CN114934089B - Biological agent for lean pork pig breeding - Google Patents
Biological agent for lean pork pig breeding Download PDFInfo
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- CN114934089B CN114934089B CN202210636477.3A CN202210636477A CN114934089B CN 114934089 B CN114934089 B CN 114934089B CN 202210636477 A CN202210636477 A CN 202210636477A CN 114934089 B CN114934089 B CN 114934089B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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Abstract
The invention provides a biological agent for lean pork pig breeding, and belongs to the technical field of pig breeding. The small green leafhopper oligopeptide is extracted from small green leafhoppers, and can remarkably inhibit lipid drop formation of pig precursor fat cells and expression of adipogenic differentiation related proteins PPARgamma and C/EBP alpha, so that the small green leafhopper oligopeptide can be used for breeding lean pigs to screen lean pigs with less fat deposition.
Description
The scheme is a divisional application, and the name of the original application is: an extract for inhibiting the differentiation of porcine precursor adipocytes, which is prepared by the following application days: 2020-10-15, the application number of the original application is: CN 202011100432.1.
Technical Field
The invention belongs to the technical field of pig breeding, and particularly relates to a biological agent for lean pig breeding.
Background
Along with the improvement of the living standard and the consumption level of people, the requirements on the quality of meat are also higher and higher, and the pork consumption is the first place in the meat consumption of the world as the country with the largest production and consumption of meat. The meat quality is an extremely important economic index in pork production, and the differentiation of fat cells and the deposition of lipid have important regulation and control effects on the meat quality of pork, and the inhibition of the differentiation of fat precursor fat cells to reduce the generation of subcutaneous fat has important value for improving the lean meat percentage of pork. Second, subcutaneous fat deposition is also an important factor affecting pig growth and meat quality, and is also of great concern in lean pig breeding. Therefore, finding a substance capable of effectively inhibiting the differentiation of pig precursor adipocytes has important research value for improving lean meat percentage of pork and pig breeding.
Insects are biological resources containing a large amount of high-quality proteins, insects are rich in various amino acids, peptides and proteins, and the existing researches prove that the insect peptide has various functions, so that it is feasible to search the insect for oligopeptides or polypeptides which can be used for pig precursor fat cells. Meanwhile, periostracum Cicadae and polypeptides thereof in the same cicada subgenera have been used for treating diseases, but little research on the same protein-rich leafhoppers is involved, so the invention selects the leafhoppers to study.
Disclosure of Invention
The invention aims to provide an extract capable of inhibiting adipogenic differentiation of porcine precursor adipocytes
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides an application of a small leafhopper extract in inhibiting adipogenic differentiation of pig precursor fat cells, wherein the small leafhopper extract is small leafhopper oligopeptide.
Preferably, the preparation method of the small leafhopper oligopeptide comprises the following steps:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain 0.02 times of the total amount, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
In addition, the invention provides application of the small green leafhopper extract in preparing an inhibitor for inhibiting adipogenic differentiation of pig precursor fat cells, wherein the small green leafhopper extract is small green leafhopper oligopeptide.
Preferably, the preparation method of the small leafhopper oligopeptide comprises the following steps:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain 0.02 times of the total amount, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
Preferably, the application comprises application of the small leafhoppers in preparing the genes PPARgamma and C/EBPalpha for inhibiting fat transformation.
In addition, the invention provides an inhibitor for inhibiting adipogenic differentiation of porcine precursor adipocytes, wherein the inhibitor is a green leafhopper oligopeptide.
Preferably, the preparation method of the small leafhopper oligopeptide comprises the following steps:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain 0.02 times of the total amount, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
In addition, the invention provides application of the small green leafhopper oligopeptide in lean pig breeding, and the preparation method of the small green leafhopper oligopeptide comprises the following steps:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain 0.02 times of the total amount, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
The invention has the beneficial effects that
The invention prepares the small green leafhopper oligopeptide from the small green leafhopper so as to recycle the insect small green leafhopper.
Secondly, the small green leafhopper oligopeptide prepared by the invention can obviously inhibit lipid drop formation of pig precursor adipocytes and can obviously inhibit expression levels of adipogenic differentiation related proteins PPARgamma and CEBP alpha, so that the small green leafhopper oligopeptide can be used for preparing inhibitors or medicines for inhibiting adipogenic differentiation of pig precursor adipocytes. Meanwhile, the method can be used for breeding lean pigs to screen lean pigs with less fat deposition.
Drawings
FIG. 1 control, 0ug/ml of the green leafhopper oligopeptide group, 150ug/ml of the green leafhopper oligopeptide group, oil red O staining results.
FIG. 2 control group, 0ug/ml of the small leafhopper oligopeptide group, 150ug/ml of the small leafhopper oligopeptide group, and the results of expression of the PPARgamma and CEBP alpha proteins of the fat transformation related proteins.
Detailed Description
In order to clearly illustrate the technical characteristics of the present solution, the present solution is described below by means of specific embodiments.
Example 1
(1) Cleaning the leafhoppers, putting the leafhoppers into a dryer for drying, and crushing the leafhoppers into powder by using a crusher;
(2) Adding 5 times of water, and fully and uniformly stirring to obtain a uniform slurry of the leafhopper;
(3) Inoculating 5% by weight of bacillus subtilis into the homogenate, fermenting for 48 hours at the pH of 7 and the temperature of 30 ℃;
(4) Sterilizing at 121deg.C under high temperature and pressure of 103.4kPa for 30min, centrifuging to obtain fermentation supernatant,
(5) Adding papain 0.02 times of the total amount, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain leafhopper peptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain a small leafhopper oligopeptide liquid;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
Example 2
Preparation of porcine precursor adipocytes
(1) Subcutaneous adipose tissue was collected from the back of 7-day-old piglets, washed 3 times with PBS containing the double antibody, and sheared into about 1mm using scissors 3 Is a block of tissue;
(2) Transferring the cut adipose tissues into a centrifuge tube, adding type I collagenase, digesting in a constant-temperature water bath at 37 ℃ for 90min, and adding a complete culture medium to stop digestion;
(3) Filtering the digestive juice by using a cell sieve of 80 meshes and a cell sieve of 200 meshes respectively, and collecting filtrate into a new sterile centrifuge tube;
(4) Placing the centrifuge tube in a centrifuge, centrifuging at 1500r/min for 10min, discarding the supernatant, adding erythrocyte lysate to the precipitate to suspend the cell precipitate, and centrifuging at 1500r/min for 5min;
(5) Removing the supernatant, adding PBS to wash the cells, and centrifuging at 1500r/min for 5min;
(6) Discarding the supernatant, adding DMEM complete medium to resuspend cells, inoculating into a cell culture dish at 37deg.C with 5% CO 2 Culturing in a cell incubator to obtain the pig precursor fat cells.
Example 3
(1) The green leafhopper oligopeptide prepared in example 1 was dissolved in DMEM to prepare 150ug/ml of green leafhopper oligopeptide solution, and the solution was subjected to sterilization filtration using a 0.22um filter membrane.
(2) Inoculating pig precursor fat cells into a 96-well plate, wherein a control group is not added with the small leafhopper oligopeptide, and an experimental group is added with 150ug/ml small leafhopper oligopeptide liquid;
(3) After 48h of incubation, OD values of the two groups of cells were determined with reference to CCK-8 instructions.
The experimental results are as follows, the OD value of the control group is 0.841+/-0.029,150, the OD value of the small leafhopper oligopeptide group is 0.875+/-0.025, and the P value is 0.426, and the difference is not statistically significant, so that the small leafhopper oligopeptide has no obvious effect on proliferation of pig precursor fat cells.
Example 4
Oil red O dyeing experiment
(1) Experimental grouping: the control group was not treated; adding 0ug/ml of the small leafhopper oligopeptide group into a lipid formation induction medium I and a lipid formation induction medium II to perform lipid formation induction, but not adding the small leafhopper oligopeptide; the 150ug/ml small leafhopper oligopeptides are used for adipogenesis induction by using an adipogenesis induction culture medium I and an adipogenesis induction culture medium II which are dissolved with 150ug/ml small leafhopper oligopeptides;
(2) Inoculating pig precursor fat cells into a cell culture plate, performing adipogenic induction culture according to experimental groups, firstly using adipogenic induction culture medium I to induce for 3 days, then using adipogenic induction culture medium II to induce for 2 days, and then changing to a complete culture medium;
(3) After adipogenic induction for 14 days, the culture medium is discarded, PBS is added for cleaning the cells for 3 times, 4% paraformaldehyde is added for cell fixation at room temperature after the excess PBS is sucked dry;
(4) After fixing for 20min, adding PBS to wash the cells for 3 times, adding oil red O staining solution, and incubating for 10min at room temperature;
(5) Cells were rinsed with PBS, observed under a microscope and photographed.
The experimental results are shown in fig. 1, and it can be seen from the graph that the quantity of the small leafhopper oligopeptides is obviously reduced compared with that of the small leafhopper oligopeptides with the ratio of 0ug/ml, so that the small leafhopper oligopeptides can inhibit the formation of lipid droplets of pig precursor fat cells, namely can inhibit the conversion of pig precursor fat cells into fat cells.
Example 5
(1) Cell grouping and lipogenic transformation were as in example 4;
(2) After the culture is finished, removing the culture medium, adding 1ml of PBS to wash the rest culture medium, discarding the PBS, adding 100ul of cell lysate, scraping off cells by using a cell scraper, and collecting the lysis mixture into an EP tube;
(3) Ultrasonically crushing cells, placing in a centrifuge, and centrifuging at 4deg.C and 12000rpm/min for 15min;
(4) After centrifugation, transferring the supernatant to a new centrifuge tube, measuring the protein concentration by using a BCA method, and adding a 5 XSDS loading buffer to adjust the protein sample concentration to 2ug/ml;
(5) Preparing 12% of separation gel and 5% of concentrated gel, adding 10ul of protein sample and protein marker, keeping constant pressure at 90V, changing into constant pressure at 120V after the separation gel, and stopping electrophoresis when bromophenol blue runs out of the separation gel;
(6) Taking out the gel, placing the film transfer clamp in an electric transfer tank according to a film transfer clamp according to a sandwich model, and electrically transferring the gel for 1.5 hours at room temperature and constant current of 250 mA;
(7) After the electric conversion is finished, taking out the PVDF film, putting the PVDF film into 5% skimmed milk powder, and sealing the PVDF film for 1h at room temperature;
(8) Membrane cutting according to the protein size, incubating PPARgamma, CEBP alpha and beta-actin, and incubating overnight at 4 ℃;
(9) Absorbing the primary antibody, washing the membrane 3 times by using TBST (Tunnel boring machine) for 10min each time, and incubating the secondary antibody for 1h at room temperature;
(10) The secondary antibody was discarded, and the membrane was washed 3 times with TBST, followed by development.
The experimental results are shown in fig. 2, and it can be seen from the graph that the small leafhopper oligopeptide can be obviously reversed to be converted into PPARgamma and CEBP alpha with increased protein expression amount caused by lipid conversion. Therefore, the green leafhopper oligopeptide can effectively inhibit the transformation of precursor adipocytes to adipocytes.
Claims (3)
1. The application of the small leafhopper oligopeptide in lean pig breeding is characterized in that the preparation method of the small leafhopper oligopeptide is as follows:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain in an amount of 0.02 times, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) Freeze-drying to obtain the finished product of the small leafhopper oligopeptide;
the green leafhopper oligopeptide can be used for screening lean pigs with less fat deposition.
2. An inhibitor for inhibiting adipogenic differentiation of porcine precursor adipocytes, wherein the inhibitor is a green leafhopper oligopeptide;
the preparation method of the green leafhopper oligopeptide comprises the following steps:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain in an amount of 0.02 times, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
3. The application of the small green leafhopper oligopeptide in preparing an inhibitor for inhibiting the expression of adipogenic differentiation related proteins in porcine precursor fat cells is characterized in that the adipogenic differentiation related proteins are PPARgamma and C/EBPalpha;
the preparation method of the green leafhopper oligopeptide comprises the following steps:
(1) Removing wings of leafhopper, drying, and pulverizing into powder;
(2) Adding 5 times of water to obtain a uniform slurry of the leafhoppers;
(3) Inoculating 5% by weight of bacillus subtilis for fermentation for 48 hours;
(4) Sterilizing at high temperature and high pressure, and centrifuging to obtain fermentation supernatant;
(5) Adding papain in an amount of 0.02 times, fermenting at pH8.5 and 55deg.C for 48 hr;
(6) Inactivating enzyme at 95deg.C for 10min, and centrifuging to obtain green leafhopper oligopeptide solution;
(7) Filtering by using an ultrafiltration membrane with the molecular weight cut-off of 1kDa to obtain the small leafhopper oligopeptide;
(8) And (5) freeze-drying to obtain the finished product of the small leafhopper oligopeptide.
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| CN105076676A (en) * | 2015-09-08 | 2015-11-25 | 南宁荣港生物科技有限公司 | Preparation method of polypeptide feed additive |
| KR20180060066A (en) * | 2016-11-28 | 2018-06-07 | 재단법인 진안홍삼연구소 | Composition for preventing production of lipid and improving lipid metabolism using active ingredient with the extract of red ginseng |
| CN109182382A (en) * | 2018-09-18 | 2019-01-11 | 嘉兴学院 | For inhibiting the substance of LGALS12 gene expression inhibiting the application in Adipocyte Differentiation |
| CN111041052A (en) * | 2019-12-30 | 2020-04-21 | 嘉兴学院 | Application of mir-206 in inhibiting triglyceride generation in pig fat cells and lean pig breeding |
| CN111454899A (en) * | 2020-04-30 | 2020-07-28 | 青岛思拓新源细胞医学有限公司 | Application of carrageenan in inhibiting mesenchymal stem cell lipogenesis transformation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105076676A (en) * | 2015-09-08 | 2015-11-25 | 南宁荣港生物科技有限公司 | Preparation method of polypeptide feed additive |
| KR20180060066A (en) * | 2016-11-28 | 2018-06-07 | 재단법인 진안홍삼연구소 | Composition for preventing production of lipid and improving lipid metabolism using active ingredient with the extract of red ginseng |
| CN109182382A (en) * | 2018-09-18 | 2019-01-11 | 嘉兴学院 | For inhibiting the substance of LGALS12 gene expression inhibiting the application in Adipocyte Differentiation |
| CN111041052A (en) * | 2019-12-30 | 2020-04-21 | 嘉兴学院 | Application of mir-206 in inhibiting triglyceride generation in pig fat cells and lean pig breeding |
| CN111454899A (en) * | 2020-04-30 | 2020-07-28 | 青岛思拓新源细胞医学有限公司 | Application of carrageenan in inhibiting mesenchymal stem cell lipogenesis transformation |
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