CN116790399B - Application of Acetobacter pasteurii in antioxidation, fat reduction and anti-aging - Google Patents
Application of Acetobacter pasteurii in antioxidation, fat reduction and anti-aging Download PDFInfo
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- CN116790399B CN116790399B CN202211676676.3A CN202211676676A CN116790399B CN 116790399 B CN116790399 B CN 116790399B CN 202211676676 A CN202211676676 A CN 202211676676A CN 116790399 B CN116790399 B CN 116790399B
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Classifications
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- 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
-
- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
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- 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
- A01K2227/703—Worms, e.g. Caenorhabdities elegans
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- 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
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- 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
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Abstract
The invention discloses an Acetobacter pasteurii WY202202 strain and application thereof. The invention separates from kefir to obtain a strain of Acetobacter pasteurism WY202202 which has the functions of resisting oxidation, reducing fat, resisting aging, prolonging the service life of animals and promoting the growth and development of animals, and the preservation number is GDMCC No:62536. the research of the invention shows that the Acetobacter pasteurii WY202202 can enhance the antioxidation capability of the caenorhabditis elegans and reduce the oxidative damage of organisms; reducing accumulation of fat, improving running power, body length and fertility of nematodes, effectively prolonging health life and slowing down body aging. The bacteria can be used as probiotics for preparing or developing products with the functions of resisting oxidation, reducing fat and resisting aging.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly relates to application of Acetobacter pasteurii in antioxidation, fat reduction and anti-aging.
Background
The study demonstrates that: probiotics such as lactobacillus plantarum (Lactobacillus plantarum), lactobacillus pentosus (Lactiplantibacillus pentosus), lactobacillus clavus (Lactiplantibacillus coryniformis), lactobacillus fermentum (Lactobacillus fermentans), lactobacillus delbrueckii (Lactobacillus delbrueckii), leuconostoc lactis (Leuconostoc lactis), lactobacillus rhamnosus (Lactobacillus rhamnosus) and the like have the effects of resisting oxidation and reducing fat, so that the health life can be effectively prolonged, and the aging of the organism can be delayed, but the effect has obvious strain specificity, so that the continuous excavation of the probiotic strain with the effects of resisting oxidation, reducing fat, resisting aging and the like has wide and practical application value.
Acetobacter pasteurii (Acetobacter pasteurianus) is a gram-negative strain, has strong acid resistance and high-efficiency ethanol and lactic acid catalyzing capacity, and is mainly used for producing table vinegar and fruit vinegar beverage. As a typical vinegar brewing microorganism, the research reports of Acetobacter pasteurii are mainly focused on the selection and selection of excellent strains, the optimization of culture medium conditions, the research of acid-resistant mechanisms, alcohol dehydrogenase, acetaldehyde dehydrogenase and other key enzymes, and the fresh reports of physiological functions such as oxidation resistance, fat reduction, aging resistance and the like are provided.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the problems and provide a Acetobacter pasteurii (Acetobacter pasteurianus) WY202202 strain and application thereof in oxidation resistance, fat reduction and aging resistance.
The first object of the present invention is to provide a Acetobacter pasteurellosis (Acetobacter pasteurianus) WY202202 strain.
The second purpose of the invention is to provide the application of the acetobacter pasteurii (Acetobacter pasteurianus) WY202202 in the aspects of prolonging the life of animals, promoting the growth and development of animals, resisting oxidation, reducing fat and resisting aging.
The above object of the present invention is achieved by the following technical scheme:
The Acetobacter pasteurellosis (Acetobacter pasteurianus) WY202202 strain provided by the invention is preserved in the microorganism strain collection of Guangdong province on the 7 th month 8 th year 2022, and the preservation number is GDMCC No:62536. the WY202202 strain was isolated in Kefir (a conventional alcoholic fermented milk beverage) and the full length of its 16S rDNA gene sequence was about 1383bp, sequenced and then aligned at NCBI using Blast, identified as Acetobacter pasteurelloides (Acetobacter pasteurianus). The strain is cultivated on an acetic acid bacteria culture medium, is a gram-negative bacterium, is rod-shaped in form, has no spores at the periphery and is arranged singly or in pairs; the colony is white, round, with a bulge in the middle and a diameter of 0.32-1.02mm, and a clear ring is arranged around the colony. The research of the invention shows that the WY202202 strain can prolong the service life, increase the body length, improve the spawning quantity and the mobility after feeding caenorhabditis elegans, enhance the oxidation resistance of the nematodes, reduce the oxidative damage and fat accumulation of organisms and delay the aging of organisms.
The invention uses the aging research model of the caenorhabditis elegans (Caenorhabditis elegans) N2, belongs to life research model animals, is a simple invertebrate model, has the advantages of short life span, simple living environment, large amount of synchronization, clear genetic background, high conservation/homology of a plurality of genes and mammals (including human beings) in evolution and the like, can research the aging mechanism from behaviors, molecules and gene levels, and is proved in nematodes in a plurality of theories at present, so the caenorhabditis elegans is a common model organism for life span and aging research, and products with the functions of prolonging the life span and resisting aging of the nematodes are generally considered to have the same efficacy on human beings.
Therefore, the invention provides the application of the Acetobacter pasteurii (Acetobacter pasteurianus) WY202202 strain or the bacterial liquid thereof in prolonging the service life of animals, promoting the growth and development of the animals, preparing products for prolonging the service life of the animals and promoting the growth and development of the animals and preparing products for resisting oxidation, reducing fat and resisting aging.
Further, the antioxidation means to improve the antioxidation capability of the organism, reduce the accumulation of oxygen free radicals in the organism and slow down the oxidative damage of the organism; the anti-aging means that the length, motility and fertility of nematodes are increased, and the damage to mitochondria is reduced; the lipid reduction refers to reduction of nematode fat accumulation.
The invention provides an antioxidant, fat-reducing and anti-aging product containing Acetobacter pasteurella (Acetobacter pasteurianus) WY202202 strain or bacterial liquid thereof.
Preferably, the concentration of the bacterial solution of the Acetobacter pasteurium (Acetobacter pasteurianus) WY202202 strain or the bacterial solution thereof is 8.4X10- 7～3.2×1011 CFU/mL.
More preferably, the concentration of Acetobacter pasteurella (Acetobacter pasteurianus) WY202202 strain is 8.4X10 9 CFU/mL.
The invention provides a method for prolonging the service life of animals and promoting the growth and development of the animals, which adopts Acetobacter pasteurium WY202202 strain or bacterial liquid thereof to feed the animals.
Preferably, the animal broadly refers to organisms belonging to the kingdom animalia, including but not limited to mammals, biologically.
Further preferably, the animal is a nematode.
Preferably, the above-mentioned products can be aimed at animals of different ages; more preferably, the method comprises the following steps: the use starts from the L1 phase.
Preferably, the product is health food, medicine, feed or additive thereof, and the product is in the form of liquid, jelly, stick, powder, tablet, granule or capsule.
The invention has the following beneficial effects:
The invention provides a novel application of Acetobacter pasteurii (Acetobacter pasteurianus) WY202202 strain in the aspects of antioxidation, fat reduction and aging resistance, and the strain can enhance the antioxidation capability of caenorhabditis elegans, reduce the oxidative damage of organisms, prolong the service life of the nematodes, increase the body length of the nematodes, improve the running power and reproductive capacity of the nematodes, reduce fat accumulation and delay the aging of the organisms. It is shown that Acetobacter pasteurii WY202202 can be used for preparing or developing antioxidant, antiaging and lipid-lowering products.
Drawings
FIG. 1 shows the Acetobacter pasteurii WY202202 as a evolutionary tree.
FIG. 2 shows the effect of Acetobacter pasteurisis WY202202 on N2 longevity of caenorhabditis elegans (wherein OP50: E.coli OP50; WY202202: acetobacter pasteurisis WY 202202; WY202202supernatant: acetobacter pasteurisis WY202202 metabolite; WY202202 heat-treatment: acetobacter pasteurisis WY202202 dead bacteria).
FIG. 3 shows the changes in body length (A), motility (B) and fertility (C) of Acetobacter pasteurella WY202202 fed to caenorhabditis elegans (wherein OP50: E.coli OP50; WY202202: acetobacter pasteurella WY202202 live bacteria).
FIG. 4 shows the effect of Acetobacter pasteurisis WY202202 on the fat content (A) and mitochondrial membrane potential (B) of insects (wherein OP50: E.coli OP50; WY202202: acetobacter pasteurisis WY202202 live bacteria).
FIG. 5 shows that Acetobacter pasteurii WY202202 affects the ROS production and antioxidant properties of nematodes (A: MDA content; B: ROS content; C: SOD activity; D: GSH content; E: CAT activity, wherein OP50 is fed E.coli OP50; WY202202 is fed Acetobacter pasteurii WY202202 viable bacteria).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Reagents or formulations employed in the following examples:
Acetic acid bacteria liquid culture medium: 10.0g/L glucose, 10.0g/L yeast powder, and 121 ℃ for 20min, 3% (V/V) absolute ethanol is added before inoculation. Acetic acid bacteria solid culture medium: agar 2% was added to the liquid medium.
Wild caenorhabditis elegans N2, E.coli OP50: purchased from nematode center CGC (Caenorhabditis GENETICS CENTER).
NGM medium: 3g of sodium chloride, 2.5g of tryptone, 17g of agar powder, 25mL of potassium phosphate buffer solution (1 mol/L), 1mL of CaCl 2 solution (1 mol/L), 1mL of MgSO 4 solution (1 mol/L), distilled water to 1000mL and autoclaved at 121 ℃ for 20min. After the temperature was reduced to about 60℃and sterilized, l mL of cholesterol solution (5 mg/mL,95% ethanol solution) was added, and after shaking, the plate was poured.
LB liquid medium: 5g of yeast powder, 10g of tryptone and 10g of NaCl, adding distilled water to 1000mL, and sterilizing at 121 ℃ for 20min.
The kit for measuring Malondialdehyde (MDA) content, superoxide dismutase (SOD) enzyme activity, reactive Oxygen Species (ROS) content, glutathione (GSH) content and Catalase (CAT) enzyme activity is from Shanghai Biyunshan Biotechnology Co.
Example 1 isolation and identification of strains
1. Isolation of strains
1ML of Kefir (Kefir) fermentation broth was diluted with 9mL of sterile physiological saline, and the dilution was performed in a gradient. Coating the diluted solution on an acetic acid bacteria solid culture medium, culturing at 30 ℃, separating and purifying plate colonies, culturing in a liquid culture medium of acetic acid bacteria, and primarily screening to obtain a strain named WY202202.
2. Traditional biological assays
Colony morphology characterization: the strain WY202202 is cultivated on an acetic acid bacteria culture medium, the strain is in a rod shape, the periphery of the strain is free of spores, and the strain WY202202 is arranged in a single or paired mode; the colony is white, round, with a bulge in the middle and a colony diameter of 0.32-1.02mm, and has a clear ring around, belonging to gram-negative bacteria.
3. Molecular biological identification
(1) The total DNA of the strain is extracted by using a DNA extraction kit, and the 16S rDNA genes of the strain are amplified by using bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') through PCR.
The PCR amplification procedure was: pre-denaturation at 98℃for 2min, denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 25s, final extension at 72℃for 2min, and 30 cycles.
The PCR amplification system is as follows: 27F 1. Mu.L, 1499R 1. Mu.L, DNA template 2. Mu.L, taq enzyme 22. Mu.L.
(2) Phylogenetic analysis
The PCR products were sequenced after 1% agarose gel detection and gel cutting recovery purification, and the 16S rDN A gene sequence with the length of about 1383bp was compared with the gene sequence recorded in Genbank and the phylogenetic tree was compared and analyzed, and as shown in FIG. 1, the WY202202 strain was found to be most similar to Acetobacter pasteurella (Acetobacter pasteurianus), and the similarity was 99.93%.
The 16S rDNA gene sequence of the strain WY202202 is specifically as follows:
GGTCGGCTGCGTCCTTGCGGTTCGCTCACCGGCTTAAGGTCAAACCAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCACCTTCATGCACTCGAGTTGCAGAGTGCAATCCGAACTGAGACGGCTTTTAGAGATCAGCATGGTGTCACCACCTAGCTTCCCACTGTCACCGCCATTGTAGCACGTGTGTAGCCCAGGACATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGCTTGTCACCGGCAGTCTCTCTAGAGTGCCCAGCCCAACCTGATGGCAACTAAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTAGAGGTCCCTTGCGGGAAACAAACATCTCTGCTTGCAGCCTCTACATTCAAGCCCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTGTGCTTAACGCGTTAACTGCGACACTGAATGACTAAGTCACCCAACATCTAGCACACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTAATGAGCCAGGTTGCCGCCTTCGCCACCGGTGTTCTTCCCAATATCTACGAATTTCACCTCTACACTGGGAATTCCACAACCCTCTCTCACACTCTAGTCTGCACGTATCAAATGCAGCTCCCAGGTTAAGCCCGGGGATTTCACATCTGACTGTACAAACCGCCTACACGCCCTTTACGCCCAGTCATTCCGAGCAACGCTAGCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCTTCTACGGGTACCGTCATCATCGTCCCCGTCGAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATTGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCATCCTCTCAAACCAGCTATTGATCATCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCAAACGCAGGCTCCTCCACAGGCGACTTGCGCCTTTGACCCTCAGGTGTCATGCGGTATTAGCACCAGTTTCCCAGTGTTATCCCCCACCCATGGATAGATACCTACGCGTTACTCACCCGTCCGCCACTAAGGCCGAAACCTTCGTGCGACTGCA.
The above morphological characteristics and 16S rDNA gene sequence results are combined, and it is shown that the bacteria selected by the present invention are classified into Acetobacter pasteurisi (Acetobacter pasteurianus) of Acetobacter (Acetobacter) and named Acetobacter pasteurisi (Acet obacter pasteurianus) WY202202 strain which was deposited at the microorganism strain deposit center (GDMCC) of Guangdong province at 7/8 of 2022, with the deposit number: GDMCC No:62536, address: building 5 of Guangzhou City martyr of Guangdong province, no. 100 of the university of road 59, post code: 510070.
EXAMPLE 2 Effect of WY202202 Strain on caenorhabditis elegans
1. Activation of bacterial species
Inoculating the Acetobacter pasteurii WY202202 100 mu L frozen at-80 ℃ into 5mL of acetic acid bacteria liquid culture medium, culturing for 20-24 h at 30 ℃ in a 180r/min shaking table, and activating the strain.
2. Culture of caenorhabditis elegans
E.coli OP50 (8.4X10 9 CFU/mL) was spread on NGM medium as nematode diet, then wild C.elegans N2 was picked up and placed on the surface of NGM medium and cultured in a biochemical incubator at 20℃where nematodes were transferred to fresh E.coli OP 50-coated NGM plates every 2 days.
3. Synchronization of caenorhabditis elegans
The nematodes in the L4 stage are collected in a centrifuge tube, 1mL of M9 buffer is added to wash out redundant escherichia coli OP50, the supernatant is removed after standing and precipitation, and the washing is repeated for 3 times. Then adding 1mL of lysate, shaking thoroughly, removing supernatant, centrifuging with M9 buffer solution (3000 r/min,1 min) for 3 times, removing supernatant and retaining ovum, transferring ovum onto new NGM plate, incubating in biochemical incubator at 20deg.C, and growing into L1 stage larva after about 16 hr.
4. Determination of caenorhabditis elegans longevity
Culturing Acetobacter pasteurii WY202202 and Escherichia coli OP50 in liquid culture medium respectively to logarithmic phase, centrifuging at 4deg.C and 8000r/min for 10min, collecting Acetobacter pasteurii WY202202 thallus and fermentation supernatant, and filtering with 0.22 μm filter membrane to obtain the metabolite of the strain to be tested. Meanwhile, inactivating the acetobacter pasteurellosis WY202202 thalli to be detected at 100 ℃ for 15min to prepare dead thalli to be detected; the metabolites of Acetobacter pasteurii WY202202 and the E.coli OP50 were each prepared according to a ratio of 1:1, re-suspending dead bacteria of Acetobacter pasteurisis WY202202 and sterile water, and live bacteria of Acetobacter pasteurisis WY202202 serving as food, and respectively feeding caenorhabditis elegans N2 by taking only escherichia coli OP50 as a control, and measuring the service life.
90L 1-stage caenorhabditis elegans N2 which are subjected to the same period as the steps are picked, respectively placed on NGM flat plates containing live bacteria, dead bacteria and metabolites thereof of Acetobacter pasteurii WY202202 and escherichia coli OP50, and placed in a biochemical incubator at 20 ℃ for culture. All the living nematodes are picked every 1 day and transferred to a fresh NGM plate coated with bacterial liquid, so that the newly hatched nematodes are prevented from affecting the experimental result. The experiment was continued until the nematodes were all dead and a nematode life curve was drawn.
5. Test results
As a result, as shown in FIG. 2, the average lives of the live bacteria, dead bacteria and metabolites thereof of Acetobacter pastoris WY202202 were significantly prolonged by 45.4%, 20.5% and 29.7% (p < 0.05), respectively, as compared with the average life span (10 d) and the maximum life span (20 d) of E.coli OP50 fed, which were prolonged by 10 days, 2 days and 5 days, respectively, indicating that the live bacteria, dead bacteria and metabolites thereof of Acetobacter pastoris WY202202 were able to significantly prolong nematode life.
EXAMPLE 3 Effect of WY202202 Strain on caenorhabditis elegans body length, motility and fertility
1. Test method
(1) To verify whether Acetobacter pasteurii WY202202 affects the body length of nematodes, nematodes cultured in synchronization to day 3 were picked, soaked in a hot water bath at 70℃for 30min to rigidify the nematode body and the worms were drawn onto a glass slide, and the body length of the nematodes was observed and measured with an optical microscope, and at least 10 nematodes were repeated for each set of experiments.
(2) Exercise capacity is one of the key indicators of health, and exercise condition and exercise capacity gradually deteriorate and weaken with aging of the body. The Acetobacter pasteurii WY202202 strain is adopted to culture caenorhabditis elegans, the culture method is the same as that of example 2, only E.coli OP50 is used as a control group, 20 nematodes are randomly picked up on a fresh NGM plate on the 3 rd day, after the nematodes freely move for 30 seconds, the head swinging times of the nematodes are observed by a stereomicroscope within 1min, and one head swinging is that the heads of the nematodes swing from one side to the other side.
(3) The selected anti-aging medicine or strain should take the precondition that the reproductive capacity of the organism is not damaged, and the reproductive capacity is one of the most critical indexes in health. Therefore, the influence of the Acetobacter pasteurella WY202202 on the reproductive capacity of the insects is verified by taking the number of offspring as an index. The caenorhabditis elegans were cultivated with the strain to be tested as described in example 2 until stage L4, and 1 nematode was randomly picked up and plated onto each of 5 parallel groups of NGM plates coated with Acetobacter pasteuris WY202202, E.coli OP 50. Transferring the oviposited adults to a new plate every 1d during the oviposition of the nematodes, continuously placing the old plate in a biochemical incubator at 20 ℃ for culturing until the eggs of the worms hatch, and counting the number of the larvae hatched by the nematodes on the plate until the oviposition of the nematodes is finished.
2. Experimental results
As a result, as shown in FIG. 3, the nematode body length of the group of Acetobacter pastoris WY202202 on day 3 was prolonged by 27.4% (FIG. 3A, p < 0.05), the head swing frequency was increased by 41.3% (FIG. 3B, p < 0.05), and the total egg yield was increased by 52.0% (FIG. 3C, p < 0.05) as compared with the control group of E.coli OP 50. The method shows that the feeding of Acetobacter pasteurii WY202202 can obviously increase the body length of nematodes, enhance the mobility and reproductive capacity of the nematodes, promote the growth and development of the nematodes and have good anti-aging performance.
EXAMPLE 4 Effect of WY202202 Strain on nematode fat content and mitochondrial Membrane potential
1. Test method
(1) Effect of Acetobacter pasteurii WY202202 on fat content in nematodes
Acetobacter pasteurii WY202202 is used for culturing caenorhabditis elegans, the culturing method is the same as that of example 2, the E.coli OP50 is fed as a control group, 20 nematodes are randomly picked up after the L4 period is continuously cultured for 3 days, and the nematode fat is measured by using an O-red oil staining method. The throat is closed by using M9 buffer solution for 3 times and 25mM levamisole hydrochloride for anesthesia, 200 mu L of 4% paraformaldehyde solution is added for standing for 15-20 min, then the solution is removed and frozen and thawed 3 times by liquid nitrogen, then the solution is dyed by 60% isopropanol red oil O, and after 4-5 h, the excess dye is washed by using M9 buffer solution and observed by using an optical microscope.
(2) Effect of Acetobacter pasteurii WY202202 on mitochondrial membrane potential in C.elegans
The Xylobacter caenorhabditis is cultivated by using Acetobacter pastoris WY202202, the cultivation method is the same as that of example 2, the E.coli OP50 is fed as a control group, 20 nematodes are randomly picked up after the L4 period is continuously cultivated for 3 days, the mitochondrial membrane potential is measured by using JC-1 dye, the nematodes and the now-prepared JC-1 dye solution are incubated for 2 hours in a dark place, the dye on the surfaces of the nematodes is washed out by using M9 buffer solution, red fluorescence (with the emission wavelength of about 590 nm) and green fluorescence (with the emission wavelength of 529 nm) are observed through a fluorescence microscope, a photographic record is carried out, and quantitative calculation is carried out on the fluorescent photograph by using Image J software.
2. Test results
As shown in FIG. 4, the red optical density of the intestinal fat of the nematodes fed by Acetobacter pastoris WY202202 is reduced by 45.7% compared with that of the control group of the Escherichia coli OP50 (FIG. 4A, p < 0.05), and the red-green fluorescence ratio of JC-1 after staining is increased by 56.2% (FIG. 4B, p < 0.05). The acetobacter pasteurii WY202202 is shown to significantly reduce fat deposition in nematodes; slowing down the decrease of mitochondrial membrane potential and reducing the oxidative damage of mitochondria.
Example 5 Effect of WY202202 Strain on antioxidant capacity of caenorhabditis elegans
1. Test method
(1) The Acetobacter pasteurii WY202202 strain is adopted to culture caenorhabditis elegans, the culture method is the same as that of example 2, only E.coli OP50 is used as a control group, the nematodes are collected after the L4 phase is continuously cultured for 3 days, and after the nematodes are washed for 3 times by M9 buffer solution, the nematodes are naturally settled. Placing the nematodes into a homogenizer, grinding on ice, centrifuging at 4 ℃ and 4 000r/min to obtain supernatant, namely protein homogenate. Malondialdehyde (MDA) content, reactive Oxygen Species (ROS) level, superoxide dismutase (SOD) enzyme activity, glutathione (GSH) content and Catalase (CAT) enzyme activity were measured according to the kit instructions.
(2) And (3) measuring the content of malondialdehyde: according to the reagent kit, TBA storage liquid (light-shielding storage) and MDA detection working liquid are prepared, a blank group (0.1mL PBS+0.2mLMDA detection working liquid) and a sample group (0.1 mL sample and 0.2mL MDA detection working liquid) are heated for 15min in a boiling water bath, 200 mu L of supernatant is taken after cooling to room temperature and added into a 96-well plate, and then absorbance is measured at 532nm by an enzyme-labeled instrument.
(3) Reactive oxygen level determination: mu.L of the protein homogenate and 50 mu.L of the now prepared 100mmol/L H-DCF-DA solution are added into a 96-well blackboard (the final concentration is 50 mmol/L) and treated for 3 hours, and the in-vivo active oxygen content is measured by a fluorescence microplate reader at an excitation wavelength of 485nm and an emission wavelength of 535 nm.
(4) Superoxide dismutase activity assay: preparing SOD detection buffer solution, WST-8/enzyme working solution and reaction starting solution, respectively preparing a sample group and a blank group according to the sample adding amount of the kit, incubating at 37 ℃ for 30min, and measuring absorbance at 450 nm.
(5) Glutathione content determination: after GSH stock solution, DTNB stock solution and NADPH stock solution were prepared according to the kit, the control group and the sample group were applied with samples, and absorbance was measured at 412nm immediately with an enzyme-labeled instrument.
(6) Measurement of catalase Activity: a catalase detection buffer, a 250mM hydrogen peroxide solution, and a catalase reaction termination solution were prepared according to the kit instructions, and the control group and the sample group were subjected to sample addition, and after incubation at 25℃for at least 15 minutes, absorbance was measured at 520 nm.
2. Test results
As shown in fig. 5, compared with the control group of escherichia coli OP50, the MDA content in the nematode fed by the acetobacter pastoris WY202202 is obviously reduced by 41.1 percent (fig. 5a, p < 0.05), the ROS level is reduced by 21.0 percent (fig. 5b, p < 0.05), the SOD activity is obviously improved by 67.6 percent (fig. 5c, p < 0.05), the GSH content is obviously improved by 65.4 percent (fig. 5d, p < 0.05), and the CAT activity is obviously improved by 87.8 percent (fig. 5e, p < 0.05), so that the acetobacter pastoris WY202202 can improve the antioxidant capacity of the nematode body by increasing the SOD enzyme activity, the CAT enzyme activity and the GSH content and reducing the MDA content and the ROS level in the body.
In conclusion, the Acetobacter pasteurella (Acetobacter pasteurianus) WY202202 strain provided by the invention can prolong the service life and the length of nematodes, increase forced power and reproductive capacity, reduce fat accumulation, reduce mitochondrial oxidative damage, improve SOD, GSH, CAT and other yields, reduce in-vivo oxygen free radical synthesis, and have the effects of resisting oxidation, reducing fat and resisting aging.
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 strain of acetobacter pasteurism (Acetobacter pasteurianus) WY202202, wherein the strain was deposited at the cantonese collection of microbiological strains under accession number GDMCC No at month 08 of 2022: 62536.
2. Use of the WY202202 strain or its bacterial liquid according to claim 1 to promote animal growth.
3. The use of WY202202 strain or its bacterial liquid in the preparation of a product for prolonging life of animals and promoting growth and development of animals according to claim 1.
4. The application of WY202202 strain or bacterial liquid thereof in preparing antioxidant, fat-reducing and anti-aging products according to claim 1.
5. The use according to claim 4, wherein the antioxidation means increases the antioxidation capacity of the body, reduces the accumulation of oxygen radicals in the body, and slows down oxidative damage of the body; the fat reduction means reduction of nematode fat accumulation; the anti-aging means increasing the length, motility and fertility of nematodes and reducing mitochondrial damage.
6. An antioxidant, lipid-reducing and anti-aging product comprising the Acetobacter pastoris (Acetobacter pasteurianus) WY202202 strain or a bacterial liquid thereof according to claim 1.
7. The product according to claim 6, wherein the Acetobacter pasteurisum (Acetobacter pasteurianus) WY202202 strain has a bacterial liquid concentration of 8.4X10 7~ 3.2×1011 CFU/mL.
8. The antioxidant, fat reducing and/or anti-aging product according to any one of claims 6 to 7, wherein said product is a health food, a pharmaceutical product, a feed or a feed additive.
9. A method for promoting growth and development of animals, which comprises feeding animals with the acetobacter pasteurii (Acetobacter pasteurianus) WY202202 strain or a bacterial solution thereof according to claim 1.
10. The use according to claim 2 or 3 or the method according to claim 9, wherein the animal is a nematode.
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