CN115725451B

CN115725451B - Enterococcus faecium FUA027 and its method for producing urolithin A and application thereof

Info

Publication number: CN115725451B
Application number: CN202211134634.7A
Authority: CN
Inventors: 刘姝; 房耀维; 杨光; 侯晓月; 卢静; 张晓萌
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2025-03-28
Anticipated expiration: 2042-09-19
Also published as: CN115725451A

Abstract

The invention belongs to the technical field of bioengineering, and provides enterococcus faecium FUA027 separated from human intestinal tracts and a method for producing urolithin A by using the enterococcus faecium FUA 027. The method has the advantages of convenient operation and high yield, and is suitable for mass production of urolithin A. The enterococcus faecium FUA027 provided by the invention has high safety and good probiotics and has potential to become novel probiotics.

Description

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

Technical Field

The invention belongs to the technical field of bioengineering, and provides enterococcus faecium FUA027 separated from intestinal tracts of healthy human bodies and a method for producing urolithin A by utilizing the enterococcus faecium FUA 027.

Background

Urolithin a (Urolithin A, UA) is a metabolite produced by the conversion of ellagic acid by the intestinal flora. Urolithin A was formally found and named in 2005 as a metabolite of Ellagic Acid (EA) and has a molecular formula of C ₁₃H₈O₄ and a relative molecular mass of 228.2. Studies have shown that urolithin A can improve cardiac function in ischemic/reperfusion mice by increasing the antioxidant activity of cardiomyocytes. Urolithin A can also induce mitochondria to perform selective autophagy by activating a PINK1/Parkin ubiquitin dependent pathway or BNIP3 receptor, thereby reducing the damage of aging to mitochondrial function. Urolithin A can regulate and control mitochondria to maintain energy steady state, and has obvious curative effect in improving chronic inflammation, cardiovascular diseases, muscle dysfunction, neurodegenerative diseases and the like. In addition, when the model organism caenorhabditis elegans is fed with urolithin A for a long period of time, compared with a control group, the urolithin A can activate mitochondrial autophagy through an AMPK signaling pathway, so that the service life of the caenorhabditis elegans is prolonged by 45.4 percent. When healthy subjects consumed strawberries enriched with urolithin a precursors daily, both their body weight and life-prolonging intestinal probiotics were significantly improved. More importantly, urolithin A plays an important role in improving human health, such as obesity resistance, intestinal diseases resistance, cancer resistance and the like.

However, urolithins is not naturally produced in every human body, and can be classified into three metabolic types, type 0 (Urolithins is not produced), type A (Uro-A is produced), type B (Uro-B or isoUro-A is produced in addition to Uro-A), due to the difference in intestinal flora among individuals, and the capability of catabolizing EA to produce final metabolites and the benefit of eating EA-rich related foods on health of individuals are also significantly different. However, the metabolic type is not constant and prolonged consumption of EA-enriched foods or high doses of EA may convert part of the metabolic type 0 population to metabolic type a or B. At present, the industrial production of Urolithins mainly uses a chemical method, and the method has the advantages of long reaction time, high cost, high energy consumption and high product price. The conversion of EA by a microbiological method to Urolithins is completed by intestinal microbial catalysis, and the microbiological method has the advantages of safety and environmental protection, but has the problems of lower product performance, harsh culture conditions and the like. Therefore, screening a strain capable of transforming EA to generate urolithin A has important significance for industrial production.

Disclosure of Invention

Aiming at the defects of the technology, the invention provides enterococcus faecium FUA027 (Enterococcus faecium) which is obtained by converting EA into Uro-A in human intestinal tracts.

Enterococcus faecium FUA027 (Enterococcus faecium) has been deposited in China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC No.24964 at day 23 of 5 of 2022. The preservation unit address is North Star Xiyu No.1, 3 of the Korean area of Beijing, china general microbiological culture Collection center. The contact phone is 010-64807850. The GenBank accession number of the strain 16S rDNA sequence is OM670243.

The enterococcus faecium FUA027 provided by the invention is used for producing urolithin A.

The screening method of enterococcus faecium FUA027 provided by the invention comprises the following steps:

1) Sample collection 7 volunteers (age: 22-27 years) without history of gastrointestinal disease were selected and ensured that no antibiotics were used 3 months prior to the study, and fecal samples were collected after they had consumed 0.7g walnut/kg body weight every day for two consecutive weeks;

2) Enrichment culture, namely inoculating a fecal sample into a liquid culture medium added with ellagic acid for enrichment culture for 72 hours;

3) Diluting and coating, namely taking the culture solution in the step 2), carrying out gradient dilution by using sterile water, and taking 50 mu L of the culture solution to coat on a solid culture medium;

4) Screening again, namely picking single colonies with different forms and sizes, inoculating the single colonies into a liquid culture medium added with ellagic acid, and culturing for 48 hours;

5) HPLC determination, namely screening out strains capable of converting ellagic acid into urolithin A by utilizing a high performance liquid chromatography;

The solid culture medium and the liquid culture medium in the step are ABB anaerobic basic broth culture medium, namely 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 pH value of 6.8+/-0.2.

In another aspect, the invention provides a method for producing urolithin a by using the enterococcus faecium FUA027, comprising the following steps:

1) Activating the strain, namely activating enterococcus faecium FUA027 kept in a refrigerator at-80 ℃ through flat plate scribing;

2) Seed culture, namely picking the activated single colony and inoculating the single colony into a seed culture medium to obtain seed liquid;

3) Fermenting culture, namely inoculating seed solution into a fermentation culture medium added with ellagic acid for fermenting culture;

4) Separating and extracting, namely centrifuging the fermentation liquor, and obtaining supernatant fluid which is urolithin A crude extract.

The seed culture medium in the step 2) is GAM anaerobic culture medium, peptone is 5.0g/L,5.0G/L of peptone, 3.0g/L of soybean peptone, 2.5g/L of yeast extract powder, 2.2g/L of beef powder, 10.0g/L of digested serum powder, 1.2g/L of beef liver extract powder, 0.5g/L of glucose, 2.5g/L of potassium dihydrogen phosphate, 3.0g/L of sodium chloride, 5.0g/L of soluble starch, 0.3g/L of L-cysteine, 1.0g/L of L-arginine, 0.2g/L of L-tryptophan, 0.3g/L of sodium thioglycolate and pH of 7.3+/-0.1.

The seed culture condition in the step 2) is that the seed is cultured for 24 hours under the anaerobic condition at 37 ℃.

The fermentation medium in the step 3) is ABB anaerobic basic broth medium, namely 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 chlorhexidine, 0.0005g/L of vitamin K, 0.5g/L of sodium thioglycolate, 1.0g/L of dithiothreitol and the pH value is 6.8+/-0.2.

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 for 10min at 4 ℃.

The invention provides an application of a composition for preparing a medicine for improving intestinal flora, wherein the composition contains enterococcus faecium FUA027.

The invention has the beneficial effects that the invention discloses the enterococcus faecium FUA027 which can convert ellagic acid to generate urolithin A, and provides a novel method for preparing the urolithin A. The enterococcus faecium FUA027 provided by the invention has high safety and good probiotics and has potential to become novel probiotics.

Drawings

FIG. 1 is a gram stain (1000) of strain FUA 027;

FIG. 2 is a colony morphology of strain FUA 027;

FIG. 3 is a phylogenetic tree of strain FUA 027;

FIG. 4 is a liquid phase diagram of the fermentation broth of strain FUA 027;

FIG. 5 is a mass spectrum of the fermentation broth of strain FUA 027;

FIG. 6 shows the transformation of ellagic acid to urolithin A by fermentation of strain FUA 027;

FIG. 7 is a graph showing the results of a hemolysis plate experiment of strain FUA 027;

FIG. 8 shows the results of acid tolerance and bile salt tolerance experiments of strain FUA027 after in vitro treatment at different pH and bile salt concentration

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

The strain FUA027 is obtained by separating healthy human body excrement samples in a university laboratory of Jiangsu ocean in Lian hong Kong, and has been preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 24964 in 2022 and 5-23. The preservation unit address is North Star Xiyu No. 1, 3 of the Korean area of Beijing, china general microbiological culture Collection center. The contact phone is 010-64807850. The GenBank accession number of the strain 16S rDNA sequence is OM670243.

The culture medium used in the screening process is ABB anaerobic basic broth culture medium, namely 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 chlorhexidine, 0.0005g/L of vitamin K, 0.5g/L of sodium thioglycolate, 1.0g/L of dithiothreitol and pH value of 6.8+/-0.2.

1 Screening of strains:

5) HPLC determination, screening out strains capable of converting ellagic acid into urolithin A by high performance liquid chromatography.

2 Morphological characteristics:

the strain was gram-positive coccus (FIG. 1). The colonies were round, milky, moist, regular-edged, halo-free, central-protruding, 0.1-0.5mm diameter, easy to pick, grown on ABB medium at 37 ℃ for 2 days (fig. 2).

3 Physiological and biochemical characteristics:

1) And (3) nitrate reduction test, namely picking a single separated colony by an inoculating loop, inoculating the single separated colony into a nitrate broth ampoule bottle, sequentially dripping a nitrate reducing reagent A and a nitrate reducing reagent B according to a ratio of 5:2 after 24 hours, and immediately observing the result, wherein the red color is positive and the color is not changed to be negative.

2) Methyl red test, namely, detecting by using a methyl red kit, culturing an MR-VP biochemical tube for 48 hours, dripping 5 drops of methyl red solution, immediately observing the result, wherein the red color is positive, and the yellow color is negative.

3) Sugar alcohol fermentation test, namely, a sugar alcohol kit is utilized, a single separated colony is selected by an inoculating loop and inoculated into a sugar fermentation tube, bromocresol purple is taken as an indicator, and after 24 hours of culture, the result is observed, yellow is positive, and purple gray or purple is negative.

The strain H ₂O₂ test, the nitrate reduction test, the methyl red test, the rhamnose test and the sorbitol test are all negative, and the mannitol, the maltose, the sucrose, the fructose and the salicin test are all positive. The results of some physiological and biochemical processes are shown in Table 1.

TABLE 1 physiological and biochemical test results of strain FUA027

Positive + & lt- & gt negative;

Amplification and analysis of the 4 Strain FUA027 16S rDNA sequence

The genome of FUA027 is obtained by extracting with Axygen kit, and the universal primer for amplifying the 16S rDNA sequence of the prokaryotic microorganism is selected to react in a PCR mix system.

The general primer for PCR reaction is 27F, 5'-AGAGTTTGATCCTGGCTCAG-3', 1490 2R, 5'-GGTTACCTTGTTACGACTT-3'.

The reaction system (50. Mu.L) was Premix (25. Mu.L), ddH ₂ O (22. Mu.L), upstream and downstream primers (1. Mu.L each), and DNA template (1. Mu.L). The reaction procedure was 94℃denaturation for 5min, 94℃denaturation for 30s,54℃annealing for 30s,72℃extension for 90s,35 cycles, 72℃final extension for 5min. GenBank (accession number: OM 670243) was submitted after sequencing. The sequence was aligned with sequences in the GenBank database and found to have 99% similarity to 16S rDNA of strain Enterococcus faecium ATCC, 19434, ^T (accession number: DQ 411813). Phylogenetic tree showed that strain FUA027 was closest to Enterococcus faecium (fig. 3).

EXAMPLE 2 Process for producing urolithin A by fermentation of Strain FUA027 and identification of urolithin A

The seed culture medium is GAM anaerobic culture medium, peptone 5.0g/L,5.0G/L of peptone, 3.0g/L of soybean peptone, 2.5g/L of yeast extract powder, 2.2g/L of beef powder, 10.0g/L of digested serum powder, 1.2g/L of beef liver extract powder, 0.5g/L of glucose, 2.5g/L of potassium dihydrogen phosphate, 3.0g/L of sodium chloride, 5.0g/L of soluble starch, 0.3g/L of L-cysteine, 1.0g/L of L-arginine, 0.2g/L of L-tryptophan, 0.3g/L of sodium thioglycolate and pH of 7.3+/-0.1.

The fermentation medium is ABB anaerobic basic broth medium, which 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 chlorhexidine, 0.0005g/L vitamin K, 0.5g/L sodium thioglycolate, 1.0g/L dithiothreitol and pH value of 6.8+/-0.2.

1. Activating the strain, namely activating enterococcus faecium FUA027 kept in a refrigerator at-80 ℃ through flat plate scribing;

2. Preparation of seed solution Single colonies of strain FUA027 were inoculated into seed medium and cultured for 24h under anaerobic conditions at 37 ℃.

3. Preparation of fermentation broth seed broth was inoculated to fermentation medium at 2% inoculum size, inoculated with 1% EA and cultured for 48h at 37 ℃ anaerobic condition.

4. Sample collection, namely centrifuging the fermentation liquor at 12000 Xg and 4 ℃ for 10min, and obtaining supernatant fluid which is urolithin A crude extract. Mixing 1.0mL of urolithin A crude extract with CH ₃CN:H₂ O H-COOH (80:19.9:0.1) solution in equal volume, sucking 1.0mL by a syringe after full mixing, filtering with a 0.22 μm filter membrane, adding into an HPLC sample bottle, and marking.

Hplc and UPLC-MS detection analysis:

HPLC analysis conditions the samples were chromatographed on a ZORBAX SB-C18 column (Agilent, USA) of 250X 4.6mm, particle size 5 μm. Acetonitrile and 1% formic acid are used as mobile phases, the sample injection amount is 20 mu L, 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. Analysis results (FIG. 4).

UPLC-MS analysis conditions were C18 column (ACQUITY UPLC BEH C, 2.1X10 mm,1.7 μm) and ACQUITY QDa ESIMS was used to analyze the sample at 150-1000 Da. The mobile phase is 0.2% formic acid and acetonitrile, the flow rate is 0.5mL/min, and the sample injection amount is 50 mu L. The gradient of elution is 0-6 min, 10-100% acetonitrile, 6-7 min, 100-100% acetonitrile, 7-8 min, 100-10% acetonitrile, 8-9 min and 10-10% acetonitrile. Analysis results (FIG. 5).

HPLC analysis results and UPLC-MS analysis results are shown in FIG. 4 and FIG. 5, and it was confirmed that the strain FUA027 was capable of producing urolithin A.

Growth kinetics and catabolism studies of strain FUA027 preparation of strain FUA027 fermentation broth, anaerobic culture at 37℃for 100H, sampling 1.0mL at intervals of 2 hours during the culture, mixing with equal volume of CH ₃CN:H₂ O: H-COOH (80:19.9:0.1) solution, sucking 1.0mL with a syringe after sufficient mixing, and HPLC measurement after passing through 0.22 μm filter membrane. The assay showed that ellagic acid catabolism and urolithin A production occurred early in the logarithmic growth phase of enterococcus faecium FUA027, with the first metabolite produced being urolithin C, which appeared at 28 hours, peaking at 40 hours, with a maximum concentration of 1.54. Mu.M. Urolithin A was detected after 40 hours and reached a maximum concentration of 10.80. Mu.M at 50 hours (FIG. 6).

EXAMPLE 3 safety and probiotic investigation of strain FUA027

1. Safety analysis of strain FUA027

The safety of strain FUA027 was studied by hemolysis plate and drug sensitive plate phenotype experiments. By comparison with Staphylococcus aureus (S.aureus) with obvious hemolysis, it was found that strain FUA027 did not produce hemolysis (FIG. 7). The nitrate reductase activity and the amino acid decarboxylase activity of the strain FUA027 were detected by the kit, and the metabolites of the test strain were found to have no nitrate reductase or lysine, ornithine, arginine, tryptophan and phenylalanine decarboxylase activities, as shown in Table 2. Strain FUA027 was found to develop resistance to 10 antibiotics in 27 drug sensitive assays, see table 3.

TABLE 2 determination of amino acid decarboxylase Activity of FUA027 Strain

TABLE 3 results of drug sensitivity test of FUA027 Strain

Note R resistance, I mediator, S sensitivity.

2. Strain FUA027 probiotic assay

The probiotics exert the promoting effect on the body health, the intestinal tract is reached by higher viable bacteria, and the low pH environment in the stomach and the antibacterial effect of the bile salt widely distributed in the intestinal cavity have great influence on the survival rate after passing through the stomach and the intestines. The probiotics of the strain FUA027 are studied by an in vitro acid-resistant and bile salt-resistant experiment. The result shows that FUA027 has stronger tolerance, and the survival rate can reach 60% when the pH value is 3.0 and the concentration of bile salt is 0.5% (figure 8), which shows that the strain can well survive in animal intestinal tracts, and can ensure to reach host intestinal tracts to play a potential role. In addition, the in vitro probiotics performance of the test strains was evaluated by measuring the bacteriostasis, the hydrophobicity and the self-coagulation rate, and from table 4, it was found that the strains had different degrees of inhibition on both pathogenic bacteria (s.aureus, e.coli).

TABLE 4 diameter of inhibition of pathogenic bacteria by FUA027 Strain

Whether a probiotic can bind to intestinal mucosal surface receptors depends on the surface properties of the strain, and strains with a high degree of hydrophobicity generally have a strong adhesion to mucosal cells. In addition, probiotics, in addition to having adhesive properties, need to be brought into sufficient quantity by self-coagulation aggregation to fully exert their probiotic effect. As can be seen from Table 5, the FUA027 strain has a stronger hydrophobicity and a higher self-coagulant property than the commercial strain Bifidobacterium longum BB536, both of which are larger than the control strain BB536. The probiotic study of the strain also included antioxidant capacity detection, and the free radical clearance of the strain was determined using an antioxidant kit. As a result, as shown in Table 6, none of the cells showed clearance, and the fermentation supernatant had good clearance. Therefore, the strain FUA027 has better probiotic activity and potential to become novel probiotics.

TABLE 5 cell surface hydrophobicity, self-agglomeration test results of Strain

Note that BB536 is a commercial strain of Bifidobacterium longum

TABLE 6 clearance of FUA027 on DPPH, OH, O ₂ ^-

Note that ND indicates no detected activity

LP001, FUA004 are respectively control strain Lactobacillus plantarum, enterococcus faecalis

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 colony after activation 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 the 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 is 7.3 ± 0.1.

5. The method for producing urolithin A according to claim 3, characterized in that the seed culture conditions in step 2) are cultured under anaerobic conditions at 37°C for 24 hours.

6. The method for producing urolithin A according to claim 3, characterized in that the fermentation medium in the step 3) is ABB anaerobic basal broth medium: 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, and 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 liquid 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 cultured under anaerobic conditions at 37°C for 48 hours.

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