Enterococcus faecium FUA027 and its method for producing urolithin A and application thereof

Abstract

The present invention belongs to the field of bioengineering technology, and provides a strain of Enterococcus faecium FUA027 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 Enterococcus faecium FUA027 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 Enterococcus faecium FUA027 separated from the intestine of a healthy human body, 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. In 2005, urolithin A was officially discovered and named as a metabolite of ellagic acid (EA), with a molecular formula of 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.

[0003] However, not everyone can naturally produce Urolithins in their body. According to the different metabolites, they can be divided into three metabolic types: type 0 (no Urolithins); type A (Uro-A); type B (Uro-B or isoUro-A in addition to Uro-A). This is due to the differences in intestinal flora between individuals, and the ability to metabolize EA to produce final metabolites and the benefits of eating EA-rich foods to individual health are also significantly different. However, the metabolic type is not static. Long-term intake of EA-rich foods or high-dose EA can convert some people with metabolic type 0 into metabolic type A or B. At present, the industrial production of Urolithins mainly uses chemical methods, which have long reaction time, high cost, high energy consumption, and expensive product prices. The microbial method converts EA to Urolithins by intestinal microorganisms. Although the microbial method has the advantages of safety and environmental protection, it has problems such as low product performance and harsh culture conditions. Therefore, screening a strain that can convert EA to urolithin A is of great significance for industrial production. Summary of the invention

[0004] In view of the shortcomings of the above-mentioned technology, the present invention provides a strain of Enterococcus faecium FUA027 (Enterococcus faecium) which is derived from human intestine and can convert EA into Uro-A.

[0005] Enterococcus faecium FUA027 (Enterococcus faecium), which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 23, 2022, with the deposit number CGMCC NO.24964. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, General Microbiology Center of China Microorganism Culture Collection Administration. The contact number is 010-64807850. The GenBank accession number of the 16S rDNA sequence of the strain is OM670243.

[0006] The Enterococcus faecium FUA027 provided by the invention is used for producing urolithin A.

[0007] The screening method of Enterococcus faecium FUA027 provided by the present invention is:

[0008] 1) Sample collection: Seven volunteers (age: 22-27 years old) with no history of gastrointestinal diseases were selected and no antibiotics were used in the three months before the study. Fecal samples were collected after they ate 0.7 g walnuts / kg body weight every day for two consecutive weeks.

[0009] 2) Enrichment culture: Fecal samples were inoculated into liquid culture medium supplemented with ellagic acid for enrichment culture for 72 h;

[0010] 3) Dilution and coating: dilute the culture solution in step 2) with sterile water, and apply 50 μL on the solid culture medium;

[0011] 4) Rescreening: Pick single colonies of different shapes and sizes and inoculate them into liquid culture medium supplemented with ellagic acid and culture for 48 hours;

[0012] 5) HPLC determination: using high performance liquid chromatography to screen out strains that can convert ellagic acid to produce urolithin A;

[0013] The solid culture medium and the liquid culture medium in the steps are both ABB anaerobic basal broth medium: 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, and pH is 6.8±0.2.

[0014] On the other hand, the present invention provides a method for using the Enterococcus faecium FUA027 to produce urolithin A, comprising the following steps:

[0015] 1) Activation of bacterial strains: Activate the Enterococcus faecium FUA027 stored in a -80°C refrigerator by streaking on a plate;

[0016] 2) Seed culture: Pick a single colony after activation and inoculate it into the seed culture medium to obtain seed liquid;

[0017] 3) Fermentation culture: inoculating the seed liquid into a fermentation medium supplemented with ellagic acid for fermentation culture;

[0018] 4) Separation and extraction: The fermentation broth is centrifuged and the resulting supernatant is the crude extract of urolithin A.

[0019] The seed culture medium in step 2) is GAM anaerobic culture medium: peptone 5.0 g / L, Peptone 5.0g / L, soy peptone 3.0g / L, yeast extract powder 2.5g / L, beef powder 2.2g / L, digested serum powder 10.0g / L, beef liver extract powder 1.2g / L, glucose 0.5g / L, potassium dihydrogen phosphate 2.5g / L, sodium chloride 3.0g / L, soluble starch 5.0g / L, L-cysteine ​​0.3g / L, L-arginine 1.0g / L, L-tryptophan 0.2g / L, sodium thioglycolate 0.3g / L, pH is 7.3±0.1.

[0020] The seed culture condition in step 2) is to culture at 37° C. under anaerobic conditions for 24 hours.

[0021] The fermentation medium in step 3) is ABB anaerobic basal broth medium: 16.0 g / L peptone, 7.0 g / L yeast extract, 5.0 g / L sodium chloride, 1.0 g / L starch, 1.0 g / L glucose, 1.0 g / L sodium pyruvate, 1.0 g / L arginine, 0.5 g / L sodium succinate, 0.5 g / L cysteine ​​hydrochloride, 0.4 g / L sodium bicarbonate, 0.5 g / L ferric pyrophosphate, 0.005 g / L hemin, 0.0005 g / L vitamin K, 0.5 g / L sodium thioglycolate, 1.0 g / L dithiothreitol, and the pH is 6.8±0.2.

[0022] The inoculation amount of the seed solution in step 3) is 2%.

[0023] The fermentation culture condition in the step 3) is cultured under anaerobic conditions at 37° C. for 48 hours.

[0024] The centrifugation condition in step 4) is centrifugation at 12000×g and 4° C. for 10 min.

[0025] The invention provides use of a composition for preparing a medicament for improving intestinal flora, wherein the composition contains Enterococcus faecium FUA027.

[0026] Beneficial effects of the present invention: The present invention discloses a strain of Enterococcus faecium FUA027 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 Enterococcus faecium FUA027 provided by the present invention has high safety and good probiotic properties, and has the potential to become a new type of probiotic. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the Gram staining image of strain FUA027 (×1000);

[0028] Figure 2 This is the colony morphology of strain FUA027;

[0029] Figure 3 is the phylogenetic tree of strain FUA027;

[0030] Figure 4 This is the liquid phase diagram of the fermentation broth of strain FUA027;

[0031] Figure 5 This is the mass spectrum of the fermentation broth of strain FUA027;

[0032] Figure 6 The conversion process of ellagic acid to urolithin A by strain FUA027;

[0033] Figure 7This is the result of hemolysis plate experiment of strain FUA027;

[0034] Figure 8 The results of the acid tolerance and bile salt tolerance experiments of strain FUA027 after treatment with different pH and bile salt concentrations in vitro DETAILED DESCRIPTION

[0035] 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.

[0036] Example 1 Screening of strains

[0037] The strain FUA027 was isolated from a healthy human fecal sample in the laboratory of Jiangsu Ocean University in Lianyungang City. The strain has been deposited in the China General Microbiological Culture Collection Center on May 23, 2022, with the deposit number CGMCCNO.24964. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, General Microbiological Center of China Microbiological Culture Collection. The contact number is 010-64807850. The GenBank accession number of the strain's 16S rDNA sequence is OM670243.

[0038] The culture medium used in the screening process was ABB anaerobic basal broth medium: 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.

[0039] 1. Screening of strains:

[0040] 1) Sample collection: Seven volunteers (age: 22-27 years old) with no history of gastrointestinal diseases were selected and no antibiotics were used in the three months before the study. Fecal samples were collected after they ate 0.7 g walnuts / kg body weight every day for two consecutive weeks.

[0041] 2) Enrichment culture: Fecal samples were inoculated into liquid culture medium supplemented with ellagic acid for enrichment culture for 72 h;

[0042] 3) Dilution and coating: dilute the culture solution in step 2) with sterile water, and apply 50 μL on the solid culture medium;

[0043] 4) Rescreening: Pick single colonies of different shapes and sizes and inoculate them into liquid culture medium supplemented with ellagic acid and culture for 48 hours;

[0044] 5) HPLC determination: High performance liquid chromatography was used to screen out strains that could convert ellagic acid into urolithin A.

[0045] 2 Morphological characteristics:

[0046] This strain is a Gram-positive cocci ( Figure 1 ). Grow on ABB medium at 37℃ for 2 days. The colonies are round, milky white, moist, with regular edges, no halo, and a central protrusion. The diameter is 0.1-0.5mm and it is easy to pick ( Figure 2 ).

[0047] 3 Physiological and biochemical characteristics:

[0048] 1) Nitrate reduction test: Use an inoculation loop to pick up a single isolated colony and inoculate it into a nitrate broth ampoule. After 24 hours, add nitrate reduction reagent A and B dropwise in a ratio of 5:2. Observe the results immediately. Red is positive, and no color change is negative.

[0049] 2) Methyl red test: Use the methyl red test kit to culture the MR-VP biochemical tube for 48 hours, add 5 drops of methyl red solution, and observe the results immediately. Red is positive and yellow is negative.

[0050] 3) Sugar alcohol fermentation test: Use a sugar alcohol kit and use an inoculation loop to pick up an isolated single colony and inoculate it into a sugar fermentation tube. Use bromocresol purple as an indicator and observe the results after 24 hours of incubation. Yellow is positive, and purple-gray or purple is negative.

[0051] The strain was negative in H2O2 test, nitrate reduction test, methyl red test, rhamnose and sorbitol test, but positive in mannitol, maltose, sucrose, fructose and salicin test. Some physiological and biochemical results are shown in Table 1.

[0052] Table 1 Physiological and biochemical test results of strain FUA027

[0053]

[0054] Note: +: positive; -: negative;

[0055] 4 Amplification and analysis of 16S rDNA sequence of strain FUA027

[0056] The genome of FUA027 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.

[0057] The universal primers used for the PCR reaction are: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0058] The reaction system (50 μL) was: Premix (25 μL), ddH2O (22 μL), upstream and downstream primers (1 μL each), DNA template (1 μL). Reaction procedure: 94°C denaturation for 5 min; 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 90 s, 35 cycles; 72°C final extension for 5 min. The sequence was submitted to GenBank (accession number: OM670243) after sequencing. The sequence was compared with the sequence in the GenBank database for homology and was found to be similar to the strain Enterococcus faecium ATCC 19434. T (Accession number: DQ411813) 16S rDNA similarity reached 99%. Phylogenetic tree showed that strain FUA027 was most closely related to Enterococcus faecium ( Figure 3 ).

[0059] Example 2 Fermentation process of strain FUA027 to produce urolithin A and identification of urolithin A

[0060] The seed culture medium is GAM anaerobic medium: peptone 5.0g / L, Peptone 5.0g / L, soy peptone 3.0g / L, yeast extract powder 2.5g / L, beef powder 2.2g / L, digested serum powder 10.0g / L, beef liver extract powder 1.2g / L, glucose 0.5g / L, potassium dihydrogen phosphate 2.5g / L, sodium chloride 3.0g / L, soluble starch 5.0g / L, L-cysteine ​​0.3g / L, L-arginine 1.0g / L, L-tryptophan 0.2g / L, sodium thioglycolate 0.3g / L, pH is 7.3±0.1.

[0061] The fermentation medium was ABB anaerobic basal broth medium: 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, and pH was 6.8±0.2.

[0062] 1. Activation of strains: Activate the Enterococcus faecium FUA027 stored in a -80℃ refrigerator by streaking on a plate;

[0063] 2. Preparation of seed solution: A single colony of strain FUA027 was inoculated into the seed culture medium and cultured at 37°C under anaerobic conditions for 24 h.

[0064] 3. Preparation of fermentation broth: Inoculate the seed liquid into the fermentation medium at a 2% inoculum, inoculate with 1% EA, and culture at 37°C under anaerobic conditions for 48 hours.

[0065] 4. Sample collection: Centrifuge the fermentation broth at 12000×g and 4℃ for 10min, and the resulting supernatant is the crude extract of urolithin A. Take 1.0mL of the crude extract of urolithin A and mix it with equal volumes of CH3CN:H2O:H-COOH (80:19.9:0.1) solution, and after thorough mixing, draw 1.0mL with a syringe, filter it through a 0.22μm filter membrane, add it to the HPLC injection bottle and mark it.

[0066] 5.HPLC and UPLC-MS detection and analysis:

[0067] HPLC analysis conditions: Chromatographic separation and analysis of samples was performed on a 250×4.6 mm, 5 μm particle size ZORBAX SB-C18 column (Agilent, USA). Acetonitrile and 1% formic acid were used as the mobile phase, the injection volume was 20 μL, and the flow rate was 1 mL / min. Recording was performed at 305 nm. The elution gradient was: 0-15 minutes, 0-20% 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. Analysis results ( Figure 4 ).

[0068] UPLC-MS analysis conditions: A C18 column (ACQUITY UPLC BEH C18, 2.1×50mm, 1.7μm) was used to analyze the samples at 150-1000Da using ACQUITY QDa ESIMS. The mobile phase was 0.2% formic acid and acetonitrile, the flow rate was 0.5mL / min, and the injection volume was 50μL. 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. Analysis results ( Figure 5 ).

[0069] The results of HPLC and UPLC-MS analysis are as follows Figure 4 and Figure 5 As shown, it was demonstrated that strain FUA027 can produce urolithin A.

[0070] 6. Study on the growth kinetics and catabolism of strain FUA027: Fermentation broth of strain FUA027 was prepared and cultured anaerobically at 37°C for 100 hours. During the culture process, 1.0 mL of the sample was taken every 2 hours and mixed with an equal volume of CH3CN:H2O:H-COOH (80:19.9:0.1) solution. After thorough mixing, 1.0 mL was drawn with a syringe and HPLC determination was performed after passing through a 0.22 μm filter membrane. The results showed that the catabolism of ellagic acid and the production of urolithin A occurred in the early logarithmic growth phase of Enterococcus faecium FUA027. The first metabolite produced was urolithin C, which appeared at 28 hours and reached a peak at 40 hours, with a maximum concentration of 1.54 μM. Urolithin A was detected after 40 hours, and reached a maximum concentration of 10.80 μM at 50 hours ( Figure 6 ).

[0071] Example 3 Study on the safety and probiotic properties of strain FUA027

[0072] 1. Safety analysis of strain FUA027

[0073] The safety of strain FUA027 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 FUA027 did not produce hemolysis ( Figure 7 ). The nitrate reductase activity and amino acid decarboxylase activity of strain FUA027 were detected by kits, and it was found that the metabolites of the test strain did not contain nitrate reductase, nor did they have lysine, ornithine, arginine, tryptophan, and phenylalanine decarboxylase activities, as shown in Table 2. In 27 drug sensitivity tests, it was found that strain FUA027 was resistant to 10 antibiotics, as shown in Table 3.

[0074] Table 2 Results of the amino acid decarboxylase activity test of FUA027 strain

[0075]

[0076] Table 3 Results of drug sensitivity test of FUA027 strain

[0077]

[0078]

[0079] Note: R: resistance; I: intermediate; S: sensitive.

[0080] 2. Analysis of the probiotic properties of strain FUA027

[0081] In order for probiotics to promote health, they must reach the intestines with a high number of viable bacteria. The low pH environment in the stomach and the antibacterial effect of bile salts widely distributed in the intestinal cavity have a great impact on their survival rate after passing through the stomach and intestines. This study studied the probiotic properties of strain FUA027 through in vitro acid resistance and bile salt resistance experiments. The results showed that FUA027 has strong tolerance, and its survival rate can reach 60% at a pH of 3.0 and a bile salt concentration of 0.5% ( Figure 8 ), indicating that the strain can survive well in the intestinal tract of animals and can ensure that it reaches the intestinal tract of the host to play a potential role. In addition, the in vitro probiotic performance of the test strain was evaluated by measuring the antibacterial property, hydrophobicity and self-aggregation rate of the test strain. It can be seen from Table 4 that the strain has different degrees of inhibitory effect on the two pathogenic bacteria (S. aureus, E. coli).

[0082] Table 4 Inhibitory diameter of FUA027 strain on pathogenic bacteria

[0083]

[0084] Whether probiotics can bind to the surface receptors of the intestinal mucosa depends on the surface properties of the strain. Strains with high hydrophobicity usually have strong adhesion to mucosal cells. In addition, in addition to adhesion, probiotics also need to reach a sufficient number through self-aggregation to fully exert their probiotic effects. It can be seen from Table 5 that compared with the commercial strain Bifidobacterium longum BB536, the FUA027 strain has strong hydrophobicity and high self-aggregation, both of which are greater than the control strain BB536. The study of the probiotic properties of the strain also includes antioxidant capacity detection, and the free radical scavenging rate of the strain is determined using an antioxidant kit. The results are shown in Table 6. The bacteria did not show scavenging ability, while the fermentation supernatant had a good scavenging effect. It can be obtained that the strain FUA027 has good probiotic activity and has the potential to become a new type of probiotic.

[0085] Table 5 Results of the strain cell surface hydrophobicity and self-aggregation test

[0086]

[0087] Note: BB536 is a commercial strain of Bifidobacterium longum

[0088] Table 6 Effects of strain FUA027 on DPPH, OH, O2 - The clearance rate

[0089]

[0090] Note: ND means no activity detected

[0091] LP001 and FUA004 are control strains Lactobacillus plantarum and Enterococcus faecalis, respectively.

Claims

1. An Enterococcus faecium FUA027, which has been deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC NO. 24964.

2. The Enterococcus faecium FUA027 according to claim 1 is used for producing urolithin A.

3. The method for producing urolithin A by using Enterococcus faecium FUA027 according to claim 1, comprising the following steps: 1) Activation of bacterial strains: Activate the Enterococcus faecium FUA027 stored in a -80℃ refrigerator by streaking on a plate; 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 into a fermentation medium supplemented with ellagic acid for fermentation culture; 4) Separation and extraction: Centrifuge the fermentation broth and the resulting supernatant is the crude extract of urolithin A.

4. The method for producing urolithin A according to claim 3, characterized in that: The seed culture medium in step 2) is GAM anaerobic culture medium: 5.0 g / L peptone, 5.0 g / L quinone, 3.0 g / L soytone, 2.5 g / L yeast extract powder, 2.2 g / L beef powder, 10.0 g / L digested serum powder, 1.2 g / L beef liver extract powder, 0.5 g / L glucose, 2.5 g / L potassium dihydrogen phosphate, 3.0 g / L sodium chloride, 5.0 g / L soluble starch, 0.3 g / L L-cysteine, 1.0 g / L L-arginine, 0.2 g / L L-tryptophan, 0.3 g / L sodium thioglycolate, and pH 7.3 ± 0.

1.

5. The method for producing urolithin A according to claim 3, characterized in that: The seed culture condition in step 2) is to culture at 37° C. under anaerobic conditions for 24 h.

6. The method for producing urolithin A according to claim 3, characterized in that: The fermentation medium in step 3) is ABB anaerobic basal broth medium: 16.0 g / L peptone, 7.0 g / L yeast extract, 5.0 g / L sodium chloride, 1.0 g / L starch, 1.0 g / L glucose, 1.0 g / L sodium pyruvate, 1.0 g / L arginine, 0.5 g / L sodium succinate, 0.5 g / L cysteine ​​hydrochloride, 0.4 g / L sodium bicarbonate, 0.5 g / L ferric pyrophosphate, 0.005 g / L hemin, 0.0005 g / L vitamin K, 0.5 g / L sodium thioglycolate, 1.0 g / L dithiothreitol, and the pH is 6.8±0.

2.

7. The method for producing urolithin A according to claim 3, characterized in that: The inoculation amount of the seed solution in step 3) is 2%.

8. The method for producing urolithin A according to claim 3, characterized in that: The fermentation culture condition in step 3) is to culture at 37°C under anaerobic conditions for 48 hours.

9. The method for producing urolithin A according to claim 3, characterized in that: The centrifugation condition in step 4) is 12000 × g, 4°C for 10 min.

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