CN116987644A - Lactobacillus mucilaginosus with antioxidation effect and application thereof - Google Patents
Lactobacillus mucilaginosus with antioxidation effect and application thereof Download PDFInfo
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- CN116987644A CN116987644A CN202311039010.1A CN202311039010A CN116987644A CN 116987644 A CN116987644 A CN 116987644A CN 202311039010 A CN202311039010 A CN 202311039010A CN 116987644 A CN116987644 A CN 116987644A
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- Prior art keywords
- lactobacillus fermentum
- lactobacillus
- mucilaginosus
- effect according
- fermentum
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention relates to a lactobacillus fermentum with an antioxidant effect, which is lactobacillus fermentum TG017 and is preserved in China center for type culture collection, and the preservation number is as follows: cctcrno: m2023513, the preservation time is [ 2023, 4, 10 ] and the address is in the university of Wuhan preservation center. The fermented lactobacillus mucilaginosus has the antioxidation effect, and can obviously remove free radicals, resist lipid peroxidation and generate antioxidase.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus mucilaginosus with an antioxidant effect and application of the lactobacillus mucilaginosus.
Background
It has been found that free radicals generally combine with more reactive oxygen species to complete the oxidation process and thereby cause damage to the interior of the human body. Therefore, if it is desired to restore normal body function, reducing the risk of free radicals, it is necessary to scavenge free radicals in the body. One of the hazards of free radicals is known as degradation and aging of human functions, but its hazard is far from it. The free base is active, can destroy cells, is extremely vulnerable to pathogenic bacteria, and weakens the resistance of the human body, such as arthritis, cardiovascular and cerebrovascular diseases, various inflammations and even cancers. We need to repair the damage to the body caused by free radicals by enhancing our defenses against free radicals by absorbing more enzymes and antioxidants.
Lipid peroxidation refers to oxidative deterioration of polyunsaturated fatty acids and lipids, which may lead to alterations in the fluidity and permeability of the cell membrane of the body, damage to DNA and proteins, and thus affect the normal function of the cell. Studies have shown that many diseases in humans, such as tumors, vascular sclerosis, aging, neurodegenerative phenomena, etc., are associated with lipid peroxidation. Lipid peroxidation can damage cells by generated free radicals, induce apoptosis, and its cytotoxicity can affect the activities of various enzymes and the synthesis of ATP. Normally, antioxidant enzymes in the natural antioxidant defense system of organisms act synergistically with antioxidants in diets or drugs to scavenge peroxides.
The metazoan is the probiotic metabolite component after the processing treatment of the probiotics, which is generally called as including thalli and metabolites, and is the microecological preparation for removing inanimate microorganisms and/or components thereof which are beneficial to the health of a host. Besides the probiotic effect, the metazoan also has the advantage that the probiotics are incomparable: on the one hand, the metazoan is more stable and has a longer shelf life than the live probiotics; on the other hand, the metazoan has higher safety, is suitable for special people such as newborns and sensitive people, and the probiotics have a certain risk for the sensitive people. The metazoan is not inhibited by the interference of antibiotics, but probiotics are difficult to use with the antibiotics at the same time, and the risk of transferring drug-resistant genes exists; in addition, the metazoan has wider action targeting and is easier to be absorbed by intestinal tracts, thereby improving the utilization rate. Therefore, the metazoan has a very wide application range, and can be used in various industries such as foods, nourishment, health care foods, living goods, cosmetics, feeds and the like.
However, it has not been found that lactobacillus fermentum has a good lipid peroxidation resistance.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the lactobacillus fermentum with the antioxidation effect and the application thereof, and the lactobacillus fermentum has the antioxidation effect, can obviously remove free radicals, resist lipid peroxidation and generate antioxidase.
The invention also aims to provide a lactobacillus fermentum with an antioxidant effect and application thereof, wherein the lactobacillus fermentum is applied as a metagen, and the metagen of the lactobacillus fermentum has strong scavenging ability to DPPH free radical and good lipid peroxidation resistance, and the bacterium is found to have very high antioxidant enzyme activity and good application safety and functional adaptability by verification liquid at a cell level.
In order to achieve the purpose, the invention adopts the following technical scheme:
a lactobacillus fermentum (limosilactobacillus fermentum) with an antioxidant effect, wherein the lactobacillus fermentum is lactobacillus fermentum TG017 and is preserved in the China center for type culture collection, and the preservation number is: [ CCTCC NO: m2023513, the preservation time is [ 2023, 4, 10 ] and the address is in the university of Wuhan preservation center.
The lactobacillus mucilaginosus TG017 is obtained by screening domestic healthy human body excrement samples, and the colony is milky white, opaque, round convex, smooth in surface and neat in edge, and has an optimal growth temperature of 37 ℃ and an optimal pH of 6 and anaerobic environment.
The gene sequence of the lactobacillus mucilaginosus TG017 is as follows:
GTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGGTTTCACATCAGAATTAAAAAAACCGCCTGGCCTCTCTTTTCGCCCCAATAAATCCGGATAACGCTTGGCACCTACGTA。
the lactobacillus mucilaginosus TG017 has the capability of producing extracellular polysaccharide with the yield of 14.02g/L.
The strain of lactobacillus mucilaginosus TG017 metaplasia has the capability of removing DPPH free radical, the clearance rate of cell-free extract is 29.28%, and the clearance rate of extracellular polysaccharide is 62.29%.
The strain of fermented lactobacillus mucilaginosus TG017 metaplasia has high antioxidant enzyme activity, the extracellular polysaccharide glutathione-peroxidase activity reaches 2890.27U/mgprot, and the supernatant fluid superoxide dismutase (SOD) enzyme activity reaches 940.26U/mgprot.
The strain of fermented lactobacillus mucilaginosus TG017 metaplasia has the capacity of resisting lipid peroxidation, the inhibition rate of cell-free extract is 22.5%, and the inhibition rate of extracellular polysaccharide is 15.51%.
The lactobacillus mucilaginosus TG017 extracellular polysaccharide has good cell anti-inflammatory effect.
Further, the lactobacillus mucilaginosus TG017 is applied to food, and the food comprises the lactobacillus mucilaginosus TG017 and is prepared by taking the lactobacillus mucilaginosus TG017 as a material.
The fermented lactobacillus mucilaginosus TG017 is applied to medicines, and the dosage forms of the medicines can be any one of powder, suppository, gel, oral liquid, hard capsule and soft capsule.
Further, the lactobacillus mucilaginosus TG017 is prepared into metaplasia for further application.
Compared with the prior art, the method has the following beneficial effects:
compared with living probiotics, the metazoan of the fermented lactobacillus mucilaginosus TG017 is more stable, has longer shelf life and is not inhibited by the interference of antibiotics. Has good application safety and functional adaptability.
The fermented lactobacillus mucilaginosus TG017 metaplasia of the invention has the functions of scavenging DPPH free radical, improving the enzyme activity of antioxidant enzyme glutathione peroxidase (GSH-Px), and resisting lipid peroxidation and inflammation of superoxide dismutase (SOD) enzyme activity.
The metagen of the lactobacillus fermentum (limosilactobacillus fermentum) has antioxidant capacity, the lactobacillus fermentum is named lactobacillus fermentum (limosilactobacillus fermentum) TG017, and the strain is preserved in China center for type culture Collection, and the preservation number is: [ CCTCC NO: m2023513, the preservation time is [ 2023, 4, 10 ] and the address is in the university of Wuhan preservation center.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the plate phenotype of Lactobacillus fermentum TG017 achieved by the present invention.
FIG. 2 is a phylogenetic tree of Lactobacillus fermentum TG017 according to the present invention.
FIG. 3 is a graph showing the results of Molisch reaction of extracellular polysaccharide of Lactobacillus mucilaginosus TG017 according to the present invention.
FIG. 4 is a graph showing the effect of DPPH clearance of Lactobacillus fermentum TG017 obtained by the present invention.
FIG. 5 is a graph showing the effect of the present invention on the lipid peroxidation resistance of Lactobacillus fermentum TG 017.
FIG. 6 is a graph showing the results of nitric oxide standard curves.
FIG. 7 is a graph showing the effect of Lactobacillus fermentum TG017 on antioxidant stress in cells.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention realizes that a strain of lactobacillus fermentum (limosilactobacillus fermentum) with an antioxidant effect is provided, wherein the lactobacillus fermentum is lactobacillus fermentum TG017 and is preserved in China center for type culture Collection, and the preservation number is: [ CCTCC NO: m2023513, the preservation time is [ 2023, 4, 10 ] and the address is in the university of Wuhan preservation center.
The lactobacillus mucilaginosus TG017 is obtained by screening domestic healthy human body excrement samples, and the colony is milky white, opaque, round convex, smooth in surface and neat in edge, and has an optimal growth temperature of 37 ℃ and an optimal pH of 6 and anaerobic environment.
The gene sequence of the lactobacillus mucilaginosus TG017 is as follows:
GTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGGTTTCACATCAGAATTAAAAAAACCGCCTGGCCTCTCTTTTCGCCCCAATAAATCCGGATAACGCTTGGCACCTACGTA。
the lactobacillus mucilaginosus TG017 has the capability of producing extracellular polysaccharide with the yield of 14.02g/L.
The strain of lactobacillus mucilaginosus TG017 metaplasia has the capability of removing DPPH free radical, the clearance rate of cell-free extract is 29.28%, and the clearance rate of extracellular polysaccharide is 62.29%.
The strain of fermented lactobacillus mucilaginosus TG017 metaplasia has high antioxidant enzyme activity, the extracellular polysaccharide glutathione-peroxidase activity reaches 2890.27U/mgprot, and the supernatant fluid superoxide dismutase (SOD) enzyme activity reaches 940.26U/mgprot.
The strain of fermented lactobacillus mucilaginosus TG017 metaplasia has the capacity of resisting lipid peroxidation, the inhibition rate of cell-free extract is 22.5%, and the inhibition rate of extracellular polysaccharide is 15.51%.
The lactobacillus mucilaginosus TG017 extracellular polysaccharide has good cell anti-inflammatory effect.
Example 1: screening of human-derived probiotic strains.
Taking healthy human feces from Fujian Xiamen area as a sample, inoculating the sample into a serum culture bottle for enriching and culturing human intestinal flora, absorbing enriched culture solution in 1, 3 and 6 days, diluting and coating on an MRS agar plate by using physiological saline gradient (MRS culture medium formula: 10g/L of casein enzyme digestate, 10g/L of beef extract powder, 4g/L of citric acid, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 2g/L of dipotassium hydrogen phosphate, 20g/L of glucose, 1.08g/L of Tween-80, and finally pH5.7+/-0.2, sterilizing for 25min at the temperature of 115 ℃), culturing in an anaerobic incubator at the temperature of 37 ℃ for 48 hours, picking single bacteria colony on the MRS agar plate, and carrying out 3 rounds of streaking and purifying to obtain the fermented lactobacillus as shown in figure 1.
Example 2: identification of strains.
Identification of strain morphology: the purified strain is inoculated to an MRS solid culture medium plate and cultured in an incubator at 37 ℃ under anaerobic conditions for 24 hours.
Single colonies were inoculated into MRS liquid medium, and after shaking culture at 37℃and 200rpm for 24-48 hours, genomic DNA was extracted using a bacterial genomic DNA rapid extraction kit.
Using the extracted genomic DNA as a template, a 16S rDNA full-length primer pair, 27F:5'-AGAGTTTGATCCTGGCTCAG-3';1492R:5'-TACGGCTACCTTGTTACTT-3' the relationship of the Lactobacillus fermentum strain (limosilactobacillus fermentum) is shown in the phylogenetic tree of FIG. 2, and the Lactobacillus fermentum strain (limosilactobacillus fermentum) is named Lactobacillus fermentum (limosilactobacillus fermentum TG 017).
Example 3: extraction of the extracellular polysaccharide of the fermented lactobacillus mucilaginosus TG 017.
Inoculating lactobacillus mucilaginosus TG017 into MRS liquid culture medium, and culturing at 37deg.C and 200rpm in shaking table until bacteria grow in the stage of platform. Centrifuging the cultured bacterial suspension at 10000Xg for 5min, collecting supernatant, filtering with 0.22 μm ultrafiltration membrane, standing the filtered culture solution at 65deg.C for 30min, and rapidly cooling to 4deg.C. The extracellular polysaccharide culture solution was concentrated ten times. Trichloroacetic acid (TCA) was added to the concentrate to a final TCA concentration of 10%, and after mixing, the mixture was allowed to stand at 4℃overnight, centrifuged at 10000Xg for 5min, and the supernatant was collected. Adding 80mL of absolute ethyl alcohol to the concentrated solution until the final concentration of the absolute ethyl alcohol is 80%, precipitating extracellular polysaccharide overnight, centrifuging 10000xg for 5min, discarding the supernatant, washing the precipitate with 30mL of 75% ethanol for 2 times, centrifuging again after each washing, balancing and centrifuging 10000xg for 5min, and discarding the supernatant. And (5) uncovering the centrifuge tube, and placing the centrifuge tube in a drying oven at 45 ℃ to dry the centrifuge tube until the weight is constant.
The extracellular polysaccharide sugar yield of the fermented lactobacillus mucilaginosus TG017 is 14.02g/L.
Identification of extracellular polysaccharide Molisch reaction: 1ml of the solution to be tested was added to the test tube. 2 drops (about 0.1 ml) of Molisch Reagent (prepared by mixing Molisch Reagent A with Molisch Reagent B in a ratio of 5g:100m and dissolving thoroughly) were added dropwise to the tube, the tube was shaken thoroughly and tilted, concentrated sulfuric acid was slowly added along the tube wall, and the tube was not shaken. And after the sulfuric acid layer is deposited at the bottom of the test tube, separating the sulfuric acid layer and the solution to be tested into two layers, and observing whether a purple ring appears outside the interface of the liquid surface.
The Molisch reaction was used to detect the reducing sugars in the extracellular polysaccharide, and when there was a clear purple ring at the interface of the liquid surface, this indicated that the extracellular polysaccharide extraction was successful. As shown in FIG. 3, a large amount of reducing sugar was present in the crude extracellular polysaccharide of Lactobacillus mucilaginosus TG 017.
Example 4: and (5) preparing a bacterial liquid metasolution required by experiments.
Extracellular polysaccharide solution formulation (EPS): 0.1g of the extracellular polysaccharide extract was weighed, dissolved in 10ml of sterile water and filtered through a 0.22. Mu.L microporous filter membrane.
Cell-free extract preparation (CFE): inoculating lactobacillus mucilaginosus TG017 into MRS liquid culture medium, and culturing at 37deg.C and 200rpm in shaking table until bacteria grow in the stage of platform. The cultured bacterial suspension was centrifuged at 10000Xg and the supernatant was collected for 5min and filtered through a 0.22 μm ultrafiltration membrane.
Preparing bacterial liquid supernatant: the fermented lactobacillus mucilaginosus TG017 is inoculated into MRS liquid culture medium, and is put into a shaking table for overnight culture at 37 ℃ and 200rpm. The cultured bacterial suspension was centrifuged at 8000rpm for 5min to obtain a supernatant.
Example 5: bacterial liquid supernatant and extracellular polysaccharide can remove DPPH free radical.
Preparing DPPH sample solution: accurately weighing 20.0mg of DPPH powder, placing into a 250mL volumetric flask, dissolving with absolute ethyl alcohol, fixing the volume to a scale, and uniformly mixing to obtain 0.2mmol/L DPPH solution, and preserving at 4 ℃ for 3.5 hours. Taking 1ml of cell-free extract, adding 1ml of DPPH with the concentration of 0.2mmol/L, uniformly mixing, standing at room temperature for 30min, taking absolute ethyl alcohol as a blank control, and measuring the absorbance change at 517 nm.
Measuring the absorbance of each sample solution at 517nm to A 1 The method comprises the steps of carrying out a first treatment on the surface of the Absorbance A was measured by using a mixture of 1.0mL of DPPH solution and 1.0mL of absolute ethyl alcohol as a negative control 2 The method comprises the steps of carrying out a first treatment on the surface of the 1.0mL of absolute ethanol solution is added to 10mL of the mixed solution of the test sample solutions with different concentrations is used as a blank control, and the absorbance A is measured 0 . According to (inhibition (%) = (1- (a) 1 -A 0 )/A 2 ) X 100%) formula calculates clearance.
DPPH is a very stable nitrogen-centered radical, and if the test substance is able to scavenge it, it indicates that the test substance has a remarkable antioxidant effect. As shown in figure 4, the clearance rate of the cell-free extract of the fermented lactobacillus mucilaginosus TG017 to DPPH free radicals reaches 29.28%, the clearance rate of the extracellular polysaccharide to DPPH free radicals reaches 62.49%, and the strain has high-quality antioxidant effect and can effectively remove the free radicals.
Example 6: and (3) performing glutathione peroxidase (GSH-Px) activity experiments on the bacterial liquid supernatant.
And taking out the fermented lactobacillus mucilaginosus TG017 from the refrigerator at the temperature of minus 80 ℃, marking and activating on an MRS agar plate, culturing in an anaerobic incubator at the temperature of 37 ℃ until single colonies grow, dipping the single colonies on the MRS agar by using an inoculating loop, inoculating the single colonies into an MRS liquid culture medium, and culturing in a shaking table at the temperature of 37 ℃ at 200rpm until the bacteria grow in the stage of the bacteria growth platform. The cultured bacterial suspension was centrifuged at 10000Xg for 5min, and the supernatant was filtered through a 0.22 μm ultrafiltration membrane, and the antioxidation result was detected by using GSH-Px kit (available from Elabscience).
The results are shown in Table 1, and after the lactobacillus mucilaginosus TG017 is cultured, the antioxidant enzyme activity of the cell-free extract reaches 70.55U/mL, and the antioxidant effect is remarkable.
Table 1 cell-free extract of TG017 enzyme Activity against oxidase
Example 7: and (3) performing an activity experiment on superoxide dismutase (SOD) of the bacterial liquid supernatant.
As shown in Table 2, WST-1 method: WST-1 can react with superoxide anions generated by catalyzing xanthine oxidase to generate water-soluble formazan dye, and SOD can catalyze superoxide anions to generate disproportionation reaction. The reaction step can be inhibited by SOD, the activity of the SOD is inversely related to the generation amount of the formazan dye, and the activity of the SOD can be calculated through calculating the colorimetric analysis of the WST-1 product. The results are shown in Table 3, and after the lactobacillus mucilaginosus TG017 is cultured, the antioxidant enzyme activity of the supernatant reaches 940.26U/mL, and the antioxidant effect is remarkable.
TABLE 2 WST-1 experiment
The calculation formula of the SOD inhibition rate: SOD inhibition (%) = [ (Δa) 1 -ΔA 2 )/ΔA 1 ]×100％；
And (3) calculating SOD activity: SOD viability (U/mL) =i/50% × (V 1 /V 2 )；
Wherein DeltaA 1 : control well OD value-control blank well OD value; ΔA 2 : measuring the OD value of the hole-the OD value of a control blank hole; i: SOD inhibition (%); v (V) 1 : total volume of reaction solution (240. Mu.L); v (V) 2 : the volume of sample (20. Mu.L) was added.
TABLE 3 anti-oxidant enzyme Activity of TG017 supernatant
Example 8: bacterial liquid supernatant and extracellular polysaccharide lipid peroxidation resistance.
(1) Preparing linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
(2) Adding 1mL of linoleic acid emulsion, 1mL of FeSO4 (1%) into 0.5mL of PBS solution (pH 7.4), adding 0.5mL of fermentation supernatant or extracellular polysaccharide solution of probiotics, carrying out water bath at 37 ℃ for 1.5h, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), carrying out water bath at 100 ℃ for 30min, cooling rapidly, centrifuging at 4000rpm/min for 15min, and collecting supernatant to measure absorbance at 532nm to obtain A; the control group was A with 0.5mL distilled water instead of the sample 0 。
Inhibition (%) = (a) 0 －A)/A 0 ×100％。
(Note: A is the absorbance of the sample group; A 0 Absorbance for control group。)
Lipid peroxidation (Lipid peroxidation) refers to oxidative deterioration of polyunsaturated fatty acids and lipids, which may lead to alterations in the fluidity and permeability of body cell membranes, damage to DNA and proteins, and thus affect the normal function of the cells. Studies have shown that many diseases in humans, such as tumors, vascular sclerosis, aging, neurodegenerative phenomena, etc., are associated with lipid peroxidation. As shown in fig. 5, lactobacillus mucilaginosus TG017 was evaluated for lipid peroxidation resistance by measuring the effect of cell-free extract and extracellular polysaccharide on lipid peroxidation of linoleic acid, the inhibition rate of CFE on lipid peroxidation was 22.50%, the inhibition rate of EPS on lipid peroxidation was 15.51%, and the metazoan were high-efficiency lipid peroxidation resistance.
Example 9: an antioxidant stress experiment of extracellular polysaccharide in cells.
50mg of exopolysaccharide was weighed and dissolved in 50ml of ultrapure water to prepare a 1mg/ml exopolysaccharide solution, and 0.22. Mu.L of microporous membrane was used for filtration. LPS was dissolved in ultrapure water to prepare a 10. Mu.g/ml LPS solution, and 0.22. Mu.L of the solution was filtered through a microporous membrane.
RAW 264.7 cells were cultured, and the plates were placed at 37℃with 5% CO 2 Incubate in incubator for 24h. After the cells had been sufficiently adherent, the old medium was discarded, the cells were washed once with PBS buffer, and 100. Mu.l of DMEM medium containing extracellular polysaccharide at a final concentration of 500. Mu.g/ml was added to each well, and the plates were placed at 37℃with 5% CO 2 After incubation for 2h in incubator, 10. Mu.l of LPS at 10. Mu.g/ml was added to each well and incubation was continued for 22h, 50. Mu.l of cell supernatant was taken in a new 96-well plate, and the NO concentration of the cell supernatant was measured using Griess kit.
Oxidative Stress (OS) refers to a state in which oxidation and antioxidant effects are unbalanced in vivo, tending to oxidize, leading to neutrophil inflammatory infiltrates, increased protease secretion, and the production of a large number of oxidation intermediates. Oxidative stress is a negative effect produced in vivo by free radicals and is considered to be an important factor in causing aging and diseases. And refers to physiological and pathological reactions of cells and tissues caused by the generation of reactive oxygen radicals (Reactive Oxygen Species, ROS) and reactive nitrogen radicals (Reactive Ntrogen Species, RNS) in the body under the harmful stimulation of the internal and external environments. Because they can oxidize or damage DNA, proteins and lipids directly or indirectly, mutation of genes, protein denaturation and lipid peroxidation can be induced, and are considered to be the most important risk factors for human aging and various important diseases such as tumor, cardiovascular and cerebrovascular diseases, neurodegenerative diseases (senile dementia), diabetes and the like.
FIG. 6 shows a standard curve of carbon monoxide, NO is an important index for oxidative stress, and is considered to inhibit the production of intracellular NO and to achieve an antioxidant emergency, and as shown in FIG. 7, the inhibitory capacity of Lactobacillus fermentum TG017 against NO is 89.45%.
Example 10: extracellular polysaccharides facilitate GSH-Px activity experiments in cells.
RAW 264.7 cells were grown in culture flasks to a density of about 80% to 90%, centrifuged (250 rpm,3 min) with pancreatin, the cells were collected and plated in 6-well plates with 2mL of cell suspension added to each well, and the cell density was about 1X 10 6 cell per well (5X 10) 5 cell/mL), the plates were placed at 37℃with 5% CO 2 Incubate in incubator for 24h.
After the cells were sufficiently adherent, the old medium was discarded, the cells were washed twice with PBS buffer, 2mL of DMEM medium containing 500. Mu.g/mL extracellular polysaccharide was added to each well, and the plates were placed at 37℃with 5% CO 2 Incubation was continued for 2h in incubator, after which 200. Mu.l, 10. Mu.g/ml LPS was added to each well and incubation was continued for 22h, and the GSH-Px (purchased from Elabscience) kit was used to test the antioxidant results.
As shown in Table 4, GSH-Px in cells after extracellular polysaccharide treatment of TG017 strain increased 2890.27U/mgprot.
TABLE 4 GSH-Px enzyme Activity in cells after extracellular polysaccharide treatment of TG017 Strain
Example 11: the survival rate of the bacteria in the gastrointestinal environment was simulated.
Preparation of simulated gastric fluid: accurately measuring 16.4mL of diluted hydrochloric acid, adding 800mL of water and 10g of pepsin, completely mixing, adding water to 1000mL of constant volume, completely mixing, and placing in a refrigerator at 4 ℃. The pH was adjusted to 2.5 with 1mol/L HCl, and after complete dissolution, the solution was filtered through a 0.22 μm microporous filter membrane for sterilization.
Preparation of simulated intestinal juice: accurately weighing 6.8g of monopotassium phosphate and adding 500mL of water. Adjusting the pH to 6.8 with 0.4% sodium hydroxide solution; and adding water into 10g of trypsin to dissolve, mixing the two solutions, and adding water to a volume of 1000mL. Placing in a refrigerator at 4 ℃. The pH value is regulated to 8.0 by 0.1mol/L NaOH, and then the solution is fully dissolved and filtered and sterilized by a microporous filter membrane with the thickness of 0.22 mu m for standby.
Activating the strain: plating line, inoculating 200 μl of bacterial suspension into 10mL MRS liquid culture medium, culturing overnight, and measuring OD 600 Diluting the bacterial liquid to OD 600 And (3) respectively taking 0.4mL of thallus suspension for standby, respectively adding the thallus suspension into 10mL of prepared simulated artificial gastric juice with pH of=2.5 and simulated artificial intestinal juice with pH of=8.0, uniformly mixing, digesting at 37 ℃, simultaneously respectively sucking 20 mu L of digestive juice for 0h and 3h for coating, counting and detecting viable bacteria after 48h, and calculating the survival rate. Wherein strain survival%o=n t /N 0 X 100%, N in 0 Represents the viable count (CFU/mL) of the strain for 0h, N t The viable count (CFU/mL) of the strain 3h was shown. As shown in Table 1, the survival rate of the lactobacillus mucilaginosus TG017 in gastric juice can reach 51.55%, and the survival rate in intestinal juice can reach 77%, which indicates that the strain can well survive and play a role in the human digestive tract.
TABLE 3 survival of TG017 in gastrointestinal fluids
In conclusion, compared with the living probiotics, the metazoan of the fermented lactobacillus mucilaginosus TG017 is more stable, has longer shelf life and is not inhibited by the interference of antibiotics. Has good application safety and functional adaptability.
The fermented lactobacillus mucilaginosus TG017 metaplasia of the invention has the functions of scavenging DPPH free radical, improving the enzyme activity of antioxidant enzyme glutathione peroxidase (GSH-Px), and resisting lipid peroxidation and inflammation of superoxide dismutase (SOD) enzyme activity.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. The lactobacillus fermentum with the antioxidation effect is characterized in that the lactobacillus fermentum is lactobacillus fermentum TG017 and is preserved in China center for type culture collection, and the preservation number is: [ CCTCC NO: m2023513, the preservation time is [ 2023, 4, 10 ] and the address is in the university of Wuhan preservation center.
2. The lactobacillus fermentum with antioxidant effect according to claim 1, wherein the lactobacillus fermentum TG017 is obtained by screening from domestic healthy human body faeces, the colony is milky white, opaque, round convex, smooth in surface and neat in edge, the optimal growth temperature is 37 ℃, and the optimal pH is 6 and anaerobic environment.
3. The lactobacillus fermentum with antioxidant effect according to claim 1, characterized in that the gene sequence of lactobacillus fermentum TG017 is:
GTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGGTTTCACATCAGAATTAAAAAAACCGCCTGGCCTCTCTTTTCGCCCCAATAAATCCGGATAACGCTTGGCACCTACGTA。
4. the lactobacillus fermentum with antioxidant effect according to claim 3, wherein one lactobacillus fermentum TG017 has the ability to produce extracellular polysaccharide in high yield.
5. The lactobacillus fermentum with antioxidant effect according to claim 3, wherein one lactobacillus fermentum TG017 has DPPH radical scavenging ability.
6. The lactobacillus fermentum with antioxidant effect according to claim 3, wherein one lactobacillus fermentum TG017 has antioxidant enzyme activity.
7. The lactobacillus fermentum with antioxidant effect according to claim 3, wherein one lactobacillus fermentum TG017 has the ability to resist lipid peroxidation.
8. The use of lactobacillus fermentum with antioxidant effect according to claim 1, further characterized in that the lactobacillus fermentum TG017 is applied to food comprising lactobacillus fermentum TG017, prepared by taking lactobacillus fermentum TG017 as a material.
9. The application of the fermented lactobacillus mucilaginosus with the antioxidation effect according to claim 1, which is characterized in that the fermented lactobacillus mucilaginosus TG017 is applied to a medicament, and the dosage form of the medicament can be any one of powder, suppository, gel, oral liquid, hard capsule and soft capsule.
10. The use of lactobacillus fermentum with antioxidant effect according to claim 1, characterized in that lactobacillus fermentum TG017 is prepared as a metazoan.
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| CN117736942A (en) * | 2024-02-20 | 2024-03-22 | 山东中科嘉亿生物工程有限公司 | Fermented lactobacillus mucilaginosus JYLF-315 for improving skin aging and metagen preparation and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117736942A (en) * | 2024-02-20 | 2024-03-22 | 山东中科嘉亿生物工程有限公司 | Fermented lactobacillus mucilaginosus JYLF-315 for improving skin aging and metagen preparation and application thereof |
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