CN117946942A - Lactobacillus plantarum MY3 and application thereof in preparing food and medicine for regulating intestines and stomach and improving constipation - Google Patents
Lactobacillus plantarum MY3 and application thereof in preparing food and medicine for regulating intestines and stomach and improving constipation Download PDFInfo
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- CN117946942A CN117946942A CN202410191870.5A CN202410191870A CN117946942A CN 117946942 A CN117946942 A CN 117946942A CN 202410191870 A CN202410191870 A CN 202410191870A CN 117946942 A CN117946942 A CN 117946942A
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- lactobacillus plantarum
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus plantarum MY3 and application thereof in preparing food and medicines for regulating intestines and stomach and improving constipation. In order to excavate more lactobacillus plantarum with a probiotic function, the invention separates and purifies a lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 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 acetylcholinesterase activity, has excellent protease activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete glutathione, can inhibit renin activity, can promote absorption of cellulose, and can improve constipation; can be used for resisting inflammation and improving intestinal flora; promoting intestinal absorption and peristalsis and other functions, and has important potential application value in the fields of regulating intestines and stomach, improving constipation and the like.
Description
Technical Field
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus plantarum MY3 and application thereof in preparing food and medicines for regulating intestines and stomach and improving constipation.
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) MY3 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 acetylcholinesterase 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 regulating intestines and stomach, improving constipation 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) MY3 strain, wherein the lactobacillus paracasei MY3 strain is deposited with the China center for type culture collection (China, with accession number: CCTCC NO: M20231157; the whole sequence of the 16S rDNA of the cheese bacillus MY3 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of a lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain according to the first aspect in the preparation of a renin inhibitor.
The study shows that the probiotics lactobacillus plantarum MY3 strain can effectively inhibit the activity of renin, which suggests that the lactobacillus plantarum MY3 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 lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain according to the first aspect in cellulase production.
The research shows that the probiotics lactobacillus plantarum MY3 strain can produce cellulase, which suggests that the lactobacillus plantarum MY3 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 bodies on proteins in foods, improving the absorption of small peptides and amino acids, resisting allergy and the like.
In a fourth aspect, the invention provides the use of a strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 according to the first aspect for the production of 3-hydroxybutyric acid.
According to research, the probiotic lactobacillus plantarum MY3 strain can produce 3-hydroxybutyric acid (3-HB), which indicates that the lactobacillus plantarum MY3 strain can be used for producing 3-HB, and the probiotic lactobacillus plantarum MY3 strain 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) MY3 strain according to the first aspect in the preparation of an acetylcholinesterase inhibitor.
The research shows that the probiotics lactobacillus plantarum MY3 strain can effectively inhibit the activity of acetylcholinesterase, and the probiotics lactobacillus plantarum MY3 strain is suggested to be used for enhancing the cognition and the memory, dilating blood vessels, exciting skeletal muscles, smooth muscles and other fields by inhibiting the activity of acetylcholinesterase.
In a sixth aspect, the invention provides use of a protease produced by a strain of lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 as described in the first aspect.
Preferably, the protease includes, but is not limited to, a protease that degrades milk proteins.
The research shows that the probiotics lactobacillus plantarum MY3 strain can produce protease, which suggests that the lactobacillus plantarum MY3 strain is expected to be used for producing 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 a lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain according to the first aspect for the production of gamma-aminobutyric acid.
The research shows that the probiotic lactobacillus plantarum MY3 strain can produce gamma-aminobutyric acid (GABA), which suggests that the lactobacillus plantarum MY3 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 by producing GABA.
According to an eighth aspect of the invention, there is provided the use of a strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 according to the first aspect in the production of reduced glutathione.
The research shows that the probiotic lactobacillus plantarum MY3 strain can produce reduced Glutathione (GSH), which suggests that the lactobacillus plantarum MY3 strain can be used for producing GSH and is used in the fields of antioxidation, whitening, aging delay, immunity enhancement, anti-tumor, antiallergic and the like by the characteristic of GSH production.
In a ninth aspect, the invention provides a probiotic functional bacterial agent, comprising the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of the first aspect.
Preferably, the microbial inoculum is a product of lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 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) MY3 strain which has a plurality of probiotics effects, including excellent cellulase activity, can generate and secrete 3-hydroxybutyric acid, can inhibit acetylcholinesterase activity, has excellent protease activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete glutathione, and can inhibit renin activity. Therefore, the lactobacillus plantarum MY3 strain has the advantages of promoting the absorption of cellulose and improving constipation; can be used for resisting inflammation and improving intestinal flora; promoting intestinal absorption and peristalsis; promoting digestion and absorption of protein food, and improving protein allergy; can be used for resisting depression and relieving hangover; has effects in resisting oxidation, whitening skin, resisting aging, relieving inflammation and resisting allergy; reducing blood pressure and protecting kidney. Therefore, the lactobacillus plantarum MY3 strain newly separated by the invention has various probiotics effects, can be used for conditioning intestines and stomach, improving constipation and other fields, for example, can be prepared into medicines for conditioning intestines and stomach and improving constipation, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Lactobacillus plantarum XY3 strains (Lactobacillus plantarum JF1 from China patent No. CN116555075B, lactobacillus plantarum JF4 from China patent No. CN116656526B, and the remaining tree building strains from the Genome database of NCBI);
FIG. 2 shows the degradation experiment (left, blank; right, experimental group) of Lactobacillus plantarum MY3 strain on cellulose plates;
FIG. 3 shows that Lactobacillus plantarum MY3 strain can produce and secrete 3-hydroxybutyric acid;
FIG. 4 shows that the secretable material of the fermentation broth of Lactobacillus plantarum MY3 significantly inhibits the activity of acetylcholinesterase;
FIG. 5 shows the degradation experiment (left, blank; right, experimental group) of Lactobacillus plantarum MY3 on milk plates;
FIG. 6 shows that Lactobacillus plantarum MY3 can produce and secrete gamma-aminobutyric acid;
FIG. 7 shows that Lactobacillus plantarum MY3 strain can produce and secrete GSH;
FIG. 8 shows that the secreted substances of Lactobacillus plantarum MY3 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: lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain was isolated from a sample of intestinal faeces of a healthy adult (BMI=19.5) in Guangzhou area of Guangdong, china by the entrusted moisturizing laboratory and was stored in a glycerol tube by cryogenic freezing 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), gamma-aminobutyric acid (GABA) detection kit (Cloud-Clone Corp., cat: CEA900 Ge), acetylcholinesterase inhibitor screening kit (abnova, cat: KA 6219), micro-reduced Glutathione (GSH) assay kit (Nanjing built, 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) MY3 Strain
Lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 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 MY3, 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 MY3 strain was subjected to molecular characterization by 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 strain MY3 has >99% homology with the known Lactobacillus plantarum (Lactiplantibacillus plantarum) 16S rDNA sequence and that it was analyzed by evolution with the homologous strain (FIG. 1) to confirm that MY3 is a different strain of Lactobacillus plantarum.
Finally, strain MY3 is preserved with the following information: preservation time: 2023, 7, 3; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: CCTCC NO: M20231157; deposit unit address: chinese university of Wuhan; classification naming: lactiplantibacillus plantarum.
Lactobacillus plantarum is a probiotic bacterial strain with wide probiotic effects such as anti-inflammatory, antihypertensive and antipathogenic bacteria, but different sources of bacterial strains have different effects, which indicates that the novel lactobacillus plantarum MY3 bacterial strain separated from a fecal sample of a healthy adult in Guangdong area can be used as a probiotic and possibly has novel effects and functions.
Lactiplantibacillus plantarum MY3 16S rDNA sequence(1442bp，SEQ ID No：1)：
TATACATGCAGTCGACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTAGAACCAGCCGCCTAAG.
Example 2 function and application of Lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 Strain
(1) Lactobacillus plantarum MY3 strain can produce cellulase
The ability of lactobacillus plantarum MY3 to degrade cellulose was identified and determined according to the agar well diffusion method using cellulose plate medium (CMC plate). In the test, 3uL of Lactobacillus plantarum MY3 bacterial solution with the concentration of 10Abs is dripped into CMC plates of the experimental group, and 3uL of blank MRS culture medium is dripped into the 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 MY3 can significantly degrade cellulose to form an obvious degradation circle (figure 2), which shows that the lactobacillus plantarum MY3 strain can produce cellulase.
Since lactobacillus plantarum MY3 strain can produce cellulase, the probiotic lactobacillus plantarum MY3 strain can play the following multiple purposes through the effect of producing cellulase: (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 MY3 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 MY3 strain can produce and secrete 3-hydroxybutyric acid (3-HB)
Lactobacillus plantarum MY3 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 MY3 was 0.51. Mu.g/mL, compared to the absence of 3-HB in the blank medium MRS, indicating that Lactobacillus plantarum MY3 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 MY3 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 lactobacillus plantarum MY3 strain can exert the functions by producing the effect of 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 MY3 strain also helps to improve intestinal flora and relieve intestinal inflammation.
(3) The fermentation liquid of the plant lactobacillus MY3 strain can effectively inhibit the activity of acetylcholinesterase
Lactobacillus plantarum MY3 cultured to a 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, fermentation broth supernatant is collected after centrifugation at 10,000 Xg and 4 ℃ for 10 minutes, and then the inhibition capacity of the fermentation broth supernatant on acetylcholinesterase (AchE) activity is measured by an acetylcholinesterase inhibitor screening kit (KA 6219). The results showed that the fermentation supernatant of strain MY3 significantly inhibited the activity of acetylcholinesterase compared to the non-inhibitory effect of the blank medium MRS, with an inhibition rate of about 26.33%, indicating that the fermentation broth of lactobacillus plantarum MY3 can effectively inhibit the activity of acetylcholinesterase (fig. 4).
Acetylcholinesterase inhibitors allow cholinergic to accumulate at the synapse under the action of acetylcholine released from the nerve fiber tip through reversible inhibition of acetylcholinesterase, excite cholinergic receptors, prolong and augment the action of acetylcholine. Acetylcholinesterase inhibitors can enhance cognitive ability and memory (improve Alzheimer's disease, improve cognitive dysfunction), dilate blood vessels (improve heart rate overspeed), excite skeletal and smooth muscles (improve constipation, postoperative abdominal distension, postoperative urinary retention).
Thus, the probiotic Lactobacillus plantarum MY3 strain inhibits acetylcholinesterase activity and excites skeletal and smooth muscles of the intestinal tract, which makes it a potential probiotic for promoting gastrointestinal absorption and peristalsis.
(4) Lactobacillus plantarum MY3 strain can produce protease
The ability of lactobacillus plantarum MY3 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 MY3 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 inverted and carried out for 3 days in a constant temperature anaerobic incubator at 37 ℃. The results show that compared with a control in which a blank culture medium is dripped, the strain MY3 can significantly degrade proteins to form an obvious degradation circle (figure 5), which shows that the strain MY3 of the lactobacillus plantarum can produce protease for degrading milk proteins.
The lactobacillus plantarum MY3 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 being 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 MY3 strain can produce and secrete gamma-aminobutyric acid (GABA)
Lactobacillus plantarum MY3 cultured with MRS liquid medium to stationary phase was expanded into new MRS liquid medium at a dilution factor 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 MY3 was significantly increased compared to the low concentration of GABA in the blank medium MRS, and the accumulated amount was 349.4pg/mL, indicating that lactobacillus plantarum MY3 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 MY3 strain can exert the above multiple purposes by the effect of producing gamma-aminobutyric acid.
(6) Lactobacillus plantarum MY3 strain can produce and secrete reduced Glutathione (GSH)
The Lactobacillus plantarum MY3 strain cultured to the stationary phase by using the 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 10,000Xg and 4 ℃ for 10 minutes, and then the concentration of GSH in the fermentation broth supernatant is measured by using a reduced Glutathione (GSH) measuring kit (A006-2-1). The results show that the concentration of GSH in the fermentation supernatant of strain MY3 was 299.17 μmol/L compared to the low concentration of GSH in the blank medium MRS, suggesting a significant increase in GSH concentration after fermentation by MY3 (< 0.0001) indicating that lactobacillus plantarum MY3 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 plantarum MY3 strain can exert the above effects by the function of the GSH produced.
(7) The fermented liquid of the plant lactobacillus MY3 strain can effectively inhibit the activity of renin
Lactobacillus plantarum MY3 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 fermentation broth supernatant was collected after centrifugation at 10,000xg at 4 ℃ for 10min, and then renin inhibitory ability of the fermentation broth supernatant was measured by a renin inhibitor screening kit (KA 1361). The results show that the fermentation supernatant of strain MY3 has the ability to inhibit renin compared to the non-inhibitory effect of the blank medium MRS, with an inhibition rate of about 12.72% (< 0.05), indicating that the fermentation broth of lactobacillus plantarum MY3 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 MY3 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 lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 of the present invention has a variety of probiotic effects: (1) has excellent cellulase activity; (2) 3-hydroxybutyric acid may be produced and secreted; (3) can inhibit acetylcholinesterase 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 MY3 strain newly separated and obtained by the invention has important application value and economic value in the fields of regulating intestines and stomach, improving constipation 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. Lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain, characterized in that the lactobacillus paracasei MY3 strain was deposited with the chinese collection of typical cultures at day 7 and 3 of 2023 under the accession number: CCTCC NO: M20231157; the whole sequence of the 16S rDNA of the cheese bacillus MY3 strain is shown in SEQ ID No: 1.
2. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of claim 1 for the preparation of renin inhibitors.
3. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of claim 1 for cellulase production.
4. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of claim 1 for the production of 3-hydroxybutyric acid.
5. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of claim 1 for the preparation of an acetylcholinesterase inhibitor.
6. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of claim 1 to produce proteases.
7. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 strain of claim 1 for the production of gamma-aminobutyric acid.
8. Use of the lactobacillus plantarum (Lactiplantibacillus plantarum) MY3 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) MY3 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) MY3 strain.
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