CN117264840A - Lactobacillus brevis XY8 and application thereof in preparation of food and medicine for resisting aging and improving gout - Google Patents
Lactobacillus brevis XY8 and application thereof in preparation of food and medicine for resisting aging and improving gout Download PDFInfo
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- CN117264840A CN117264840A CN202311322726.2A CN202311322726A CN117264840A CN 117264840 A CN117264840 A CN 117264840A CN 202311322726 A CN202311322726 A CN 202311322726A CN 117264840 A CN117264840 A CN 117264840A
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/24—Lactobacillus brevis
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus brevis XY8 and application thereof in preparation of food and drugs for resisting aging and improving gout. The invention separates and purifies the fecal sample of a healthy adult in Guangdong area of China to obtain a Lactobacillus brevis (Levilactobacillus brevis) XY8 strain, and the strain has various probiotics effects, including excellent protease activity, 3-hydroxybutyric acid generation, xanthine oxidase inhibition, gamma-aminobutyric acid generation and secretion, hyaluronic acid generation and secretion, glutathione generation and secretion, DPPH free radical elimination, hydroxyl free radical elimination, superoxide dismutase activity and cholesterol degradation. Therefore, the Lactobacillus brevis XY8 strain can be used in the fields of anti-aging, gout improvement and the like, and has important application value and economic value.
Description
Technical Field
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus brevis XY8 and application thereof in preparation of food and drugs for resisting aging and improving gout.
Background
Lactobacillus brevis (Levilactobacillus brevis) are widely distributed, are usually attached to the surface of stems and leaves in plants, are more common in kimchi, and are most in the small intestine in the digestive system of humans and animals. The Lactobacillus brevis cells are rod-shaped, do not form spores, and are gram-positive; facultative anaerobism; can ferment glucose, sodium gluconate, arabinose, fructose, ribose, lactose, maltose, melibiose and galactose to produce acid; can grow in an environment close to the concentration of the salt in the small intestine of the human body; good growth around pH 6.0 and good acid resistance even in an environment of pH 3.0. Lactobacillus brevis can produce acid with high yield by pentose phosphate ketal enzyme (PPK) way, wherein glucose or pentose can be used as carbon source.
Lactobacillus brevis is safe for consumption, widely found in the intestinal tract of humans and animals and excreted with faeces. Lactobacillus is a part of the normal flora of the human body, which is an important component of the normal microbial system of the intestinal tract and accompanies the host for life, and has an important effect on maintaining the intestinal microecological balance. In recent years, lactobacillus brevis has become a hot spot of research in recent years as a very potential probiotic lactobacillus, and is being continuously made into probiotic preparations suitable for human and animals.
At present, lactobacillus brevis has been reported to have a variety of different probiotic functions. For example, it can alleviate influenza virus infection symptoms, and has antiviral and immunity regulating activities; can relieve constipation, irritable bowel syndrome and inflammatory bowel disease, and improve intestinal health; can improve sleep; can reduce anti-allergen antibody and inhibit systemic anaphylaxis of mice; can reduce cholesterol and inhibit fat production; can be used for improving diabetes and liver injury.
The sources of lactobacillus brevis are diverse, leading to a diversity of genes and functions. However, there are still few studies on the isolation and identification, the probiotic properties and the metabolic mechanism of lactobacillus brevis, and it has been found that lactobacillus brevis with probiotic functions are not very much, and although some research progress has been made on the functions and applications of lactobacillus brevis at present, the potential efficacy of lactobacillus brevis has not yet been fully developed. Therefore, it is necessary to excavate more lactobacillus brevis with probiotic functions and further excavate the functions according to different sources of lactobacillus brevis so as to make the lactobacillus brevis function better, for example, determining the efficacy according to the functions of strains or probiotic metabolites and defining the application prospect of the lactobacillus brevis. In conclusion, the probiotics lactobacillus brevis and the application thereof have wide development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention separates and purifies a fecal sample of a healthy adult in Guangdong area of China to obtain a Lactobacillus brevis (Levilactobacillus brevis) XY8 strain, and the strain has multiple functions of producing 3-hydroxybutyric acid, inhibiting Xanthine Oxidase (XOD) activity, producing reduced Glutathione (GSH) and the like, and has important potential application value.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a Lactobacillus brevis (Levilactobacillus brevis) XY8 strain, wherein the Lactobacillus brevis XY8 strain is deposited with the China center for type culture Collection, with the accession number: cctccc No. M20231167; the 16S rDNA complete sequence of the Lactobacillus brevis XY8 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of a strain of Lactobacillus brevis (Levilactobacillus brevis) XY8 as described in the first aspect for the production of 3-hydroxybutyric acid.
According to research, the probiotic lactobacillus brevis XY8 strain can produce 3-hydroxybutyric acid (3-HB), and the probiotic lactobacillus brevis XY8 strain is suggested to be expected to be used for producing 3-HB, and is applied to the fields of supplying energy for various activities of the body, resisting osteoporosis, preventing and treating chronic syndromes, improving brain cognitive functions, improving lipid metabolism and the like through the characteristic of producing 3-HB.
In a third aspect, the invention provides the use of a strain of lactobacillus brevis (Levilactobacillus brevis) XY8 as described in the first aspect for the preparation of a xanthine oxidase inhibitor.
The research shows that the probiotic lactobacillus brevis XY8 strain can inhibit Xanthine Oxidase (XOD) activity, and suggests that the lactobacillus brevis XY8 strain is expected to be applied to the fields of reducing purine in vivo and uric acid generation, controlling uric acid level, preventing gout attack and the like by inhibiting the XOD activity.
In a fourth aspect the invention provides the use of a strain of lactobacillus brevis (Levilactobacillus brevis) XY8 as described in the first aspect for the production of reduced Glutathione (GSH).
The research shows that the probiotic lactobacillus brevis XY8 strain can produce reduced Glutathione (GSH), which suggests that the lactobacillus brevis XY8 strain is expected to be used for producing GSH, and the probiotic lactobacillus brevis XY8 strain is applied to the fields of antioxidation, whitening, aging delay, immunity enhancement, anti-tumor, antiallergic and the like through the characteristic of GSH production.
In a fifth aspect, the invention provides the use of a strain of lactobacillus brevis (Levilactobacillus brevis) XY8 as described in the first aspect for producing proteases.
The research shows that the probiotic lactobacillus brevis XY8 strain can produce protease, which suggests that the lactobacillus brevis XY8 strain is expected to be used for producing protease, and the characteristic of producing protease is applied to the fields of promoting the digestion and absorption of human body to protein in food, resisting allergy, helping the digestion and absorption of nutrition of animals, and the like.
In a sixth aspect, the invention provides the use of a strain of lactobacillus brevis (Levilactobacillus brevis) XY8 as described in the first aspect for the production of gamma-aminobutyric acid.
The research shows that the probiotic lactobacillus brevis XY8 strain can produce gamma-aminobutyric acid (GABA), which suggests that the lactobacillus brevis XY8 strain is expected to be used for producing GABA, and can be applied to the fields of improving the sleep quality of organisms, resisting depression, resisting anxiety, reducing blood pressure, improving lipid metabolism, enhancing memory and brain activity, accelerating brain metabolism, strengthening liver and kidney, promoting ethanol metabolism, improving climacteric syndrome and the like through the characteristic of producing GABA.
In a seventh aspect, the invention provides the use of a lactobacillus brevis (Levilactobacillus brevis) XY8 strain according to the first aspect for the production of hyaluronic acid.
The research shows that the probiotics lactobacillus brevis XY8 strain can produce Hyaluronic Acid (HA), which suggests that the lactobacillus brevis XY8 strain is hopeful to be used for producing HA, and the characteristics of producing HA are applied to the fields of anti-inflammation, anti-angiogenesis, anti-aging, moisturizing, wrinkle smoothing and the like.
According to an eighth aspect of the present invention there is provided the use of a strain of Lactobacillus brevis (Levilactobacillus brevis) XY8 as described in the first aspect for the preparation of an antioxidant, said strain of Lactobacillus brevis (Levilactobacillus brevis) XY8 exerting an antioxidant effect by scavenging DPPH free radicals, scavenging hydroxyl free radicals and superoxide dismutase activity.
The probiotic Lactobacillus brevis XY8 strain can remove DPPH free radical and hydroxyl free radical (OH) to avoid the harm of the two free radicals to the organism, and can also be applied to the fields of antioxidation, anti-inflammatory, immunity enhancement, aging resistance, blood lipid reduction, liver function enhancement, vision improvement, blood sugar reduction and the like through superoxide dismutase (SOD) activity. Of course, the Lactobacillus brevis (Levilactobacillus brevis) XY8 strain of the present invention can also be used as DPPH radical scavenger, hydroxyl radical scavenger, superoxide dismutase (SOD) activity enhancer, etc.
In a ninth aspect, the invention provides the use of a lactobacillus brevis (Levilactobacillus brevis) XY8 strain according to the first aspect for the preparation of a cholesterol-degrading or lowering formulation.
The research shows that the probiotic lactobacillus brevis XY8 strain can degrade cholesterol, which suggests that the lactobacillus brevis XY8 strain is expected to be used for preparing preparations for degrading or reducing cholesterol, and the damage of excessive cholesterol to organisms is avoided through the function of degrading the cholesterol.
According to a tenth aspect of the present invention, there is provided a probiotic functional bacterial agent, characterized in that the bacterial agent comprises the Lactobacillus brevis (Levilactobacillus brevis) XY8 strain according to the first aspect.
Preferably, the microbial inoculum is a fermented product of the lactobacillus brevis (Levilactobacillus brevis) XY8 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 auxiliary materials comprise a carrier and an excipient, wherein the excipient refers to a diluent, a binder, a lubricant, a disintegrating agent, a cosolvent, a stabilizer 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 injection, tablet, granule, capsule, dripping pill, sustained release agent and oral liquid preparation.
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 the fecal sample of a healthy adult in Guangdong area of China to obtain a Lactobacillus brevis (Levilactobacillus brevis) XY8 strain, wherein the strain XY8 has various probiotics effects, including excellent protease activity, 3-hydroxybutyric acid generation, xanthine oxidase inhibition, gamma-aminobutyric acid generation and secretion, hyaluronic acid generation and secretion, glutathione generation and secretion, DPPH free radical elimination, hydroxyl free radical elimination, superoxide dismutase activity and cholesterol degradation. Therefore, lactobacillus brevis XY8 strain has the functions of promoting digestion and absorption of protein food and improving protein allergy; can improve intestinal flora; can prevent and relieve hyperuricemia and gout; can resist depression and promote sleep; anti-wrinkle and whitening; antiallergic; antioxidant and anti-inflammatory; anti-aging; enhancing immunity and protecting liver; reducing cholesterol and improving cardiovascular health. Therefore, the lactobacillus brevis XY8 strain newly separated by the invention has various probiotics effects, can be used in the fields of anti-aging, gout improving and the like, for example, can be prepared into anti-aging and gout improving medicines, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Lactobacillus brevis XY8 strain (strain is derived from Genome database of NCBI, wherein Levilactobacillus brevis JT is another strain previously declared by the inventors, with the preservation number of CCTCC NO: M20221508, patent publication number of CN 116555074A);
FIG. 2 shows the degradation experiment (left, blank; right, experimental group) of Lactobacillus brevis XY8 strain on milk flat plate;
FIG. 3 shows that Lactobacillus brevis XY8 strain can produce 3-hydroxybutyric acid;
FIG. 4 shows that Lactobacillus brevis XY8 strain inhibits XOD activity;
FIG. 5 shows that Lactobacillus brevis XY8 strain can produce and secrete gamma-aminobutyric acid;
FIG. 6 shows that Lactobacillus brevis XY8 strain can produce and secrete hyaluronic acid;
FIG. 7 shows that Lactobacillus brevis XY8 strain can produce and secrete GSH;
FIG. 8 shows that Lactobacillus brevis XY8 strain can scavenge DPPH free radicals;
FIG. 9 shows that Lactobacillus brevis XY8 strain can scavenge hydroxyl radicals;
FIG. 10 shows that fermentation broth of Lactobacillus brevis XY8 strain has SOD activity;
FIG. 11 is a standard curve of cholesterol;
FIG. 12 shows cholesterol lowering effect of Lactobacillus brevis XY8 strain.
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) Lactobacillus brevis (Levilactobacillus brevis) XY8 strain isolated from a sample of intestinal faeces of a healthy adult (BMI=18.8) in Guangzhou, china was placed in glycerol tubes for cryopreservation at-80 ℃. In general, it is inoculated on the surface of a plate of MRS solid medium and cultured upside down in a constant temperature anaerobic incubator at 37℃for 24 hours to obtain colonies, or cultured in a constant temperature anaerobic incubator at 37℃in a liquid MRS medium for 24-48 hours with shaking to obtain bacterial cells and fermentation broth.
(2) The kit comprises: 3-hydroxybutyric acid (3-HB) detection kit (Cloud-Clone Corp., cat: CEB022 Ge), xanthine oxidase activity assay kit (Box manufacture, cat: AKAO 006M), gamma-aminobutyric acid (GABA) detection kit (Cloud-Clone Corp., cat: CEA900 Ge), hyaluronic acid (also known as hyaluronic acid, HA) detection kit (Cloud-Clone Corp., cat: CEA182 Ge), micro-reduced Glutathione (GSH) assay kit (Nanjing, cat: A006-2-1), DPPH free radical scavenging ability kit (Nanjing, cat: A153-1-1), hydroxyl radical (& OH) assay kit (Nanjing, cat: A018-1-1), superoxide dismutase (SOD) assay kit (Nanjing, cat: A001-3).
(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 Lactobacillus brevis (Levilactobacillus brevis) XY8 Strain
The lactobacillus brevis Levilactobacillus brevis XY strain was isolated from a sample of intestinal faeces of a healthy adult (bmi=18.8) in guangzhou, 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 XY8, and bacterial colonies of the strain are observed to be flat, convex in the middle, milky white and irregular in edges after 24 hours of culture.
Next, the isolated XY8 strain was subjected to molecular characterization by a 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACTT), and then the XY8 strain was subjected to whole genome sequencing by Beijing Baimeike Biotechnology Co. The resulting 16S rDNA sequence (SEQ ID No: 1) was subjected to BLAST alignment at NCBI' S Genome database. The results showed that the XY8 strain had >99% homology with the known Lactobacillus brevis (Levilactobacillus brevis) 16S rDNA sequence and was analyzed by evolution with the homologous strain (FIG. 1) to confirm that XY8 was a homologous, different strain of Lactobacillus brevis.
Finally, strain XY8 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. M20231167; deposit unit address: chinese university of Wuhan; classification naming: levilactobacillus brevis.
Lactobacillus brevis is a probiotic strain which can be used in foods and has wide probiotic effects, such as antivirus, constipation relieving, intestinal health improving, sleep improving, cholesterol reducing, diabetes improving and the like, but different strains from different sources have different effects, which shows that a novel Lactobacillus brevis XY8 isolated from the intestinal tract of Drosophila can be used as a probiotic and possibly has novel effects and functions.
Levilactobacillus brevis XY8 16S rDNA sequence(1432bp，SEQ ID No：1)：
GTCGAACGAGCTTCCGTTGAATGACGTGCTTGCACTGATTTCAACAATGAAGCGAGTGGCGAACTGGTGAGTAACACGTGGGAAATCTGCCCAGAAGCAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTTGTTTGAAAGGTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGGTGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACACCTTTGAGAGTAACTGTTCAAGGGTTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTAGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAATCTTAGAGATAAGACGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTA。
Example 2 function and application of Lactobacillus brevis (Levilactobacillus brevis) XY8 strain
(1) Protease capable of producing degradable milk protein by lactobacillus brevis XY8 strain
The ability of Lactobacillus brevis XY8 strain to secrete proteolytic proteins was determined according to the agar well diffusion assay using skim milk plate medium (MP plate). In the test, 3uL of Lactobacillus brevis XY8 bacterial liquid with the concentration of 10Abs is dripped into an MP plate of an experimental group, and 3uL of blank MRS culture medium is dripped into a control group. And cultured upside down in an anaerobic incubator at 37℃for 3 days. The results show that XY8 can significantly degrade protein and form a distinct degradation circle (FIG. 2) compared with the control in which the blank medium was added dropwise, indicating that Lactobacillus brevis XY8 strain can produce proteases that degrade milk protein.
Therefore, the lactobacillus brevis XY8 strain can produce protease, and can promote the digestion and absorption of protein in food by human body and improve the absorption of small peptide and amino acid when used as a probiotic 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) Lactobacillus brevis XY8 strain can produce 3-hydroxybutyric acid (3-HB)
Lactobacillus brevis XY8 cultured in MRS liquid medium to stationary phase is spread in new MRS liquid medium at dilution ratio of 1:30, bacterial suspension is collected at stationary phase for 24 hr, cultured thallus is collected after centrifugation at 10,000Xg and 4deg.C for 10min, and obtained thallus is obtained by buffer PBS (8 g NaCl, 0.2g KCl, 1.44g Na are weighed 2 HPO 4 、0.24gKH 2 PO 4 Dissolving in 800mL distilled water, regulating the solution to 7.2 with HCl, adding distilled water to a volume of 1L to obtain PBS buffer solution, and then performing cleavage to prepare a thallus lysate, and measuring the concentration of 3-HB of the cultured thallus by using 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 XY8 was 216.18. Mu.g/mL as compared with the cell lysis buffer PBS, indicating that Lactobacillus brevis XY8 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 Lactobacillus brevis XY8 can be used as 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 Lactobacillus brevis XY8 strain can exert the above multiple uses by virtue of producing 3-hydroxybutyric acid.
In addition, 3-hydroxybutyric acid is an endogenous small molecule substance naturally produced by the body, has an important role in maintaining the integrity of colorectal tissues, and has the functions of maintaining intestinal health, preventing colonic diseases and diminishing inflammation and productivity. The 3-HB treatment can promote the proliferation of beneficial intestinal bacteria, relieve the symptoms of multiple sclerosis, and has great potential in regulating flora and improving health. Therefore, the probiotic Lactobacillus brevis XY8 strain is also helpful for improving intestinal flora and relieving intestinal inflammation.
(3) Lactobacillus brevis XY8 strain can inhibit Xanthine Oxidase (XOD) activity
Lactobacillus brevis XY8 strain cultured in MRS liquid culture medium to stationary phase is spread 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 XY8 had a significant inhibition of xanthine oxidase activity compared to the blank medium MRS without inhibition of xanthine oxidase activity with a rate of 89.73% (< 0.05), indicating that lactobacillus brevis XY8 can produce and secrete metabolites to inhibit the activity of Xanthine Oxidase (XOD) during stationary phase (fig. 4).
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 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, and has questioned safety. It has been used until now because of its excellent inhibitory effect on xanthine oxidase. Therefore, the research of new low-toxicity and high-efficiency xanthine oxidase inhibitors is of great significance.
Thus, the probiotic Lactobacillus brevis XY8 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.
(4) Lactobacillus brevis XY8 strain can produce gamma-aminobutyric acid (GABA)
Lactobacillus brevis XY8 cultured in MRS liquid medium to stationary phase is spread in new MRS liquid medium at dilution ratio of 1:30, bacterial suspension is collected at stationary phase for 24 hr, cultured thallus is collected after centrifugation at 10,000Xg and 4deg.C for 10min, and obtained thallus is obtained by buffer PBS (8 g NaCl, 0.2g KCl, 1.44g Na are weighed 2 HPO 4 、0.24gKH 2 PO 4 Dissolving in 800mL distilled water, regulating the solution to 7.2 with HCl, adding distilled water to a volume of 1L to obtain PBS buffer solution, performing lysis to obtain thallus lysate, and measuring GABA concentration in thallus after fermentation culture by using GABA specific ELISA kit (CEA 900 Ge). The results showed that GABA concentration in the cell lysate of the strain XY8 was significantly increased compared to the cell lysate buffer PBS, and the accumulated amount was 289.73pg/mL, indicating that Lactobacillus brevis XY8 was able to produce gamma-aminobutyric acid in the stationary phase (FIG. 5).
Gamma-aminobutyric acid is an important central nervous system inhibitory neurotransmitter, and is widely present in animals, plants and microorganisms. It has been demonstrated that GABA, a small molecular weight non-protein amino acid, is food safe and can be used as a food additive. Research shows that the intake of a certain amount of GABA has the physiological effects of improving the sleep quality of organisms, resisting depression, resisting anxiety, reducing blood pressure, improving lipid metabolism, enhancing memory and brain activity, accelerating brain metabolism, strengthening liver and kidney, promoting ethanol metabolism (dispelling the effects of alcohol), improving climacteric syndrome and the like.
Thus, the probiotic Lactobacillus brevis XY8 strain can exert the above multiple uses by virtue of the production of gamma-aminobutyric acid.
(5) Lactobacillus brevis XY8 strain can produce and secrete Hyaluronic Acid (HA)
Lactobacillus brevis XY8 cultured in MRS liquid medium to stationary phase was expanded into new MRS liquid medium at dilution ratio of 1:30, bacterial suspension was collected at stationary phase 24h, supernatant of fermentation broth was collected after centrifugation at 10,000Xg at 4℃for 10min, and HA concentration of supernatant of fermentation broth was measured by hyaluronic acid (also known as hyaluronic acid, HA) specific ELISA kit (CEA 182 Ge). The results showed that the concentration of HA in the fermentation supernatant of strain XY8 was significantly increased compared to the MRS control, and the accumulated amount was 119.02ng/mL, indicating that lactobacillus brevis XY8 can produce and secrete hyaluronic acid in the stationary phase (fig. 6).
Hyaluronic acid, also known as hyaluronic acid, is a biodegradable, biocompatible, non-toxic, non-allergenic polymer with a variety of biological functions. Has anti-inflammatory and anti-angiogenesis effects, and has strong anti-aging, moisturizing and wrinkle smoothing abilities. The anti-wrinkle agent is beneficial to skin anti-wrinkle, promotes wound anti-inflammation and healing, can be used as an anti-wrinkle agent, and has the potential of developing skin cosmetics. In addition, HA HAs high lubricating, water absorbing and retaining ability, and can affect various cell functions such as migration, adhesion and proliferation, so that HA is also widely used in biomedical fields such as ophthalmic surgery, arthritis treatment, wound healing scaffolds, tissue engineering, implant materials, and the like.
Thus, the probiotic Lactobacillus brevis XY8 strain can exert the above multiple uses by virtue of hyaluronic acid production.
(6) Lactobacillus brevis XY8 strain can produce and secrete reduced Glutathione (GSH)
Lactobacillus brevis XY8 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 XY8 was 281.60 μmol/L, and that the concentration of GSH after XY8 fermentation was significantly increased (< 0.001) compared to the MRS control, indicating that lactobacillus brevis XY8 can produce and secrete Glutathione (GSH) during the stationary phase (fig. 7).
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 Lactobacillus brevis XY8 strain can exert the above multiple effects by the function of the GSH produced.
(7) Lactobacillus brevis XY8 strain capable of scavenging DPPH free radical
Lactobacillus brevis XY8 strain cultured to stationary phase by MRS liquid culture medium 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 DPPH scavenging ability of fermentation broth supernatant is measured by DPPH free radical scavenging ability kit (Nanjing built, cat: A153-1-1). The results showed that fermentation supernatant of strain XY8 had a DPPH radical scavenging rate of 15.28% (×p < 0.01) compared to the blank MRS, demonstrating that lactobacillus brevis XY8 has a better DPPH radical scavenging ability, exhibiting a good antioxidant capacity (fig. 8).
The term "radical" is also referred to as "radical" in chemistry, and refers to an atom or group having unpaired electrons formed by homolytic cleavage of a molecule of a compound under external conditions such as photothermal. Since free radicals contain unpaired electrons, they are extremely unstable (particularly hydroxyl radicals) and therefore abstract electrons from neighboring molecules (including fat, protein and DNA) and leave themselves in a stable state. In this way, the adjacent molecules become a new radical and then abstract electrons. Such a linkage reaction may damage the structure of the cell, resulting in loss of cell function, gene mutation, and even death.
Free radicals have a number of disadvantages. Such as: (1) Weakening the resistance of cells, and making the body susceptible to bacterial and germ infection; (2) Generating cell-destroying chemicals, forming carcinogens; (3) Preventing the normal development of cells, interfering the recovery function of the cells and leading the update rate of the cells to be lower than the withering rate; (4) Disrupting genetic (DNA) tissue in the body, disrupting cellular operation and regenerative function, causing genetic mutations, and evolving into cancer; (5) Disruption of mitochondria (energy storage) within cells, causing oxidative fatigue; (6) Destroying the cell membrane, interfering with the metabolism of the cell, so that the cell membrane loses the function of protecting the cell; (7) Amino acids necessary for invasion of tissues and hormones interfere with the operation of the in vivo system, resulting in vicious circle, so that more free radicals are generated, which chain reaction causes the free radicals to endanger the whole body; (8) Disrupting proteins, disrupting enzymes in the body, leading to inflammation and aging; (9) Destroying fat to cause lipid peroxidation, which leads to atherosclerosis and cardiovascular and cerebrovascular diseases; (10) Breaking down carbohydrates, degrading hyaluronic acid, leading to arthritis, etc.
Thus, the probiotic lactobacillus brevis XY8 strain can avoid the harm of the free radical to the organism by eliminating the function of DPPH free radical.
(8) Lactobacillus brevis XY8 strain can scavenge hydroxyl radical (OH)
Lactobacillus brevis XY8 strain cultured to stationary phase by MRS liquid culture medium is expanded and cultured to 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 capability of the fermentation broth supernatant for scavenging hydroxyl free radicals is measured by a hydroxyl free radical (OH) measuring kit (Nanjing built, cat: A018-1-1). The results showed that the fermentation supernatant of strain XY8 had a hydroxyl radical clearance of 23.08% (. Times.p < 0.001) compared to the blank MRS, indicating that lactobacillus brevis XY8 had better ability to scavenge hydroxyl radicals, exhibiting good antioxidant capacity (fig. 9).
Hydroxyl radicals (. OH) are an important active oxygen species, which, from the molecular formula, are formed by the loss of one electron from the hydroxyl radical (OH). Hydroxyl radical has extremely strong electron-obtaining capability, namely oxidizing capability, oxidation potential is 2.8V, and the hydroxyl radical is an oxidant which is second to fluorine in nature.
Hydroxyl radicals can destroy almost all types of macromolecules, including carbohydrates, nucleic acids (mutations), lipids (lipid peroxidation), and amino acids (e.g., phenylalanine to meta-tyrosine and ortho-tyrosine), etc. The attack of hydroxyl radicals on the human body begins with the cell membrane, which causes the cell membrane to lose its elasticity and function, causing diseases of the cardiovascular system. A large amount of data prove that the occurrence mechanism of various diseases such as inflammation, tumor, aging, hematopathy, heart, liver, spleen, lung, skin and the like has close relation with the excessive generation of free radicals in the body or the reduced capability of scavenging the free radicals.
Thus, the probiotic Lactobacillus brevis XY8 strain can avoid the harm of the free radical to the organism by eliminating the function of the hydroxyl free radical.
(9) Lactobacillus brevis XY8 strain has superoxide dismutase (SOD) activity
Lactobacillus brevis XY8 strain cultured to a stationary phase by using an MRS liquid culture medium is expanded and cultivated into a new MRS liquid culture medium at a dilution ratio of 1:30, bacterial suspension is collected when the culture is carried out to the stationary phase for 24 hours, fermentation broth supernatant is collected after centrifugation at 10,000 Xg and 4 ℃ for 10 minutes, and then SOD activity of the fermentation broth supernatant is measured by a superoxide dismutase (SOD) measuring kit (Nanjing built, cat: A001-3). Wherein, when the SOD inhibition rate in each milliliter of reaction solution reaches 50 percent, the corresponding SOD amount is one SOD activity unit (U). The results showed that the SOD activity of the fermentation supernatant of strain XY8 was 9.52U/mg compared to the control MRS, indicating that the Lactobacillus brevis XY8 fermentation broth had better SOD activity and exhibited good antioxidant capacity (FIG. 10).
Superoxide dismutase (SOD) is an antioxidant enzyme, and has antioxidant, antiinflammatory, and immunity enhancing effects. The main expression is as follows: (1) antioxidant: SOD can remove free radical in cells, relieve damage of oxidative stress to cells, and protect cells from free radical attack. (2) anti-inflammatory: SOD can inhibit inflammatory response, reduce inflammatory cell release, and reduce the degree and duration of inflammation. (3) enhancing immunity: SOD can promote the production of immune cells, improve the immunity of the body, prevent the occurrence of diseases and restore the health of the body. (4) anti-aging: can prevent lipid peroxidation, effectively remove free radicals, keep cells active, and prevent aging. (5) reducing blood fat: can remove intravascular resistance, soften blood vessel, reduce blood viscosity, and gradually restore blood lipid level. (6) enhancing liver function: can reduce toxin in blood and reduce damage of oxygen free radical to liver. (7) improving vision: is helpful for scavenging excessive free radicals in eyes, improving vision, and preventing cataract. (8) reducing blood sugar: can restore activity of islet cells, accelerate division speed, and further achieve the purpose of reducing blood sugar.
Thus, the probiotic Lactobacillus brevis XY8 strain can exert the above effects by having a function of superoxide dismutase (SOD) activity.
(10) Lactobacillus brevis XY8 strain degradable cholesterol
Preparation of a phthalic dicarboxaldehyde solution: accurately weighing 0.05g of phthalaldehyde reagent, dissolving with glacial acetic acid, and fixing the volume to 100mL for later use. Simultaneously, a standard stock solution of cholesterol (1.0 mg/mL) was prepared and refrigerated at 4℃for further use. Diluting cholesterol stock solution according to 2-time gradient, adding glacial acetic acid to 2mL, adding 4mL of phthalic aldehyde solution, standing at room temperature for 10min, slowly adding 4mL of 18.4M concentrated sulfuric acid into cold water bath, vortex mixing, naturally cooling, standing for 10min, measuring absorbance value at 550nm within 20min, and constructing a standard curve of cholesterol (figure 11).
Lactobacillus brevis XY8 strain cultured with MRS liquid medium to stationary phase is expanded into new MRS-cholesterol (0.2 mg/mL) medium at dilution ratio of 1:30, bacterial suspension is collected at stationary phase for 24h, fermentation broth supernatant is collected after centrifugation at 10,000Xg and 4deg.C for 10min, absorbance value of supernatant at 550nm is measured, and cholesterol content in supernatant is calculated according to standard curve of cholesterol. The results showed that the clearance of cholesterol from the fermentation supernatant of strain XY8 compared to the blank MRS was 8.92% (< P < 0.001), indicating that lactobacillus brevis XY8 has the ability to degrade cholesterol (fig. 12).
Cholesterol is an important component of body tissue, and excessive cholesterol intake has become an important factor in inducing cardiovascular and cerebrovascular diseases such as coronary heart disease, atherosclerosis, and cerebral apoplexy. A large scale of research results have shown that controlling elevated cholesterol levels not only reduces the risk of heart disease, but also helps to avoid kidney disease such as renal failure.
The total cholesterol is too high, which can lead to various diseases. The main expression is as follows: (1) cholesterol stones: the high secretion of liver cholesterol can lead to supersaturation of cholesterol, and the crystallization and the overgrowth of the cholesterol can further destroy the balance of the liver and intestine circulatory system of bile acid, thereby leading to the formation of cholesterol stones in the organism. (2) lung cancer: high cholesterol diets may be associated with an increased risk of developing lung cancer, but the amount of cholesterol intake in the diet is affected by the constitution of the individual, the composition of the diet, or other high fat diet, and does not accurately reflect the actual level of cholesterol in the body, which is more closely related to the risk of developing lung cancer. (3) coronary arteriosclerosis: elevated serum LDL and triacylglycerol levels are important causes of atherosclerotic lesion formation. Lowering LDL cholesterol levels has become an important goal in reducing the complications of atherosclerotic lesions and the resultant cardiovascular and cerebrovascular diseases. Atherosclerotic lesions begin with localized accumulation of LDL, which is sequestered under the endothelium by adherence to proteoglycan-rich extracellular matrix proteins and accumulated by mechanisms such as changes in endothelial permeability, intercellular transport, and active receptor-mediated cell migration. (4) hypercholesterolemia: the risk of hypercholesterolemia is obviously increased for men and women with excessive intake of dietary cholesterol (more than or equal to 300 mg/d). (5) coronary heart disease and ischemic stroke: elevated cholesterol or low density lipoprotein cholesterol is an independent risk factor for coronary heart disease and ischemic stroke. Plasma high density lipoprotein cholesterol may be associated with diseases such as inflammation, thrombosis, etc.
Thus, the probiotic Lactobacillus brevis XY8 strain can avoid the damage to the organism caused by excessive cholesterol by degrading the function of cholesterol.
Taken together, the newly isolated lactobacillus brevis (Levilactobacillus brevis) XY8 strain of the present invention has various probiotic effects: (1) has excellent protease activity; (2) 3-hydroxybutyric acid may be produced; (3) can inhibit xanthine oxidase activity; (4) gamma-aminobutyric acid can be produced and secreted; (5) can produce and secrete hyaluronic acid; (6) glutathione can be produced and secreted; (7) DPPH free radical can be eliminated; (8) scavenging hydroxy radicals; (9) has superoxide dismutase activity; (10) degradable cholesterol. Therefore, the lactobacillus brevis XY8 strain obtained by the new separation of the invention has important application value and economic value.
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 (10)
1. A lactobacillus brevis (Levilactobacillus brevis) XY8 strain, wherein the lactobacillus brevis XY8 strain was deposited at the chinese collection for 7 and 3 days at 2023 under the accession number: cctccc No. M20231167; the 16S rDNA complete sequence of the Lactobacillus brevis XY8 strain is shown in SEQ ID No: 1.
2. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for producing 3-hydroxybutyric acid.
3. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for the preparation of a xanthine oxidase inhibitor.
4. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for producing reduced glutathione.
5. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for producing proteases.
6. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for producing gamma-aminobutyric acid.
7. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for producing hyaluronic acid.
8. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for the preparation of an antioxidant, wherein the lactobacillus brevis (Levilactobacillus brevis) XY8 strain exerts an antioxidant effect by scavenging DPPH radicals, scavenging hydroxyl radicals and superoxide dismutase activity.
9. Use of the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1 for the preparation of a cholesterol-degrading or lowering formulation.
10. A probiotic functional bacterial agent, characterized in that it comprises the lactobacillus brevis (Levilactobacillus brevis) XY8 strain of claim 1.
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| CN117384788B (en) * | 2023-10-10 | 2024-03-22 | 广东悦创生物科技有限公司 | Saliva combined lactobacillus SM4 and application thereof in preparation of whitening and cholesterol lowering foods and medicines |
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