CN117866841A - Enterococcus faecalis XY2 and application thereof in preparing food and medicine for coordinating intestines and stomach and improving gout - Google Patents
Enterococcus faecalis XY2 and application thereof in preparing food and medicine for coordinating intestines and stomach and improving gout Download PDFInfo
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- CN117866841A CN117866841A CN202410070327.XA CN202410070327A CN117866841A CN 117866841 A CN117866841 A CN 117866841A CN 202410070327 A CN202410070327 A CN 202410070327A CN 117866841 A CN117866841 A CN 117866841A
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- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to enterococcus faecalis XY2 and application thereof in preparing foods and medicines for regulating intestines and stomach and improving gout. 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 of China to obtain a enterococcus faecalis (Enterococcus faecalis) XY2 strain, and the strain has excellent protease activity, can inhibit alpha-glucosidase activity, can inhibit acetylcholinesterase activity, can inhibit xanthine oxidase activity, is hopeful to be applied to the fields of promoting digestion and absorption of protein foods, improving protein allergy, reducing blood sugar and weight, promoting intestinal absorption and peristalsis, preventing and relieving hyperuricemia, gout and the like, and has important application prospects in the fields of regulating intestines and stomach, 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 XY2 and application thereof in preparing foods and medicines for regulating intestines and stomach and improving gout.
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) XY2 strain is obtained by separating and purifying faecal samples of a healthy adult in Guangdong area of China, has excellent protease activity, can inhibit alpha-glucosidase activity, can inhibit acetylcholinesterase activity and xanthine oxidase activity, and has important application prospects in the fields of regulating intestines and stomach, improving gout 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) XY2 strain, wherein the enterococcus faecalis XY2 strain is preserved in China center for type culture Collection (China, with the accession number: cctccc No. M20221504; the 16S rDNA complete sequence of the enterococcus faecalis XY2 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY2 strain according to the first aspect in the production of a protease.
The research shows that the probiotics enterococcus faecalis XY2 strain can produce protease, which suggests that the enterococcus faecalis XY2 strain is hopeful to be used for producing the protease, and the characteristics of the protease can be used for promoting the digestion and absorption of human body to protein in food, resisting allergy, helping animals digest and absorb nutrition and other fields.
Preferably, the protease includes, but is not limited to, a protease that degrades milk proteins.
In a third aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY2 strain according to the first aspect for the preparation of an alpha-glucosidase inhibitor.
Through researches, the probiotic enterococcus faecalis XY2 strain can effectively inhibit the activity of alpha-glucosidase, and the alpha-glucosidase is related to type 2 diabetes, and the inhibition of the alpha-glucosidase is one of methods for controlling postprandial hyperglycemia, which suggests that the enterococcus faecalis XY2 strain is expected to be applied to the fields of reducing blood sugar, inhibiting obesity and the like.
In a fourth aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY2 strain according to the first aspect for the preparation of an acetylcholinesterase inhibitor.
The study shows that the probiotics enterococcus faecalis XY2 strain can effectively inhibit the activity of acetylcholinesterase, and the probiotics enterococcus faecalis XY2 strain can be used for enhancing the cognitive ability and the memory, dilating blood vessels, exciting skeletal muscles, smooth muscles and other fields by inhibiting the activity of acetylcholinesterase.
In a fifth aspect, the invention provides the use of an enterococcus faecalis (Enterococcus faecalis) XY2 strain according to the first aspect for the preparation of a xanthine oxidase inhibitor.
The study shows that the probiotic enterococcus faecalis XY2 strain can inhibit Xanthine Oxidase (XOD) activity, and the probiotic enterococcus faecalis XY2 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) XY2 strain of the first aspect.
Preferably, the microbial agent is the product of fermentation of the enterococcus faecalis (Enterococcus faecalis) XY2 strain of claim 1.
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 of China to obtain a enterococcus faecalis (Enterococcus faecalis) XY2 strain which has various probiotics effects including excellent protease activity, alpha-glucosidase activity inhibition, acetylcholinesterase activity inhibition and xanthine oxidase activity inhibition. Therefore, the enterococcus faecalis XY2 strain has the effects of promoting digestion and absorption of protein food and improving protein allergy; reducing blood glucose and weight; promoting intestinal absorption and peristalsis; preventing and relieving hyperuricemia, gout and other functions. Therefore, the enterococcus faecalis XY2 strain newly separated by the invention has various probiotics effects, can be used for conditioning intestines and stomach, improving gout and other fields, for example, can be prepared into medicines for conditioning intestines and stomach and improving gout, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of enterococcus faecalis XY2 strains (the built strain is derived from the Genome database of NCBI);
FIG. 2 shows the degradation of milk plates by enterococcus faecalis XY2 strain (left, blank; right, experimental group);
FIG. 3 shows that the secretable material of enterococcus faecalis XY2 fermentation broth significantly inhibited alpha-glucosidase activity;
FIG. 4 shows that the secretable material of enterococcus faecalis XY2 fermentation broth significantly inhibited acetylcholinesterase activity;
FIG. 5 shows that enterococcus faecalis XY2 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) XY2 strain was isolated from a gut fecal sample of a healthy adult (BMI=18.9) from a gut microbiome study moisturizing laboratory in Guangzhou area of Guangdong, china 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 inversely cultured for 24 hours in a constant temperature anaerobic incubator at 37 ℃ to obtain a bacterial colony, or is shake-cultured for 24-48 hours in an MRS liquid culture medium in the constant temperature anaerobic incubator at 37 ℃ to obtain a fermentation broth.
(2) The kit comprises: alpha-glucosidase inhibitor screening kit (abcam, cat: ab 284520), acetylcholinesterase inhibitor screening kit (abnova, cat: KA 6219), xanthine oxidase activity assay kit (box manufacturer, 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) XY2 Strain
Enterococcus faecalis (Enterococcus faecalis) XY2 strain was isolated from faeces of a healthy adult in Guangdong province of China 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 "Burjie's Manual of identification of bacteria" (eighth edition) and the "manual of identification of classification of fungi", which identify the strains belonging to bacteria first, and then observe the growth of the colonies: growth morphology, presence or absence of hyphae, uniformity or absence of color, colony individual surface and edge. Finally, a purified strain is obtained through preliminary separation, the strain number is XY2, 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 XY2 strain was subjected to molecular characterization by a 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACTT), and then the XY2 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 strain XY2 was identified as a different strain of enterococcus faecalis by evolutionary analysis with homologous strains (FIG. 1).
Finally, strain XY2 was deposited with the following information: preservation time: 2022, 9, 27; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: cctccc No. M20221504; 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 XY2 separated from human faeces can be used as a probiotic and possibly has novel effects and functions.
Enterococcus faecalis XY2 16S rDNA sequence(1431bp，SEQ ID No：1)：
GCAGTCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAA。
Example 2 Functions of enterococcus faecalis (Enterococcus faecalis) XY2 Strain and uses thereof
(1) Protease capable of degrading milk protein by enterococcus faecalis XY2 strain
The ability of enterococcus faecalis XY2 to secrete proteolytic proteins was identified and measured using skim milk plate medium (MP plates) according to the agar well diffusion assay. In the test, 3uL of enterococcus faecalis XY2 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 in an anaerobic incubator with constant temperature at 37 ℃ for 3 days in an inverted mode. The results show that compared with a control in which a blank culture medium is dripped, the strain XY2 can significantly degrade proteins to form an obvious degradation circle (figure 2), which shows that the enterococcus faecalis XY2 strain can produce protease for degrading milk proteins.
Enterococcus faecalis XY2 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 is applied to the production in the protease fields of 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) The fermentation liquor of enterococcus faecalis XY2 strain can effectively inhibit the activity of alpha-glucosidase
Enterococcus faecalis XY2 cultured in MRS liquid culture medium to stationary phase is spread 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 then the influence of fermentation broth supernatant on enzyme activity ability of alpha-glucosidase to hydrolyze glucose is measured through an alpha-glucosidase inhibitor screening kit (ab 284520). The results showed that the fermentation supernatant of strain XY2 significantly inhibited the ability of alpha-glucosidase to hydrolyze glucose compared to the non-inhibitory effect of the blank medium MRS, with an inhibition rate of about 15.91%, indicating that the fermentation broth of enterococcus faecalis XY2 can effectively inhibit the activity of alpha-glucosidase (FIG. 3).
a-glucosidase, an enzyme that plays a role in carbohydrate breakdown, is associated with type 2 diabetes, and inhibition of α -glucosidase is one of the methods of controlling postprandial hyperglycemia, thereby contributing to the treatment of diabetes. Thus, α -glucosidase inhibitors help to maintain blood glucose levels and can improve diabetic complications. Furthermore, inhibition of α -glucosidase activity may control obesity.
Thus, the probiotic enterococcus faecalis XY2 strain has the function of inhibiting a-glucosidase activity, which makes it a potential hypoglycemic and obesity inhibiting probiotic.
(3) Enterococcus faecalis XY2 strain fermentation liquor can effectively inhibit activity of acetylcholinesterase
Enterococcus faecalis XY2 cultured in MRS liquid culture medium to stationary phase is spread 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 then the inhibitory capacity of fermentation broth supernatant on acetylcholinesterase (AchE) activity is measured by acetylcholinesterase inhibitor screening kit (KA 6219). The results showed that the fermentation supernatant of strain XY2 significantly inhibited the activity of acetylcholinesterase compared to the non-inhibitory effect of the blank medium MRS, with an inhibition rate of about 26.02%, indicating that the fermentation broth of enterococcus faecalis XY2 can effectively inhibit the activity of acetylcholinesterase (fig. 4).
The acetylcholinesterase inhibitor can continuously accumulate at synapses under the action of acetylcholine released by nerve fiber tips through reversible inhibition of acetylcholinesterase, excite cholinergic receptors, and prolong and increase the action of acetylcholine. Acetylcholinesterase inhibitors can enhance cognitive ability and memory (improve Alzheimer's disease, improve cognitive dysfunction), dilate blood vessels (improve heart rate overspeed), excite skeletal and smooth muscles (improve constipation, postoperative abdominal distension, postoperative urinary retention). Thus, the probiotic enterococcus faecalis XY2 strain can inhibit acetylcholinesterase activity, and can excite skeletal muscle and smooth muscle of intestinal tract, which makes it a potential probiotic for promoting gastrointestinal absorption and peristalsis.
(4) Enterococcus faecalis XY2 strain can inhibit Xanthine Oxidase (XOD) activity
Enterococcus faecalis XY2 strain cultured in MRS liquid culture medium to stationary phase is spread 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 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 XY2 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 23.06% (< 0.05), indicating that enterococcus faecalis XY2 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 free radicals. 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 XY2 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 (Enterococcus faecalis) XY2 strain of the present invention has a variety of probiotic effects: (1) has excellent protease activity; (2) can inhibit alpha-glucosidase activity; (3) can inhibit acetylcholinesterase activity; (4) xanthine oxidase activity can be inhibited. Therefore, the enterococcus faecalis XY2 strain obtained by the novel separation has important application value and economic value in the fields of regulating intestines and stomach, improving gout 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 (9)
1. An enterococcus faecalis (Enterococcus faecalis) XY2 strain, wherein said enterococcus faecalis XY2 strain was deposited with the chinese collection at 2022, 9 and 27, under the accession number: cctccc No. M20221504; the 16S rDNA complete sequence of the enterococcus faecalis XY2 strain is shown in SEQ ID No: 1.
2. Use of the enterococcus faecalis (Enterococcus faecalis) XY2 strain of claim 1 for producing protease.
3. The use according to claim 2, wherein the protease comprises a protease that degrades milk proteins.
4. Use of the enterococcus faecalis (Enterococcus faecalis) XY2 strain of claim 1 for the preparation of an alpha-glucosidase inhibitor.
5. Use of the enterococcus faecalis (Enterococcus faecalis) XY2 strain of claim 1 in the preparation of an acetylcholinesterase inhibitor.
6. Use of the enterococcus faecalis (Enterococcus faecalis) XY2 strain of claim 1 in the preparation of a xanthine oxidase inhibitor.
7. A probiotic functional bacterial agent, characterized in that it comprises the enterococcus faecalis (Enterococcus faecalis) XY2 strain of claim 1.
8. The probiotic functional microbial agent of claim 7, wherein the microbial agent is a fermented product of enterococcus faecalis (Enterococcus faecalis) XY2 strain.
9. The probiotic functional microbial agent of claim 7, wherein the microbial agent further comprises pharmaceutically acceptable excipients in the field of pharmaceutical applications.
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