CN118146998A - Bifidobacterium longum subspecies longum KS1 and application thereof in preparation of anti-aging and sleep-aiding food and medicine - Google Patents
Bifidobacterium longum subspecies longum KS1 and application thereof in preparation of anti-aging and sleep-aiding food and medicine Download PDFInfo
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- CN118146998A CN118146998A CN202410295182.3A CN202410295182A CN118146998A CN 118146998 A CN118146998 A CN 118146998A CN 202410295182 A CN202410295182 A CN 202410295182A CN 118146998 A CN118146998 A CN 118146998A
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to bifidobacterium longum subspecies longum KS1 and application thereof in preparation of anti-aging and sleep-aiding food and drugs. In order to excavate more bifidobacterium longum subspecies with the probiotic function, the invention separates and purifies an intestinal fecal sample of a healthy adult in Guangzhou area to obtain a bifidobacterium subspecies (Bifidobacterium longum subsp.longum) KS1 strain which can produce 3-hydroxybutyric acid, can produce and secrete hyaluronic acid, can produce and secrete glutathione, has excellent protease activity, can produce and secrete gamma-aminobutyric acid, can inhibit xanthine oxidase activity and can inhibit renin activity. Has the functions of resisting inflammation and improving intestinal flora; anti-aging and anti-wrinkle; the product has the functions of resisting oxidation, whitening skin, delaying aging, resisting inflammation, resisting allergy 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 bifidobacterium longum subspecies longum KS1 and application thereof in preparation of anti-aging and sleep-aiding food and drugs.
Background
Bifidobacterium longum (Bifidobacterium longum subsp. Longum) is a subspecies of Bifidobacterium longum, a gram-positive, anaerobic branching rod-shaped bacterium, naturally occurring in the human gastrointestinal tract, most abundant in the adult human intestinal tract.
The bifidobacterium longum subspecies longum is an edible probiotic and has high safety. Long bifidobacteria subspecies are widely present in the intestinal tract of humans and animals and excreted with faeces, are part of the normal flora of the human body, and are important for maintaining the intestinal microecological balance as an important component of the normal microbial system of the intestinal tract and as a lifelong companion host. Meanwhile, the bifidobacterium longum subspecies are also edible probiotics serving as probiotics in intestinal flora, so that the digestion capacity of a human body can be enhanced, and the immunity can be improved. In recent years, long subspecies of bifidobacterium longum have become a hotspot for research as a probiotic lactobacillus with great potential and are being continuously used to make probiotic preparations suitable for humans and animals.
It was found that different strains of bifidobacterium longum subspecies longum have different probiotic functions, such as: (1) improving oxidative stress: studies show that bifidobacterium longum subspecies longum K5 has strong oxidation resistance. (2) degrading cholesterol: studies have shown that Bifidobacterium longum subspecies BCBL-583 strain can lower 86.31 + -1.85% cholesterol. (3) ameliorating symptoms associated with the disease by modulating the immune system. It has been found that oral administration of 1×109CFU/mL of bifidobacterium longum subspecies 51A in a cancer therapeutic drug irinotecan-induced intestinal mucositis mouse model can reduce intestinal permeability, inflammation and oxidative damage, increase production of secretory immunoglobulin a (IgA) in intestinal fluid of mucositis mice, and further alleviate intestinal damage caused by irinotecan; it has also been studied to find that bifidobacterium longum subspecies longum BL21 can improve type 2 diabetes by modulating glucose-related metabolism and regulating intestinal microbiota in a mouse model of type 2 diabetes. (4) has stronger antiallergic ability: the study shows that oral administration of Bifidobacterium longum subspecies 51A can reduce IgE protein of anti-ovalbumin in serum and slgA in intestinal juice, and can also reduce recruitment of eosinophils and neutrophils, thereby improving symptoms of allergy; in addition, bifidobacterium longum subspecies longum CCM7952 and bifidobacterium subspecies longum BB536 may also improve allergic reactions. (5) Has the functions of inhibiting obesity and improving metabolic diseases caused by obesity: researches show that bifidobacterium longum subspecies BL21 can remarkably improve weight increase induced by high-fat diet, improve intestinal flora disorder caused by high-fat diet, increase the abundance of Ackermansia with obesity inhibiting effect, reduce serum TC, TG and LDL-C levels, and reduce epididymal fat accumulation and liver injury; bifidobacterium longum subspecies BCBL-583 can reduce total cholesterol and LDL-cholesterol in blood, reduce the abundance of related lactococcus for obesity, and have cholesterol reducing and anti-obesity effects; in addition, bifidobacterium longum subspecies OLP-01 in combination with exercise training can be used as a strategy for the treatment of obesity.
Since the long subspecies of the bifidobacterium longum have source diversity, the long subspecies of the bifidobacterium longum have gene diversity and functional diversity, and different strains of the long subspecies of the bifidobacterium longum have different probiotic functions. At present, although a small part of researches begin to pay attention to the development and utilization of bifidobacterium longum subspecies, the researches on the separation and identification, the probiotics characteristics and the metabolic mechanism of the bifidobacterium subspecies still remain small, and the development and the utilization of the bifidobacterium subspecies are affected to a certain extent. Therefore, it is necessary to develop the probiotic function deeply according to different sources of bifidobacterium longum subspecies, define the application prospect, and further enrich the number of the bifidobacterium subspecies so as to make the bifidobacterium subspecies play a larger role in human health. In conclusion, the research and application of the probiotic bifidobacterium longum subspecies longum have wider development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention separates and purifies intestinal fecal samples of a healthy adult in Guangzhou area of China to obtain a bifidobacterium longum subspecies (Bifidobacterium longum subsp. Longum) KS1 strain which can produce 3-hydroxybutyrate, can produce and secrete hyaluronic acid, can produce and secrete glutathione, has excellent protease activity, can produce and secrete gamma-aminobutyric acid, can inhibit xanthine oxidase activity, can inhibit renin activity, and has important potential application value in the fields of anti-aging, sleep aiding 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 present invention provides a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain, said bifidobacterium subspecies longum KS1 strain having been deposited in the chinese collection of typical cultures at 9 months of 2022 under the accession number: CCTCC NO: M20221509; the 16S rDNA complete sequence of the bifidobacterium longum subspecies longum KS1 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect in the preparation of a renin inhibitor.
The research shows that the probiotic bifidobacterium longum subspecies KS1 strain can effectively inhibit the activity of renin, which suggests that the bifidobacterium subspecies longum KS1 strain can be used as a renin inhibitor for inhibiting the activity of renin, thereby preventing and treating hypertension, other cardiovascular diseases and kidney diseases.
In a third aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect in the production of reduced glutathione.
Through researches, the probiotic bifidobacterium longum subspecies KS1 strain can produce reduced Glutathione (GSH), which indicates that the bifidobacterium subspecies KS1 strain can be used for producing GSH, and can be used in the fields of antioxidation, whitening, aging delay, immunity enhancement, anti-tumor, antiallergic and the like through the characteristic of GSH production.
In a fourth aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect for the production of 3-hydroxybutyric acid.
According to research, the probiotic bifidobacterium longum subspecies KS1 strain can produce 3-hydroxybutyric acid (3-HB), and the bifidobacterium longum subspecies KS1 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, relieving intestinal inflammation and other fields through the characteristic of producing 3-HB.
In a fifth aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect for the production of hyaluronic acid.
Through researches, the bifidobacterium longum subspecies KS1 strain of probiotics can produce Hyaluronic Acid (HA), which suggests that the bifidobacterium subspecies KS1 strain of the longum is expected to be used for producing the HA, and the characteristics of the HA are applied to the fields of anti-inflammatory and anti-angiogenesis effects, anti-aging, moisturizing, wrinkle smoothing, wound diminishing inflammation and healing and the like.
In a sixth aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect in the production of a protease.
Preferably, the protease includes, but is not limited to, a protease that degrades milk proteins.
The research shows that the strain KS1 of the bifidobacterium longum subspecies longum of the probiotics can produce protease, which suggests that the strain KS1 of the bifidobacterium subspecies longum is expected to be used for producing the protease, and the characteristics of the protease are applied to the fields of promoting the digestion and absorption of the protein in the food, resisting allergy, helping the digestion and absorption of the nutrition of animals and the like.
In a seventh aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect for the production of gamma-aminobutyric acid.
The research shows that the probiotic bifidobacterium longum subspecies KS1 strain can produce gamma-aminobutyric acid (GABA), which suggests that the bifidobacterium subspecies KS1 strain can be used for producing GABA and can be used for improving the sleeping quality of organisms, resisting depression, resisting anxiety, reducing blood pressure, improving lipid metabolism and other fields by producing the GABA.
According to an eighth aspect of the present invention there is provided the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to the first aspect in the preparation of a xanthine oxidase inhibitor.
The research shows that the probiotic bifidobacterium longum subspecies KS1 strain can inhibit Xanthine Oxidase (XOD) activity, and the bifidobacterium longum subspecies KS1 strain can be used for inhibiting xanthine oxidase activity, reducing purine in vivo and uric acid generation, thereby controlling uric acid level and preventing gout attack through XOD activity.
In a ninth aspect, the present invention provides a probiotic functional bacterial agent comprising a bifidobacterium longum strain KS1 according to the first aspect.
Preferably, the microbial inoculum is a product of fermentation of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain.
Preferably, the microbial inoculum further comprises 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 intestinal canal fecal sample of a healthy adult in Guangzhou area of China to obtain a bifidobacterium longum subspecies (Bifidobacterium longum subsp. Longum) KS1 strain which has various probiotics effects including 3-hydroxybutyric acid production, hyaluronic acid production and secretion, glutathione production and secretion, excellent protease activity, gamma-aminobutyric acid production and secretion, xanthine oxidase activity inhibition and renin activity inhibition. Thus, bifidobacterium longum subspecies longum KS1 strain has a potential to be anti-inflammatory and improve intestinal flora; anti-aging and anti-wrinkle; has effects in resisting oxidation, whitening skin, resisting aging, relieving inflammation and resisting allergy; promoting digestion and absorption of protein food, and improving protein allergy; can be used for relieving depression, relieving hangover, and improving sleep; can prevent and relieve hyperuricemia and gout; lowering blood pressure and protecting kidney. Therefore, the bifidobacterium longum subspecies KS1 strain newly separated by the invention has various probiotics effects, can be used in the fields of anti-aging, sleep-aiding and the like, for example, can be prepared into anti-aging and sleep-aiding medicines, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Bifidobacterium longum subspecies KS1 strain (Bifidobacterium longum subspecies MY1 from China patent No. CN116555076B ", bifidobacterium subspecies KS2 from China patent No. CN 117286045A), the remaining established strains all being from the Genome database of NCBI);
FIG. 2 shows that bifidobacterium longum subspecies longum KS1 strain produces 3-hydroxybutyric acid;
FIG. 3 shows that bifidobacterium longum subspecies longum KS1 strain produces and secretes hyaluronic acid;
FIG. 4 shows that bifidobacterium longum subspecies longum KS1 strain can produce and secrete GSH;
FIG. 5 shows the degradation experiment of bifidobacterium longum subspecies longum KS1 on milk plates (left, blank; right, experimental group);
FIG. 6 shows that bifidobacterium longum subspecies longum KS1 can produce and secrete gamma-aminobutyric acid;
FIG. 7 shows that bifidobacterium longum subspecies longum KS1 strain inhibits XOD activity;
FIG. 8 shows that the secretive material of the Bifidobacterium longum subspecies KS1 fermentation broth significantly inhibited renin 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) Strains: the bifidobacterium longum subsp (Bifidobacterium longum subsp. Longum) KS1 strain was isolated from a sample of intestinal faeces from a healthy adult (bmi=22.8) in guangzhou, china by the entrusted moisturizing laboratory and placed in a glycerol tube for cryogenic storage at-80 ℃. In general, the strain is inoculated on the surface of a 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 bacterial colonies, or is cultured for 24-48 hours in a shaking way in a MRS liquid culture medium in a constant temperature anaerobic incubator at 37 ℃ to obtain bacterial bodies and fermentation liquor.
(2) The kit comprises: 3-hydroxybutyric acid (3-HB) detection kit (Cloud-Clone Corp., cat: CEB022 Ge), hyaluronic acid (also known as hyaluronic acid, HA) detection kit (Cloud-Clone Corp., cat: CEA182 Ge), micro-reduced Glutathione (GSH) detection kit (Nanjing, cat: A006-2-1), gamma-aminobutyric acid (GABA) detection kit (Cloud-Clone Corp., cat: CEA900 Ge), xanthine oxidase activity detection kit (Box manufacturing, cat: AKAO 006M), renin (Renin) inhibitor screening kit (abnova, cat: KA 1361).
(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, 1L of ddH 2 O, 6.2-6.6 of pH value, and autoclaving at 121 ℃ for 20min to prepare an 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, supplementing ddH 2 O to 1L, adjusting the pH to 6.2-6.6, and carrying out high-pressure sterilization at 121 ℃ for 20min to prepare the 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, 1L of ddH 2 O, pH adjustment to 6.2-6.6, and high-pressure sterilization at 121 ℃ for 20min, thus preparing an MP plate.
EXAMPLE 1 isolation and identification of Bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 Strain
The bifidobacterium longum subspecies (Bifidobacterium longum subsp. Longum) KS1 strain was isolated from a fecal sample from 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", which identify strains belonging to bacteria first. A purified strain is obtained by preliminary separation, the strain number is KS1, and after 48 hours of culture, the bacterial colony of the strain is observed to be milky white, round, convex, smooth and neat in edge.
Next, the isolated KS1 strain was subjected to molecular characterization by a 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACTT), and then subjected to whole genome sequencing by Beijing Baimaike 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 KS1 strain had >99% homology with the known bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) 16S rDNA sequence and that evolution analysis was performed with the homologous strain (FIG. 1) confirming that KS1 was a different strain of bifidobacterium subspecies longum.
Finally, strain KS1 was deposited with the following information: preservation time: 2022, 9, 27; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: CCTCC NO: M20221509; deposit unit address: chinese university of Wuhan; classification naming: bifidobacterium longum subsp.
The bifidobacterium longum subspecies longum is a probiotic bacterial strain which has wide probiotic effects, such as antivirus, constipation relieving, intestinal health improving, sleep improving, cholesterol reducing, diabetes improving and the like, but different sources of bacterial strains have different effects, which shows that a novel bifidobacterium subspecies longum KS1 separated from human excrement can be used as probiotic bacteria and possibly has novel effects and functions.
Bifidobacterium longum subsp.Longum KS1 16S rDNA sequence(1436bp，SEQ ID No：1)：
ACGCGCGGGTGCTTACCATGCAAGTCGAACGGGATCCATCAGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCGCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGCGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCGGTCTAAGGGAGGCTCGAGTG.
Example 2 Functions of Bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 Strain and uses thereof
(1) The bifidobacterium longum subspecies longum KS1 strain can produce 3-hydroxybutyric acid (3-HB)
The bifidobacterium longum subspecies KS1 cultured to the stationary phase by using an MRS liquid culture medium is expanded and cultured 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, bacterial cells are collected after centrifugation at 10,000 Xg and 4 ℃ for 10 minutes, 1OD 600 of the obtained bacterial cells is taken and dissolved in 800mL of distilled water by using 500uL buffer PBS (8 g of NaCl, 0.2g of KCl and 1.44g of Na 2HPO4、0.24g KH2 PO4 are weighed, the solution is adjusted to 7.2 by using HCl, finally distilled water is added to reach the volume of 1L, and the concentration of 3-HB of the bacterial cells after the culture is determined 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 KS1 was 94.34. Mu.g/mL as compared with the cell lysate buffer PBS, indicating that the bifidobacterium longum subspecies KS1 was able to produce 3-hydroxybutyric acid during the stationary phase (FIG. 2).
3-HB can supply energy for various physical activities and is a potential energy/functional food that has been added to athlete drinks, so the probiotic bifidobacterium longum subspecies KS1 strain 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. The probiotic bifidobacterium longum subspecies longum KS1 strain may serve several purposes as described above by providing 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. 3-HB can promote the proliferation of beneficial intestinal bacteria and relieve the symptoms of multiple sclerosis mice through treatment, and has great potential in regulating flora and improving health.
Thus, the probiotic bifidobacterium longum subspecies KS1 strain also helps to improve intestinal flora and alleviate intestinal inflammation.
(2) The bifidobacterium longum subspecies longum KS1 strain can produce and secrete Hyaluronic Acid (HA)
The bifidobacterium longum subspecies longum KS1 cultured with MRS broth to stationary phase was expanded into new MRS broth at a dilution factor of 1:30, bacterial suspension was harvested at 24h of culture to stationary phase, the supernatant of the broth was harvested after centrifugation at 10,000xg at 4 ℃ for 10min, and the HA concentration of the supernatant of the broth was then determined by means of a 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 KS1 was significantly increased compared to the low concentration of HA in the blank medium MRS, with an accumulated amount of 31.52ng/mL, indicating that bifidobacterium longum subspecies KS1 can produce and secrete gamma-aminobutyric acid during the stationary phase (fig. 3).
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 bifidobacterium longum subspecies KS1 strain may serve multiple purposes as described above by virtue of hyaluronic acid production.
(3) Bifidobacterium longum subspecies longum KS1 strain can produce and secrete reduced Glutathione (GSH)
The strain KS1 of Bifidobacterium longum grown subspecies cultivated 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 24h of stationary phase, and after centrifugation at 10,000Xg and 4 ℃ for 10min, supernatant of fermentation broth was collected, and GSH concentration of supernatant of fermentation broth was measured by reduced Glutathione (GSH) measuring kit (A006-2-1). The results show that the concentration of GSH in the fermentation supernatant of KS1 is 274.94 μmol/L, and that the concentration of GSH after KS1 fermentation is significantly increased (< P < 0.01) compared to the low concentration of GSH in the blank medium MRS, indicating that bifidobacterium longum subspecies longum KS1 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 bifidobacterium longum subspecies KS1 strain may exert the above multiple effects by virtue of the function of GSH produced.
(4) Bifidobacterium longum subspecies KS1 strain can produce protease
The ability of bifidobacterium longum subspecies KS1 to secrete protease hydrolyzed protein was identified and measured according to the agar well diffusion assay using skim milk plate medium (MP plate). In the test, 3uL of bifidobacterium longum subspecies longum KS1 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 the culture is inverted and carried out for 3 days in a constant temperature anaerobic incubator at 37 ℃. The results show that strain KS1 can significantly degrade proteins and form a distinct degradation circle (FIG. 5) compared to the control with the blank medium, indicating that strain KS1 of Bifidobacterium longum can produce proteases that degrade milk proteins.
The bifidobacterium longum subspecies KS1 can produce protease, and can promote the digestion and absorption of human body to protein in food and improve the 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.
(5) Bifidobacterium longum subspecies longum KS1 strain produces gamma-aminobutyric acid (GABA)
The bifidobacterium longum subspecies KS1 cultured to the stationary phase by using an MRS liquid culture medium is expanded and cultured 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, cultured thalli are collected after centrifugation at 10,000 Xg and 4 ℃ for 10 minutes, 1OD600 of the obtained thalli is taken and dissolved in 800mL of distilled water by using 500uL buffer PBS (8 g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 are weighed, the solution is adjusted to 7.2 by using HCl, finally distilled water is added to a volume of 1L, and the concentration of GABA in the thalli after fermentation culture is determined by using a GABA specific ELISA kit (CEA 900 Ge) after the preparation of the bacterial lysate is obtained. The results showed that the concentration of GABA in the cell lysate of the strain KS1 was significantly increased compared to the cell lysate buffer PBS, and the accumulated amount was 63.32pg/mL, indicating that the bifidobacterium longum subspecies KS1 were able to produce gamma-aminobutyric acid in the stationary phase (FIG. 6).
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 intake of a certain amount of GABA has the physiological effects of improving sleeping 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 alcohol effect), improving climacteric syndrome and the like.
Thus, the probiotic bifidobacterium longum subspecies longum KS1 strain may serve several purposes as described above by virtue of producing gamma-aminobutyric acid.
(6) The strain KS1 of Bifidobacterium longum subspecies longum can inhibit Xanthine Oxidase (XOD) activity
The strain KS1 of Bifidobacterium longum grown subspecies cultivated 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 24h of cultivation to stationary phase, and supernatant of fermentation broth was collected after centrifugation at 10,000Xg and 4℃for 10min, and then activity of xanthine oxidase in supernatant of fermentation broth was measured by xanthine oxidase activity measuring kit (box manufacturing, cat: AKAO 006M). The results showed that the fermentation supernatant of strain KS1 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 100% (< 0.05), indicating that bifidobacterium longum subspecies longum KS1 can produce and secrete metabolites during stationary phase to inhibit the activity of Xanthine Oxidase (XOD) (fig. 7).
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.
Therefore, the probiotic bifidobacterium longum subspecies KS1 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.
(7) The fermentation liquor of the bifidobacterium longum subspecies longum KS1 strain can effectively inhibit the activity of renin
The bifidobacterium longum subspecies KS1 cultured in the MRS liquid culture medium to the stationary phase are expanded into a new MRS liquid culture medium at a dilution ratio of 1:30, bacterial suspension is collected when the culture medium is cultured to the stationary phase for 24 hours, fermentation broth supernatant is collected after centrifugation at 10,000Xg and 4 ℃ for 10 minutes, and the renin inhibition capacity of the fermentation broth supernatant is measured by a renin inhibitor screening kit (KA 1361). The results show that the fermentation supernatant of strain KS1 has the ability to inhibit renin compared to the non-inhibitory effect of the blank medium MRS with an inhibition ratio of about 17.90% (< 0.05), indicating that the fermentation broth of bifidobacterium longum subspecies KS1 can effectively inhibit renin (fig. 8).
Renin is an aspartic protease of about 40kDa that converts angiotensinogen to angiotensin I. Angiotensin Converting Enzyme (ACE) is a monomeric zinc metalloenzyme found in vascular endothelium that converts angiotensin i to angiotensin ii, which is the final active messenger of the renin-angiotensin system (RAS) pathway. Angiotensin II can inhibit renin secretion by acting directly on glomerular cells. Angiotensin II has many physiological roles, the most important of which is that it can act as a powerful vasoconstrictor, increasing blood pressure by changing the resistance of the surrounding blood vessels. Since renin is the only known renin substrate, cleavage of renin by renin is critical to the ultimate activity of the RAS pathway, inhibition of renin would be an attractive strategy to control hypertension. In addition, renin inhibitors may prevent the formation of angiotensin I and angiogenin. Thus, the effect of renin inhibitors on inhibiting renin activity can be utilized to prevent and treat hypertension and other cardiovascular and renal diseases.
It can be seen that the probiotic bifidobacterium longum subspecies KS1 strain has the ability to inhibit renin, which makes it a potential probiotic for lowering blood pressure and protecting the kidneys.
Taken together, the novel isolated bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain of the present invention has a variety of probiotic efficacy: (1) 3-hydroxybutyric acid may be produced; (2) can produce and secrete hyaluronic acid; (3) glutathione can be produced and secreted; (4) has superior protease activity; (5) gamma-aminobutyric acid can be produced and secreted; (6) can inhibit xanthine oxidase activity; (7) renin activity can be inhibited. Therefore, the bifidobacterium longum subspecies KS1 strain obtained by the new separation has important application value and economic value in the fields of anti-aging, sleep aiding 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 (10)
1. A bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain, wherein the bifidobacterium subspecies longum KS1 strain was deposited with the chinese collection of typical cultures at 2022, 9 months and 27 days, under the accession number: CCTCC NO: M20221509; the 16S rDNA complete sequence of the bifidobacterium longum subspecies longum KS1 strain is shown in SEQ ID No: 1.
2. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 in the preparation of a renin inhibitor.
3. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 for the production of reduced glutathione.
4. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 for the production of 3-hydroxybutyric acid.
5. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 for the production of hyaluronic acid.
6. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 in the production of a protease.
7. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 for the production of gamma-aminobutyric acid.
8. Use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain as claimed in claim 1 in the preparation of a xanthine oxidase inhibitor.
9. A probiotic functional bacterial agent, characterized in that it comprises a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain according to claim 1.
10. The probiotic functional microbial agent according to claim 9, characterized in that it is a fermented product of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) KS1 strain.
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