CN112210529A - Extract for inhibiting differentiation of pig precursor fat cells - Google Patents
Extract for inhibiting differentiation of pig precursor fat cells Download PDFInfo
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- CN112210529A CN112210529A CN202011100432.1A CN202011100432A CN112210529A CN 112210529 A CN112210529 A CN 112210529A CN 202011100432 A CN202011100432 A CN 202011100432A CN 112210529 A CN112210529 A CN 112210529A
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Abstract
The invention provides an extract for inhibiting differentiation of pig precursor adipocytes, belonging to the technical field of pig breeding. The lesser leafhopper oligopeptide is extracted from the lesser leafhopper, can obviously inhibit lipid drop formation of the pig precursor fat cell and expression of adipogenic differentiation related proteins PPAR gamma and C/EBP alpha, and can be used for preparing an inhibitor or a medicament for inhibiting adipogenic differentiation of the pig precursor fat cell. Meanwhile, the method can be used for lean pig breeding to screen lean pigs with less fat deposition.
Description
Technical Field
The invention belongs to the technical field of pig breeding, and particularly relates to an extract for differentiating pig precursor fat cells.
Background
With the improvement of the living standard and consumption standard of people, the requirements on the meat quality are higher and higher, and as the world with the largest meat production and consumption, pork consumption accounts for the first place in meat consumption in China. The meat quality is an extremely important economic index in pork production, adipocyte differentiation and lipid deposition have important regulation and control functions on the meat quality of pork, and the inhibition of the differentiation of lipocyte precursor to reduce the generation of subcutaneous fat has important value on improving the lean meat percentage of pork. Secondly, subcutaneous fat deposition is also an important factor affecting pig growth and meat quality, and is also of great interest in lean pig breeding. Therefore, the search for substances capable of effectively inhibiting the differentiation of the pig precursor adipocytes has important research value for improving the lean meat percentage of pork and breeding pigs.
Insects are biological resources containing a large amount of high-quality protein, the insect bodies of the insects are rich in various amino acids, peptides and proteins, and the prior researches prove that insect peptides have various functions, so that the search of oligopeptides or polypeptides which can be used for porcine preadipocytes from the insects is feasible. Meanwhile, cicada slough of cicada and polypeptide thereof in the same suborder cicada are used for treating diseases, while lesser research on lesser green leafhoppers containing rich protein is involved, so that the green leafhoppers are selected to be researched.
Disclosure of Invention
The invention aims to provide an extract capable of inhibiting adipogenic differentiation of pig preadipocytes
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides an application of an lesser leafhopper extract in inhibiting adipogenic differentiation of pig precursor fat cells, wherein the lesser leafhopper extract is a lesser leafhopper oligopeptide.
Preferably, the preparation method of the lesser leafhopper oligopeptide comprises the following steps:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
In addition, the invention provides application of the lesser leafhopper extract in preparing an inhibitor for inhibiting adipogenic differentiation of pig precursor fat cells, wherein the lesser leafhopper extract is lesser leafhopper oligopeptide.
Preferably, the preparation method of the lesser leafhopper oligopeptide comprises the following steps:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
Preferably, the application comprises the application of the lesser leafhopper in preparing genes related to fat transformation inhibition, namely PPAR gamma and C/EBP alpha.
In addition, the invention provides an inhibitor for inhibiting adipogenic differentiation of pig precursor adipocytes, wherein the inhibitor is lesser leafhopper oligopeptide.
Preferably, the preparation method of the lesser leafhopper oligopeptide comprises the following steps:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
In addition, the invention provides application of the lesser leafhopper oligopeptide in lean pork breeding, and the preparation method of the lesser leafhopper oligopeptide comprises the following steps:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
The invention has the beneficial effects that
The lesser leafhopper oligopeptide is prepared from the lesser leafhopper, so that the pest lesser leafhopper is recycled.
Secondly, the lesser leafhopper oligopeptide prepared by the invention can obviously inhibit the lipid drop formation of the pig precursor fat cells and can obviously inhibit the expression quantity of adipogenic differentiation related proteins PPAR gamma and CEBP alpha protein, so that the lesser leafhopper oligopeptide can be used for preparing the inhibitor or the medicine for inhibiting the adipogenic differentiation of the pig precursor fat cells. Meanwhile, the method can be used for lean pig breeding to screen lean pigs with less fat deposition.
Drawings
FIG. 1 shows the results of oil red O staining in the control group, 0ug/ml Oligopeptide group, and 150ug/ml Oligopeptide group.
FIG. 2 shows the results of the protein expression of fat transformation related proteins PPAR gamma and CEBP alpha in a control group, a 0ug/ml lesser leafhopper oligopeptide group and a 150ug/ml lesser leafhopper oligopeptide group.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is described below by way of specific embodiments.
Example 1
(1) Removing wings of lesser leafhoppers, cleaning, putting the lesser leafhoppers in a dryer for drying, and crushing the lesser leafhoppers into powder by using a crusher;
(2) adding 5 times of water, and fully and uniformly stirring to obtain uniform serous fluid of lesser leafhoppers;
(3) inoculating 5 wt% of bacillus subtilis in the homogenate, carrying out fermentation for 48h at the temperature of 30 ℃ and at the pH of 7;
(4) sterilizing at 121 deg.C under high temperature and high pressure of 103.4kPa for 30min, centrifuging to obtain fermentation supernatant,
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain cicada peptide solution;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain oligopeptide solution of lesser leafhopper;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
Example 2
Preparation of porcine preadipocytes
(1) Collecting subcutaneous adipose tissue of back of 7-day-old piglet, washing with PBS containing double antibody for 3 times, and cutting the adipose tissue to about 1mm with scissors3The tissue mass of (a);
(2) transferring the cut adipose tissues into a centrifuge tube, adding type I collagen protease, digesting in a constant-temperature water bath kettle at 37 ℃, adding a complete culture medium after digesting for 90min, and stopping digesting;
(3) filtering the digestive juice by using 80-mesh and 200-mesh cell sieves respectively, and collecting filtrate to a new sterile centrifuge tube;
(4) placing the centrifugal tube in a centrifugal machine, centrifuging for 10min at 1500r/min, discarding supernatant, adding erythrocyte lysate into the precipitate to suspend cell precipitation, and centrifuging for 5min at 1500 r/min;
(5) discarding the supernatant, adding PBS to wash the cells, and centrifuging at 1500r/min for 5 min;
(6) discarding supernatant, adding DMEM to suspend cells completely, inoculating in cell culture dish, 37 deg.C, 5% CO2Culturing in a cell culture box to obtain the pig precursor fat cells.
Example 3
(1) The cicada oligopeptide prepared in example 1 was dissolved in DMEM to prepare a 150ug/ml cicada oligopeptide solution, and then sterilized and filtered using a 0.22um filter membrane.
(2) Inoculating the pig precursor fat cells into a 96-well plate, adding no lesser leafhopper oligopeptide into a control group, and adding 150ug/ml lesser leafhopper oligopeptide solution into an experimental group;
(3) after 48h of culture, OD values of both groups of cells were determined with reference to CCK-8 instructions.
The experimental results show that the control group with OD value of 0.841 +/-0.029,150 had OD value of 0.875 +/-0.025 and P value of 0.426, and the difference between the two values was not statistically significant, so that the lesser leafhopper oligopeptide had no significant effect on the proliferation of the pig precursor adipocytes.
Example 4
Oil red O staining experiment
(1) Grouping experiments: the control group was not treated; adding 0ug/ml of lesser leafhopper oligopeptide group into a adipogenic induction culture medium I and an adipogenic induction culture medium II for adipogenic induction, but not adding the lesser leafhopper oligopeptide; the 150ug/ml lesser leafhopper oligopeptide group is subjected to adipogenesis induction by using an adipogenesis induction culture medium I and an adipogenesis induction culture medium II dissolved with 150ug/ml lesser leafhopper oligopeptide;
(2) inoculating pig precursor fat cells into a cell culture plate, performing adipogenesis induction culture according to experimental groups, firstly inducing for 3 days by using an adipogenesis induction culture medium I, then inducing for 2 days by using an adipogenesis induction culture medium II, and then replacing with a complete culture medium;
(3) after adipogenic induction for 14 days, removing the culture medium, adding PBS to wash the cells for 3 times, sucking off redundant PBS, and adding 4% paraformaldehyde to fix the cells at room temperature;
(4) fixing for 20min, adding PBS to wash cells for 3 times, adding oil red O staining solution, and incubating at room temperature for 10 min;
(5) cells were rinsed with PBS, placed under a microscope for observation and photographed.
The experimental result is shown in fig. 1, and it can be seen from the figure that the number of lipid droplets is obviously reduced compared with 0ug/ml of lesser leafhopper oligopeptide group after the lesser leafhopper oligopeptide is added, which indicates that the lesser leafhopper oligopeptide can inhibit the formation of lipid droplets of the pig precursor fat cells, i.e. can inhibit the conversion of the pig precursor fat cells into fat cells.
Example 5
(1) Cell grouping and adipogenic transformation as in example 4;
(2) after the culture is finished, removing the culture medium, adding 1ml of PBS to clean the residual culture medium, removing the PBS, adding 100ul of cell lysis solution, scraping the cells by using a cell scraper, and collecting lysis mixed solution to an EP tube;
(3) ultrasonically breaking cells, placing in a centrifuge, centrifuging at 12000rpm/min at 4 deg.C for 15 min;
(4) after the centrifugation is finished, transferring the supernatant into a new centrifuge tube, measuring the protein concentration by using a BCA method, and adding a 5 xSDS loading buffer solution to adjust the protein sample concentration to be 2 ug/ml;
(5) preparing 12% separation gel and 5% concentration gel, adding 10ul of protein sample and protein marker, keeping constant voltage at 90V, changing to constant voltage at 120V after separation gel, and stopping electrophoresis when bromophenol blue runs out of the separation gel;
(6) taking out the gel, placing the gel in an electric rotating tank according to a sandwich model and a rotating film clamp, and electrically rotating at room temperature and constant current of 250mA for 1.5 h;
(7) after the electrotransformation is finished, taking out the PVDF membrane, putting the PVDF membrane into 5% skimmed milk powder, and sealing for 1h at room temperature;
(8) cutting the membrane according to the size of the protein, incubating PPAR gamma, CEBP alpha and beta-actin, and incubating overnight at 4 ℃;
(9) absorbing primary antibody, washing the membrane for 3 times by using TBST (tert-butyl ether-tert-butyl ether) for 10min each time, incubating the secondary antibody, and incubating at room temperature for 1 h;
(10) the secondary antibody was discarded, and the membrane was washed 3 times with TBST and developed.
The experimental results are shown in fig. 2, and it can be seen from the figure that the leafhopper oligopeptide can be significantly inverted into lipid, so that the protein expression levels of PPAR γ and CEBP α are increased. Therefore, the lesser leafhopper oligopeptide can effectively inhibit the transformation of precursor fat cells into fat cells.
Claims (8)
1. The application of the lesser leafhopper extract in inhibiting adipogenic differentiation of pig precursor fat cells is characterized in that the lesser leafhopper extract is lesser leafhopper oligopeptide.
2. The use of claim 1, wherein the cicada oligopeptide is prepared by the following method:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
3. The application of the lesser leafhopper extract in preparing the inhibitor for inhibiting the adipogenic differentiation of the pig precursor fat cells is characterized in that the lesser leafhopper extract is lesser leafhopper oligopeptide.
4. The use of claim 3, wherein the cicada oligopeptide is prepared by the following method:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
5. The use according to claim 4, wherein the use comprises the use of leafhopper for preparing genes PPAR γ and C/EBP α associated with the inhibition of fat transformation.
6. An inhibitor for inhibiting adipogenic differentiation of pig precursor adipocytes, which is an oligopeptide of lesser leafhopper.
7. The inhibitor according to claim 6, wherein the cicada oligopeptide is prepared by the following method:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
8. The application of the lesser leafhopper oligopeptide in the breeding of lean pork pigs is characterized in that the preparation method of the lesser leafhopper oligopeptide comprises the following steps:
(1) removing wings of lesser leafhopper, drying and pulverizing into powder;
(2) adding 5 times of water to obtain homogenate of lesser leafhopper;
(3) inoculating 5 wt% of bacillus subtilis and fermenting for 48 h;
(4) sterilizing at high temperature and high pressure, centrifuging and taking fermentation supernatant;
(5) adding papain in an amount which is 0.02 times that of the mixture, fermenting for 48 hours at the pH of 8.5 and the temperature of 55 ℃;
(6) inactivating enzyme at 95 deg.C for 10min, and centrifuging to obtain oligopeptide solution of lesser leafhopper;
(7) filtering with ultrafiltration membrane with cut-off molecular weight of 1KDa to obtain lesser leafhopper oligopeptide;
(8) and (5) freeze-drying to obtain the finished product of the lesser leafhopper oligopeptide.
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| JP2000083656A (en) * | 1998-09-09 | 2000-03-28 | Meiji Milk Prod Co Ltd | Precursor adipocyte strain |
| CN106069459A (en) * | 2016-06-07 | 2016-11-09 | 王燕 | The cultural method in Flos persicae labyrinth, southern high-temperature height Humid Area |
| 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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| CN105076676B (en) * | 2015-09-08 | 2018-11-20 | 南宁荣港生物科技有限公司 | The preparation method of additive of polypeptide feed |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000083656A (en) * | 1998-09-09 | 2000-03-28 | Meiji Milk Prod Co Ltd | Precursor adipocyte strain |
| CN106069459A (en) * | 2016-06-07 | 2016-11-09 | 王燕 | The cultural method in Flos persicae labyrinth, southern high-temperature height Humid Area |
| 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 |
| CN113088487A (en) * | 2020-04-30 | 2021-07-09 | 青岛思拓新源细胞医学有限公司 | Mesenchymal stem cell adipogenic transformation inhibitor |
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