CN117866839A - Enterococcus faecalis XY7 and application thereof in preparation of anti-inflammatory and gout-improving foods and medicines - Google Patents
Enterococcus faecalis XY7 and application thereof in preparation of anti-inflammatory and gout-improving foods and medicines Download PDFInfo
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- CN117866839A CN117866839A CN202410069480.0A CN202410069480A CN117866839A CN 117866839 A CN117866839 A CN 117866839A CN 202410069480 A CN202410069480 A CN 202410069480A CN 117866839 A CN117866839 A CN 117866839A
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to enterococcus faecalis XY7 and application thereof in preparation of anti-inflammatory and gout-improving foods and medicines. In order to further develop and utilize the probiotics function of enterococcus faecalis, the invention separates and purifies a fecal sample of a healthy adult in Guangdong area to obtain a enterococcus faecalis (Enterococcus faecalis) XY7 strain, the strain XY7 has excellent protease activity, can produce and secrete 3-hydroxybutyric acid, can produce and secrete glutathione, can inhibit xanthine oxidase activity, is hopeful to be applied to promote digestion and absorption of protein foods, improve protein allergy, can resist inflammation and improve intestinal flora, can resist oxidation and whiten, delay aging, resist inflammation and allergy, can prevent and relieve hyperuricemia, gout and the like, and has important application value and economic value in the fields of resisting inflammation, improving gout and the like.
Description
Technical Field
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to enterococcus faecalis XY7 and application thereof in preparation of anti-inflammatory and gout-improving foods and medicines.
Background
Enterococcus faecalis (Enterococcus faecalis) is a gram-positive bacterium and a hydrogen peroxide-negative bacterium, and is one of the main flora in the human and animal intestinal tracts. Enterococcus faecalis (E.faecalis) is a facultative anaerobic gram-positive lactic acid bacterium, the form of the bacterium is spherical or chain, most of the bacterium is arranged in double or short chain, the bacterium is usually not moving, the diameter is 0.5-1.0 microns, the bacterium is free of capsules and spores, the bacterium has strong adaptability and resistance to the environment, and the bacterium can resist various antibiotics such as tetracycline, kanamycin and gentamicin, and the requirement on growth conditions is not strict. In addition, enterococcus faecalis can use arginine as an energy source to ferment sorbitol, but cannot ferment arabinose.
Enterococcus faecalis is a normal colonising bacteria of the human intestinal tract and is present in approximately 90-95% of the human body. This bacterium is often one of the first microorganisms to colonize the human gastrointestinal tract early in life and plays an important role in the development of the intestinal immune system. The feed additive catalog (2013) divides enterococcus faecalis into species that can be added to feed, which can form a biofilm in the animal's gut to adhere to the mucosa of the animal's gut and develop, grow and reproduce thereon. In addition, enterococcus faecalis can soften fibers in the feed and improve the conversion rate of the feed.
Enterococcus faecalis is one of main flora in intestinal tracts of human beings and animals, can generate natural antibiotics, and is beneficial to body health; bacteriostasis substances such as bacteriocin and the like can be produced, the growth of pathogenic bacteria such as escherichia coli, salmonella and the like can be inhibited, and the intestinal microenvironment can be improved; can inhibit the proliferation of urease-producing bacteria and putrefying bacteria in intestinal tract, reduce the content of urease and endotoxin in intestinal tract, and reduce the content of ammonia and endotoxin in blood. Meanwhile, enterococcus faecalis is a type of microorganism normally existing in the digestive tract, has strong tolerance and colonization capacity in intestinal mucosa, is facultative anaerobic lactobacillus, is suitable for production and application, is also a good feeding microorganism, and has wide application in aquatic products and livestock and poultry. In addition, enterococcus faecalis is not only a normal flora in human and animal intestinal tracts, but also an important lactobacillus, has better intestinal adhesion capability, can generate lactic acid and some antibacterial substances, inhibit the reproduction of intestinal putrefying bacteria and pathogenic bacteria and intestinal infection, reduce diarrhea rate, promote the digestion and absorption of nutrient substances, enhance immunity, improve feed intake and feed conversion rate, promote animal health, and is one of the probiotic bacteria used first choice in livestock production and food industry. According to researches of scholars, enterococcus faecalis and cellulase are added into the corn straw silage, so that the color, smell, texture and the like of the silage can be obviously improved, the disappearance rate of dry matters is improved by 8%, the mass ratio of ammoniacal nitrogen to total nitrogen is reduced by 33%, the mass fraction of butyric acid is reduced by 82%, and meanwhile, the contents of neutral washing fibers and acidic washing fibers are respectively reduced by 10% and 7%, and the quality of the silage is obviously improved. Meanwhile, researches prove that enterococcus faecalis can generate a plurality of antibacterial substances, and the antibacterial substances have good inhibition effects on pathogenic bacteria such as salmonella, escherichia coli, staphylococcus aureus and the like. Research has also shown that enterococcus faecalis is capable of producing vodkamycin and can effectively inhibit the growth and reproduction of listeria, staphylococcus aureus and spoilage microorganisms. Enterococcus faecalis also enhances the activity of macrophages, promotes the immune response in animals, and increases antibody levels. Enterococcus faecalis can form a biological film in the intestinal tract of an animal to be adhered to the intestinal mucosa of the animal, and can form a lactobacillus barrier to resist the negative effects of external germ viruses, mycotoxins and the like during development, growth and reproduction.
A great deal of research shows that different strains of enterococcus faecalis have different probiotic functions. Such as: (1) Enterococcus faecalis strain T-110 was used as A probiotic (BIO-THREE) in combination with Bacillus subtilis TO-A and Clostridium butyricum TO-A. Together, these three strains can reduce post-operative infections in surgical patients and shorten hospitalization times in infants suffering from acute diarrhea. (2) A crossover study found that a single administration of enterococcus faecalis YM0831 strain to healthy human subjects significantly improved the response of blood glucose to sucrose tolerance tests.
Since enterococcus faecalis sources are diverse, genes thereof are diverse and functionally diverse. However, the separation and identification, the probiotics and the metabolism mechanism of enterococcus faecalis are still less studied at present, and the development and the utilization of the enterococcus faecalis are affected to a certain extent. Therefore, more enterococcus faecalis with a probiotic function needs to be excavated, and different functions of the enterococcus faecalis need to be further excavated according to different sources of the enterococcus faecalis, so that the enterococcus faecalis can better play roles, for example, the efficacy of the enterococcus faecalis is determined according to the functions of the strain or the probiotic metabolic products, and the application prospect of the enterococcus faecalis is clear. In conclusion, the research and application of enterococcus faecalis have a very wide development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the enterococcus faecalis (Enterococcus faecalis) XY7 strain is obtained by separating and purifying faecal samples of a healthy adult in Guangdong region, has excellent protease activity, can generate and secrete 3-hydroxybutyric acid, can generate and secrete glutathione, can inhibit xanthine oxidase activity, and has important potential application value in the fields of anti-inflammatory, gout improvement and the like.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides an enterococcus faecalis (Enterococcus faecalis) XY7 strain, wherein the enterococcus faecalis XY7 strain is preserved in China center for type culture Collection (China, with the accession number: cctccc No. M20231166; the 16S rDNA complete sequence of the enterococcus faecalis XY7 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY7 strain according to the first aspect for the production of a protease for the degradation of milk proteins.
The research shows that the probiotics enterococcus faecalis XY7 strain can produce protease, which suggests that the enterococcus faecalis XY7 strain can be used for producing the protease, and the characteristic of producing the protease is used for degrading the cell wall of pathogenic fungi to control diseases; decomposing cellulose in the compost; preparing livestock and poultry breeding feed and the like.
In a third aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY7 strain according to the first aspect for the production of 3-hydroxybutyric acid.
Through researches, the probiotics enterococcus faecalis XY7 strain can produce 3-hydroxybutyric acid (3-HB), and the probiotics enterococcus faecalis XY7 strain is suggested to be used for producing 3-HB, and is used for providing energy for various activities of the body, resisting osteoporosis, preventing and treating chronic syndrome, improving brain cognitive function, improving lipid metabolism and other fields through the characteristic of producing 3-HB.
In a fourth aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY7 strain according to the first aspect for the production of reduced glutathione.
The research shows that the probiotics enterococcus faecalis XY7 strain can produce reduced Glutathione (GSH), which suggests that the enterococcus faecalis XY7 strain can be used for producing GSH, and the characteristics of GSH production can be used in the fields of antioxidation, whitening, aging delay, immunity enhancement, anti-tumor, antiallergic and the like.
In a fifth aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY7 strain according to the first aspect for the preparation of a xanthine oxidase inhibitor.
The study shows that the probiotic enterococcus faecalis XY7 strain can inhibit Xanthine Oxidase (XOD) activity, and the probiotic enterococcus faecalis XY7 strain can be used for inhibiting xanthine oxidase activity through XOD activity, reducing purine in vivo and uric acid generation, thereby controlling uric acid level and preventing gout attack.
In a sixth aspect, the invention provides a probiotic functional bacterial agent comprising the enterococcus faecalis (Enterococcus faecalis) XY7 strain of the first aspect.
Preferably, the microbial inoculum is a product of fermentation of the enterococcus faecalis (Enterococcus faecalis) XY7 strain according to the first aspect.
Preferably, in the field of medical application, the microbial inoculum further comprises pharmaceutically acceptable auxiliary materials.
More preferably, the adjuvant comprises a carrier and an excipient. The excipient refers to diluents, binders, lubricants, disintegrants, cosolvents, stabilizers and the like which can be used in the pharmaceutical field and some medicinal matrixes. The carrier is a functional pharmaceutical adjuvant available in the pharmaceutical field and comprises a surfactant, a suspending agent, an emulsifying agent and a plurality of novel pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid and the like.
Preferably, in the field of medical application, the dosage forms of the microbial inoculum comprise tablets, granules, capsules, dripping pills, sustained release agents, oral liquid preparations and injections.
More preferably, the above-mentioned dosage forms refer to clinically usual dosage forms. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if some drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.
Compared with the prior art, the invention has the beneficial effects that:
the invention separates and purifies faeces samples of a healthy adult in Guangdong area to obtain a strain of enterococcus faecalis (Enterococcus faecalis) XY7, wherein the strain XY7 has various probiotics effects, including excellent protease activity, can produce and secrete 3-hydroxybutyric acid, can produce and secrete glutathione, and can inhibit xanthine oxidase activity. Therefore, the enterococcus faecalis XY7 strain has the advantages of promoting digestion and absorption of protein food and improving protein allergy; can be used for resisting inflammation and improving intestinal flora; has effects in resisting oxidation, whitening skin, resisting aging, relieving inflammation and resisting allergy; can prevent and relieve hyperuricemia, gout and other functions. Therefore, the enterococcus faecalis XY7 strain newly separated by the invention has various probiotics effects, can be used in the fields of anti-inflammatory and gout improvement, for example, can be prepared into medicines for anti-inflammatory and gout improvement, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of enterococcus faecalis XY7 strains (the built strain is derived from the Genome database of NCBI);
FIG. 2 shows the degradation of milk plates by enterococcus faecalis XY7 strain (left, blank; right, experimental group);
FIG. 3 shows that enterococcus faecalis XY7 strain may produce 3-hydroxybutyric acid;
FIG. 4 shows that enterococcus faecalis XY7 strain may produce and secrete GSH;
FIG. 5 shows that enterococcus faecalis XY7 strain inhibits XOD activity.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The following examples relate to the following experimental materials:
(1) Enterococcus faecalis (Enterococcus faecalis) XY7 strain was isolated from a sample of intestinal faeces of a healthy adult (BMI=19.5) in Guangzhou area of China by an intestinal microbiome study moisturizing laboratory and placed in a glycerol tube for cryogenic storage at-80 ℃. In general, the strain is inoculated on the surface of an MRS solid culture medium flat plate and is cultured for 24 hours in an inverted way in a constant temperature anaerobic incubator at 37 ℃ to obtain a bacterial colony, or is cultured for 24-48 hours in an MRS liquid culture medium in an oscillation way in the constant temperature anaerobic incubator at 37 ℃ to obtain a fermentation liquor.
(2) The kit comprises: 3-hydroxybutyric acid (3-HB) detection kit (Cloud-Clone Corp., cat: CEB022 Ge), micro-reduced Glutathione (GSH) determination kit (Nanjing built, cat: A006-2-1), xanthine oxidase activity determination kit (Box manufacturing, cat: AKAO 006M).
(3) MRS plate: 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar and ddH 2 Filling O to 1L, adjusting pH to 6.2-6.6, and autoclaving at 121deg.C for 20min to obtain MRS plate.
(4) MRS liquid medium: 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and ddH 2 Adding O to 1L, adjusting pH to 6.2-6.6, and autoclaving at 121deg.C for 20min to obtain MRS liquid culture medium.
(5) MP plate: 10g of skimmed milk powder, 1g of sodium chloride, 10g of beef extract, 10g of peptone, 5g of yeast extract, 20g of glucose, 2g of tri-ammonium citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar and ddH 2 Filling O to 1L, adjusting pH to 6.2-6.6, autoclaving at 121deg.C for 20min, and making into MP plate.
EXAMPLE 1 isolation and identification of enterococcus faecalis (Enterococcus faecalis) XY7 Strain
Enterococcus faecalis (Enterococcus faecalis) XY7 strain was isolated from faeces of a healthy adult in Guangdong province of China, and was specifically as follows:
the fecal sample was repeatedly washed 3 times with sterile water, placed in a mortar, 500uL of sterile water was added per 100mg of fecal sample, thoroughly ground to homogenate, and an appropriate amount of the grinding fluid was pipetted, spread on an MRS plate, and incubated at room temperature for 3 days. Colonies to be streaked and purified in the separation assay plates were then numbered with a marker and strain numbers were marked on the plates accordingly. After labelling, colonies were picked and inoculated onto MRS plates and the strains were purified by plate streaking. If the strain cannot be separated by the method, colonies need to be picked from the enrichment plate, and the colonies are coated on a culture medium after being subjected to gradient dilution by MRS liquid culture medium. Finally, reference is made to the "Berger's Manual of bacteria identification" (eighth edition) and the "manual of fungus classification identification", first to distinguish strains belonging to bacteria, and then to observe the growth conditions of colonies. The primary separation is carried out to obtain a purified strain, the strain number is XY7, and after 24 hours of culture, bacterial colonies of the strain are observed to be round or oval, moist, convex, smooth in edge and flat in surface.
Next, the isolated XY7 strain was subjected to molecular characterization by a 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACTT), and then the XY7 strain was subjected to whole genome sequencing by Beijing Baimeike Biotechnology Co. The resulting sequence 16S rDNA sequence (SEQ ID No: 1) was subjected to BLAST alignment at NCBI' S Genome database. The results showed that the homology with the known 16S rDNA sequence of enterococcus faecalis (Enterococcus faecalis) was >99% and that the evolution analysis was performed with the homologous strain (FIG. 1) confirming that XY7 was an enterococcus faecalis of a different species.
Finally, strain XY7 was deposited with the following information: preservation time: 2023, 7, 3; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: cctccc No. M20231166; deposit unit address: chinese university of Wuhan; classification naming: enterococcus faecalis.
Enterococcus faecalis has wide probiotic effects such as anti-inflammatory, anti-aging and cholesterol reducing effects, but strains from different sources have different effects, which indicates that a novel enterococcus faecalis XY7 separated from human faeces can be used as a probiotic and possibly has novel effects and functions.
Enterococcus faecalis XY7 16S rDNA sequence(1425bp，SEQ ID No：1)：
GCTATACTGCAGTCGAACGCTTCTTTCCTCCCGAGTGCTTGCNCTCNATTGGAAAGAGGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTACCCATCAGAGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATAACAGTTTATGCCGCATGGCATAAGAGTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGACGTTAGTAACTGAACGTCCCCTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCTAGACCGCGAGGTCATGCAAATCTCTTAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGG。
Example 2 Functions of enterococcus faecalis (Enterococcus faecalis) XY7 Strain and uses thereof
(1) Protease capable of degrading milk protein by enterococcus faecalis XY7 strain
The ability of enterococcus faecalis XY7 to secrete protease proteolytic proteins was identified and measured according to the agar well diffusion assay using skim milk plate medium (MP plate). In the experiment, 3uL of enterococcus faecalis XY7 bacterial liquid with the concentration of 10Abs is dripped into an MP plate of an experimental group, 3uL of blank MRS culture medium is dripped into a control group, and the culture is performed for 3 days in an anaerobic incubator with constant temperature at 37 ℃. The results show that strain XY7 can significantly degrade protein and form a significant degradation circle (FIG. 2) compared with the control in which the blank medium is added dropwise, indicating that the enterococcus faecalis XY7 strain can produce protease that degrades milk protein.
Enterococcus faecalis XY7 can produce protease, and can promote digestion and absorption of protein in food by human body and improve absorption of small peptide and amino acid when used as a probiotic bacterial strain. And can be used for resisting allergy (improving food allergy caused by protein dyspepsia or non-absorption). In addition, the method can also be used for extracting protease and can be applied to the production of protease in food industry, washing industry and the like; can also be used in microbial feed to help animals digest and absorb nutrition, and improve the utilization rate of the feed.
(2) Enterococcus faecalis XY7 strain may produce 3-hydroxybutyric acid (3-HB)
The enterococcus faecalis XY7 cultured by MRS liquid culture medium to stationary phase is expanded and cultured into new MRS liquid culture medium at dilution ratio of 1:30, thallus is collected when culturing to stationary phase for 24h, thallus after centrifugation is carried out at 10,000Xg and 4 ℃ for 10min, and 1OD is obtained 600 The bacterial lysate was prepared by lysis with 500uL buffer PBS (8 g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4 were dissolved in 800mL distilled water, the solution was adjusted to 7.2 with HCl, and finally distilled water was added to a volume of 1L to obtain PBS buffer), and then the 3-HB concentration of the bacterial lysate was measured by a 3-HB specific ELISA kit (CEB 022 Ge). As a result, it was found that the concentration of 3-HB in the cell lysate of the strain XY7 was 164.70. Mu.g/mL, compared with the absence of 3-HB in the cell lysis buffer PBS, indicating that enterococcus faecalis XY7 can produce 3-hydroxybutyric acid in the stationary phase (FIG. 3).
3-HB can provide energy for various physical activities and is a potential energy/functional food that has been added to athlete drinks, so the probiotic enterococcus faecalis XY7 can be an additive to energy foods. Meanwhile, in view of the fact that 3-HB can effectively resist osteoporosis, prevent and treat chronic syndromes (hypertension, alcoholic fatty liver, enteritis and intestinal cancer), improve brain cognitive functions (improving learning and memory capacity, protecting glial cells and improving Alzheimer's disease), and improve lipid metabolism. Thus, the probiotic enterococcus faecalis XY7 strain may serve several of the above purposes by having the effect of producing 3-hydroxybutyric acid.
In addition, 3-HB is an endogenous small molecule naturally occurring in the body, has an important role in maintaining colorectal tissue integrity, and has the effects of maintaining intestinal health, preventing colonic disease and diminishing inflammation. The 3-HB treatment can promote the proliferation of beneficial intestinal bacteria, relieve the symptoms of multiple sclerosis mice, and has great potential in the aspects of regulating flora and improving health. Therefore, the probiotic enterococcus faecalis XY7 strain is also beneficial for improving intestinal flora and relieving intestinal inflammation.
(3) Enterococcus faecalis XY7 strain capable of producing and secreting reduced Glutathione (GSH)
Enterococcus faecalis XY7 strain cultured in MRS liquid culture medium to stationary phase is expanded into new MRS liquid culture medium at dilution ratio of 1:30, bacterial suspension is collected when culturing to stationary phase for 24h, fermentation broth supernatant is collected after centrifugation at 10,000Xg and 4 ℃ for 10min, and concentration of GSH in fermentation broth supernatant is measured by reduced Glutathione (GSH) measuring kit (A006-2-1). The results showed that the concentration of GSH in the fermentation supernatant of strain XY7 was 265.03 μmol/L compared to the low concentration of GSH in the blank medium MRS, indicating a significant increase in GSH concentration after XY7 fermentation (< 0.01), demonstrating that enterococcus faecalis XY7 can produce and secrete Glutathione (GSH) during stationary phase (fig. 4).
Glutathione (GSH) is a tripeptide consisting of glutamic acid, cysteine and glycine, and containing gamma-amide bond and mercapto group, and has antioxidant effect and integrated detoxification effect. The sulfhydryl group on cysteine is a glutathione reactive group (so glutathione is often abbreviated as GSH). Glutathione helps to maintain normal immune system function, has antioxidant and integrated detoxification effects, and plays an important role in various cell biochemical processes, such as free radical neutralization, detoxification, cysteine transport and storage, maintenance of cell redox, ascorbic acid and vitamin E regeneration, and the like. Mainly comprises the following aspects:
(1) detoxification: combined with poison or medicine to eliminate its toxic action;
(2) participate in the oxidation-reduction reaction: as an important reducing agent, participate in various oxidation-reduction reactions in the body;
(3) protection of thiol enzyme activity: maintaining the active group (-SH) of the sulfhydryl enzyme in a reduced state;
(4) maintenance of the stabilization of erythrocyte membrane structure: eliminating the damage of oxidant to erythrocyte membrane structure.
Thus, the various biological functions of GSH confer a variety of efficacy and utility, primarily represented by:
1) Antioxidant: scavenging free radicals in human bodies, protecting sulfhydryl groups in molecules such as a plurality of proteins, enzymes and the like from being oxidized by harmful substances, thereby ensuring the normal exertion of physiological functions of the proteins, the enzymes and the like; the content of glutathione in human erythrocytes is great, which has important significance for protecting the sulfhydryl group of protein on erythrocyte membrane in a reduced state and preventing hemolysis; it also has effects in preventing skin aging and pigmentation, reducing melanin formation, improving skin antioxidant capacity, and making skin luster.
2) Clinical medicine: the sulfhydryl chelates toxins such as heavy metals, fluoride, mustard gas and the like to prevent poisoning; can also be used as a medicament for treatment or adjuvant therapy in the aspects of hepatitis, hemolytic diseases, keratitis, cataract, retina diseases and the like; can also correct unbalance of acetylcholinesterase and cholinesterase, and has antiallergic effect.
3) Food additives: strengthening food nutrition, stabilizing vitamin C, and strengthening flavor.
In conclusion, the glutathione can be used for medicines and can be used as a base material of functional foods, and has wide application value in the fields of the functional foods such as antioxidation, whitening, aging delaying, immunity enhancing, anti-tumor, antiallergic and the like.
Thus, the probiotic enterococcus faecalis XY7 strain may exert several of the above effects by virtue of the function of the GSH produced.
(4) Enterococcus faecalis XY7 strain can inhibit Xanthine Oxidase (XOD) activity
Enterococcus faecalis XY7 strain cultured in MRS liquid culture medium to stationary phase is expanded and cultured in new MRS liquid culture medium at dilution ratio of 1:30, bacterial suspension is collected when culturing to stationary phase for 24h, fermentation broth supernatant is collected after centrifugation at 10,000Xg and 4 ℃ for 10min, and then xanthine oxidase activity in fermentation broth supernatant is measured by xanthine oxidase activity measuring kit (box manufacturing, cat: AKAO 006M). The results showed that the fermentation supernatant of strain XY7 had a significant inhibition of xanthine oxidase activity compared to the blank medium MRS without inhibition of xanthine oxidase activity, with an inhibition rate of 21.28% (< 0.05), indicating that enterococcus faecalis XY7 can produce and secrete metabolites during stationary phase to inhibit the activity of Xanthine Oxidase (XOD) (fig. 5).
Xanthine oxidase is a key enzyme in the catabolism of purines, and can catalyze the direct production of uric acid from hypoxanthine and xanthine. Thus, when xanthine oxidase activity is abnormally active in the body, it leads to the production of a large amount of uric acid, thereby causing hyperuricemia or gout.
Xanthine oxidase inhibitors such as allopurinol inhibit xanthine oxidase activity and prevent the metabolism of hypoxanthine and xanthine into uric acid, thereby reducing uric acid production and improving gout and hyperuricemia. Xanthine oxidase inhibitors can also reduce stress response and damage to tissues caused by free radicals, and are expected to be clinically used for treating gout and diseases caused by peroxide. At present, allopurinol is one of main medicines for treating hyperuricemia and gout, and is also the only chemical medicine for inhibiting uric acid generation clinically, but the medicine has a plurality of side effects, can cause fever, allergic rash abdominal pain, diarrhea, leucocyte and thrombocytopenia and multiple organ damage, even has reports of death, has questioned safety, and has long been used until now because of good inhibition effect on xanthine oxidase. Therefore, the research of new low-toxicity and high-efficiency xanthine oxidase inhibitors is of great significance.
Therefore, the probiotic enterococcus faecalis XY7 strain is expected to reduce in-vivo purine and uric acid generation by inhibiting the activity of xanthine oxidase, thereby controlling uric acid level and preventing gout flares.
Taken together, the newly isolated enterococcus faecalis XY7 strain of the present invention has a variety of probiotic effects: (1) has excellent protease activity; (2) 3-hydroxybutyric acid may be produced and secreted; (3) glutathione can be produced and secreted; (4) xanthine oxidase activity can be inhibited. Therefore, the enterococcus faecalis XY7 strain obtained by the novel separation has important application value and economic value in the fields of anti-inflammatory, gout improving and the like.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (8)
1. An enterococcus faecalis (Enterococcus faecalis) XY7 strain, wherein said enterococcus faecalis XY7 strain was deposited with the chinese collection at month 7 and 3 of 2023 under the accession number: cctccc No. M20231166; the 16S rDNA complete sequence of the enterococcus faecalis XY7 strain is shown in SEQ ID No: 1.
2. Use of the enterococcus faecalis (Enterococcus faecalis) XY7 strain of claim 1 for the production of a protease that degrades milk proteins.
3. Use of the enterococcus faecalis (Enterococcus faecalis) XY7 strain of claim 1 for the production of 3-hydroxybutyric acid.
4. Use of the enterococcus faecalis (Enterococcus faecalis) XY7 strain of claim 1 for producing reduced glutathione.
5. Use of the enterococcus faecalis (Enterococcus faecalis) XY7 strain of claim 1 in the preparation of a xanthine oxidase inhibitor.
6. A probiotic functional bacterial agent, characterized in that it comprises the enterococcus faecalis (Enterococcus faecalis) XY7 strain of claim 1.
7. The probiotic functional microbial agent according to claim 6, characterized in that the microbial agent is a fermented product of the enterococcus faecalis (Enterococcus faecalis) XY7 strain according to claim 1.
8. The probiotic functional microbial agent of claim 6, wherein the microbial agent further comprises pharmaceutically acceptable excipients in the field of pharmaceutical applications.
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