CN113980841A - Microbial agent for promoting fat deposition and separation method and application thereof - Google Patents
Microbial agent for promoting fat deposition and separation method and application thereof Download PDFInfo
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- CN113980841A CN113980841A CN202111222820.1A CN202111222820A CN113980841A CN 113980841 A CN113980841 A CN 113980841A CN 202111222820 A CN202111222820 A CN 202111222820A CN 113980841 A CN113980841 A CN 113980841A
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- 230000001737 promoting effect Effects 0.000 title claims abstract description 31
- 230000000813 microbial effect Effects 0.000 title claims abstract description 30
- 238000000926 separation method Methods 0.000 title abstract description 7
- 238000000151 deposition Methods 0.000 title description 24
- 241001468157 Lactobacillus johnsonii Species 0.000 claims abstract description 29
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 29
- 241000699670 Mus sp. Species 0.000 claims abstract description 18
- 210000003608 fece Anatomy 0.000 claims abstract description 17
- 239000010871 livestock manure Substances 0.000 claims abstract description 17
- 238000003304 gavage Methods 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 8
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- 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
- A01K67/02—Breeding vertebrates
-
- 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
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention belongs to the technical field of microbial agents, and discloses a microbial agent for promoting fat deposition, a separation method and application thereof, wherein the microbial agent for promoting fat deposition is lactobacillus johnsonii (the preservation number is CCTCC M20211241) separated from Ningxiang pig manure; the method for verifying the microbial agent for promoting fat deposition comprises the following steps: ICR male mice with a weight of 36.5g and similar health conditions were selected and divided into 3 groups: control group, high-fat group and high-fat + lactobacillus johnsonii gavage group, 10 mice per group; the lactobacillus johnsonii gavage group was 9 a.m. daily: 00, the stomach filling amount is 0.3ml per tube, and the bacterial liquid concentration is 1 multiplied by 10^9 CFU/ml; the mouse room adopts a management method of 12h illumination and 12h dark circulation, has proper temperature and humidity, and can drink water and eat food freely. Experimental results show that the Ningxiang pig manure source lactobacillus johnsonii provided by the invention can promote fat deposition in organisms.
Description
Technical Field
The invention belongs to the technical field of microbial agents, and particularly relates to a microbial agent for promoting fat deposition and a separation method and application thereof.
Background
At present, China is a big country for pork production and consumption, and live pig breeding has an important basic strategic position in national economic development. With the development of 40 years of innovation, the living standard of people is improved, the demand of the market for pork is continuously increased, and higher requirements on the quality of the pork are also provided. And intramuscular fat is an important influence factor affecting meat quality and taste. In recent years, commercial pigs mostly pursue slaughtering performance and lean meat percentage, the lean meat percentage is as high as more than 60%, the intramuscular fat content is less than 2.5%, and the pork quality and the mouthfeel are greatly influenced. In addition, the role of the intestinal microorganisms in lipid metabolism is more and more concerned, and researches show that the intestinal microorganisms participate in pork quality and lipid metabolism and can be used as potential targets for regulating and controlling the pork lipid metabolism and the pork quality. However, no microbial agent for promoting fat deposition is reported in the prior art. Therefore, a new microbial agent for promoting fat deposition is needed to make up for the blank of the prior art.
Through the above analysis, the problems and defects of the prior art are as follows: at present, the research on promoting the fat deposition of animals is less, and no report on the microbial agent for promoting the fat deposition is found in the prior art.
The difficulty in solving the above problems and defects is: the microbial inoculum is screened from the intestine of Ningxiang pigs by using a 16S rDNA sequencing technology, is subjected to correlation analysis with serum lipid, and is screened for differential strains to carry out experiments. The experimental difficulty lies in the mastering and data analysis of the 16S rDNA sequencing technology, Pearson correlation analysis and other technologies.
The significance of solving the problems and the defects is as follows: the microbial inoculum can improve fat content of organism and lipid content in blood serum. The result can be used for culturing lean type pig, and can promote fat deposition of lean type pig, and improve meat quality and taste.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a microbial agent for promoting fat deposition and a separation method thereof.
The invention is realized by the following steps that the microbial agent for promoting fat deposition is lactobacillus johnsonii separated from Ningxiang pig manure, and the preservation unit is as follows: wuhan university strain preservation center (CCTCC), preservation date: 2021, 9, 29 days, accession no: CCTCC M20211241. The dosage is 1 × 109CFU/ml。
Another object of the present invention is to provide a method for separating the microbial agent for promoting fat deposition. Taking 0.1g of healthy Ningxiang pig manure, dissolving the pig manure with 5mL of sterile PBS, uniformly mixing the pig manure with the PBS by oscillation, and diluting the pig manure to 10 degrees in a gradient manner-7Draw 100. mu.L gradient at 10-2～10-7The diluted solution is coated on an MRS plate added with 0.5 percent of L-cysteine and is placed in a constant temperature incubator with 5 percent of carbon dioxide and 37 ℃ for culturing for 48 hours. Selecting a plate with an appropriate dilution gradient, picking single colonies with different forms, and carrying out colony PCR identification by using universal primers for amplifying the 16S rDNA gene of the bacteria, namely 27-FAGAGTTTGATCCTGGCTCAG and 1492R-GGTTACCTTGTTACGACTT.
The invention also aims to provide application of the microbial agent for promoting fat deposition in preparation of a composition for promoting fat deposition.
Another object of the present invention is to provide an animal model construction method for verifying the efficacy of the microbial agent for promoting fat deposition, the animal model construction method comprising:
(1) 30 ICR male mice with similar body weight and health condition are selected and divided into 3 groups: control group, high-fat group and high-fat + lactobacillus johnsonii gavage group, 10 mice per group;
(2) the lactobacillus johnsonii gavage group was 9 a.m. daily: 00, the stomach filling amount is 0.3ml per tube, and the bacterial liquid concentration is 1 multiplied by 10^9 CFU/ml;
(3) the mouse room adopts a management method of 12h illumination and 12h dark circulation, has proper temperature and humidity, and can drink water and eat food freely.
Further, in the step (1), the weight of the ICR male mouse is 36.5 +/-0.5 g.
By combining all the technical schemes, the invention has the advantages and positive effects that: the microbial agent for promoting fat deposition provided by the invention is lactobacillus johnsonii, and the lactobacillus johnsonii can promote the deposition of fat in organisms to a certain extent. The experimental result shows that the lactobacillus johnsonii does not significantly increase the body weight of the mice, but the HFD + Lj group significantly increases the subcutaneous fat content of the mice (P < 0.01), compared with the control group, the HFD + Lj group significantly increases the epididymal fat of ICR mice (P < 0.01), and the HFD + Lj group also has the tendency of increasing abdominal fat, perirenal fat and total fat. Therefore, the invention continues to measure the lipid content in serum and further explores the effect of lactobacillus johnsonii on serum lipids. The Lactobacillus johnsonii group significantly increased the serum heavy bile acid (P < 0.001), cholesterol (P < 0.001), high density lipoprotein (P < 0.01) and low density lipoprotein ((P < 0.05), but significantly decreased the triglyceride ((P < 0.01) and had a tendency to increase the serum glucose level, as compared to the control group.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method for verifying a microbial agent for promoting fat deposition provided by an embodiment of the invention.
FIG. 2 is a schematic diagram showing the result of Lactobacillus johnsonii promoting fat deposition in mice according to the present invention.
FIG. 3 is a graph showing the results of the enhancement of serum lipid levels by Lactobacillus johnsonii according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a microbial agent for promoting fat deposition and a separation method and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
The invention provides a microbial agent for promoting fat deposition, which is lactobacillus johnsonii separated from Ningxiang pig manure, and the microbial agent has the following preservation number: CCTCC M20211241.
The dosage of the Lactobacillus johnsonii is 1 × 109CFU/ml。
As shown in fig. 1, the method for verifying the microbial agent for promoting fat deposition provided by the embodiment of the invention comprises the following steps:
s101, selecting 30 ICR male mice with the weight of 36.5 +/-0.5 g and similar health conditions, and dividing the ICR male mice into 3 groups: control group Cont, high-fat group HFD and high-fat + lactobacillus johnsonii gavage group HFD + Lj, 10 mice per group;
s102, lactobacillus johnsonii gavage group was 9: 00, the stomach filling amount is 0.3ml per tube, and the bacterial liquid concentration is 1 multiplied by 10^9 CFU/ml;
and S103, adopting a management method of 12h illumination and 12h dark circulation in a mouse room, wherein the temperature and the humidity are proper, and water drinking and food intake are free.
The invention also provides a separation method of the microbial agent for promoting fat deposition. Taking 0.1g of healthy Ningxiang pig manure, dissolving the pig manure with 5mL of sterile PBS, uniformly mixing the pig manure with the PBS by oscillation, and diluting the pig manure to 10 degrees in a gradient manner-7Draw 100. mu.L gradient at 10-2～10-7The diluted solution is coated on an MRS plate added with 0.5 percent of L-cysteine and is placed in a constant temperature incubator with 5 percent of carbon dioxide and 37 ℃ for culturing for 48 hours. Selecting a plate with proper dilution gradient, picking single colonies with different forms, and amplifying the 16S rDNA gene of the bacteria by using a universal primer of 27-FAGAGTTTGATCCTGGCTCAG and 1492R-GGTTACCTTGTTACGACTT were used for colony PCR identification.
The present invention will be further described with reference to the following examples.
The invention discloses a microbial agent, namely lactobacillus johnsonii, capable of promoting the deposition of integrated fat.
1. Materials and methods
In the experiment, 30 ICR male mice with similar body weights (36.5 +/-0.5 g) and health conditions are selected and divided into 3 groups: control group (Cont), high-fat group (HFD and high-fat + Lactobacillus johnsonii gavage group (HFD + Lj), 10 mice per group, Lactobacillus johnsonii gavage group was performed at 9: 00 am every day, gavage amount was 0.3 ml/mouse, bacterial liquid concentration was 1 x 10^9CFU/ml, rat room was managed by 12h light and 12h dark cycle, temperature and humidity were appropriate, and water and food were freely drunk.
2. Results
As can be seen from FIG. 2, Lactobacillus johnsonii did not significantly increase the body weight of mice, but the HFD + Lj group significantly increased the subcutaneous fat content of mice (P < 0.01), compared to the control group, the HFD + Lj group significantly increased the epididymal fat of ICR mice (P < 0.01), and the HFD + Lj group also had a tendency to increase abdominal fat, perirenal fat and total fat. Therefore, the invention continues to measure the content of heavy serum lipids and further explore the influence of lactobacillus johnsonii on serum lipids.
As can be seen from FIG. 3, the Lactobacillus johnsonii group significantly increased the serum heavy bile acids (P < 0.001), cholesterol (P < 0.001), high density lipoproteins (P < 0.01) and low density lipoproteins ((P < 0.05), but significantly decreased the triglyceride content ((P < 0.01) and had a tendency to increase the serum glucose content, as compared to the control group.
3. Conclusion
Lactobacillus johnsonii can promote the deposition of fat in the body to some extent.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. The microbial agent for promoting fat deposition is lactobacillus johnsonii separated from Ningxiang pig manure, and the preservation number is as follows: CCTCC M20211241.
2. The microbial agent for promoting fat deposition according to claim 1, wherein the lactobacillus johnsonii is used in a dose of 1 x 109CFU/ml。
3. Use of the microbial agent for promoting fat deposition according to claim 1 in the preparation of a composition for promoting fat deposition.
4. An animal model construction method for verifying the efficacy of the microbial agent for promoting fat deposition according to claim 1, wherein the animal model construction method comprises:
(1) 30 ICR male mice with similar body weight and health condition are selected and divided into 3 groups: control group, high-fat group and high-fat + lactobacillus johnsonii gavage group, 10 mice per group;
(2) the lactobacillus johnsonii gavage group was 9 a.m. daily: 00, the stomach filling amount is 0.3ml per tube, and the bacterial liquid concentration is 1 multiplied by 10^9 CFU/ml;
(3) the mouse room adopts a management method of 12h illumination and 12h dark circulation, has proper temperature and humidity, and can drink water and eat food freely.
5. The method for constructing animal model according to claim 4, wherein the weight of said ICR male mouse in step (1) is 36.5 g.
6. The method for separating a microbial agent for promoting fat deposition according to claim 1, wherein the method for separating a microbial agent for promoting fat deposition comprises:
taking 0.1g of healthy Ningxiang pig manure, dissolving the pig manure with 5mL of sterile PBS, uniformly mixing the pig manure with the PBS by oscillation, and diluting the pig manure to 10 degrees in a gradient manner-7Draw 100. mu.L gradient at 10-2～10-7The diluted solution is coated on an MRS plate added with 0.5 percent of L-cysteine and is placed in a 5 percent carbon dioxide constant temperature incubator at 37 ℃ for culturing for 48 hours; selecting a plate with an appropriate dilution gradient, picking single colonies with different forms, and carrying out colony PCR identification by using universal primers for amplifying the 16S rDNA gene of the bacteria, namely 27-FAGAGTTTGATCCTGGCTCAG and 1492R-GGTTACCTTGTTACGACTT.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911335A (en) * | 2014-04-18 | 2014-07-09 | 四川农业大学 | Lactobacillus johnsonii BS15 and application thereof in preventing and treating non-alcoholic fatty liver |
| CN111254089A (en) * | 2019-12-30 | 2020-06-09 | 杭州娃哈哈科技有限公司 | Lactobacillus plantarum with weight losing function and application thereof |
| CN113234643A (en) * | 2021-06-30 | 2021-08-10 | 江南大学 | Lactobacillus johnsonii capable of relieving ETEC (enterotoxigenic) diarrhea and application thereof |
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- 2021-10-20 CN CN202111222820.1A patent/CN113980841A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911335A (en) * | 2014-04-18 | 2014-07-09 | 四川农业大学 | Lactobacillus johnsonii BS15 and application thereof in preventing and treating non-alcoholic fatty liver |
| CN111254089A (en) * | 2019-12-30 | 2020-06-09 | 杭州娃哈哈科技有限公司 | Lactobacillus plantarum with weight losing function and application thereof |
| CN113234643A (en) * | 2021-06-30 | 2021-08-10 | 江南大学 | Lactobacillus johnsonii capable of relieving ETEC (enterotoxigenic) diarrhea and application thereof |
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