Lactococcus garvei FUA009 and method for producing urolithin A thereof

Abstract

The present invention belongs to the field of bioengineering technology, and provides a strain of Lactococcus garvei FUA009 isolated from human intestinal tract, and a method for producing urolithin A using the strain. The method is easy to operate, has high yield, and is suitable for mass production of urolithin A. The Lactococcus garvei FUA009 provided by the present invention has high safety, good probiotic properties, and has the potential to become a new type of probiotic.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and provides a strain of Lactococcus garvieae FUA009 separated from human intestinal tract, and a method for producing urolithin A by using the strain. Background Art

[0002] Urolithin A (UA) is a metabolite produced by intestinal flora converting ellagic acid. Urolithin A was officially discovered and named as a metabolite of ellagic acid in 2005, and its molecular formula is C 13 H8O4, with a relative molecular mass of 228.2. Studies have shown that urolithin A can improve the cardiac function of ischemia / reperfusion mice by increasing the antioxidant activity of cardiomyocytes. Urolithin A can also induce selective autophagy in mitochondria by activating the PINK1 / Parkin ubiquitin-dependent pathway or the BNIP3 receptor, thereby reducing the damage of mitochondrial function caused by aging. Urolithin A can not only regulate mitochondria to maintain energy homeostasis, but also has significant therapeutic effects in improving chronic inflammation, cardiovascular disease, muscle dysfunction, neurodegenerative diseases, etc. In addition, when the model organism Caenorhabditis elegans was fed with urolithin A for a long time, it was found that urolithin A can activate mitochondrial autophagy through the AMPK signaling pathway, extending the lifespan of Caenorhabditis elegans by 45.4% compared with the control group. When healthy subjects ate strawberries rich in urolithin A precursors every day, their body weight and intestinal probiotics related to longevity were significantly improved. More importantly, urolithin A also plays an important role in improving human health, such as anti-obesity, intestinal diseases, and anti-cancer. It is particularly important to develop a method for producing chitosanase with high activity, high stability and low cost.

[0003] At present, urolithin A is a hot topic in the study of polyphenol metabolites, and how to obtain urolithin A efficiently and safely has attracted the attention of scholars. At present, urolithin A is mainly obtained by traditional chemical processes, which is not only too expensive but also pollutes the environment. However, urolithin A can also be obtained by intestinal flora transforming ellagitannins in vivo or in vitro, but not all human bodies can produce urolithin substances. Studies have found that the human body is divided into three metabolic types: type A, type B and type 0. Type A people can transform ellagitannins to only produce urolithin A, accounting for 67.1% of the total population. In addition to urolithin A, type B people also produce urolithin B and (or) isourolithin A. Type 0 people do not produce any urolithin metabolites in their bodies. The production of the above three metabolic types depends on the composition of the human intestinal flora. Therefore, screening a strain that can produce urolithin A in vivo and in vitro is of great significance to industrial production and human health research. Summary of the invention

[0004] In view of the shortcomings of the above-mentioned technologies, the present invention provides a strain of Lactococcus garvieae FUA009 (Lactococcus garvieae) producing urolithin A from human intestine.

[0005] A strain of Lactococcus gasseri FUA009, which was deposited in the General Microbiology Center of China Culture Collection Administration on May 23, 2022, with the deposit number CGMCC NO.24965.

[0006] The Lactococcus garvieae FUA009 provided by the present invention is used for producing urolithin A.

[0007] i) The screening method of Lactococcus gasseri FUA009 provided by the present invention is: 1.0 g of fecal sample is obtained from a volunteer who has been verified to produce urolithin A, after diluting 10 times with normal saline, 1.0 mL is added to 50 mL of ABB liquid culture medium, and cultured under anaerobic conditions at 37° C. for 48 hours;

[0008] ii) Then, 1.0 mL of liquid was obtained from the cell suspension by plate screening method, diluted and spread on ABB solid medium, and cultured under anaerobic conditions at 37° C. for 48 hours to obtain more than 50 microbial colonies;

[0009] iii) Each colony was inoculated into ABB liquid culture medium containing 1% ellagic acid, and fermented under anaerobic conditions at 37°C for 48 hours. Finally, the strain producing urolithin A was verified and found by liquid chromatography-mass spectrometry and named FUA009.

[0010] On the other hand, the present invention provides a method for producing urolithin A by using the Lactococcus garvieae FUA009, comprising the following steps:

[0011] 1) Activation of bacterial strains: Lactococcus garvieae FUA009 stored in a -80°C refrigerator was activated by streaking on a plate;

[0012] 2) Seed culture: Pick a single activated colony and inoculate it into a seed culture medium to obtain seed liquid;

[0013] 3) Fermentation culture: inoculating the seed liquid at an inoculum amount of 2% into a fermentation medium supplemented with 1% ellagic acid for fermentation culture;

[0014] 4) Separation and extraction: The fermentation broth is centrifuged and the resulting supernatant is the crude extract of urolithin A.

[0015] The seed culture medium in step 2) is ABB anaerobic basal broth medium, with the formula: peptone 16.0 g / L, yeast extract 7.0 g / L, sodium chloride 5.0 g / L, starch 1.0 g / L, glucose 1.0 g / L, sodium pyruvate 1.0 g / L, arginine 1.0 g / L, sodium succinate 0.5 g / L, cysteine ​​hydrochloride 0.5 g / L, sodium bicarbonate 0.4 g / L, ferric pyrophosphate 0.5 g / L, hemin chloride 0.005 g / L, vitamin K 0.0005 g / L, sodium thioglycolate 0.5 g / L, dithiothreitol 1.0 g / L, pH 6.8±0.2;

[0016] The culture condition in step 2) is culturing at 37° C. under anaerobic conditions for 24 hours.

[0017] The fermentation medium in step 3) is a seed medium containing 1% ellagic acid.

[0018] The inoculation amount of the seed solution in step 3) is 2%.

[0019] The fermentation culture condition in the step 3) is cultured under anaerobic conditions at 37° C. for 48 hours.

[0020] The centrifugation condition in step 4) is centrifugation at 12000×g and 4° C. for 10 min.

[0021] The present invention provides use of a composition for preparing a medicament for improving intestinal flora, wherein the composition contains Lactococcus garvieae FUA009.

[0022] Beneficial effects of the present invention: The present invention discloses a strain of Lactococcus garvieae FUA009 that can convert ellagic acid to produce urolithin A, and provides a new method for preparing urolithin A. The method is easy to operate, has a high yield, and is suitable for mass production of urolithin A. The Lactococcus garvieae FUA009 provided by the present invention has high safety, good probiotic properties, and has the potential to become a new type of probiotic. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Gram staining of strain FUA009 (×1000)

[0024] Figure 2 Phylogenetic tree of strain FUA009

[0025] Figure 3 The liquid phase and mass spectrometry of the fermentation broth of strain FUA009. The numbers in Figure A are 1: urolithin D, 2: ellagic acid, 3: urolithin M6, 4: urolithin C, 5: isoureolithin A, 6: urolithin A, 7: urolithin B; the numbers in Figure B are 1: ellagic acid; 2: urolithin A

[0026] Figure 4The results of the hemolysis plate experiment of strain FUA009

[0027] Figure 5 Results of the acid tolerance and bile salt tolerance experiments of strain FUA009 after treatment with different pH and bile salt concentrations in vitro DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0029] Example 1 Screening of strains

[0030] Strain FUA009 is Lactococcus garvieae FUA009 isolated from human intestines. The strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 23, 2022, with the deposit number CGMCCNO.24965. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The contact number is 010-64807355. The GenBank accession number of the 16S rDNA sequence of the strain is NR326725.

[0031] The culture medium used in the screening process was: ABB anaerobic basal broth medium, formula: peptone 16.0 g / L, yeast extract powder 7.0 g / L, sodium chloride 5.0 g / L, starch 1.0 g / L, glucose 1.0 g / L, sodium pyruvate 1.0 g / L, arginine 1.0 g / L, sodium succinate 0.5 g / L, cysteine ​​hydrochloride 0.5 g / L, sodium bicarbonate 0.4 g / L, ferric pyrophosphate 0.5 g / L, hemin chloride 0.005 g / L, vitamin K 0.0005 g / L, sodium thioglycolate 0.5 g / L, dithiothreitol 1.0 g / L, pH 6.8±0.2.

[0032] 1. Screening of strains:

[0033] 1.0g of fecal sample was obtained from a volunteer who had been verified to produce urolithin A. After diluting 10 times with normal saline, 1.0mL was added to 50mL ABB liquid culture medium and cultured at 37°C for 48 hours. Then, 1.0mL of liquid was obtained from the cell suspension by plate screening, diluted and spread on ABB solid culture medium, and cultured under anaerobic conditions at 37°C for 48 hours to obtain more than 50 microbial colonies. Each colony was inoculated in ABB liquid culture medium containing 1% ellagic acid, and fermented and cultured under anaerobic conditions at 37°C for 48 hours. Finally, the strain FUA009 that produces urolithin A was verified and found by liquid chromatography-mass spectrometry.

[0034] 2 Morphological characteristics:

[0035] This strain is a Gram-positive coccus. After growing in ABB anaerobic basal broth at 37°C for 48 hours, the colonies are round, milky white and translucent, with a moist surface, regular edges, no halo, and a central protrusion. The diameter is 0.5-1.0 mm, and it is easy to pick. Figure 1 .

[0036] 3 Physiological and biochemical characteristics:

[0037] (1) Decomposition test

[0038] Take the well-growing isolate FUA009 and inoculate it into a microbiological tube containing lactose, mannitol, esculin, salicin, sorbitol, arginine dihydrolase, and ribose, and culture at 37℃ for 2-5 days to observe the results, and set up a negative control. If there is an obvious color change, it is judged as positive.

[0039] (2) VP reaction

[0040] Take the isolated bacteria pure culture and inoculate it into the glucose peptone water culture medium, and culture it at 37℃ for 48-72h. First add 0.6mL of VP reagent A solution to the culture medium, then add 0.2mL of B solution, mix well, and let it stand for 15min before judging the result. If the culture medium turns red, it is positive, and if it does not change, it is negative.

[0041] (3) Catalase test

[0042] Take an appropriate amount of well-grown isolate FUA009 and place it on a clean glass slide, then drop a drop of 3% H2O2 solution and observe the result immediately. If bubbles are generated within 20 seconds, it means that the isolate can produce catalase, which means the result is positive.

[0043] Experimental results: The lactose test, mannitol test, esculin test, salicin test and arginine dihydrolase test of the strain FUA009 were positive, and the catalase test, sorbitol, ribose and VP reactions were all negative. Some physiological and biochemical results are shown in Table 1.

[0044] Table 1 Physiological and biochemical test results of strain FUA009

[0045]

[0046] Note: +: positive; -: negative;

[0047] 4 Amplification and analysis of 16S rDNA sequence of strain FUA009

[0048] The genome of FUA009 was extracted using an Axygen kit, and universal primers for amplifying the 16S rDNA sequence of prokaryotic microorganisms were used to react in a PCR mix system.

[0049] The universal primers used for the PCR reaction were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0050] The reaction system (50 μL) is: Premix (25 μL), ddH2O (22 μL), upstream and downstream primers (1 μL each), DNA template (1 μL). Reaction procedure: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 90 s, 35 cycles; final extension at 72°C for 5 min. After sequencing, the sequence was submitted to GenBank (accession number: NR326725). The sequence was compared with the sequence in the GenBank database for homology, and it was found that the similarity with the 16S rDNA of the strain Lactococcus garvieae (accession number: NR113268) was 98%. The phylogenetic tree shows that the strain FUA009 is most closely related to Lactococcus garvieae, see Figure 2 .

[0051] The GenBank accession number of the 16S rDNA sequence of Lactococcus garvei FUA009 is NR326725, and the specific sequence is as follows:

[0052]

[0053] Example 2 Fermentation process of strain FUA009 to produce urolithin A and identification of urolithin A

[0054] The seed culture medium is ABB anaerobic basal broth medium with the following formula: peptone 16.0 g / L, yeast extract powder 7.0 g / L, sodium chloride 5.0 g / L, starch 1.0 g / L, glucose 1.0 g / L, sodium pyruvate 1.0 g / L, arginine 1.0 g / L, sodium succinate 0.5 g / L, cysteine ​​hydrochloride 0.5 g / L, sodium bicarbonate 0.4 g / L, ferric pyrophosphate 0.5 g / L, hemin chloride 0.005 g / L, vitamin K 0.0005 g / L, sodium thioglycolate 0.5 g / L, dithiothreitol 1.0 g / L, pH 6.8±0.2.

[0055] The fermentation medium was ABB anaerobic basal broth medium containing 1% ellagic acid.

[0056] 1. Activate the Lactococcus garvieae FUA009 stored in a -80℃ refrigerator by streaking on a plate;

[0057] 2. Preparation of seed solution: A single colony of strain FUA009 was inoculated into ABB anaerobic basal broth medium and cultured at 37°C under anaerobic conditions for 24 h;

[0058] 3. Preparation of fermentation broth: Inoculate the seed liquid into the fermentation medium at a 2% inoculum amount and culture at 37°C under anaerobic conditions for 48 h.

[0059] 4. Sample Collection

[0060] After anaerobic culture for 48 hours, the fermentation liquid was centrifuged at 12000×g and 4°C for 10 minutes, and the supernatant was the crude extract of urolithin A.

[0061] 5HPLC and UPLC-MS analysis

[0062] Take 1.0 mL of fermentation broth and mix it with equal volumes of C2H3N:H2O:H-COOH (80:19.9:0.1) solution. After thorough mixing, draw 1.0 mL with a syringe, pass it through a 0.22 μm filter membrane, add it to the HPLC injection bottle and mark it.

[0063] HPLC analysis conditions: Chromatographic separation and analysis of samples were performed on a 250×4.6 mm, 5 μm particle size ZORBAX SB-C18 column (Agilent, USA). Acetonitrile and 1% methanol were used as the mobile phase, the injection volume was 5 μl, and the flow rate was 1 ml / min. Recording was performed at 305 nm. The elution gradient was: 0-15 minutes, 20-20% acetonitrile; 15-20 minutes, 20-70% acetonitrile; 20-21 minutes, 70-95% acetonitrile; 21-24 minutes, 95-100% acetonitrile; 24-25 minutes, 100-20% acetonitrile.

[0064] UPLC-MS analysis conditions: A C18 column (ACQUITY UPLC BEH C18, 2.1×50 mm, 1.7 μm) was used to analyze the samples at 150-1000 Da using ACQUITY QDa ESI MS. The mobile phase was 0.2% formic acid and acetonitrile, the flow rate was 0.5 ml / min, and the injection volume was 50 μl. The elution gradient was: 0-6 minutes, 10-100% acetonitrile; 6-7 minutes, 100-100% acetonitrile; 7-8 minutes, 100-10% acetonitrile; 8-9 minutes, 10-10% acetonitrile.

[0065] By comparing with urolithin A, B, C, D, M6, isourolitin A and ellagic acid standards, it was verified that FUA009 can produce urolithin A. The analysis results are shown in Figure 3 .

[0066] Example 3 Study on the safety and probiotic properties of strain FUA009

[0067] 1 Safety analysis of strain FUA009

[0068] The safety of strain FUA009 was studied by hemolysis plate and drug sensitivity tablet phenotype experiments. By comparing with Staphylococcus aureus (S. aureus), which has obvious hemolysis, it was found that strain FUA009 did not produce hemolysis. Figure 4 In the 27 drug sensitivity test, strain FUA009 was found to be resistant to 7 antibiotics, see Table 2.

[0069] Table 2 Results of drug sensitivity test of strain FUA009

[0070]

[0071]

[0072] Note: R: resistance; I: intermediate; S: sensitive.

[0073] 2 Analysis of the probiotic properties of strain FUA009

[0074] This study used acid tolerance to evaluate the survival rate of the screened strains after passing through the stomach. At the same time, combined with the physiological parameters of gastric juice and gastric emptying time of healthy people and the fact that probiotic products are usually recommended to be consumed 0.5 hours after a meal, the acid tolerance of the screened strains was determined using pH 2.0, 2.5, 3.0, 3.5 and 4.0, and retention times of 1, 2 and 3 hours. The results showed that with the decrease in pH and the extension of culture time, the survival rate of the strains decreased to varying degrees, but the survival rate of FUA009 after 3 hours of treatment under different pH conditions was above 55%. In order for probiotic strains to play their probiotic role in the human intestine, they must be able to tolerate the antibacterial effect of bile salts widely distributed in the intestinal cavity. Therefore, the strain's tolerance to bile salts is one of the important criteria for screening probiotics. The concentration of bile salts changes with the time of feeding and different parts of the intestine. Within 1 hour after feeding, the bile salt concentration is between 1.5%-2% (w / v), and then decreases to about 0.3%. A large number of studies have selected a concentration range of 0.1%-0.5% when evaluating the bile salt tolerance of probiotics. Therefore, this study compared the tolerance of the screened strains to 0.1%-0.5% bile. The survival rate of strain FUA009 was less than 60% after culturing in 0.4% and 0.5% bile salt culture media for 3 hours, while the survival rates in 0.3%, 0.2% and 0.1% bile salt culture media were 62%, 64% and 66%, respectively. This study demonstrated through in vitro acid resistance and bile salt resistance experiments that strain FUA009 has good probiotic activity and has the potential to become a new type of probiotic, see Figure 5 .

Claims

1. A Lactococcus gasseri FUA009, which has been deposited in the China General Microbiological Culture Collection Center on June 15, 2022, with a deposit number of CGMCC NO. 24965.

2. The method for producing urolithin A by using Lactococcus garvei FUA009 according to claim 1, comprising the following steps: 1) Activation of bacterial strains: Lactococcus garvieae FUA009 stored in a -80°C refrigerator was activated by streaking on plates; 2) Seed culture: Pick a single activated colony and inoculate it into the seed culture medium to obtain seed liquid; 3) Fermentation culture: Inoculate the seed liquid at an inoculum rate of 2% into a fermentation medium supplemented with 1% ellagic acid for fermentation culture; 4) Separation and extraction: Centrifuge the fermentation broth and the resulting supernatant is the crude extract of urolithin A.

3. The method according to claim 2, characterized in that The seed culture medium in step 2) is ABB anaerobic basal broth medium, and the formula is: peptone 16.0 g / L, yeast extract powder 7.0 g / L, sodium chloride 5.0 g / L, starch 1.0 g / L, glucose 1.0 g / L, sodium pyruvate 1.0 g / L, arginine 1.0 g / L, sodium succinate 0.5 g / L, cysteine ​​hydrochloride 0.5 g / L, sodium bicarbonate 0.4 g / L, ferric pyrophosphate 0.5 g / L, hemin chloride 0.005 g / L, vitamin K 0.0005 g / L, sodium thioglycolate 0.5 g / L, and dithiothreitol 1.0 g / L.

4. The method according to claim 2, characterized in that The culture condition in step 2) is culturing at 37° C. under anaerobic conditions for 24 h.

5. The method according to claim 2, characterized in that The fermentation medium in step 3) is a seed medium containing 1% ellagic acid, and the fermentation culture condition in step 3) is cultured at 37° C. under anaerobic conditions for 48 h.

6. The method according to claim 2, characterized in that The inoculation amount of the seed solution in step 3) is 2%.

7. The method according to claim 2, characterized in that The centrifugation condition in step 4) is 12000×g and 4° C. for 10 min.

8. The Lactococcus garvei FUA009 of claim 1 is used for producing urolithin A.

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