阅读说明：本技术 一种大鲵低聚肽及其制备方法和应用 (Andrias davidianus oligopeptide and preparation method and application thereof ) 是由 肖慰南 贾绍辉 于 2021-06-24 设计创作，主要内容包括：本发明涉及大鲵肽技术领域,具体涉及一种大鲵低聚肽及其制备方法和应用,所述制备方法包括大鲵蛋白提取；大鲵蛋白酶解：将上述所得脱脂大鲵粉加水搅拌,然后加入复合酶进行酶解,酶解条件为：pH值7.2～7.5,酶解温度50～60℃,酶解时间5～7小时,酶解完成后升温至90～110℃随后使复合水解酶灭活；以及大鲵肽分离纯化：将所得酶解产物经过截留1000道尔顿分子量的可再生纤维素透析膜进行过滤,收集滤液,滤液经过疏水柱进一步分离得分离液,分离液冷冻干燥得大鲵低聚肽；所得的大鲵低聚肽,能够降低体重、调节血脂,显著性减少肥胖模型鼠食欲,减少血清中甘油三脂、总胆固醇及提高低密度脂蛋白蛋白的含量。(The invention relates to the technical field of giant salamander peptides, in particular to giant salamander oligopeptide and a preparation method and application thereof, wherein the preparation method comprises the steps of extracting giant salamander protein; proteolysis of giant salamanders: adding water into the obtained degreased giant salamander powder, stirring, and then adding a complex enzyme for enzymolysis, wherein the enzymolysis conditions are as follows: the pH value is 7.2-7.5, the enzymolysis temperature is 50-60 ℃, the enzymolysis time is 5-7 hours, and after the enzymolysis is finished, the temperature is raised to 90-110 ℃, and then the compound hydrolase is inactivated; and separating and purifying giant salamander peptide: filtering the obtained enzymolysis product by a reproducible cellulose dialysis membrane with the molecular weight of 1000 daltons, collecting filtrate, further separating the filtrate by a hydrophobic column to obtain a separation solution, and freeze-drying the separation solution to obtain the giant salamander oligopeptide; the obtained giant salamander oligopeptide can reduce weight, regulate blood fat, remarkably reduce appetite of obese model mice, reduce triglyceride and total cholesterol in serum and improve the content of low-density lipoprotein.)

1. A preparation method of giant salamander oligopeptide is characterized by comprising the following steps:

(1) giant salamander protein extraction

Crushing fresh giant salamander meat, homogenizing, freezing and centrifuging, performing freeze drying treatment, and then crushing the giant salamander meat to below 60 meshes to obtain giant salamander meat powder; leaching to remove fat, and volatilizing the solvent to obtain degreased giant salamander powder;

(2) giant salamander proteolysis

Adding water into the obtained degreased giant salamander powder, stirring, and then adding a complex enzyme for enzymolysis, wherein the enzymolysis conditions are as follows: the pH value is 7.2-7.5, the enzymolysis temperature is 50-60 ℃, the enzymolysis time is 5-7 hours, and after the enzymolysis is finished, the temperature is raised to 90-110 ℃, and then the compound hydrolase is inactivated;

(3) giant salamander peptide separation and purification

And (3) filtering the enzymolysis product obtained in the step (2) through a reproducible cellulose dialysis membrane with the molecular weight of 1000 daltons being intercepted, collecting filtrate, further separating the filtrate through a hydrophobic column to obtain separation liquid, and freeze-drying the separation liquid to obtain the giant salamander oligopeptide.

2. The method according to claim 1, wherein the step (1) of freeze-centrifugation is performed at a temperature of 3 to 5 ℃ and a rotation speed of 1000 to 1200r/min for 10 to 30 min.

3. The preparation method of claim 1, wherein the freeze-drying process in step (1) is freeze-drying at-20 ℃ to-45 ℃ and under 25Pa to 30Pa for 24h to 36 h.

4. The method according to claim 1, wherein the leaching agent used in the leaching in step (1) is food grade n-hexane.

5. The preparation method according to claim 1, wherein the complex enzyme in the step (2) is composed of alkaline protease, neutral protease and trypsin according to a mass ratio of 1: 2-2.5: 3-3.5.

6. The method according to claim 5, wherein the mass ratio of the alkaline protease, the neutral protease and the trypsin is 1:2: 3.5.

7. The giant salamander oligopeptide prepared according to the preparation method of any one of claims 1 to 6.

8. The application of the giant salamander oligopeptide according to claim 7 in preparation of fat-regulating and weight-reducing products.

Technical Field

The invention relates to the technical field of giant salamander peptides, and particularly relates to giant salamander oligopeptide and a preparation method and application thereof.

Background

Obesity refers to a condition of excess accumulation of body fat, especially triglycerides, due to a degree of significant overweight and an excessively thick fat layer. It is difficult to suppress obesity because excessive fat accumulation in the body due to excessive food intake or metabolic changes of the body causes excessive weight gain and causes pathological, physiological changes or latency of the body. The nutrition control and the exercise intervention are considered as the most effective weight-losing means, and a safe and effective weight-losing product with the weight-losing effect is lacked in the market at present.

Giant salamanders, also called giant salamanders, are amphibian animals with high nutritional value, are known as 'underwater ginseng', chicken, skin, viscera and the like of the giant salamanders contain rich protein, amino acid, fatty acid, trace elements and the like, and the giant salamander protein is deeply processed to obtain a series of giant salamander bioactive peptides which are beneficial to human life activities. At present, the giant salamander bioactive peptides mostly take the muscle, skin, internal organs, fat, blood and the like of the giant salamander as raw materials, and the preparation method mainly comprises acid hydrolysis, alkali hydrolysis and enzymolysis, but little attention is paid to the bioactive peptides with specific functions. In recent years, related reports disclose a preparation method and effects of giant salamander peptide, the giant salamander collagen peptide has the physiological activity functions of primary oxidation resistance, aging resistance, tumor resistance, blood pressure reduction, stomach mucosa repair, bone and ligament health care, hair care, alopecia prevention and the like, and has the special effects of repairing epithelial cells, promoting wound healing, protecting liver injury and enhancing immunity. At present, related researches are carried out to obtain giant salamander collagen peptide with the lipid-lowering function as a target, a large number of proteolysis methods of the giant salamander collagen peptide are tried, and the giant salamander collagen peptide with ideal lipid-regulating and weight-reducing effects is still difficult to obtain.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the giant salamander oligopeptide and the preparation method and the application thereof, the giant salamander oligopeptide with the molecular weight of less than 1000 daltons is obtained through enzymolysis, separation and purification steps, and the giant salamander oligopeptide has obvious lipid regulating and weight losing effects.

The invention provides a preparation method of giant salamander oligopeptide, which comprises the following steps:

(1) giant salamander protein extraction

Crushing fresh giant salamander meat, homogenizing, freezing and centrifuging, performing freeze drying treatment, and then crushing the giant salamander meat to below 60 meshes to obtain giant salamander meat powder; leaching to remove fat, and volatilizing the solvent to obtain degreased giant salamander powder;

(2) giant salamander proteolysis

Adding water into the obtained degreased giant salamander powder, stirring, and then adding a complex enzyme for enzymolysis, wherein the enzymolysis conditions are as follows: the pH value is 7.2-7.5, the enzymolysis temperature is 50-60 ℃, the enzymolysis time is 5-7 hours, and after the enzymolysis is finished, the temperature is raised to 90-110 ℃, and then the compound hydrolase is inactivated;

(3) giant salamander peptide separation and purification

And (3) filtering the enzymolysis product obtained in the step (2) through a reproducible cellulose dialysis membrane with the molecular weight of 1000 daltons being intercepted, collecting filtrate, further separating the filtrate through a hydrophobic column to obtain separation liquid, and freeze-drying the separation liquid to obtain the giant salamander oligopeptide.

Preferably, the freezing and centrifuging process in the step (1) is freezing and centrifuging for 10-30 min at the temperature of 3-5 ℃ and the rotating speed of 1000-1200 r/min.

Preferably, the freeze-drying process in the step (1) is to freeze-dry for 24-36 hours at the temperature of-20 ℃ to-45 ℃ and under the pressure of 25-30 Pa. The freeze drying can avoid the degradation and structural change of the protein in the drying process, and can maintain the activity of the protein to the maximum extent.

Preferably, the leaching agent used in the leaching process in the step (1) is food grade n-hexane.

Preferably, the mass volume ratio of the giant salamander powder to the water in the step (2) is 1: 3-15.

Preferably, the complex enzyme in the step (2) is composed of alkaline protease, neutral protease and trypsin according to the mass ratio of 1: 2-2.5: 3-3.5; more preferably, the mass ratio of the alkaline protease, the neutral protease and the trypsin is 1:2: 3.5. The probability that the tail end of the product obtained by enzymolysis of the compound enzyme according to the proportion is hydrophobic amino acid is high, so that the bioactivity of the giant salamander peptide is increased.

Preferably, the step (3) further comprises the steps of filling a chromatography column with the inner diameter of 5.5cm and the length of 20cm by using a sepharose hydrophobic chromatography medium, after the hydrophobic chromatography column is filled, balancing by using 2M NaCl buffer solution, wherein the balanced volume is 3 times of the column volume, after the balancing is finished, slowly adding the filtrate into the chromatography column, standing for 1h at room temperature, then performing sample elution by using a NaCl solution by using a gradient elution method, collecting eluent when the salt ion concentration is highest, desalting by using a desalting column, and freeze-drying the separation solution.

The invention also provides the giant salamander oligopeptide obtained by the preparation method.

The invention also provides an application of the giant salamander oligopeptide in preparing a lipid-regulating and weight-losing product, and the application specifically relates to the application of the giant salamander oligopeptide in reducing weight, regulating blood fat, remarkably reducing appetite of obese model mice, reducing triglyceride and total cholesterol in serum and improving the content of low-density lipoprotein.

The application also comprises the combination of the fat-regulating and weight-reducing product and aerobic exercise, in particular to the aerobic exercise while eating the fat-regulating and weight-reducing product.

The beneficial effect of the invention is that,

(1) the giant salamander oligopeptide finally obtained in the giant salamander peptide separation and purification process has the characteristic of small molecular weight, and the absorption efficiency is superior to that of protein and macromolecular peptide; in addition, the prepared giant salamander oligopeptide is hydrophobic small molecular peptide, so that the giant salamander oligopeptide has stronger biological activity;

(2) the giant salamander oligopeptide prepared by the preparation method provided by the invention can reduce the weight, regulate the blood fat, remarkably reduce the appetite of obese model mice, reduce triglyceride and total cholesterol in serum and improve the content of low-density lipoprotein.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a graph showing the body weight changes of experimental rats in a control group and a high fat model group according to the present invention;

FIG. 2 shows four blood lipid measurements before and after modeling of a control group and a high lipid model group according to the present invention;

in the figure, A is the four detection results of blood fat at week 1 and week 6 of the control group; b is four detection results of blood fat of the high fat model group at week 1 and week 6;

FIG. 3 is a graph comparing the results of the A-E group to the weight loss of obese rats provided by the present invention;

FIG. 4 is a graph comparing the four results of the effects of groups A to E on the blood fat of obese rats;

in the figure, I: TG content in serum; II: TC content in serum; III: HDL content in serum; IV: LDL content in serum;

FIG. 5 is a graph comparing the results of the A-E group provided by the present invention on the effect of the food intake of obese rats;

FIG. 6 is a graph comparing the results of the A-E groups provided by the present invention on the effect of cytokines on the regulation of energy metabolism and appetite in obese rats;

in the figure, I: irisin concentration, II: FGF15 concentration, III: leptin concentration, IV: the NPY concentration;

FIG. 7 is a graph comparing the results of the A-E groups provided by the present invention on the effect of cytokines on the regulation of energy metabolism and appetite in obese rats;

in the figure, I: western blot detection of energy metabolism-related proteins, II: quantifying PGC-1 alpha expression, III: quantification of p-AMPK expression, IV: quantifying the Irisin expression;

FIG. 8 is a graph comparing the results of the effects of groups A to E on the expression of proteins involved in regulating the energy metabolism and appetite of obese rats;

in the figure, I: western blot detection of appetite regulation-related proteins, II: quantification of NPY expression, III: POMC expression was quantified.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A group is a control group, B group is a high-fat model group, C group is a high-fat model exercise group, D group is a high-fat model giant salamander peptide group, and E group is a high-fat model giant salamander peptide combined exercise group;

p <0.05 indicates significant difference from the control group, and # p <0.05 indicates significant difference from the model group.

Example 1

A preparation method of giant salamander oligopeptide comprises the following steps:

(1) giant salamander protein extraction

Crushing fresh giant salamander meat by a crusher, homogenizing, freezing and centrifuging (the rotating speed is 1000 revolutions and the temperature is 4 ℃) for 10min, collecting supernate, putting the supernate into a precooling steel plate (the thickness is 0.3cm), putting the precooled supernate into a freeze dryer, and then freezing and drying the supernate, wherein the parameters of freeze drying are as follows: the temperature is 0 ℃, the pressure is 25Pa, and the time is 36 h; after freeze drying treatment, crushing the giant salamander meat to below 60 meshes to obtain giant salamander meat powder; extracting with food grade n-hexane to remove fat, and volatilizing the solvent to obtain defatted giant salamander powder;

(2) giant salamander proteolysis

Adding water into the obtained degreased giant salamander powder according to the mass-volume ratio of 1:15, stirring, and then adding a complex enzyme (the mass ratio is that alkaline protease: neutral protease: trypsin is 1:2:3.5), wherein the enzymolysis conditions are as follows: the pH value is 7.5, the enzymolysis temperature is 55 ℃, the enzymolysis time is 6 hours, the temperature is raised to 100 ℃ after the enzymolysis is finished, and the complex enzyme is inactivated for 20 min; the probability that the tail end of a product obtained by enzymolysis of the compound enzyme according to the proportion is hydrophobic amino acid is higher, and the bioactivity of the giant salamander peptide is increased;

(3) giant salamander peptide separation and purification

Filtering the enzymolysis product by a reproducible cellulose dialysis membrane with the molecular weight of 1000 daltons, collecting filtrate, adopting a sepharose hydrophobic chromatography medium, filling the sepharose hydrophobic chromatography medium into a chromatography column with the inner diameter of 5.5cm and the length of 20cm, after the hydrophobic chromatography column is filled, balancing by adopting 2M NaCl buffer solution, wherein the balance volume is 3 times of the column volume, slowly adding the filtrate into the chromatography column after the balance is finished, standing for 1h at room temperature, then carrying out sample elution by adopting a gradient elution method by using NaCl solution, collecting eluent when the salt ion concentration is the highest, desalting by a desalting column, and freeze-drying the separation solution to obtain the giant salamander oligopeptide.

Example 2

A preparation method of giant salamander oligopeptide comprises the following steps:

(1) giant salamander protein extraction

Crushing fresh giant salamander meat by a crusher, homogenizing, freezing and centrifuging (the rotating speed is 1000 revolutions and the temperature is 4 ℃) for 10min, collecting supernate, putting the supernate into a precooling steel plate (the thickness is 0.3cm), putting the precooled supernate into a freeze dryer, and then freezing and drying the supernate, wherein the parameters of freeze drying are as follows: the temperature is minus 45 ℃, the pressure is 30Pa, and the time is 24 h; after freeze drying treatment, crushing the giant salamander meat to below 60 meshes to obtain giant salamander meat powder; extracting with food grade n-hexane to remove fat, and volatilizing the solvent to obtain defatted giant salamander powder;

(2) giant salamander proteolysis

Adding water into the obtained degreased giant salamander powder according to the mass-volume ratio of 1:3, stirring, and then adding a complex enzyme (the mass ratio is that alkaline protease: neutral protease: trypsin is 1:2.5:3), wherein the enzymolysis conditions are as follows: the pH value is 7.2, the enzymolysis temperature is 60 ℃, the enzymolysis time is 6 hours, the temperature is raised to 90 ℃ after the enzymolysis is finished, and the complex enzyme is inactivated for 20 min; the probability that the tail end of a product obtained by enzymolysis of the compound enzyme according to the proportion is hydrophobic amino acid is higher, and the bioactivity of the giant salamander peptide is increased;

(3) giant salamander peptide separation and purification

Filtering the enzymolysis product by a reproducible cellulose dialysis membrane with the molecular weight of 1000 daltons, collecting filtrate, adopting a sepharose hydrophobic chromatography medium, filling the sepharose hydrophobic chromatography medium into a chromatography column with the inner diameter of 5.5cm and the length of 20cm, after the hydrophobic chromatography column is filled, balancing by adopting 2M NaCl buffer solution, wherein the balance volume is 3 times of the column volume, slowly adding the filtrate into the chromatography column after the balance is finished, standing for 1h at room temperature, then carrying out sample elution by adopting a gradient elution method by using NaCl solution, collecting eluent when the salt ion concentration is the highest, desalting by a desalting column, and freeze-drying the separation solution to obtain the giant salamander oligopeptide.

Example 3

A preparation method of giant salamander oligopeptide comprises the following steps:

(1) giant salamander protein extraction

Crushing fresh giant salamander meat by a crusher, homogenizing, freezing and centrifuging (the rotating speed is 1000 revolutions and the temperature is 4 ℃) for 10min, collecting supernate, putting the supernate into a precooling steel plate (the thickness is 0.3cm), putting the precooled supernate into a freeze dryer, and then freezing and drying the supernate, wherein the parameters of freeze drying are as follows: the temperature is-20 ℃, the pressure is 30Pa, and the time is 30 h; after freeze drying treatment, crushing the giant salamander meat to below 60 meshes to obtain giant salamander meat powder; extracting with food grade n-hexane to remove fat, and volatilizing the solvent to obtain defatted giant salamander powder;

(2) giant salamander proteolysis

Adding water into the obtained degreased giant salamander powder according to the mass-volume ratio of 1:8, stirring, and then adding a complex enzyme (the mass ratio is alkaline protease: neutral protease: trypsin is 1:2:3.5), wherein the enzymolysis conditions are as follows: the pH value is 7.2, the enzymolysis temperature is 60 ℃, the enzymolysis time is 6 hours, the temperature is raised to 110 ℃ after the enzymolysis is finished, and the complex enzyme is inactivated for 20 min; the probability that the tail end of a product obtained by enzymolysis of the compound enzyme according to the proportion is hydrophobic amino acid is higher, and the bioactivity of the giant salamander peptide is increased;

(3) giant salamander peptide separation and purification

Filtering the enzymolysis product by a reproducible cellulose dialysis membrane with the molecular weight of 1000 daltons, collecting filtrate, adopting a sepharose hydrophobic chromatography medium, filling the sepharose hydrophobic chromatography medium into a chromatography column with the inner diameter of 5.5cm and the length of 20cm, after the hydrophobic chromatography column is filled, balancing by adopting 2M NaCl buffer solution, wherein the balance volume is 3 times of the column volume, slowly adding the filtrate into the chromatography column after the balance is finished, standing for 1h at room temperature, then carrying out sample elution by adopting a gradient elution method by using NaCl solution, collecting eluent when the salt ion concentration is the highest, desalting by a desalting column, and freeze-drying the separation solution to obtain the giant salamander oligopeptide.

Example 4 evaluation of giant salamander peptide for lipid-regulating and weight-reducing

1. Construction of obese model rat

(1) The high-fat feed contains three nutrients, namely 58.3% of fat, 21% of protein and 20.7% of carbohydrate according to the energy supply ratio. The specific formula of the high-fat feed comprises: comprises 5 percent of sucrose, 18 percent of lard oil, 15 percent of yolk powder, 0.5 percent of cholate, 1 percent of cholesterol and 60.5 percent of basal feed in percentage by mass.

(2) Constructing an obesity model rat: the experimental rat is fed in an SPF animal room for 6 weeks by adopting the formula feed, four blood lipids of the experimental rat are detected until the experimental rat reaches an obesity model, the feeding condition is that the room temperature is maintained at 18-24 ℃, the pressure is maintained at 10-20 Kpa, the relative humidity is maintained at 40-70%, and drinking water is normal.

2. Grouping and testing of animals

The SPF SD rats are purchased from the animal experiment center of Sanxia university, Hubei province, the weight of the rats is 200-250 g, after adaptive feeding for 1 week, the animals are firstly divided into two groups, one group is a normal control group (group A), and 10 SD rats are purchased; the other group is an experimental group, high fat feeding is carried out firstly, an obesity model is constructed, after the obesity model is constructed successfully, the experimental group is randomly divided into 4 groups, and each group is 20, namely a high fat model group (B group), a high fat model exercise group (C group), a high fat model giant salamander peptide group (D group) and a high fat model giant salamander peptide combined exercise group (E group); in the experiment, the experimental rats in the group A and the experimental rats in the group B are both a quiet group, the group C is subjected to bench-running training for 6 weeks, the experimental rats in the group D are gavaged daily and are given 0.4g/kg of giant salamander oligopeptide (prepared in example 1), the experimental rats in the group E are gavaged daily and are given 0.4g/kg of giant salamander oligopeptide (prepared in example 1) and are simultaneously subjected to bench-running exercise training, and the training scheme is consistent with that of the experimental rats in the group C.

3. Results of the experiment

(1) Weight change of rats fed with high-fat feed

Through weekly weight measurement of experimental rats (see fig. 1), we found that there was no significant difference between the body weight of the control group and the body weight of the experimental rats in the high fat model group in the first week of molding, and that the body weight of the experimental rats in the high fat model group was not significantly increased compared to the body weight of the control group after three weeks of high fat diet feeding. The body weights of the experimental rats of the high fat model group are 373.46 +/-12.92 g, 418.43 +/-11.79 g and 441.75 +/-15.32 g respectively at week 4 and week 5 and week 6, and are significantly higher than those of the rats of the contemporary control group (312.31 +/-11.87 g, 336.57 +/-12.64 g and 359.66 +/-14.75 g). The above results indicate that the obese rat model was successfully constructed.

(2) Four detection results of blood fat of rats fed with high-fat feed

ELISA results (FIG. 2) showed that after 6 weeks of high-fat diet feeding, Triglyceride (TG), Total Cholesterol (TC) and low-density lipoprotein (LDL) in the serum of model rats were significantly increased to 1.61. + -. 0.22mmol/l, 5.11. + -. 0.42mmol/l and 1.85. + -. 0.23mmol/l from 0.52. + -. 0.13mmol/l, 1.35. + -. 0.23mmol/l and 0.30. + -. 0.08mmol/l, respectively; while the High Density Lipoprotein (HDL) is reduced from 0.39 + -0.12 mmol/l to 0.29 + -0.07 mmol/l, but there is no significant difference. The control group corresponding to this showed significant changes in TG, TC, HDL and LDL in the serum after 6 weeks of feeding with the normal diet. Combining the above results, we considered the obesity model rat to be successfully constructed.

(3) Giant salamander peptide for promoting weight loss of obese rats

After the modeling is successful, the experimental rats are subjected to 6-week intervention according to the experimental animal training scheme, and the weight of the rats in a control group (group A) and a high-fat model group (group B) maintains the trend of slowly increasing, and the weight of the rats in the 6 th week is remarkably increased compared with the weight of the rats in the first week; the body weight of the rats in the simple high fat model exercise group (group C) remained slightly increased in the first two weeks and gradually decreased from the third week, and the body weight was significantly decreased in the 6 th week compared with the second week; the weight of the rats in the high-fat model giant salamander peptide group (group D) which are fed with the giant salamander oligopeptide of 0.4g/kg body weight only slightly increases in the first three weeks, the weight slightly decreases in the fourth week, the descending trend is slower than that of the experimental rats in the group C, and the descending curve is more stable; the results of the group E of experimental rats which were given 0.4g/kg of giant salamander oligopeptide and simultaneously underwent running training showed that the body weight of the group of experimental rats remained decreasing and the decrease was significantly larger and more steep than that of the group C and group D (see fig. 3), unlike the other groups of experimental rats.

(4) Four effects of giant salamander peptide on fat rat blood fat

Four items of blood lipids including Triglycerides (TG), Total Cholesterol (TC), Low Density Lipoprotein (LDL), and High Density Lipoprotein (HDL) are classic indicators for the assessment of obesity. In the experiment, the influence of the giant salamander peptide and the giant salamander peptide combined with aerobic exercise on the blood fat of the obese rat is respectively examined. The results show that the concentrations of TG, TC, HDL and LDL of rats in the high-fat model group and the control group have no significant difference before and after the experiment, while the concentrations of TG, TC and LDL in the serum of the rats in the exercise group are significantly lower than those before the exercise intervention after 6 weeks of the exercise intervention, and the concentration of HDL is significantly higher than that before the exercise intervention. The serum TG, TC, HDL and LDL concentrations of the experimental rats orally administered with the giant salamander peptide alone varied in the same manner before and after the experiment as those of the exercise intervention group, except that the variation range was slightly smaller than that of the exercise group. The giant salamander peptide-coupled exercise intervention showed the ability to significantly alter TG, TC, HDL and LDL concentrations more than that of the giant salamander peptide administered alone with exercise and alone orally (see fig. 4). In addition, the concentrations of TG, TC, HDL and LDL in the serum of each experimental rat group before the experiment are not significantly different, but the concentrations of TG, TC, HDL and LDL in the serum of the experimental rats of the exercise group and the combined intervention group after the experiment are significantly changed compared with the model group (# p < 0.05).

(5) Giant salamander peptide for reducing appetite of obese model rats

Energy intake is also a key factor for evaluating weight loss effect, so we next examined the change of food intake of rats in each experimental group, as shown in the results of fig. 5, the food intake of the rats in the control group (group a), the high fat model group (group B) and the high fat model exercise group (group C) did not change significantly within 6 weeks of intervention, while the food intake of the rats given with giant salamander peptide alone was significantly reduced after 3 weeks of intervention, and the food intake began to stabilize at about 5 and 6 weeks. Similarly, the food intake of the high-fat model giant salamander peptide-combined sports group experimental rats also decreased significantly at 3 weeks after the intervention, but the food intake was higher than that of the experimental rats orally administered giant salamander peptide alone at the same time.

(6) Influence of giant salamander peptide combined with aerobic exercise on energy metabolism and appetite regulation related cytokines

After examining the concentration of cytokines related to energy metabolism and appetite regulation in serum of each experimental group, the concentration (118.74 +/-16.65 ng/ml) of Irisin in serum of rats in a high-fat model group is lower than that of a control group (257.43 +/-21.78 ng/ml) significantly, and after 6 weeks of bench exercise intervention, the concentration of Irisin in serum is obviously increased (187.58 +/-18.52 ng/ml), although the experimental rats which are orally administered with giant salamander peptide only have certain increase in serum of lipid models with higher concentration of Irisin, the increase is obviously smaller than that of the exercise intervention group. The concentration of the Irisin in the serum of the experimental rats of the high-fat model giant salamander peptide combined exercise intervention group is greatly increased compared with that of the experimental rats of the model group, and the concentration of the Irisin is higher than that of the experimental rats of the simple exercise intervention group. Meanwhile, the concentration of FGF15 in serum of 21.52 +/-2.83 pg/ml in the model group is remarkably increased compared with that of a control group (7.35 +/-1.36 pg/ml), the concentration of FGF in serum of experimental rats of the exercise group and the oral giant salamander peptide group (18.47 +/-1.66 pg/ml) is reduced compared with that of the control group, but no remarkable difference exists (see figure 6), but the concentration of FGF15 in serum of experimental rats of the high-fat model giant salamander peptide combined exercise group (14.23 +/-1.45) is remarkably reduced compared with that of the model group. Similarly, the serum contents of Leptin (Leptin) and Neuropeptide (NPY) in the experimental rats of the model group (1.33 +/-0.41 ng/ml and 2.82 +/-0.43 ng/ml respectively) are obviously higher than those in the control group, and the serum concentrations of Leptin and NPY in the experimental rats of the simple exercise group and the oral giant salamander peptide group are reduced to a certain degree but have no significant difference. And the concentrations of the two cytokines in the serum of the experimental rat of the giant salamander peptide combined high-fat model sports group are remarkably reduced compared with the concentration of the two cytokines in the model group (respectively 0.97 +/-0.28 ng/ml and 1.89 +/-0.27 ng/ml).

(7) Influence of giant salamander peptide combined with aerobic exercise on energy metabolism related signal pathway

As shown in fig. 7, the PGC-1 α expression level in skeletal muscle of the obese model rat is significantly decreased compared to the control group, the PGC-1 α expression level in skeletal muscle of the experimental rat of the simple exercise group is significantly increased compared to the high-fat model group, and the PGC-1 α expression level in skeletal muscle of the experimental rat orally administered the giant salamander peptide alone is not significantly changed compared to the high-fat model group, but the PGC-1 α expression level in skeletal muscle of the rat of the high-fat model giant salamander peptide combined exercise group is also significantly increased, and the increase ratio is greater than that of the experimental rat of the high-fat model exercise group. We also find that the p-AMPK expression level in skeletal muscle of an experimental rat of a high-fat model group is not significantly changed compared with that of a control group, the p-AMPK expression level in skeletal muscle of the experimental rat of a high-fat model exercise group and a high-fat model giant salamander peptide combined exercise group is obviously increased compared with that of the model group, and the p-AMPK expression level in skeletal muscle of the experimental rat of the high-fat model giant salamander peptide combined exercise group is higher. In addition, the change of the expression level of the Irisin in skeletal muscle of each group of experimental rats is detected, and the expression level of the Irisin in the high-fat model group is found to be in a descending trend compared with that in a control group, and the exercise intervention, particularly the oral giant salamander peptide combined exercise intervention, can increase the expression level of the Irisin in the skeletal muscle of the experimental rats, but the oral giant salamander peptide alone does not seem to cause the change of the expression level of the Irisin (see figure 7).

(8) Influence of giant salamander peptide combined with aerobic exercise on appetite regulation related protein expression of experimental rat

Based on the above experiments, oral giant salamander peptide has the effect of reducing the appetite of experimental rats, and in the present study, we also preliminarily investigated the expression of appetite-regulating related proteins of obese rats by various intervention means. As shown in fig. 8II, compared to the control group, the expression level of Neuropeptide (NPY) in hypothalamus of the experimental rats in the high-fat model group was dramatically increased, the expression of NPY in hypothalamus of the experimental rats in the simple high-fat model exercise group and the oral giant salamander peptide group was significantly decreased compared to the model group, and the expression level of NPY in hypothalamus of the obese rats was more greatly decreased by the combined exercise of the high-fat model giant salamander peptide. In contrast, we found that the expression level of pro-melanocortin (POMC) in hypothalamus of experimental rats in the high-fat model group was significantly decreased compared with that of the control group, and both exercise intervention, oral administration of giant salamander peptide and combined treadmill exercise of giant salamander peptide significantly increased the expression level of POMC in hypothalamus of obese rats, and the effect of oral administration of giant salamander peptide alone on the expression level of POMC in hypothalamus was more significant (see fig. 8 III).

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.