CN117946939A - Lactobacillus plantarum SM2 and application thereof in preparation of cholesterol-lowering and sleep-aiding foods and medicines - Google Patents
Lactobacillus plantarum SM2 and application thereof in preparation of cholesterol-lowering and sleep-aiding foods and medicines Download PDFInfo
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- CN117946939A CN117946939A CN202410191644.7A CN202410191644A CN117946939A CN 117946939 A CN117946939 A CN 117946939A CN 202410191644 A CN202410191644 A CN 202410191644A CN 117946939 A CN117946939 A CN 117946939A
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus plantarum SM2 and application thereof in preparation of cholesterol-lowering and sleep-assisting foods and medicines. In order to excavate more lactobacillus plantarum with a probiotic function, the invention separates and purifies a lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain from a fecal sample of a healthy adult in Guangdong area, the strain has excellent cellulase activity, can generate and secrete 3-hydroxybutyric acid, can inhibit xanthine oxidase activity, has excellent protease activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete glutathione, can inhibit renin activity, and has the functions of promoting absorption of cellulose, improving constipation and reducing cholesterol; can resist depression, sober up, assist sleep and other functions, and has important application value and economic value in the fields of cholesterol reduction, sleep assistance and the like.
Description
Technical Field
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus plantarum SM2 and application thereof in preparation of cholesterol-lowering and sleep-assisting foods and medicines.
Background
Lactobacillus plantarum (Lactiplantibacillus plantarum), also known as Lactobacillus plantarum, belongs to the genus Lactobacillus of the family Lactobacillus, and is either anaerobic or facultative anaerobic, with an optimal culture pH of 6.5, and the cells are in the form of straight or curved rods, singly, sometimes in pairs or in chains. Lactobacillus plantarum is a probiotic in human gastrointestinal tract, can produce various natural antibacterial substances such as organic acid, bacteriocin, hydrogen peroxide, diethyl phthalide and the like, and has various functions of maintaining balance of intestinal flora, reducing cholesterol level, improving organism immunity, promoting nutrient absorption and the like.
Lactobacillus plantarum is an edible lactobacillus, is closely related to human life, is commonly used in dairy products, meat and fermented products of a plurality of vegetables, can play a beneficial role by being planted in intestinal tracts through stomach, has important influence on intestinal microorganisms, and has wide application in the fields of food fermentation, industrial lactic acid fermentation, medical care and the like. It is well known that lactobacillus is a part of the normal flora of the human body, and is an important component of the normal microbial system of the intestinal tract and is accompanied by a host for life, and has important significance for maintaining the microecological balance of the intestinal tract. The lactobacillus plantarum is also an important intestinal probiotics serving as edible lactobacillus, can play a role in assisting intestinal peristalsis, can also enhance the digestion capacity of a human body, improve immunity and the like. In recent years, lactobacillus plantarum has become a hotspot for research as a probiotic lactobacillus with great potential, and is being continuously made into probiotic preparations suitable for human and animals.
It was found that different strains of lactobacillus plantarum have different probiotic functions, such as: (1) anti-pathogenic bacteria: it has been found that Lactobacillus plantarum has a strong inhibitory effect on Salmonella and on helicobacter pylori. (2) modulating immunity: studies show that the lactobacillus plantarum HK-LP has strong induction effect on IL-12 of mice, and the administration of the HK-LP can inhibit the generation of immunoglobulin, resist food allergy of the mice caused by natural diet, and inhibit the growth of tumors and the transplantation of tumor cells in the bodies of the mice. (3) cadmium removal effect: the research shows that the lactobacillus plantarum CCFM8610 has acid resistance, has good tolerance function and adsorption capacity to cadmium ions in vitro, can reduce the cadmium content in the liver and kidney of the cadmium-exposed mice, and has the effect of promoting the cadmium discharge of the cadmium-exposed mice through feces. (4) inhibition of Escherichia coli: the research shows that the lactobacillus plantarum N13 has acid resistance and cholate resistance, has very high adhesion to the intestinal epithelial cell strain HT-29 cells, can inhibit the growth of intestinal invasive escherichia coli, and can prevent the invasion of the intestinal epithelial cells to HT-29. (5) has proteolytic activity: studies show that the lactobacillus plantarum CW006 has proteolytic activity, and can ferment raw milk to generate angiotensin converting enzyme inhibitory peptide, thereby achieving the antihypertensive effect.
Since lactobacillus plantarum has source diversity, the lactobacillus plantarum has gene diversity and functional diversity, and different lactobacillus plantarum strains have different probiotic functions. At present, although a small part of researches start to pay attention to the development and utilization of lactobacillus plantarum, the researches on the separation and identification, the probiotics characteristics and the metabolic mechanism of the lactobacillus plantarum are still less, and the development and the utilization of the lactobacillus plantarum are affected to a certain extent. Therefore, it is necessary to develop the probiotic function deeply according to different sources of lactobacillus plantarum, so as to define the application prospect, further enrich the number of lactobacillus plantarum and make the lactobacillus plantarum play a larger role in human health. In conclusion, the research and application of the probiotic lactobacillus plantarum have a relatively wide development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention separates and purifies a plant lactobacillus (Lactiplantibacillus plantarum) SM2 strain from a fecal sample of a healthy adult in Guangdong area of China, the strain has excellent cellulase activity, can generate and secrete 3-hydroxybutyric acid, can inhibit xanthine oxidase activity, has excellent protease activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete glutathione, can inhibit renin activity, and has important potential application value in the fields of cholesterol reduction, 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 invention provides a lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain, wherein the lactobacillus paracasei SM2 strain is preserved in China center for type culture collection (China, with the preservation number: CCTCC NO: M20221506; the whole sequence of the 16S rDNA of the Lactobacillus paracasei SM2 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of the first aspect for the preparation of a renin inhibitor.
The study shows that the probiotics lactobacillus plantarum SM2 strain can effectively inhibit the renin activity, and the lactobacillus plantarum SM2 strain is suggested to be used as a renin inhibitor for inhibiting the renin activity, so that hypertension, other cardiovascular diseases and kidney diseases can be prevented and treated.
In a third aspect, the invention provides the use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of the first aspect for producing cellulase.
The research shows that the probiotics lactobacillus plantarum SM2 strain can produce cellulase, which suggests that the lactobacillus plantarum SM2 strain can be used for producing the cellulase, and the characteristics of producing the cellulase are used for promoting the digestion and absorption of human body to protein in food, improving the absorption of small peptide and amino acid, resisting allergy and other fields.
In a fourth aspect, the invention provides the use of a lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain according to the first aspect for the production of 3-hydroxybutyric acid.
The research shows that the probiotic lactobacillus plantarum SM2 strain can produce 3-hydroxybutyric acid (3-HB), which suggests that the lactobacillus plantarum SM2 strain can be used for producing 3-HB, and can be used for providing energy for various activities of the body, resisting osteoporosis, preventing and treating chronic syndromes, improving brain cognitive functions, 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 lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain according to the first aspect for the preparation of xanthine oxidase inhibitors.
Through researches, the probiotic lactobacillus plantarum SM2 strain can inhibit Xanthine Oxidase (XOD) activity, which indicates that the lactobacillus plantarum SM2 strain can be used for inhibiting xanthine oxidase activity through XOD activity, reducing purine in vivo and uric acid generation, thereby controlling uric acid level and preventing gout attack.
In a sixth aspect, the invention provides the use of a lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain according to the first aspect for the production of proteases.
Preferably, the protease includes, but is not limited to, a protease that degrades milk proteins.
The research shows that the probiotics lactobacillus plantarum SM2 strain can produce protease, which suggests that the lactobacillus plantarum SM2 strain is hopeful 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 human bodies to proteins in foods, resisting allergy, helping animals digest and absorb nutrition and the like.
In a seventh aspect, the invention provides the use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of the first aspect for the production of gamma-aminobutyric acid.
The research shows that the probiotic lactobacillus plantarum SM2 strain can produce gamma-aminobutyric acid (GABA), which suggests that the lactobacillus plantarum SM2 strain can be used for producing GABA and is used for improving the sleeping quality of organisms, resisting depression, resisting anxiety, reducing blood pressure, improving lipid metabolism and other fields through the characteristic of producing GABA.
According to an eighth aspect of the present invention, there is provided the use of the Lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of the first aspect for the production of reduced glutathione.
The research shows that the probiotics lactobacillus plantarum SM2 strain can produce reduced Glutathione (GSH), which suggests that the lactobacillus plantarum SM2 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 ninth aspect, the invention provides a probiotic functional bacterial agent, comprising the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of the first aspect.
Preferably, the microbial inoculum is a product of lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain after fermentation.
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 the excrement sample of a healthy adult in Guangdong area of China to obtain a lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain which has a plurality of probiotics effects, including excellent cellulase activity, can generate and secrete 3-hydroxybutyric acid, can inhibit xanthine oxidase activity, has excellent protease activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete glutathione, and can inhibit renin activity. Therefore, lactobacillus plantarum SM2 strain has the effects of promoting the absorption of cellulose, improving constipation and reducing cholesterol; can be used for relieving inflammation, improving intestinal flora and improving lipid metabolism; can prevent and relieve hyperuricemia and gout; promoting digestion and absorption of protein food, and improving protein allergy; can be used for relieving depression, relieving hangover, and improving sleep; has effects in resisting oxidation, whitening skin, resisting aging, relieving inflammation and resisting allergy; reducing blood pressure and protecting kidney. Therefore, the lactobacillus plantarum SM2 strain newly separated has various probiotics, can be used in the fields of cholesterol reduction, sleep aiding and the like, for example, can be manufactured into medicines for cholesterol reduction and sleep aiding, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Lactobacillus plantarum SM2 strain (Lactobacillus plantarum JF1 is from Chinese invention patent "CN116555075B", lactobacillus plantarum JF4 is from Chinese invention patent "CN116656526B", and the remaining tree building strains are all from Genome database of NCBI);
FIG. 2 shows the degradation experiment (left, blank; right, experimental group) of Lactobacillus plantarum SM2 strain on cellulose plates;
FIG. 3 shows that Lactobacillus plantarum SM2 strain can produce and secrete 3-hydroxybutyric acid;
FIG. 4 Lactobacillus plantarum SM2 strain can inhibit XOD activity;
FIG. 5 shows the degradation experiment (left, blank control; right, experimental group) of Lactobacillus plantarum SM2 strain on milk plates;
FIG. 6 shows that Lactobacillus plantarum SM2 strain can produce and secrete gamma-aminobutyric acid;
FIG. 7 shows that Lactobacillus plantarum SM2 strain can produce and secrete GSH;
fig. 8 shows that lactobacillus plantarum SM2 fermentation broth secretable substances significantly inhibit 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: lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain was committed to a gut microbiome study moisturizing laboratory from a gut stool sample isolated from a healthy adult (BMI=23.0) in Guangzhou, china 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 manner in a constant temperature anaerobic incubator at 37 ℃ to obtain a bacterial colony, or is cultured for 24-48 hours in a shaking manner in a MRS liquid culture medium in a constant temperature anaerobic incubator at 37 ℃ to obtain a 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 manufacturing, cat: AKAO M), gamma-aminobutyric acid (GABA) detection kit (Cloud-Clone Corp., cat: CEA900 Ge), micro-reduced Glutathione (GSH) assay kit (Nanjing, cat: A006-2-1), 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.
(6) CMC plate: 10g of sodium carboxymethyl cellulose, 1.5g of ammonium sulfate, 0.3g of manganese sulfate, 0.2g of calcium chloride, 5g of sodium chloride, 0.3g of urea, 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, 6.2-6.6, and autoclaving at 121 ℃ for 20min, and preparing into CMC plates.
EXAMPLE 1 isolation and identification of Lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 Strain
The lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain is isolated from a fecal sample of a healthy adult in Guangdong area of China, and is 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. The primary separation is carried out to obtain a purified strain with the strain number of SM2, and after 24 hours of culture, white round colony and convex center are observed, and the colony surface is fine and smooth.
Next, the isolated SM2 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 the SM2 strain had >99% homology with the known Lactobacillus plantarum (Lactiplantibacillus plantarum) 16S rDNA sequence and was analyzed by evolution with the homologous strain (FIG. 1) to confirm that SM2 was a different strain of Lactobacillus plantarum.
Finally, strain SM2 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: M20221506; deposit unit address: chinese university of Wuhan; classification naming: lactiplantibacillus plantarum.
Lactobacillus plantarum is a probiotic bacterial strain which can be used for food, has wide probiotic effects, such as anti-inflammatory, antihypertensive and antipathogenic bacteria, and the like, but different sources of bacterial strains have different effects, which shows that the novel lactobacillus plantarum SM2 bacterial strain separated from a fecal sample of a healthy adult in Guangdong area can be used as the probiotic, and possibly has novel effects and functions.
Lactiplantibacillus plantarum SM2 16S rDNA sequence(1436bp，SEQ ID No：1)：
TTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCTCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACT.
Example 2 function of Lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 Strain and use thereof
(1) Lactobacillus plantarum SM2 strain can produce cellulase
The ability of lactobacillus plantarum SM2 to degrade cellulose was identified and determined according to the agar well diffusion method using cellulose plate medium (CMC plate). In the experiment, 3uL of lactobacillus plantarum SM2 bacterial solution with the concentration of 10Abs is dripped into a CMC plate of an experimental group, and 3uL of blank MRS culture medium is dripped into a control group. Congo red staining was performed after 3 days of inversion culture in an anaerobic incubator at 37 ℃. The results show that compared with a control in which a blank culture medium is dripped, the strain SM2 can significantly degrade cellulose to form an obvious degradation circle (figure 2), which shows that the lactobacillus plantarum SM2 strain can produce cellulase.
Since lactobacillus plantarum SM2 strains can produce cellulases, the probiotic lactobacillus plantarum SM2 strains can exert the following multiple purposes through the effect of producing cellulases: (1) for promoting the digestive absorption of dietary components; (2) The constipation is improved, and probiotics decompose cellulose in intestinal tracts to generate certain moisture, so that the constipation relieving tea has the effect of softening stool; (3) Cholesterol reduction is the most important effect of cellulose, because the soluble fiber can inhibit the absorption of cholesterol by human body after being absorbed by human body, can be combined with cholesterol in intestinal tract, quickens the metabolism of cholesterol in human body, prevents human body from inducing hyperlipidemia due to hypercholesteremia, and can reduce the incidence rate of arteriosclerosis and coronary heart disease; (4) The non-sweet 'sugar' of cellulose can slow down the absorption of glucose by blood, balance the concentration of blood sugar and promote the sensitivity of muscle and fat cells to insulin, thereby preventing and assisting in treating diabetes.
In addition, the lactobacillus plantarum SM2 strain can also be applied to fermentation extraction of cellulase; applied to degrading cell walls of pathogenic fungi and controlling diseases; the method is applied to the decomposition of cellulose in compost; the method is applied to preparing livestock and poultry raising feeds, such as monogastric animal feeds for pigs, chickens and the like, so as to overcome the defect that cellulose cannot be utilized.
(2) Lactobacillus plantarum SM2 strain can produce and secrete 3-hydroxybutyric acid (3-HB)
Lactobacillus plantarum SM2 strain cultured with 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 fermentation broth supernatant was collected after centrifugation at 10,000Xg and 4℃for 10min, and then 3-HB concentration of fermentation broth supernatant was measured by 3-HB specific ELISA kit (CEB 022 Ge). The results showed that the concentration of 3-HB in the supernatant of strain SM2 was 1.14. Mu.g/mL, compared to the absence of 3-HB in the blank medium MRS, indicating that Lactobacillus plantarum SM2 can produce and secrete 3-hydroxybutyric acid during the stationary phase (FIG. 3).
3-Hydroxybutyric acid (3-HB) can provide energy for various activities of the body, is a potential energy/functional food, has been added to athlete drinks, and thus the probiotic Lactobacillus plantarum SM2 can be used as an additive for 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 lactobacillus plantarum SM2 strain can exert the functions through the effect of producing 3-hydroxybutyric acid.
In addition, 3-HB is an endogenous small molecule naturally occurring in the body, has an important role in maintaining colorectal tissue integrity, and has the effects of maintaining intestinal health, preventing colonic disease and diminishing inflammation. The 3-HB treatment can promote the proliferation of beneficial intestinal bacteria, relieve the symptoms of multiple sclerosis mice, and has great potential in the aspects of regulating flora and improving health. Therefore, the probiotic lactobacillus plantarum SM2 strain also helps to improve intestinal flora and alleviate intestinal inflammation.
(3) Lactobacillus plantarum SM2 strain can inhibit Xanthine Oxidase (XOD) activity
Lactobacillus plantarum SM2 strain cultured with MRS liquid medium to stationary phase is spread into new MRS liquid 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 SM2 had a significant inhibition of xanthine oxidase activity compared to the absence of inhibition of xanthine oxidase activity by the blank medium MRS with a rate of 61.53% (< 0.05), indicating that lactobacillus plantarum SM2 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.
Therefore, the probiotic lactobacillus plantarum SM2 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 plantarum SM2 strain can produce protease
The ability of lactobacillus plantarum SM2 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 lactobacillus plantarum SM2 bacterial solution 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 performed for 3 days in an anaerobic incubator with constant temperature at 37 ℃. The results show that the strain SM2 can significantly degrade proteins and form a significant degradation circle (FIG. 5) compared with a control in which a blank medium is dripped, which indicates that the Lactobacillus plantarum SM2 strain can produce proteases that degrade milk proteins.
The lactobacillus plantarum SM2 can produce protease, and can promote digestion and absorption of protein in food by human body and improve absorption of small peptide and amino acid when used as a probiotic bacterial strain. And can be used for resisting allergy (improving food allergy caused by protein dyspepsia or non-absorption). In addition, the method can also be used for extracting protease and can be applied to the production of protease in food industry, washing industry and the like; can also be used in microbial feed to help animals digest and absorb nutrition, and improve the utilization rate of the feed.
(5) Lactobacillus plantarum SM2 strain can produce and secrete gamma-aminobutyric acid (GABA)
Lactobacillus plantarum SM2 cultured with MRS liquid medium to stationary phase was expanded into new MRS liquid medium at a dilution ratio of 1:30, bacterial suspension was collected at 24h of stationary phase, and after centrifugation at 10,000×g at 4 ℃ for 10min, the supernatant of the fermentation broth was collected, and then GABA concentration of the supernatant of the fermentation broth was measured by GABA specific ELISA kit (CEA 900 Ge). The results showed that the concentration of GABA in the fermentation supernatant of strain SM2 was significantly increased compared to the low concentration of GABA in the blank medium MRS, and the accumulated amount was 173.60pg/mL, indicating that lactobacillus plantarum SM2 can produce and secrete 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 Lactobacillus plantarum SM2 strain can exert the above multiple uses by the effect of producing gamma-aminobutyric acid.
(6) Lactobacillus plantarum SM2 strain can produce and secrete reduced Glutathione (GSH)
Lactobacillus plantarum SM2 strain cultured with MRS liquid medium to stationary phase is expanded and cultivated into new MRS liquid medium at dilution ratio of 1:30, bacterial suspension is collected when the strain is cultivated to stationary phase for 24 hours, fermentation broth supernatant is collected after centrifugation at 10,000Xg and 4 ℃ for 10 minutes, and then concentration of GSH in the 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 SM2 was 261.93 μmol/L compared to the low concentration of GSH in the blank medium MRS, suggesting a significant increase in GSH concentration after fermentation with SM2 (< 0.01), indicating that lactobacillus plantarum SM2 can produce and secrete Glutathione (GSH) during 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, fluorides 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 plantarum SM2 strain may exert the above multiple effects by the function of the GSH produced.
(7) The fermentation liquid of the lactobacillus plantarum SM2 strain can effectively inhibit the activity of renin
Lactobacillus plantarum SM2 cultured in an MRS liquid culture medium to a stationary phase is expanded and cultured in 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 then renin inhibition capacity of the fermentation broth supernatant is measured through a renin inhibitor screening kit (KA 1361). The results show that the fermentation supernatant of strain SM2 has the ability to inhibit renin compared to the non-inhibitory effect of the blank medium MRS, with an inhibition rate of about 14.28% (< 0.05), indicating that the fermentation broth of lactobacillus plantarum SM2 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 lactobacillus plantarum SM2 strain has the ability to inhibit renin, which makes it a potential probiotic for lowering blood pressure and protecting the kidneys.
In summary, the newly isolated lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of the present invention has various probiotic effects: (1) has excellent cellulase activity; (2) 3-hydroxybutyric acid may be produced and secreted; (3) can inhibit xanthine oxidase activity; (4) has superior protease activity; (5) gamma-aminobutyric acid can be produced and secreted; (6) glutathione can be produced and secreted; (7) renin activity can be inhibited. Therefore, the lactobacillus plantarum SM2 strain obtained by the new separation has important application value and economic value in the fields of cholesterol reduction, 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 lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain, characterized in that the lactobacillus paracasei SM2 strain was deposited with the chinese collection of typical cultures at 9 and 27 of 2022 under the accession number: CCTCC NO: M20221506; the whole sequence of the 16S rDNA of the Lactobacillus paracasei SM2 strain is shown in SEQ ID No: 1.
2. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the preparation of renin inhibitors.
3. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the production of cellulases.
4. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the production of 3-hydroxybutyric acid.
5. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the preparation of xanthine oxidase inhibitors.
6. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the production of proteases.
7. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the production of gamma-aminobutyric acid.
8. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1 for the production of reduced glutathione.
9. A probiotic functional bacterial agent, characterized in that it comprises the lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain of claim 1.
10. The probiotic functional microbial agent of claim 9, wherein the microbial agent is a fermented product of lactobacillus plantarum (Lactiplantibacillus plantarum) SM2 strain.
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