CN117721033B - Lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines - Google Patents
Lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines Download PDFInfo
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- CN117721033B CN117721033B CN202311321763.1A CN202311321763A CN117721033B CN 117721033 B CN117721033 B CN 117721033B CN 202311321763 A CN202311321763 A CN 202311321763A CN 117721033 B CN117721033 B CN 117721033B
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to a lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines. The invention separates and purifies intestinal canal fecal sample of a healthy adult in Guangzhou area of China to obtain a fermented lactobacillus mucilaginosus (Limosilactobacillus fermentum) KS6 strain which has various probiotics effects, including excellent protease activity, can generate 3-hydroxybutyric acid, can inhibit xanthine oxidase activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete hyaluronic acid, can generate and secrete glutathione, can remove DPPH free radical, can remove hydroxyl free radical, has superoxide dismutase activity, can degrade cholesterol, can be used in the fields of anti-inflammatory and sleep-aiding, and the like, can be manufactured into anti-inflammatory and sleep-aiding medicines, 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 a lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines.
Background
Lactobacillus mucilaginosus (Limosilactobacillusfermentum), also known as lactobacillus fermentum, gram-positive rod-shaped bacteria, facultative anaerobism. The lactobacillus fermentum has higher tolerance and can adapt to the environment with low acid and high bile salt in human intestinal tracts. In addition, the lactobacillus fermentum also has higher safety and genetic stability, is one of the earliest planted probiotics in human intestinal tracts, can continuously change along with the growth of human ages and the change of human bodies, and can influence the health of human bodies when the number of the probiotics is lower than a normal value.
Lactobacillus fermentum is widely present in the intestinal tract of humans and animals and is excreted with faeces. Lactobacillus mucilaginosus is a part of the normal flora of the human body, which is one of the important components of the normal microbial system of the intestinal tract and accompanies the host for life, and has important significance for maintaining the microecological balance of the intestinal tract. Meanwhile, as probiotics in intestinal flora, the fermented mucus lactobacillus is also edible lactobacillus, has no toxic or side effect, can play a role in helping intestinal peristalsis, and can also enhance the digestion capability of human bodies, improve immunity and the like. At present, fermented lactobacillus mucilaginosus has become a hot spot of research as a probiotic lactobacillus mucilaginosus with great potential and is being continuously used to make probiotic preparations suitable for human and animals.
Studies have shown that different strains of lactobacillus fermentum have different probiotic functions, such as: (1) enhancing host immunity. For example, the lactobacillus mucilaginosus CECT5716 can effectively prevent, relieve and treat common uncomfortable symptoms of pregnant women and lactating women, help the fetus to pre-establish an intestinal immune protection system before birth, and remarkably improve the immunity and the resistance of infants. (2) antibacterial: preventing respiratory tract infection, reducing incidence of common cold, cough, etc., and preventing vaginal infection. (3) improving intestinal health: promoting health of gastrointestinal digestive system, reducing harmful substances in intestinal tract, and relieving constipation. (4) Helping the fetus build an autoimmune system, and laying a foundation for the innate immunity of the baby. (5) Effectively preventing and treating mastitis, relieving mammitis, relieving breast soreness, relieving pain, promoting breast health, and relieving tension and anxiety.
Lactobacillus fermentum has genetic and functional diversity due to its diversity in sources. However, there are still few studies on the isolation and identification, the probiotic characteristics and the metabolic mechanism of lactobacillus fermentum at present, and it has been found that lactobacillus fermentum with probiotic functions is not very much, and although some research progress has been made on the functions and applications of lactobacillus fermentum at present, the potential efficacy of lactobacillus fermentum has not yet been fully developed. Therefore, it is necessary to excavate more lactobacillus fermentum with probiotic functions and further excavate the functions of lactobacillus fermentum according to different sources of lactobacillus fermentum so as to make the lactobacillus fermentum function better, for example, determining the efficacy of lactobacillus fermentum according to the functions of the strain or the probiotic metabolites, and defining the application prospect of lactobacillus fermentum. In conclusion, the probiotic fermented lactobacillus mucilaginosus and the application thereof have wide development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention separates and purifies intestinal fecal samples of a healthy adult in Guangzhou area of China to obtain a fermented lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain which has the functions of producing 3-hydroxybutyric acid, inhibiting xanthine oxidase activity, producing and secreting gamma-aminobutyric acid, producing glutathione and the like, and has important potential application value.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The first aspect of the invention provides a lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain, wherein the lactobacillus fermentum KS6 strain is deposited with the China center for type culture collection, with accession number: CCTCC NO: M20231158; the 16S rDNA complete sequence of the lactobacillus mucilaginosus KS6 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of a lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the production of 3-hydroxybutyric acid.
The research shows that the probiotic fermentation lactobacillus mucilaginosus KS6 strain can produce 3-hydroxybutyric acid (3-HB), which suggests that the fermentation lactobacillus mucilaginosus KS6 strain is expected to be used for producing 3-HB, and can be applied to the fields of providing energy for various activities of the body, resisting osteoporosis, preventing and treating chronic syndrome, improving brain cognitive function, improving lipid metabolism and the like through the characteristic of producing 3-HB.
In a third aspect the invention provides the use of a lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the production of reduced Glutathione (GSH).
Through researches, the probiotic fermented lactobacillus mucilaginosus KS6 strain can produce reduced Glutathione (GSH), which suggests that the fermented lactobacillus mucilaginosus KS6 strain is expected to be used for producing GSH, and is applied to the fields of antioxidation, whitening, aging delay, immunity enhancement, anti-tumor, antiallergic and the like through the characteristic of GSH production.
In a fourth aspect, the invention provides the use of a lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the preparation of a xanthine oxidase inhibitor.
The research shows that the probiotic fermented lactobacillus mucilaginosus KS6 strain can inhibit Xanthine Oxidase (XOD) activity, and suggests that the fermented lactobacillus mucilaginosus KS6 strain is expected to be applied to the fields of reducing purine in vivo and uric acid generation, controlling uric acid level, preventing gout attack and the like by inhibiting the XOD activity.
In a fifth aspect, the invention provides the use of a lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to the first aspect in the production of a protease.
The research shows that the probiotics fermentation lactobacillus mucilaginosus KS6 strain can produce protease, which suggests that the fermentation lactobacillus mucilaginosus KS6 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 sixth aspect, the invention provides the use of a lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the production of gamma-aminobutyric acid.
The research shows that the probiotic fermented lactobacillus mucilaginosus KS6 strain can produce gamma-aminobutyric acid (GABA), and the probiotic fermented lactobacillus mucilaginosus KS6 strain is expected to be used for producing GABA, and can be applied to the fields of improving the 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, improving climacteric syndrome and the like through the characteristic of producing GABA.
In a seventh aspect, the invention provides the use of a lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the production of hyaluronic acid.
The research shows that the probiotics fermentation lactobacillus mucilaginosus KS6 strain can produce Hyaluronic Acid (HA), which suggests that the fermentation lactobacillus mucilaginosus KS6 strain is hopeful to be used for producing HA, and is applied to the fields of anti-inflammation, anti-angiogenesis, anti-aging, moisturizing, wrinkle smoothing and the like through the characteristic of producing the HA.
According to an eighth aspect of the present invention, there is provided the use of the Lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the preparation of an antioxidant, said Lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain exerting an antioxidant effect by scavenging DPPH free radicals, scavenging hydroxyl free radicals and superoxide dismutase activity.
The probiotic fermented lactobacillus mucilaginosus KS6 strain can remove DPPH free radical and hydroxyl free radical (OH) so as to avoid the harm of the two free radicals to the organism, and can also be applied to the fields of antioxidation, anti-inflammatory, immunity enhancement, aging resistance, blood fat reduction, liver function enhancement, vision improvement, blood sugar reduction and the like through superoxide dismutase (SOD) activity. Of course, the Lactobacillus mucilaginosus KS6 strain of the present invention may be used as DPPH radical scavenger, hydroxyl radical scavenger, superoxide dismutase (SOD) activity enhancer, etc.
In a ninth aspect, the invention provides the use of a lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to the first aspect for the preparation of a cholesterol-degrading or lowering formulation.
The research shows that the probiotic fermented lactobacillus mucilaginosus KS6 strain can degrade cholesterol, which suggests that the fermented lactobacillus mucilaginosus KS6 strain is hopeful to be used for preparing preparations for degrading or reducing cholesterol, and the harm to organisms caused by excessive cholesterol is avoided through the function of degrading the cholesterol.
In a tenth aspect, the invention provides a probiotic functional bacterial agent comprising the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain of the first aspect.
Preferably, the microbial inoculum is a fermented product of the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to the first aspect.
Preferably, in the field of pharmaceutical application, the microbial agent further comprises a pharmaceutically acceptable carrier and/or excipient.
More preferably, the above excipients refer to diluents, binders, lubricants, disintegrants, co-solvents, stabilizers, etc. and some pharmaceutically acceptable bases which are useful in the pharmaceutical field. 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 fermented lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain, wherein the strain KS6 has various probiotics effects, including excellent protease activity, can generate 3-hydroxybutyric acid, inhibit xanthine oxidase activity, can generate and secrete gamma-aminobutyric acid, can generate and secrete hyaluronic acid, can generate and secrete glutathione, can remove DPPH free radical, can remove hydroxyl free radical, has superoxide dismutase activity and can degrade cholesterol. Thus, the lactobacillus mucilaginosus KS6 strain has the functions of promoting the digestion and absorption of protein foods and improving protein allergy; can improve intestinal flora; can prevent and relieve hyperuricemia and gout; can resist depression and promote sleep; anti-wrinkle and whitening; antiallergic; antioxidant and anti-inflammatory; anti-aging; enhancing immunity and protecting liver; reducing cholesterol and improving cardiovascular health. Therefore, the newly separated lactobacillus mucilaginosus KS6 strain has various probiotics effects, can be used in the fields of anti-inflammatory and sleep-aiding, and the like, for example, can be prepared into anti-inflammatory and sleep-aiding medicines, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Lactobacillus mucilaginosus KS6 strain (strain is derived from Genome database of NCBI, wherein Limosilactobacillusfermentum JF is another strain previously declared by the inventors and has a preservation number of CCTCC NO: M20221511 and a patent publication number of CN 116814464A);
FIG. 2 shows the degradation experiment (left, blank; right, experimental group) of Lactobacillus mucilaginosus KS6 strain on milk plates;
FIG. 3 shows that Lactobacillus mucilaginosus KS6 strain produces 3-hydroxybutyric acid;
FIG. 4 shows that Lactobacillus mucilaginosus KS6 strain inhibits XOD activity;
FIG. 5 shows that Lactobacillus mucilaginosus KS6 strain produces gamma-aminobutyric acid;
FIG. 6 shows that Lactobacillus mucilaginosus KS6 strain can produce and secrete hyaluronic acid;
FIG. 7 shows that Lactobacillus mucilaginosus KS6 strain can produce and secrete GSH;
FIG. 8 shows that Lactobacillus mucilaginosus KS6 strain can scavenge DPPH free radicals;
FIG. 9 shows that Lactobacillus mucilaginosus KS6 strain can scavenge hydroxyl radicals;
FIG. 10 shows that fermentation broth of Lactobacillus mucilaginosus KS6 strain has SOD activity;
FIG. 11 is a standard curve of cholesterol;
FIG. 12 shows cholesterol lowering by Lactobacillus mucilaginosus KS6 strain.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The following examples relate to the following experimental materials:
(1) Lactobacillus mucilaginosus (Limosilactobacillus fermentum) KS6 strain was isolated from a intestinal fecal sample of a healthy adult (bmi=19.5) 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 reversely cultured for 24 hours in a constant temperature anaerobic incubator at 37 ℃ to obtain a bacterial colony, or is shake cultured for 24-48 hours in a MRS liquid culture medium in the 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 maker, cat: AKAO M), GABA detection kit (CEA 900 Ge), hyaluronic acid detection kit (CEA 182 Ge), reduced Glutathione (GSH) assay kit (A006-2-1), DPPH radical scavenging ability kit (Nanjing, cat: A153-1-1), hydroxyl radical (. OH) assay kit (Nanjing, cat: A018-1-1), superoxide dismutase (SOD) assay kit (Nanjing, cat: A001-3).
(3) MRS plate: 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar, 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 Lactobacillus mucilaginosus (Limosilactobacillus fermentum) KS6 Strain
The lactobacillus mucilaginosus Limosilactobacillusfermentum KS strain was isolated from a sample of intestinal faeces of a healthy adult (bmi=19.5) in guangzhou, china as follows:
the fecal sample was repeatedly washed 3 times with sterile water, placed in a mortar, 500uL of sterile water was added per 100mg of fecal sample, thoroughly ground to homogenate, and an appropriate amount of the grinding fluid was pipetted, spread on an MRS plate, and incubated at room temperature for 3 days. Colonies to be streaked and purified in the separation assay plates were then numbered with a marker and strain numbers were marked on the plates accordingly. After labelling, colonies were picked and inoculated onto MRS plates and the strains were purified by plate streaking. If the strain cannot be separated by the method, colonies need to be picked from the enrichment plate, and the colonies are coated on a culture medium after being subjected to gradient dilution by MRS liquid culture medium. Finally, reference is made to the "Burjie's Manual of identification of bacteria" (eighth edition) and the "manual of identification of classification of fungi", which identify the strains belonging to bacteria first, and then observe the growth of the colonies: growth morphology, presence or absence of hyphae, uniformity or absence of color, colony individual surface and edge. The primary separation is carried out to obtain a purified strain, and after 24 hours of culture, bacterial colony of the strain is observed to be round, milky white, smooth in surface, raised and neat in edge, and the strain number is KS6.
Next, the isolated KS6 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 KS6 strain had >99% homology with the known 16S rDNA sequence of Lactobacillus fermentum (Limosilactobacillusfermentum) and was analyzed by evolution with the homologous strain (FIG. 1), confirming that the KS6 strain was a different strain of Lactobacillus fermentum.
Finally, strain KS6 was deposited with the following information: preservation time: 2023, 7, 3; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: CCTCC NO: M20231158; deposit unit address: chinese university of Wuhan; classification naming: limosilactobacillusfermentum.
Lactobacillus mucilaginosus is a state approved probiotic strain for food, which has wide probiotic effects such as antibacterial, anti-inflammatory and improving health of mother and infant, but different sources of strains have different effects, which means that a new lactobacillus mucilaginosus KS6 isolated from kimchi samples according to the present invention can be used as a probiotic and may have new effects and functions.
Limosilactobacillusfermentum KS616S rDNA sequence(1450bp，SEQ ID No：1)：
TACTGCAGTCGACGCGTTGGCCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACANCGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAANGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGAACACCCAAAGTCGGTGGGGTAACCTTTAGGAGCCAGCCGCCTAAGTG.
Example 2 function and use of Lactobacillus mucilaginosus (Limosilactobacillus fermentum) KS6 Strain
(1) Protease capable of producing degradable milk protein by fermenting lactobacillus mucilaginosus KS6 strain
The ability of lactobacillus mucilaginosus KS6 to secrete proteolytic proteins was determined according to the agar well diffusion assay using skim milk plate medium (MP plate). In the test, 3uL of lactobacillus mucilaginosus KS6 bacteria solution with the concentration of 10Abs is dripped into an MP plate of the experimental group, and 3uL of blank MRS culture medium is dripped into a control group. And cultured upside down in an anaerobic incubator at 37℃for 3 days. The results show that strain KS6 can significantly degrade proteins and form a significant degradation circle (FIG. 2) compared with the control in which the blank medium is added dropwise, indicating that the strain KS6 of Lactobacillus mucilaginosus can produce proteases that degrade milk proteins.
It can be seen that lactobacillus mucilaginosus KS6 produces protease, and when used as a probiotic bacterial strain, can promote digestion and absorption of protein in food by human body, and improve absorption of small peptide and amino acid. And can be used for resisting allergy (improving food allergy caused by protein dyspepsia or non-absorption). In addition, the method can also be used for extracting protease and is applied to the production in the protease fields of food industry, washing industry and the like; can also be used in microbial feed to help animals digest and absorb nutrition, and improve the utilization rate of the feed.
(2) Lactobacillus mucilaginosus KS6 strain produces 3-hydroxybutyric acid (3-HB)
The lactobacillus mucilaginosus KS6 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, the obtained thalli are dissolved in 800mL distilled water by using buffer PBS (8 g NaCl, 0.2g KCl and 1.44g Na 2HPO4、0.24gKH2PO4 are weighed, the solution is regulated to 7.2 by using HCl, finally distilled water is added to reach a volume of 1L, and the PBS buffer solution is obtained), and after the thalli are cracked to prepare a thalli lysate, the concentration of 3-HB of the thalli is measured 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 KS6 was 290.71. Mu.g/mL as compared with the cell lysate buffer PBS, indicating that Lactobacillus fermentum KS6 can produce 3-hydroxybutyric acid in the stationary phase (FIG. 3).
3-HB can provide energy for various physical activities and is a potential energy/functional food that has been added to athlete drinks, so that probiotic-fermented Lactobacillus mucilaginosus KS6 can be an additive to energy foods. Meanwhile, in view of the fact that 3-HB can effectively resist osteoporosis, prevent and treat chronic syndromes (hypertension, alcoholic fatty liver, enteritis and intestinal cancer), improve brain cognitive functions (improving learning and memory capacity, protecting glial cells and improving Alzheimer's disease), and improve lipid metabolism. Thus, the probiotic fermented lactobacillus mucilaginosus KS6 strain can exert the above multiple uses by the effect of producing 3-hydroxybutyric acid.
In addition, 3-hydroxybutyric acid is an endogenous small molecule substance naturally produced by the body, has an important role in maintaining the integrity of colorectal tissues, and has the functions of maintaining intestinal health, preventing colonic diseases and diminishing inflammation and productivity. The 3-HB treatment can promote the proliferation of beneficial intestinal bacteria, relieve the symptoms of multiple sclerosis, and has great potential in regulating flora and improving health. Thus, probiotic fermented lactobacillus mucilaginosus KS6 strain also helps to improve intestinal flora and alleviate intestinal inflammation.
(3) Lactobacillus mucilaginosus KS6 strain inhibits Xanthine Oxidase (XOD) activity
Lactobacillus mucilaginosus KS6 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 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 M). The results showed that the fermentation supernatant of strain KS6 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 lactobacillus mucilaginosus KS6 can produce and secrete metabolites to inhibit Xanthine Oxidase (XOD) activity 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. At present, allopurinol is one of main medicines for treating hyperuricemia and gout, and is the only chemical medicine for inhibiting uric acid generation clinically, but the medicine has a plurality of side effects, can cause fever, allergic rash abdominal pain, diarrhea, leucocyte and thrombocytopenia and multiple organ damage, even has reports of death, and has questioned safety. It has been used until now because of its excellent inhibitory effect on xanthine oxidase. Therefore, the research of new low-toxicity and high-efficiency xanthine oxidase inhibitors is of great significance.
Thus, the probiotic fermented lactobacillus mucilaginosus KS6 strain is expected to reduce in-vivo purine and uric acid generation by inhibiting xanthine oxidase activity, thereby controlling uric acid level and preventing gout flares.
(4) Lactobacillus mucilaginosus KS6 strain produces and secretes gamma-aminobutyric acid (GABA)
Lactobacillus fermentum KS6 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 stationary phase, and after centrifugation at 10,000×g at 4 ℃ for 10min, the broth supernatant was harvested, and then GABA concentration of the broth supernatant was determined by GABA specific ELISA kit (CEA 900 Ge). The results showed that the concentration of GABA in the fermentation supernatant of strain KS6 was significantly increased compared to the blank MRS control, with an accumulated amount of 85.80pg/mL, indicating that lactobacillus mucilaginosus KS6 can produce and secrete γ -aminobutyric acid during the stationary phase (fig. 5).
Gamma-aminobutyric acid is an important central nervous system inhibitory neurotransmitter, and is widely present in animals, plants and microorganisms. It has been demonstrated that GABA, a small molecular weight non-protein amino acid, is food safe and can be used as a food additive. Research shows that 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 fermented lactobacillus mucilaginosus KS6 strain can exert the above multiple uses by the effect of producing gamma-aminobutyric acid.
(5) The lactobacillus mucilaginosus KS6 strain can produce and secrete Hyaluronic Acid (HA)
Lactobacillus fermentum KS6 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 10,000×g at 4 ℃ for 10min, and then the HA concentration of the supernatant of the broth was determined by means of a hyaluronic acid (also known as hyaluronic acid, HA) specific ELISA kit (CEA 182 Ge). The results showed a significant increase in the concentration of HA in the fermentation supernatant of strain KS6 compared to the blank MRS control, with an accumulated amount of 76.12ng/mL, indicating that lactobacillus mucilaginosus KS6 can produce and secrete hyaluronic acid during stationary phase (fig. 6).
Hyaluronic acid, also known as hyaluronic acid, is a biodegradable, biocompatible, non-toxic, non-allergenic polymer with a variety of biological functions. Has anti-inflammatory and anti-angiogenesis effects, and has strong anti-aging, moisturizing and wrinkle smoothing abilities. The anti-wrinkle agent is beneficial to skin anti-wrinkle, promotes wound anti-inflammation and healing, can be used as an anti-wrinkle agent, and has the potential of developing skin cosmetics. In addition, HA HAs high lubricating, water absorbing and retaining ability, and can affect various cell functions such as migration, adhesion and proliferation, so that HA is also widely used in biomedical fields such as ophthalmic surgery, arthritis treatment, wound healing scaffolds, tissue engineering, implant materials, and the like.
Thus, the probiotic fermented lactobacillus mucilaginosus KS6 strain can exert the above multiple uses by the efficacy of hyaluronic acid production.
(6) Lactobacillus mucilaginosus KS6 strain can produce and secrete reduced Glutathione (GSH)
Lactobacillus mucilaginosus KS6 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 after centrifugation at 10,000Xg and 4℃for 10min, fermentation broth supernatant was collected, and then concentration of GSH in fermentation broth supernatant was measured by reduced Glutathione (GSH) measuring kit (A006-2-1). The results showed that the concentration of GSH in the fermentation supernatant of strain KS6 was 301.47 μmol/L compared to the blank MRS control, and that the concentration of GSH after KS6 fermentation was significantly increased (< 0.0001) indicating that lactobacillus mucilaginosus KS6 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 fermented lactobacillus mucilaginosus KS6 strain can exert the above multiple effects by the function of the generated GSH.
(7) The fermented lactobacillus mucilaginosus KS6 strain can remove DPPH free radical
Lactobacillus mucilaginosus KS6 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 DPPH scavenging ability of fermentation broth supernatant is measured by DPPH free radical scavenging ability kit (Nanjing built, cat: A153-1-1). The results showed that the fermentation supernatant of strain KS6 had a DPPH radical scavenging rate of 12.34% (×p < 0.01) compared to the blank MRS, demonstrating that lactobacillus mucilaginosus KS6 has a better DPPH radical scavenging ability, exhibiting a good antioxidant capacity (fig. 8).
The term "radical" is also referred to as "radical" in chemistry, and refers to an atom or group having unpaired electrons formed by homolytic cleavage of a molecule of a compound under external conditions such as photothermal. Since free radicals contain unpaired electrons, they are extremely unstable (particularly hydroxyl radicals) and therefore abstract electrons from neighboring molecules (including fat, protein and DNA) and leave themselves in a stable state. In this way, the adjacent molecules become a new radical and then abstract electrons. Such a linkage reaction may damage the structure of the cell, resulting in loss of cell function, gene mutation, and even death.
Free radicals have a number of disadvantages. Such as: (1) Weakening the resistance of cells, and making the body susceptible to bacterial and germ infection; (2) Generating cell-destroying chemicals, forming carcinogens; (3) Preventing the normal development of cells, interfering the recovery function of the cells and leading the update rate of the cells to be lower than the withering rate; (4) Disrupting genetic (DNA) tissue in the body, disrupting cellular operation and regenerative function, causing genetic mutations, and evolving into cancer; (5) Disruption of mitochondria (energy storage) within cells, causing oxidative fatigue; (6) Destroying the cell membrane, interfering with the metabolism of the cell, so that the cell membrane loses the function of protecting the cell; (7) Amino acids necessary for invasion of tissues and hormones interfere with the operation of the in vivo system, resulting in vicious circle, so that more free radicals are generated, which chain reaction causes the free radicals to endanger the whole body; (8) Disrupting proteins, disrupting enzymes in the body, leading to inflammation and aging; (9) Destroying fat to cause lipid peroxidation, which leads to atherosclerosis and cardiovascular and cerebrovascular diseases; (10) Breaking down carbohydrates, degrading hyaluronic acid, leading to arthritis, etc.
Thus, the lactobacillus mucilaginosus KS6 strain can avoid the harm of the free radical to the organism by eliminating the function of DPPH free radical.
(8) Lactobacillus mucilaginosus KS6 strain can scavenge hydroxyl radicals (OH)
Lactobacillus mucilaginosus KS6 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 supernatant of fermentation broth was collected after centrifugation at 10,000Xg and 4℃for 10min, and then the scavenging ability of supernatant of fermentation broth to hydroxyl radical was measured by a hydroxyl radical (. OH) measuring kit (Nanjing built, cat: A018-1-1). The results show that the fermentation supernatant of strain KS6 had a hydroxyl radical clearance of 24.76% (< P < 0.01) compared to the blank MRS, indicating that lactobacillus mucilaginosus KS6 has a better ability to scavenge hydroxyl radicals, exhibiting a good antioxidant capacity (fig. 9).
Hydroxyl radicals (. OH) are an important active oxygen species, which, from the molecular formula, are formed by the loss of one electron from the hydroxyl radical (OH). Hydroxyl radical has extremely strong electron-obtaining capability, namely oxidizing capability, oxidation potential is 2.8V, and the hydroxyl radical is an oxidant which is second to fluorine in nature.
Hydroxyl radicals can destroy almost all types of macromolecules, including carbohydrates, nucleic acids (mutations), lipids (lipid peroxidation), and amino acids (e.g., phenylalanine to meta-tyrosine and ortho-tyrosine), etc. The attack of hydroxyl radicals on the human body begins with the cell membrane, which causes the cell membrane to lose its elasticity and function, causing diseases of the cardiovascular system. A large amount of data prove that the occurrence mechanism of various diseases such as inflammation, tumor, aging, hematopathy, heart, liver, spleen, lung, skin and the like has close relation with the excessive generation of free radicals in the body or the reduced capability of scavenging the free radicals.
Thus, the lactobacillus mucilaginosus KS6 strain can avoid the harm of the free radical to the organism by removing the function of the hydroxyl free radical.
(9) The fermented lactobacillus mucilaginosus KS6 strain has superoxide dismutase (SOD) activity
Lactobacillus mucilaginosus KS6 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 supernatant of fermentation broth was collected after centrifugation at 10,000Xg and 4℃for 10min, and then SOD activity of supernatant of fermentation broth was measured by superoxide dismutase (SOD) measuring kit (Nanjing built, cat: A001-3). Wherein, when the SOD inhibition rate in each milliliter of reaction solution reaches 50 percent, the corresponding SOD amount is one SOD activity unit (U). The results showed that the SOD activity of the fermentation supernatant of strain KS6 was 9.52U/mg compared with the blank MRS, indicating that the fermentation broth of Lactobacillus mucilaginosus KS6 had better SOD activity and exhibited good antioxidant capacity (FIG. 10).
Superoxide dismutase (SOD) is an antioxidant enzyme, and has antioxidant, antiinflammatory, and immunity enhancing effects. The main expression is as follows: (1) antioxidant: SOD can remove free radical in cells, relieve damage of oxidative stress to cells, and protect cells from free radical attack. (2) anti-inflammatory: SOD can inhibit inflammatory response, reduce inflammatory cell release, and reduce the degree and duration of inflammation. (3) enhancing immunity: SOD can promote the production of immune cells, improve the immunity of the body, prevent the occurrence of diseases and restore the health of the body. (4) anti-aging: can prevent lipid peroxidation, effectively remove free radicals, keep cells active, and prevent aging. (5) reducing blood fat: can remove intravascular resistance, soften blood vessel, reduce blood viscosity, and gradually restore blood lipid level. (6) enhancing liver function: can reduce toxin in blood and reduce damage of oxygen free radical to liver. (7) improving vision: is helpful for scavenging excessive free radicals in eyes, improving vision, and preventing cataract. (8) reducing blood sugar: can restore activity of islet cells, accelerate division speed, and further achieve the purpose of reducing blood sugar.
Thus, the probiotic fermented lactobacillus mucilaginosus KS6 strain can exert the above effects by having the function of superoxide dismutase (SOD) activity.
(10) Degradable cholesterol of lactobacillus mucilaginosus KS6 strain
Preparation of a phthalic dicarboxaldehyde solution: accurately weighing 0.05g of phthalaldehyde reagent, dissolving with glacial acetic acid, and fixing the volume to 100mL for later use. Simultaneously, a standard stock solution of cholesterol (0.1 mg/mL) was prepared and refrigerated at 4℃for further use. Diluting cholesterol stock solution according to 2-time gradient, adding glacial acetic acid to 2mL, adding 4mL of phthalic aldehyde solution, standing at room temperature for 10min, slowly adding 4mL of 18.4M concentrated sulfuric acid into cold water bath, vortex mixing, naturally cooling, standing for 10min, measuring absorbance value at 550nm within 20min, and constructing a standard curve of cholesterol (figure 11).
Lactobacillus mucilaginosus KS6 strain cultured with MRS liquid medium to stationary phase was expanded into new MRS-cholesterol medium at dilution ratio of 1:30, bacterial suspension was collected at 24h of stationary phase, supernatant of fermentation broth was collected after centrifugation at 10,000Xg and 4 ℃ for 10min, absorbance value of supernatant at 550nm was then measured, and cholesterol content in supernatant was calculated according to standard curve of cholesterol. The results showed that the clearance of cholesterol from the fermentation supernatant of strain KS6 was 16.20% (< P < 0.001) compared to the blank MRS, indicating that lactobacillus mucilaginosus KS6 has the ability to degrade cholesterol (fig. 12).
Cholesterol is an important component of body tissue, and excessive cholesterol intake has become an important factor in inducing cardiovascular and cerebrovascular diseases such as coronary heart disease, atherosclerosis, and cerebral apoplexy. A large scale of research results have shown that controlling elevated cholesterol levels not only reduces the risk of heart disease, but also helps to avoid kidney disease such as renal failure.
The total cholesterol is too high, which can lead to various diseases. The main expression is as follows: (1) cholesterol stones: the high secretion of liver cholesterol can lead to supersaturation of cholesterol, and the crystallization and the overgrowth of the cholesterol can further destroy the balance of the liver and intestine circulatory system of bile acid, thereby leading to the formation of cholesterol stones in the organism. (2) lung cancer: high cholesterol diets may be associated with an increased risk of developing lung cancer, but the amount of cholesterol intake in the diet is affected by the constitution of the individual, the composition of the diet, or other high fat diet, and does not accurately reflect the actual level of cholesterol in the body, which is more closely related to the risk of developing lung cancer. (3) coronary arteriosclerosis: elevated serum LDL and triacylglycerol levels are important causes of atherosclerotic lesion formation. Lowering LDL cholesterol levels has become an important goal in reducing the complications of atherosclerotic lesions and the resultant cardiovascular and cerebrovascular diseases. Atherosclerotic lesions begin with localized accumulation of LDL, which is sequestered under the endothelium by adherence to proteoglycan-rich extracellular matrix proteins and accumulated by mechanisms such as changes in endothelial permeability, intercellular transport, and active receptor-mediated cell migration. (4) hypercholesterolemia: the risk of hypercholesterolemia is obviously increased for men and women with excessive intake of dietary cholesterol (more than or equal to 300 mg/d). (5) coronary heart disease and ischemic stroke: elevated cholesterol or low density lipoprotein cholesterol is an independent risk factor for coronary heart disease and ischemic stroke. Plasma high density lipoprotein cholesterol may be associated with diseases such as inflammation, thrombosis, etc.
Thus, the lactobacillus mucilaginosus KS6 strain can avoid the damage of excessive cholesterol to the organism by degrading the function of cholesterol.
Taken together, the novel isolated lactobacillus mucilaginosus KS6 strain of the present invention has a variety of probiotic effects: (1) has excellent protease activity; (2) 3-hydroxybutyric acid may be produced; (3) can inhibit xanthine oxidase activity; (4) gamma-aminobutyric acid can be produced and secreted; (5) can produce and secrete hyaluronic acid; (6) glutathione can be produced and secreted; (7) DPPH free radical can be eliminated; (8) scavenging hydroxy radicals; (9) has superoxide dismutase activity; (10) degradable cholesterol. Therefore, the lactobacillus mucilaginosus KS6 strain obtained by the new separation has important application value and economic value.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (10)
1. A lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain, wherein the lactobacillus fermentum KS6 strain was deposited with the chinese collection of typical cultures at day 7 and 3 of 2023 under the accession number: CCTCCNO: M20231158; the 16S rDNA complete sequence of the lactobacillus mucilaginosus KS6 strain is shown in SEQ ID No: 1.
2. Use of the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to claim 1 for the production of 3-hydroxybutyric acid.
3. Use of the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to claim 1 for the production of reduced glutathione.
4. Use of the lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain of claim 1 in the preparation of a xanthine oxidase inhibitor.
5. Use of a strain of lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 as claimed in claim 1 in the production of a protease, wherein the protease is a protease that degrades milk proteins.
6. Use of the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to claim 1 for the production of gamma-aminobutyric acid.
7. Use of the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain of claim 1 for producing hyaluronic acid.
8. Use of the lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain according to claim 1 for the preparation of an antioxidant, wherein the lactobacillus fermentum (Limosilactobacillusfermentum) KS6 strain exerts an antioxidant effect by scavenging DPPH radicals, scavenging hydroxyl radicals and superoxide dismutase activity.
9. Use of the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to claim 1 for the preparation of a cholesterol-degrading or lowering formulation.
10. A probiotic functional bacterial agent, characterized in that it comprises the lactobacillus mucilaginosus (Limosilactobacillusfermentum) KS6 strain according to claim 1.
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| CN112877241A (en) * | 2021-02-02 | 2021-06-01 | 浙江大学 | Human lactobacillus fermentum ZJUIDS06 and application thereof |
| CN114657095A (en) * | 2022-03-22 | 2022-06-24 | 南昌大学 | Lactobacillus fermentum NCU001464 |
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| WO2023118729A1 (en) * | 2021-12-21 | 2023-06-29 | Lesaffre Et Compagnie | Use of a limosilactobacillus fermentum bacterial strain alone or in combination with saccharomyces cerevisiae for preventing and/or treating vaginal candidiasis |
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| CN112877241A (en) * | 2021-02-02 | 2021-06-01 | 浙江大学 | Human lactobacillus fermentum ZJUIDS06 and application thereof |
| WO2023118729A1 (en) * | 2021-12-21 | 2023-06-29 | Lesaffre Et Compagnie | Use of a limosilactobacillus fermentum bacterial strain alone or in combination with saccharomyces cerevisiae for preventing and/or treating vaginal candidiasis |
| CN114657095A (en) * | 2022-03-22 | 2022-06-24 | 南昌大学 | Lactobacillus fermentum NCU001464 |
| CN116173078A (en) * | 2022-12-06 | 2023-05-30 | 完美（广东）日用品有限公司 | Application of fermented lactobacillus mucilaginosus in preparation of products for preventing or delaying huntington chorea |
| CN115960778A (en) * | 2022-12-12 | 2023-04-14 | 长沙理工大学 | Lactobacillus fermentum and application thereof in regulating and controlling flavor of rice product |
| CN116286468A (en) * | 2022-12-30 | 2023-06-23 | 上海交大昂立股份有限公司 | Lactobacillus mucilaginosus LF-ONLLY with antioxidant function and application thereof in fermented food |
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