CN119015203A - A fermented Lactobacillus mucilaginosus strain A21038 with antioxidant, anti-aging and anti-tumor activities and its application - Google Patents
A fermented Lactobacillus mucilaginosus strain A21038 with antioxidant, anti-aging and anti-tumor activities and its application Download PDFInfo
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Abstract
本发明提供了一种具有抗氧化、抗衰老和抗肿瘤活性的发酵粘液乳杆菌(Limosilactobacillusfermentum)菌株A21038及其应用，属于功能微生物技术领域。所述菌株A21038是从百岁健康老人肠道粪便中分离筛选获得，经过形态鉴定和分子鉴定，属于发酵粘液乳杆菌。所述菌株A21038具有良好的耐胃肠液和牛胆碱能力。本发明提供的发酵粘液乳杆菌菌株A21038兼具抑菌、抗炎症、抗氧化和抗肿瘤活性，为临床相关疾病的治疗和药物开发以及体外制剂或日用品的制备提供了新途径。
The present invention provides a fermented mucus lactobacillus (Limosilactobacillusfermentum) strain A21038 with antioxidant, anti-aging and anti-tumor activities and its application, belonging to the field of functional microbial technology. The strain A21038 is obtained by separation and screening from the intestinal feces of a healthy centenarian, and belongs to fermented mucus lactobacillus through morphological identification and molecular identification. The strain A21038 has good resistance to gastrointestinal fluid and bovine choline. The fermented mucus lactobacillus strain A21038 provided by the present invention has antibacterial, anti-inflammatory, antioxidant and anti-tumor activities, and provides a new approach for the treatment of clinically related diseases and drug development, as well as the preparation of in vitro preparations or daily necessities.
Description
Technical Field
The invention belongs to the technical field of functional microorganisms, and particularly relates to a fermented lactobacillus mucilaginosus (Limosilactobacillusfermentum) strain A21038 with antioxidant, anti-aging and antitumor activities and application thereof.
Background
The intestinal microorganisms play a non-negligible role in the life of people, can maintain the health of the intestinal tract, can regulate and control various signal paths through metabolites, and are closely related to the occurrence and treatment of various diseases. For example, lactobacillus symbiotic with human intestinal tracts is a beneficial bacterium, and plays an important role in inhibiting invasion of pathogenic bacteria, relieving intestinal inflammation and injury, delaying aging and even resisting the development and development of tumor. Lactobacillus fermentum is a class of food-acceptable probiotics approved by the health department that can colonise the human intestinal tract by altering the host flora composition and producing beneficial effects on the host. The timely addition of some active probiotics to the diet is of exceptional importance for people presenting with relevant symptoms or diseases. For example, publication No. CN113234612A discloses Lactobacillus fermentum ZS40 having a prophylactic effect on inflammation, which can be used to better alleviate inflammation by inhibiting the activation of NF- κB and MAPK signaling pathways.
The oxidation-reduction reaction does not occur in the human body at any time, the skin is the first barrier of the human body, and the aging damage is unavoidable. Patent publication No. CN117363524A discloses a Lactobacillus gasseri MY4 and application thereof in preparing sleep-aiding and whitening food and medicine, and the strain has the effects of resisting oxidation, whitening and delaying aging. In addition, luo et al reported a strain of Lactobacillus reuteri FLRE K1, which was shown to significantly reduce the number of melanoma cells in mice, and to have anti-melanoma activity against non-cancerous tissues without toxic or side effects. It can be seen that lactobacillus has wide application in antioxidation and anti-tumor aspects as probiotics. However, there is no report on lactobacillus having antioxidant, antiaging and antitumor activities at present.
Disclosure of Invention
In view of the above, the invention aims to provide an application of lactobacillus mucilaginosus in preparing antioxidant, anti-aging and anti-tumor products.
The invention also aims to provide a novel lactobacillus mucilaginosus (Limosilactobacillus fermentum) strain A21038 which has the functions of antioxidation, anti-aging and anti-tumor.
The invention provides the use of lactobacillus mucilaginosus (Limosilactobacillusfermentum) for the preparation of a product having at least two functions: antioxidant, antiaging, antibacterial and antitumor.
Preferably, the antioxidant is expressed in terms of scavenging ability to at least one of: DPPH radicals, hydroxy radicals, superoxide anions and H 2O2.
Preferably, the anti-aging is manifested by superoxide dismutase activity and/or glutathione peroxidase activity.
Preferably, when preparing an antioxidant and/or anti-aging product, the product comprises at least one of the following: medicine, health product, cosmetic and food.
Preferably, the bacteria in the bacteriostasis include escherichia coli.
Preferably, the tumor or cancer in the anti-tumor comprises colon cancer and/or melanoma.
Preferably, when the antibacterial and/or antitumor product is prepared, the product comprises a medicine.
The invention provides a lactobacillus mucilaginosus strain A21038, the preservation number is GDMCC No:64675.
The invention provides a probiotic, and an active ingredient comprises the lactobacillus mucilaginosus strain A21038.
The invention provides application of the lactobacillus mucilaginosus strain A21038 or the probiotics in preparing products with at least one of antioxidation, anti-aging, bacteriostasis and anti-tumor.
The invention provides the use of lactobacillus mucilaginosus (Limosilactobacillusfermentum) for the preparation of a product having at least two functions: antioxidant, antiaging, antibacterial and antitumor. The invention respectively develops the experiments of antioxidation, anti-aging, bacteriostasis and anti-tumor under the in vitro condition by fermenting the lactobacillus mucilaginosus strain. The fermented lactobacillus mucilaginosus strain has good inhibition capability on pathogenic bacteria such as escherichia coli and/or escherichia coli, which indicates that the fermented lactobacillus mucilaginosus strain has good antibacterial property. The invention respectively uses DPPH free radical, hydroxyl free radical (OH -), superoxide anion and hydrogen peroxide (H 2O2) as indexes to show that the fermentation lactobacillus mucilaginosus has more than 98 percent of clearance rate to DPPH free radical, can still grow after being cultured for 12 hours in H 2O2 with high concentration of 0.5mM, has good tolerance to H 2O2, and can play an antioxidant role. The results of the detection on the anti-aging aspect show that the lactobacillus fermentum has superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity, and the effect on delaying skin aging is equivalent to that of the lactobacillus rhamnosus LGG which is a commercial strain. Meanwhile, the anti-tumor effect of the fermented lactobacillus mucilaginosus is verified by taking human colon cancer cells HCT116 and mouse colon cancer cells MC38 as experimental objects, and the result shows that the bacteria, whether living bacteria or inactivated bacteria, have the performance of inhibiting the proliferation of colon cancer cells, and the inhibition effect is related to time. Meanwhile, the anti-tumor effect of the fermented lactobacillus mucilaginosus is verified by taking the mouse melanoma cell B16 as an experimental object, and the result is the same as the colon cancer inhibition result and also has the effect of inhibiting the growth of melanoma. Therefore, the invention proves that the fermented lactobacillus mucilaginosus has the activities of antioxidation, anti-aging, bacteriostasis and anti-tumor at the same time for the first time, and has extremely high application value in the aspects of preparing anti-tumor drugs, bacteriostasis drugs, anti-aging and/or antioxidation drugs, health care products and cosmetics.
The invention also provides a lactobacillus mucilaginosus strain A21038 with the preservation number of GDMCCNo:64675. the strain A21038 is separated from intestinal fecal samples of the hundred-year-old healthy people, and belongs to the fermentation lactobacillus mucilaginosus through morphological and molecular identification. The strain A21038 has high growth speed, has the capacity of resisting gastrointestinal fluid and bovine bile salt, and can play a role in intestinal tract colonisation through the gastrointestinal tract. The strain A21038 has good inhibition effect on escherichia coli pathogenic bacteria, has scavenging capability on free radicals, has tolerance on high-concentration hydrogen peroxide, and has good antioxidation capability. In addition, the strain A21038 has superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity, and has the capacity of delaying aging. In addition, the live bacteria or the inactivated bacteria of the strain A21038 have the effect of inhibiting the cell proliferation of colon cancer and melanoma. Therefore, the strain A21038 has the characteristics of oxidation resistance, aging resistance, bacteriostasis and tumor resistance, and has extremely high application value in industrial production.
Drawings
FIG. 1 is a morphology of Lactobacillus mucilaginosus strain A21038;
FIG. 2 is a graph showing the growth of Lactobacillus fermentum strain A21038;
FIG. 3 is a graph showing the results of the test of the tolerance of Lactobacillus fermentum strain A21038 to artificial gastrointestinal fluids;
FIG. 4 is a graph showing the results of a test for the resistance of Lactobacillus fermentum strain A21038 to bovine bile salts;
FIG. 5 is a graph showing the results of the test for the ability of Lactobacillus fermentum strain A21038 to tolerate H 2O2;
FIG. 6 is a graph showing the results of the detection of the inhibition of HCT116 growth of human colon cancer cells by Lactobacillus fermentum strain A21038;
FIG. 7 is a graph showing the results of detection of the inhibition of the growth of MC38, a mouse colon cancer cell by Lactobacillus fermentum strain A21038;
FIG. 8 is a graph showing the results of the test for inhibiting the growth of mouse melanoma cells B16 by Lactobacillus fermentum strain A21038.
Biological material preservation information
Lactobacillus mucilaginosus (Limosilactobacillusfermentum) strain A21038 is deposited with the microorganism strain collection in Guangdong province at the year 2024, month 05 and 23, the unit is GDMCC, the address is building 5 of No. 59 of Dai 100 in Guangzhou city martyr, and the deposit number is GDMCC No:64675.
Detailed Description
The invention provides an application of fermented lactobacillus mucilaginosus in preparing products with at least two functions: antioxidant, antiaging, antibacterial and antitumor.
In the present invention, the lactobacillus mucilaginosus preferably includes thalli and/or spores. The fermented lactobacillus mucilaginosus thallus refers to solid-phase thallus which is obtained by performing amplification culture, solid-liquid separation and collection on activated fermented lactobacillus mucilaginosus. The lactobacillus plantarum spores refer to that after lactobacillus mucilaginosus grows and develops to a certain stage, a circular or elliptic stress-resistant dormancy body is formed in cells.
In the present invention, the form of the lactobacillus fermentum preferably includes at least one of the following: a culture solution of the fermentation lactobacillus mucilaginosus, a culture supernatant of the fermentation lactobacillus mucilaginosus and a bacterial suspension of the fermentation lactobacillus mucilaginosus. The preparation method of the culture solution of the fermentation lactobacillus mucilaginosus is to inoculate the activated fermentation lactobacillus mucilaginosus into MRS liquid culture medium for culture to obtain the culture solution. The temperature of the culture is preferably 36 to 38℃and more preferably 37 ℃. The time of the culture is 12 to 16 hours, more preferably 14 hours. The inoculation amount of the lactobacillus mucilaginosus is preferably 5-10%, more preferably 8%. In the preparation method of the culture supernatant of the fermented lactobacillus mucilaginosus and the bacterial suspension of the fermented lactobacillus mucilaginosus, preferably, the culture solution obtained by the preparation is subjected to solid-liquid separation, the liquid phase is the culture supernatant, and the solid phase is collected for resuspension to obtain the bacterial suspension.
In the present invention, the strain of lactobacillus fermentum preferably includes lactobacillus fermentum strain a21038. The lactobacillus mucilaginosus strain A21038 has a preservation number of GDMCC No:64675.
In the present invention, the use when preparing a product comprising two functions preferably includes use in preparing an antioxidant and anti-aging product and/or use in preparing a bacteriostatic and anti-tumor product; or in the preparation of antioxidant and antitumor products and/or in the preparation of bacteriostatic and anti-aging products; or in the preparation of anti-aging and anti-tumor products and/or in the preparation of products with antioxidant and antibacterial properties.
In the present invention, the use when preparing a product comprising three functions preferably includes use in preparing a product that is antioxidant, anti-aging and bacteriostatic; or in preparing products with oxidation resistance, bacteriostasis and tumor resistance; or in the preparation of anti-aging, antibacterial and anti-tumor products; or in the preparation of antioxidant, antiaging and antitumor products. The use when preparing a product comprising four functions preferably includes use in preparing products that are antioxidative, anti-ageing, bacteriostatic and anti-tumour.
In the present invention, the antioxidant is preferably expressed in terms of scavenging ability to at least one of: DPPH radicals, hydroxyl radicals, superoxide anions and H 2O2, more preferably DPPH radicals and H 2O2. The anti-aging is preferably manifested in having superoxide dismutase activity and/or glutathione peroxidase activity. When preparing an antioxidant and/or anti-aging product, the product preferably comprises at least one of the following: medicine, health product, cosmetic and food. The bacteria in the bacteriostasis preferably include escherichia coli. The tumor or cancer in the anti-tumor preferably comprises colon cancer and/or melanoma. When the bacteriostatic and/or antitumor product is prepared, the product preferably comprises a pharmaceutical product. When the antibacterial product is prepared, the product can also be used for preparing a bacteriostatic agent used under in-vitro conditions. The method of preparing the product having the above functions is not particularly limited, and the product may be prepared by methods well known in the art, for example, by mixing a culture solution, supernatant or fermentation solution of lactobacillus mucilaginosus as an active ingredient with an auxiliary material.
The invention provides a lactobacillus mucilaginosus strain A21038, the preservation number is GDMCC No:64675.
In the present invention, the strain A21038 is isolated from a intestinal fecal sample of a hundred year old healthy person. Bacterial colony of the strain A21038 on the MRS flat plate is round, and has the advantages of regular edge, opacity, raised middle, smooth surface and light yellow, and belongs to gram-positive bacteria. The nucleotide sequence of the 16S rDNA of the strain A21038 is shown as SEQ ID NO. 1. The strain A21038 belongs to the lactobacillus mucilaginosus through morphological identification and molecular identification. The growth curve of the strain A21038 shows that the strain A21038 has the characteristic of high growth speed, 2 hours enter a logarithmic phase, 6 to 24 hours are a stationary phase, and no decay phase occurs in 24 hours.
In the invention, the strain A21038 is treated in artificial gastric juice for 3 hours, and the survival rate is 100%. The survival rate is maintained to be more than 70% after the treatment for 7 hours in the artificial intestinal juice, and the effect is better than that of a control strain A20014 screened in the same batch. The survival rate of the strain A21038 in the ox gall salt with the concentration of 0.5g/L for 24 hours is 100 percent. The above results demonstrate that the strain A21038 has the ability to resist gastrointestinal fluids and bovine bile salts and can act through colonisation of the gastrointestinal tract in the intestinal tract.
In the invention, the strain A21038 has good inhibition effect on escherichia coli pathogenic bacteria. The E.coli pathogenic bacteria preferably include E.coli and/or E.coli. In the embodiment of the invention, the antibacterial property of the strain A21038 is illustrated by taking the escherichia coli strain O157:H27 (ATCC 35150) and escherichia coli (ATCC 25922) as pathogenic bacteria, and the result shows that the antibacterial ring of the strain A21038 on the escherichia coli strain O157:H27 is 21.33mm, and the antibacterial effect is superior to that of a control strain A20014 screened in the same batch; the antibacterial circle of the strain A21038 on the escherichia coli strain is 25.00mm, and the antibacterial effect is equivalent to that of a control strain A20014 screened in the same batch.
In the invention, the strain A21038 has good scavenging ability to free radicals, and also has good tolerance to high-concentration hydrogen peroxide and good oxidation resistance. The radicals preferably include DPPH radicals, hydroxyl radicals (OH -) and superoxide anions (O 2 -). The DPPH free radical is a stable nitrogen center free radical, is one of important indexes of the antioxidant capacity of a sample, and is widely applied to the research of antioxidant foods, health-care products and medicines. Hydroxyl radicals (OH-) are one of the active oxygen species and can kill red blood cells, degrade DNA, cell membranes and polysaccharide compounds, and in turn find that many of the deleterious effects caused by them are significantly reduced when a scavenger of hydroxyl radicals is added. Superoxide anions are also an index for detecting oxidation resistance, and when an organism is subjected to external stress, active oxygen such as superoxide anions in the organism is generated and accumulated in a large amount, and can be used as a signal of the oxidation stress of the organism. Therefore, the generation of superoxide anion free radicals in organisms under the stress condition can indirectly reflect the damaged condition and the resistance strength of tissue cells. Hydrogen peroxide (H 2O2) is the most common active oxygen molecule in living body, is a byproduct of active oxygen metabolism, is mainly produced by catalysis of superoxide dismutase (SOD), xanthine Oxidase (XOD) and the like, and is degraded by catalysis of Catalase (CAT), active enzyme (POD) and the like. The concentration of H 2O2 in normal human body is at a lower level, typically 10-100. Mu.M. The high concentration of H 2O2 can induce the increase of in vivo oxidative stress reaction, directly or indirectly oxidize biomacromolecules such as nucleic acid, protein and the like in cells, and damage cell membranes, thereby accelerating the aging and disintegration of cells, generating toxic action on human cells, and even causing abnormal cell functions and organ damage, and causing related diseases.
In one embodiment of the invention, the clearance rate of the strain A21038 to DPPH is more than 98%, and the effect is obviously better than that of the control strain A20014 (15%). The clearance rate of the strain A21038 to the hydroxyl radicals is 20%, and the effect is obviously better than that of a control strain A20014 (10%). The clearance rate of the strain A21038 to superoxide anions is 33%, and the effect is obviously better than that of the control strain A20014 (24%). In another example of the invention, the resistance of strain A21038 to high concentrations of hydrogen peroxide was determined, strain A21038 still grew when treated in 0.5mM H 2O2 for 12H, with good resistance to H 2O2, whereas control strain A20014 failed to reproduce under the same conditions, with no resistance to H 2O2.
In the invention, the strain A21038 has superoxide dismutase activity and glutathione peroxidase (GSH-Px) activity and has the capacity of delaying aging. Superoxide dismutase can catalyze superoxide anions to perform disproportionation to generate hydrogen peroxide (H 2O2) and oxygen (O 2), is an important antioxidant enzyme in organisms, and plays a role in preventing skin aging and injury. Glutathione peroxidase exclusively uses Glutathione (GSH) to reduce peroxides, especially hydrogen peroxide. The glutathione is composed of glutamic acid, cysteine and glycine, and has the functions of antioxidation and integration detoxification. The glutathione not only can be used for medical drugs, but also has the whitening effect of reducing melanin growth and delaying aging. In one embodiment of the invention, the strain A21038 has the capability of secreting superoxide dismutase and glutathione peroxidase, wherein the activity of the superoxide dismutase is 0.27U/10 4 CFU, and the activity of the glutathione peroxidase is 0.77nmol/min/mL.
In the invention, the live bacteria or the inactivated bacteria of the strain A21038 have the effect of inhibiting the cell proliferation of colon cancer and melanoma. The preparation method of the inactivated strain A21038 is preferably to treat the thallus for 30min at 70 ℃. In one embodiment of the invention, the live bacteria of strain a21038 are superior to the inactivated bacteria in inhibiting tumor cell growth, with the same results in commercial strain LGG.
The invention provides a probiotic, and an active ingredient comprises the lactobacillus mucilaginosus strain A21038.
In the invention, the probiotics means to supplement beneficial bacteria in intestinal tracts, so that the beneficial bacteria have sufficient quantity and exert unique biological effects, thereby achieving the aims of health care or disease prevention and treatment. The viable bacteria concentration of the Lactobacillus fermentum strain A21038 is preferably 1X 10 9CFU/mg～1×1011 CFU/mg or 1X 10 9CFU/mL～1×1011 CFU/mL, more preferably 5X 10 9CFU/mg～5×1010 CFU/mg or 5X 10 9CFU/mL～5×1010 CFU/mL, still more preferably 8X 10 9CFU/mg～2×1011 CFU/mg or 8X 10 9CFU/mL～2×1011 CFU/mL, most preferably 1X 10 10 CFU/mg or 1X 10 10 CFU/mL. The formulation of the probiotic is preferably at least one of the following: tablets, drops, capsules and powders. The powder is obtained by processing a low-temperature freeze-drying technology, so that the activity of probiotics is ensured. The adjuvants of the powder preferably include a freeze-drying agent, such as skimmed milk powder, etc. The powder is suitable for warm water infusion and infants. The tablet is prepared by mixing and tabletting the fermented lactobacillus mucilaginosus strain A21038 and auxiliary materials, and is suitable for chewing and taking. The auxiliary materials preferably comprise sweetener or cocoa powder and the like. The capsule is preferably packaged in capsule shell as capsule core by mixing lactobacillus mucilaginosus strain A21038 alone or with adjuvants. The drops are prepared by mixing lactobacillus mucilaginosus strain A21038 with edible oil to obtain air and water-isolated drops, and squeezing the encapsulated oily substances into the mouth of the drops when taking.
In view of the multiple functions of the lactobacillus fermentum strain a21038, the invention provides the application of the lactobacillus fermentum strain a21038 or the probiotic in preparing a product with at least one of antioxidation, anti-aging, bacteriostasis and anti-tumor.
The invention is not particularly limited to the specific type of product in question, but may be of the type known in the art for its specific efficacy.
The present invention provides lactobacillus fermentum strain a21038 with antioxidant, anti-aging and antitumor activities and its application in the following detailed description, but they should not be construed as limiting the scope of the invention.
Example 1
Isolation and characterization of Lactobacillus mucilaginosus Strain A21038
Intestinal fecal samples of the century old are diluted by a multiple ratio, spread on an MRS plate, and cultured for 48 hours at 37 ℃ in a biochemical incubator to obtain single colonies. Picking single colony, inoculating to MRS liquid culture medium, and shake culturing at 37 deg.C for 12-16 hr. The bacterial liquid is streaked on an MRS plate, and single bacterial colony is continuously cultured. After culturing for 24 hours, single colony is selected and streaked again, and single colony is continuously cultured, thus obtaining the pure isolated lactobacillus. The isolated single colony of lactobacillus is selected, gram staining, 16S rDNA PCR amplification and 16S rDNA sequencing are carried out, and the result is obtained through NCBI database comparison.
The growth state of the strain A21038 in an MRS plate is shown in fig. 1, and the bacterial colony is round, neat in edge, opaque, convex in the middle, smooth in surface and pale yellow, and belongs to gram-positive bacteria.
The 16SrDNA sequence of the strain A21038 is GGCCTAATACATGCAGTCGAACGC GTTGGCCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAGCGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAGCCAGCCGCGTAAGGGGACAGA(SEQ ID NO:1)., and the similarity of the sequence of the strain A21038 and the sequence of the strain Lactobacillusfermentum strain 562516S is 99.93 percent, so that the strain A21038 is lactobacillus mucilaginosus. The strain A21038 was deposited with the Guangdong province microorganism strain collection under accession number GDMCCNo 64675.
Example 2
Growth curve of lactobacillus mucilaginosus strain A21038
The strain is taken out of a glycerol preservation tube, streaked in an MRS solid flat plate, placed in a 37 ℃ incubator for culture for 24 hours, picked up and monoclone to 2mL of MRS liquid culture medium for culture for 12-16 hours, the steps are repeated for 1 time, so that the strain is fully activated, then 0.2mL of bacterial suspension is sucked into a 96-well plate, 3 repeated holes are made, the enzyme-labeled instrument is used for detecting the growth condition in 24 hours, the detection wavelength is 600nm, and statistics and drawing are carried out after the detection is finished.
FIG. 2 shows the growth curve of Lactobacillus fermentum A21038. The strain A21038 has a relatively high growth speed, enters a logarithmic growth phase after 2 hours, and is in a stationary phase after 6-24 hours, and the strain grows stably without a decay phase within 24 hours.
Example 3
Artificial gastrointestinal fluid resistant function of fermentation lactobacillus mucilaginosus strain A21038
After thawing glycerol storage tubes of the strain A21038 and other strains of fermented lactobacillus mucilaginosus A20014 (serving as control strains) separated from the faeces of the centenarian, respectively, carrying out plate streaking by using an MRS solid culture medium, placing the plates in a 37 ℃ incubator for overnight culture, respectively picking up monoclonal colonies after 24 hours, inoculating the monoclonal colonies into the MRS liquid culture medium, and carrying out shake culture at 37 ℃ for 12-16 hours. The step is repeated for 1 time, so that the bacterial strain is fully activated, and bacterial liquid is obtained. Inoculating bacterial liquid into artificial gastric juice with pH of 2.5 (formula is pepsin 10g, hydrochloric acid 16.4ml, distilled water constant volume to 100 ml) according to 10% inoculum size, setting 3 time points (0 h, 1h, 3 h), respectively taking 100 μl, coating onto MRS plate, culturing at 37 ℃ in a biochemical incubator for 48h, and counting the number of single bacterial colonies; bacterial liquid after 3h of artificial gastric juice treatment is inoculated into artificial intestinal juice with pH of 6.8 according to an inoculum size of 10 percent (the formula is pancreatin 10g, monopotassium phosphate 6.8g, distilled water is fixed to volume of 1000ml, naOH is used for adjusting pH value), 4 time points (0 h, 2h, 4h and 8 h) are set, 100 mu l of bacterial liquid is respectively coated on an MRS plate, after the bacterial liquid is cultured for 48h at 37 ℃ in a biochemical incubator, the number of single bacterial colonies is counted, and the survival rate is calculated according to a formula I.
Survival (%) = (number of single colonies at different time points/number of single colonies at 0 h) ×100% formula I
The results are shown in fig. 3, wherein the survival rate of the control strain a20014 in the artificial gastric juice with the pH of 2.5 reaches 100%, and the survival rate in intestinal juice is reduced with the time, and the survival rate in intestinal juice for 7h is lower than 20%. Compared with the control strain A20014, the survival rate of the strain A21038 reaches 100% in artificial gastric juice for 3h, and the survival rate of the strain A21038 still keeps more than 70% after intestinal juice for 7h, which is obviously superior to the control strain A20014. It was demonstrated that strain A21038 has excellent gastrointestinal tolerance and can successfully pass gastrointestinal fluid stimulation to reach intestinal tract for action.
Example 4
Determination of bovine bile salt resistance of fermented lactobacillus mucilaginosus A21038
After thawing glycerol storage tubes of the strain A21038 and other strains of fermented lactobacillus mucilaginosus A20014 (serving as control strains) separated from the faeces of the centenarian, respectively, carrying out plate streaking by using an MRS solid culture medium, placing the plates in a 37 ℃ incubator for overnight culture, respectively picking up monoclonal colonies after 24 hours, inoculating the monoclonal colonies into the MRS liquid culture medium, and carrying out shake culture at 37 ℃ for 12-16 hours. The step is repeated for 1 time, so that the bacterial strain is fully activated, and bacterial liquid is obtained. 1mL of the mixed solution is added into MRS culture medium respectively containing 0.3g/L or 0.5g/L of ox gall salt, anaerobic culture is carried out for 18h at 37 ℃, 100uL of culture solution after 0h, 2h and 24h of addition is respectively taken for gradient dilution coating, and the mixed solution is placed into anaerobic condition at 37 ℃ for culturing for 1-2 days, and the number of viable bacteria is counted. Calculating the bile salt-resistant survival rate of the strain according to a formula II:
Bile salt-tolerant survival rate= (N h viable count/0 h viable count) ×100% formula II
Wherein N represents 2h or 24h.
As a result, as shown in FIG. 4, the survival rate of the strain A21038 in bovine bile salt at a concentration of 0.5g/L for 24 hours was still more than 100% as compared with the control strain A20014, indicating that the strain A21038 has excellent bile salt tolerance.
Example 5
Lactobacillus mucilaginosus strain A21038 inhibits pathogenic bacteria function
Pathogenic bacteria in logarithmic growth phase (concentration 1X 10 9 CFU/mL) such as Escherichia coli O157: H7 (ATCC 35150) and Escherichia coli (ATCC 25922) were applied by pipetting 0.1mL onto LB plates and placing oxford cups.
According to the method of example 2 for activating the strain, an activated bacterial suspension was obtained, OD 600 was adjusted to 0.5 (concentration 1X 10 6 CFU/mL), 200. Mu.l of the bacterial suspension was inoculated into oxford cups, 3 replicates were performed, and the effect on pathogenic bacteria was observed overnight. The size statistics mode of the inhibition zone is that the size of the inhibition zone is measured by a ruler, and an average value of 3 times of repetition is taken. The results are shown in Table 1 below.
TABLE 1 detection results of pathogenic bacteria inhibition by strains
The results show that the lactobacillus mucilaginosus strain A21038 has the capability of remarkably inhibiting the growth of pathogenic bacteria and is superior to the control strain A20014.
Example 6
Antioxidant function of lactobacillus mucilaginosus strain A21038
Activated bacterial suspensions were obtained as in example 2 for activating strain A21038, and control strain A20014 bacterial suspension was prepared by adjusting OD 600 to 0.5 (concentration 1X 10 6 CFU/mL) for use.
In the presence of an antioxidant, DPPH free radicals are scavenged, the color of the solution becomes light, the absorbance at 515nm is reduced, and the change of the absorbance is proportional to the scavenging degree of the free radicals in a certain range. The ability of the sample to scavenge DPPH radicals is reflected by the extent of the decrease in absorbance. Two strains were tested according to the DPPH radical scavenging ability kit instructions (Jiang Lai organisms).
2-Deoxyribose is oxidized to a malondialdehyde analog in the presence of hydroxyl radical (OH -), and then condensed with thiobarbituric acid (TBA) to produce a colored product, and the hydroxyl radical (OH -) content is calculated by measuring the maximum absorption peak of the colored product at 532 nm. Two strains were tested according to the specification of the hydroxyl radical (OH -) assay kit (Jiang Lai organism).
The reaction of superoxide anion (O 2 -) with hydroxylamine produces NO 2 －,NO2 －, which under the action of sulfanilic acid and alpha-naphthylamine produces a pink azo dye with maximum light absorption at 540nm, from which the O 2 - content in the sample can be calculated from the A540 value. Two strains were tested according to the protocol of the superoxide anion (O 2 -) kit (Grignard organism).
The test results are shown in Table 2.
Table 2 results of antioxidant tests of two strains
The results show that the lactobacillus mucilaginosus strain A21038 has good antioxidant capacity and is obviously superior to the control strain A20014.
Example 7
Determination of the ability of Lactobacillus mucilaginosus Strain A21038 to withstand H 2O2
Hydrogen peroxide (H 2O2) is the most common active oxygen molecule in organisms, is a byproduct of active oxygen metabolism, is mainly produced by catalysis of SOD, XOD and the like, and is degraded by catalysis of CAT, POD and the like. The concentration of H 2O2 in normal human body is at a lower level, typically 10-100. Mu.M. The high concentration of H 2O2 can induce the increase of in vivo oxidative stress reaction, directly or indirectly oxidize biomacromolecules such as nucleic acid, protein and the like in cells, and damage cell membranes, thereby accelerating the aging and disintegration of cells, generating toxic action on human cells, and even causing abnormal cell functions and organ damage, and causing related diseases.
The strain A21038 and the control strain A20014 are taken out from a glycerol storage tube and streaked for 3 times, monoclone is selected to MRS liquid culture medium for culture, H 2O2 with different concentrations of 0.5mM, 1.0mM and 2.0mM is added, and the growth condition is detected by using an enzyme-labeled instrument, and after the measurement is finished, the drawing is carried out.
As a result, as shown in FIG. 5, lactobacillus mucilaginosus A21038 (FIG. 5A) was still grown in H 2O2 at a high concentration of 0.5mM for 12 hours, and had a good tolerance to H 2O2, while the control strain A20014 was unable to reproduce under the same conditions (FIG. 5B) and had no tolerance to H 2O2. The strain A21038 can still stably grow under the in-vivo high oxidative stress environment, and plays an antioxidant role.
Example 8
Fermented lactobacillus mucilaginosus strain A21038 has senescence delaying function
Superoxide dismutase (SOD) can catalyze superoxide anions to perform disproportionation to generate hydrogen peroxide (H 2O2) and oxygen (O 2), is an important antioxidant enzyme in organisms, and plays a role in preventing skin aging and injury. Glutathione (GSH) is composed of glutamic acid, cysteine and glycine, and has antioxidant effect and integrated detoxification effect. The glutathione not only can be used for medical drugs, but also has the whitening effect of reducing melanin growth and delaying aging.
The commercial strain lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) strain LGG is used as beneficial bacteria in human intestinal tracts, and can enhance the immunity of organisms, improve the gastrointestinal functions and delay the aging process of organisms. According to the research results of the embodiment, each performance of the strain A21038 is obviously superior to that of the control strain A20014 of the same genus, so that LGG is selected as the control strain in the following embodiment, and the anti-aging function of the strain A21038 is researched.
(1) Strain A21038 has the activity detection of superoxide dismutase (SOD)
The concentration of the strain A21038 and the concentration of the commercial strain lactobacillus rhamnosus LGG strain liquid are respectively regulated to be 5 multiplied by 10 6 CFU/mL, supernatant is removed by centrifugation, and the activity of the strain containing SOD is measured according to the operation flow of an SOD activity detection kit (Jiang Lai organism). The result is defined as that when the SOD inhibition rate of each 10 4 CFU single colony in the reaction system reaches 50%, the corresponding SOD amount is one SOD activity unit.
(2) Strain A21038 has glutathione peroxidase (GSH-Px) activity detection
The bacterial liquid concentrations of the strain A21038 and the control strain LGG are respectively regulated to 5 multiplied by 10 6 CFU/mL, the supernatant is removed by centrifugation, and the GSH-Px content in the strain is measured according to the operation flow of a GSH-Px detection kit (provided by Grignard organisms). The results are defined as 1nmol of GSH per 10 4 cells per minute oxidized to 1 enzyme activity unit under 25℃reaction conditions.
The enzyme activities contained in the lactobacillus mucilaginosus A21038 are shown in the following Table 3.
TABLE 3 enzyme activity test results
| Strain | SOD activity (U/10 4 CFU) | GSH-Px enzyme activity (nmol/min/mL) |
| A21038 | 0.27 | 0.77 |
| LGG | 0.51 | 0.77 |
The results show that the lactobacillus mucilaginosus strain A21038 has good glutathione peroxidase activity, can play a role in delaying skin aging, and has the effect equivalent to that of the control strain LGG.
Example 9
Inhibition of human colon cancer cell HCT116 by Lactobacillus fermentum strain A21038
HCT116 cell line was inoculated in RPMI1640 medium (TransGen) containing 10% fetal bovine serum, and when cells were grown to logarithmic phase, cells were counted by a hemocytometer after digestion with pancreatin, and cells were uniformly inoculated in 96-well plates at 5000 cells per well and placed in a three-gas incubator at 37 ℃ for overnight culture.
There are prior art reports that the commercial strain lactobacillus rhamnosus LGG has an anti-tumor effect, and thus LGG strain was selected as a positive control of this example. The strain A21038 and the control strain LGG are taken out of a glycerol preservation tube, streaked in an MRS solid flat plate, placed in a 37 ℃ incubator for culture, and then picked up and monoclone to 2mL of MRS liquid culture medium for culture for 12-16 hours after 24 hours, the step is repeated for 1 time, so that the strain is fully activated to obtain bacterial suspension, the bacterial suspension OD 600 =0.5 is regulated, the concentration is 1×10 6 CFU/mL, after the bacterial suspension is washed 3 times by PBS, the bacterial suspension is divided into living bacteria and inactivated bacteria (30 minutes at 70 ℃), 0.2mL of the bacterial suspension per hole is sequentially added into a 96-well plate, the living bacteria and the cells are incubated for 2 hours, and the inactivated bacteria and the cells are incubated for 72 hours after inactivation. HCT116 cell proliferation was detected using CCK-8 reagent (solebao).
The results are shown in FIG. 6. The strain A21038 viable bacteria can obviously inhibit the proliferation of HCT116 cells (P < 0.0001), and the effect of the strain A21038 viable bacteria is equivalent to that of a control strain LGG. The strain a21038 showed a significant inhibitory effect (P < 0.001) within 48h after inactivation and was superior to the control strain LGG. The effect of the inactivated strain is obviously weaker than that of the live bacteria, and the potential anti-tumor cell growth effect can be exerted after the strain is inactivated, probably because the inactivated mycoprotein plays a role, which needs to be further studied.
Example 10
Inhibition of mouse colon cancer cell MC38 by Lactobacillus fermentum strain A21038
The MC38 cell line was inoculated in DMEM medium (TransGen) containing 10% fetal bovine serum, and after cells were grown to logarithmic phase, they were counted by a hemocytometer after digestion with pancreatin, and the cells were inoculated uniformly into 96-well plates at 5000 cells per well and placed in a three-gas incubator at 37℃for overnight culture.
The strain A21038 and the control strain LGG are respectively taken out of a glycerol preservation tube, streaked in an MRS solid plate, placed in a 37 ℃ incubator for culture, and then picked up and monoclone until 2mL of MRS liquid culture medium is cultured for 12-16 hours after 24 hours, the step is repeated for 1 time, the strain is fully activated, bacterial suspension is obtained, the bacterial suspension OD 600 =0.5 is regulated, the concentration is 1×10 6 CFU/mL, after the bacterial suspension is washed 3 times by PBS, the bacterial suspension is divided into living bacteria and inactivated bacteria (30 minutes at 70 ℃), and 0.2mL of the bacterial suspension per hole is sequentially added into a 96-well plate and respectively incubated with cells for 24 hours, 48 hours and 72 hours. MC38 cell proliferation was detected using CCK-8 reagent (Soxhobao).
As shown in fig. 7, both the strain a21038 and the commercial strain LGG can significantly inhibit the proliferation of colon cancer cells, and the more significant the inhibition effect of viable bacteria (P < 0.0001) is over time, the half inhibition effect of the strain a21038 is reached at 72h after inactivation, which is significantly weaker than the viable bacteria effect. Therefore, it is speculated that the potential anti-colon cancer cell growth ability of the strain A21038 is probably mainly dependent on the living bacteria or the metabolites generated by the living bacteria, and the bacterial cells play a weak role after inactivation.
Example 11
Inhibition of murine melanoma cell B16 by Lactobacillus fermentum strain A21038
B16 cell lines were inoculated in DMEM medium (TransGen) containing 10% fetal bovine serum, and when cells were grown to log phase, cells were counted by a hemocytometer after digestion with pancreatin, and cells were uniformly inoculated in 96-well plates at 5000 cells per well and incubated overnight in a three-gas incubator at 37 ℃.
The strain A21038 and the control strain LGG are taken out of a glycerol preservation tube, streaked in an MRS solid plate, placed in a 37 ℃ incubator for culture, and then picked up and monoclone until 2mL of MRS liquid culture medium is cultured for 12-16 hours after 24 hours, the step is repeated for 1 time, the strain is fully activated to obtain bacterial suspension, the bacterial suspension OD 600 to 0.5 (the concentration is 1X 10 6 CFU/mL), after being washed 3 times by PBS, the bacterial suspension is divided into living bacteria and inactivated bacteria (30 min at 70 ℃), and 0.2mL of the bacterial suspension per hole is sequentially added into a 96-well plate to be respectively incubated with cells for 24 hours. Cell proliferation was detected using the CCK-8 reagent (Soxhobao).
As shown in fig. 8, the strain a21038 can significantly inhibit proliferation of melanoma cells (P < 0.0001), and the effect of the strain is equivalent to that of the control strain LGG. The effect of inactivation was weaker than that of live bacteria, either of A21038 or commercial strain LGG. Therefore, it is presumed that the potential anti-melanoma cell growth ability of the strain A21038 may depend on the living bacterium itself or the metabolite produced thereby, and the inactivated bacterial cells exert a weaker effect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019023555A1 (en) * | 2017-07-28 | 2019-01-31 | Evelo Biosciences, Inc. | Polymer compositions |
| WO2022145901A1 (en) * | 2020-12-28 | 2022-07-07 | 주식회사 리스큐어바이오사이언시스 | Pharmaceutical composition for preventing or treating cancer comprising lactobacillus fermentum strain-derived nanovesicles as active ingredient |
| CN116004455A (en) * | 2022-12-28 | 2023-04-25 | 广西爱生生命科技有限公司 | Lactobacillus fermentum strain A21196 with function of secreting 5-hydroxytryptamine and application thereof |
| CN116656578A (en) * | 2023-07-26 | 2023-08-29 | 杭州微致生物科技有限公司 | A kind of Lactobacillus mucus fermentum VB216 and its application |
| CN117402768A (en) * | 2023-03-13 | 2024-01-16 | 广东悦创生物科技有限公司 | Lactobacillus reuteri KA1 and application thereof in preparation of anti-inflammatory and anti-tumor food and drug |
| CN117721033A (en) * | 2023-10-13 | 2024-03-19 | 广东悦创生物科技有限公司 | Lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019023555A1 (en) * | 2017-07-28 | 2019-01-31 | Evelo Biosciences, Inc. | Polymer compositions |
| WO2022145901A1 (en) * | 2020-12-28 | 2022-07-07 | 주식회사 리스큐어바이오사이언시스 | Pharmaceutical composition for preventing or treating cancer comprising lactobacillus fermentum strain-derived nanovesicles as active ingredient |
| CN116004455A (en) * | 2022-12-28 | 2023-04-25 | 广西爱生生命科技有限公司 | Lactobacillus fermentum strain A21196 with function of secreting 5-hydroxytryptamine and application thereof |
| CN117402768A (en) * | 2023-03-13 | 2024-01-16 | 广东悦创生物科技有限公司 | Lactobacillus reuteri KA1 and application thereof in preparation of anti-inflammatory and anti-tumor food and drug |
| CN116656578A (en) * | 2023-07-26 | 2023-08-29 | 杭州微致生物科技有限公司 | A kind of Lactobacillus mucus fermentum VB216 and its application |
| CN117721033A (en) * | 2023-10-13 | 2024-03-19 | 广东悦创生物科技有限公司 | Lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines |
Non-Patent Citations (1)
| Title |
|---|
| H CHEN: ""Exploration of the Molecular Mechanisms Underlying the Anti-Photoaging Effect of Limosilactobacillus fermentum XJC60"", 《FRONTIERS IN CELLULAR》, 31 December 2022 (2022-12-31), pages 163 - 164 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120272367A (en) * | 2025-04-10 | 2025-07-08 | 渤海大学 | Lactobacillus mucilaginosus BL-SWN1 and application thereof |
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