalotin, cefotaxime, ceftriaxone, ceftazidime, piperacillin, ampicillin, oxacillin, penicillin G, aztreonam, compound neonomine, nitrofurantoin, chloramphenicol, polymyxin B, clindamycin, kanamycin, gentamicin, streptomycin, vancomycin) according to a drug sensitive kit (Hangzhou beach microbial agent Co., ltd.). The strain is activated and cultured to logarithmic phase, 100 μl of 10 is taken -3 The concentrated bacterial solution was spread on ABB solid medium. The drug sensitive paper sheets containing different concentrations are placed on an ABB solid flat plate, after anaerobic culture is carried out for 48 hours at 37 ℃, the size of a bacteriostasis zone is measured, each group of tests are repeated for 3 times to calculate the average value, and the sensitivity of the strain to different drugs is judged by referring to the performance standard of the sensitivity test of the antibacterial drug paper sheet method.
The safety of the strain FUA329 is studied by a hemolytic activity experiment, a drug sensitive test paper experiment and the like. By comparison with beta-hemolytic active staphylococcus aureus (s.aureus), strain FUA329 was found not to produce hemolysis, as shown in fig. 3. Strain FUA329 was found to develop resistance to 9 antibiotics in 27 drug sensitive assays, see table 2 below.
TABLE 2 results of FUA329 drug sensitivity test of Strain
Note that: r: resistance; i: an intermediary; s: sensitivity.
2. Strain FUA329 probiotic assay
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 activated strain FUA329 into the ABB liquid culture media with different pH values according to an inoculum size of 1%, performing anaerobic culture at 37 ℃ and sampling at 0h, 1h, 2h and 3h respectively. The number of viable bacteria in each group of tests was calculated by a dilution-coated plate method, the original ABB liquid medium results with pH value of 6.8+ -0.2 were recorded as control groups, and each group of tests was repeated three times. The tolerance of the strain to the acid environment was calculated according to the following formula.
Survival (%) =a 1 /A 0 ×100％
Wherein: a is that 0 、A 1 Viable count of control and experimental groups, respectively.
The probiotics reach the stomach through the alimentary canal after being ingested, so the size of the tolerance to gastric juice is an important index for developing and utilizing the resources of the probiotics, the pH of human gastric juice is generally 2.0-3.0, the gastric emptying time is about 2-3 h, the probiotics products are usually recommended to be eaten after meal for 0.5h, the pH is 2.0, 3.0, 4.0, 5.0 and 6.0, the survival rate of the acid tolerance of the screening strains is evaluated by adopting the pH of 1, 2 and 3h, and the tolerance of the strains to the extremely acidic environment in gastric juice is tested. The results showed that the viability of the strain decreased to varying degrees with decreasing pH and prolonged incubation time, but that the viability of FUA329 after 3h treatment at different pH was above 58% as shown in figure 8.
Bile salt resistance experiment: 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 the ABB liquid culture medium respectively at 1% inoculation amount, anaerobic culturing at 37deg.C, and sampling at 0h, 1h, 2h, and 3h respectively. The number of viable bacteria in each group of test is calculated by adopting a dilution coating flat plate method, the results of the original ABB liquid culture medium without bile salts are recorded by log CFU/ml and are used as a control group, each group of test is repeated three times, and the tolerance rate of the strain to the bile salt environment is calculated according to the following formula.
Survival (%) =b 1 /B 0 ×100％
Wherein: b (B) 0 、B 1 Viable count of control and experimental groups, respectively.
In order for a probiotic strain to exert its probiotic effect in the human intestinal tract, it is necessary to be able to tolerate the bacteriostatic effect of bile salts widely distributed in the intestinal lumen, and therefore the ability of the strain to tolerate bile salts is also one of the important criteria for screening probiotics. Thus, the present study compares the ability of the screened strain to tolerate 0.1% -0.5% bile, with the survival rate of strain FUA329 after 3h of culture in 0.4% and 0.5% bile salt medium being less than 60% and the survival rates after 3h of culture in 0.3%, 0.2% and 0.1% bile salt medium being 61%, 70.9% and 77.7%, respectively. The in vitro acid resistance and bile salt resistance experiments prove that the strain FUA329 has better probiotic activity and potential to become novel probiotics, and the figure is 8.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention.

Claims (8)

1. Streptococcus thermophilus (Streptococcus thermophilus) FUA329 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 24963 in 2022, 5 and 23.
2. Use of streptococcus thermophilus FUA329 according to claim 1 for the production of urolithin a.
3. The method of using streptococcus thermophilus FUA329 for producing urolithin a according to claim 1, comprising the steps of:
1) Activating strains: taking FUA329 preserved in a glycerol tube in a refrigerator at the temperature of minus 80 ℃ to carry out three-area lineation on an ABB solid culture medium to obtain a single colony;
2) Seed culture: inoculating single colony of activated strain FUA329 into a seed culture medium for culturing to obtain seed solution;
3) Fermentation culture: inoculating the seed solution into a fermentation medium added with ellagic acid for fermentation culture;
4) Separating and extracting: and centrifuging the fermentation liquor, and obtaining supernatant fluid which is the urolithin A crude extract.
4. A method for producing urolithin a according to claim 3, wherein said ABB solid media in step 1) is formulated as ABB anaerobic basal broth supplemented with 2% agar.
5. A method for producing urolithin a according to claim 3, wherein said seed medium in step 2) is ABB anaerobic basal broth medium, ph6.8±0.2, comprising: 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 hemin, 0.0005g/l vitamin K, 0.5g/l sodium thioglycolate, 1.0g/l dithiothreitol; the culture condition in the step 2) is that the culture is performed under anaerobic condition at 37 ℃ for 24 hours.
6. A method for producing urolithin a according to claim 3, wherein said fermentation medium in step 3) is ABB anaerobic basal broth medium containing 20 μm ellagic acid.
7. A method for producing urolithin a according to claim 3, wherein said seed solution in step 3) is inoculated in an amount of 2% and the fermentation culture condition is anaerobic culture at 37 ℃ for 48 hours.
8. A method for producing urolithin a according to claim 3, wherein the centrifugation conditions in step 4) are centrifugation at 12000 x g 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
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