CN106632605B

CN106632605B - Active peptide prepared from tuna leftovers and having liver injury repair effect

Info

Publication number: CN106632605B
Application number: CN201611201846.7A
Authority: CN
Inventors: 丁国芳; 艾杨洋; 黄芳芳; 杨最素; 李小娟; 许丹
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2016-12-22
Filing date: 2016-12-22
Publication date: 2020-06-16
Anticipated expiration: 2036-12-22
Also published as: CN106632605A

Abstract

The invention discloses a bioactive peptide with liver injury repairing function prepared by tuna leftovers, which has the amino acid sequence as follows: Met-Thr-His-Asp-Asp-Val-Asp-Glu. The method takes the leftover of the tuna which is a byproduct of aquatic product processing as a raw material, extracts the collagen peptide with the liver cell repairing effect from the raw material, is simple and easy to implement, has low production cost, can obviously improve the value of the aquatic product processing byproduct, avoids waste, protects the environment and provides an early basis for developing related functional foods.

Description

Active peptide prepared from tuna leftovers and having liver injury repair effect

Technical Field

The invention relates to an active peptide, in particular to an active peptide with a liver injury repairing effect, which is prepared by using tuna leftovers.

Background

The ocean area accounts for about 71 percent of the earth surface, the organisms living in the ocean are up to 20 thousands, and the unique living environment and living mode of the marine organisms determine the complexity, diversity and particularity of marine products, so that the extraction and separation of effective bioactive substances from the marine organisms have important significance, and the extraction of effective bioactive substances from marine plants, marine animals and marine microorganisms is more and more concerned by experts and scholars. At present, the most studied are marine biotoxins, anti-tumor factors, anti-oxidation factors, cardiovascular active peptides and the like. The life of human beings is deeply influenced by marine toxins, wherein toxic organisms still threaten the marine life and production of people, and the marine toxins which are found comprise tetrodotoxin, stinging tail toxin, jellyfish toxin, saxitoxin and the like. The marine biotoxin can be directly developed into a natural medicine or further used as a lead compound for the design of innovative medicines. With the rapid development of tuna fishery in China, tuna processing has become the central importance of economic development of ocean fishery. Tuna products are mainly processed into cans, and the produced leftovers account for 50-70% of the total weight. Mainly takes viscera, minced meat and fish heads as main materials, and leftovers not only contain high-quality protein, but also are rich in a large amount of bioactive substances. However, the existing tuna leftovers are simply utilized and are relatively wasted.

Non-alcoholic fatty liver disease is a pathological syndrome unrelated to alcohol, and is mainly characterized by fat accumulation and steatosis in liver cells, NAFLD can develop into non-alcoholic steatohepatitis, and finally, the liver cirrhosis and liver cancer can be possibly degraded. The incidence of the NAFLD is high in developed countries, the incidence of the NAFLD is up to 20% -30%, the incidence of the NAFLD of adults in developed regions of China is about 15%, the life of people is seriously threatened, but the incidence of the NAFLD is low in laggard regions of China. In recent years, with the improvement of the living standard of Chinese people, the aging of population and the prevalence of obesity, and the gradual increase of fat and grease ingested by people, NAFLD patients gradually increase in China in recent years and become the most common liver diseases affecting the body health of people.

With respect to the pathogenesis of NAFLD, it is most accepted by the "secondary stroke" theory proposed by Day that the accumulation of lipids in hepatocytes constitutes the first stroke, which is the oxidative stress and lipid peroxidation that occurs on this basis. The occurrence of NAFLD includes insulin and leptin resistance, excessive accumulation of fat in the liver, inflammatory reactions in liver tissue and cells, and the like. The primary function of insulin is to reduce blood glucose levels by up-regulating the concentration of glucose transporters to facilitate glucose absorption. When cells metabolize, glucose taken in by the body is converted into hepatic glycogen to be stored in the liver, the other action of insulin can store lipid and inhibit lipolysis, and intracellular insulin resistance can hydrolyze stored triglyceride to increase the content of free fatty acid in plasma, so that the accumulation of fat in the liver cells is caused.

The harm of NAFLD mainly includes the following aspects: the traditional Chinese medicine composition can promote atherosclerosis, so patients are often accompanied with hyperlipidemia, the blood viscosity of the patients is increased, and due to the small molecular weight of low-density lipoprotein, the low-density lipoprotein easily causes arteries deposited on blood vessel walls, so that the elasticity of the arteries is reduced, the flexibility of the arteries is reduced, blood circulation disorder is finally caused, and the life is threatened; NAFLD also induces hypertension and coronary heart disease, and is very likely to cause myocardial infarction and death; NAFLD also impairs digestion, and proteins, carbohydrates and lipids ingested by the body must be metabolized by the liver, but the liver of patients with NAFLD is injured and tends to involve the digestive system. NAFLD can exacerbate liver damage. Accumulation of fat in the liver. The liver cells are enlarged and denatured, the cell nucleus is extruded to one side, and the burden of mitochondria can be further increased, so that the metabolism of other nutrients, vitamins and hormones is influenced. Long-term hepatocyte degeneration accelerates damage to hepatocytes, which may further progress to liver fibrosis and even liver cancer, and NAFLD may induce or exacerbate diabetes.

At present, no effective treatment method for NAFLD exists, non-basic treatment methods such as drug treatment and surgical treatment are mostly adopted, and the non-basic treatment mainly comprises treatment for improving insulin resistance, treatment for NAFLD-related basic diseases and metabolic syndrome, anti-oxidation and anti-inflammation and liver protection treatment, weight-losing surgical treatment and the like. Research has proved that biguanides and thiazolidines can significantly improve collective insulin resistance, but the side effects present therein are not of great concern. The treatment of NAFLD-related basic diseases and metabolic symptoms mainly includes weight loss treatment, lipid lowering treatment and angiotensin converting enzyme inhibitor treatment. The fat-reducing exercise including diet exercise and aerobic exercise can reduce the fat content in the body to a certain extent, thereby relieving the body pressure and achieving the effect of relieving. Lipid lowering therapy has the same principle as weight reducing therapy, but the effect and safety of lipid lowering drugs are yet to be studied.

Disclosure of Invention

The invention aims to provide the active peptide with the liver injury repairing function prepared by utilizing the tuna leftovers, which is prepared by taking the tuna leftovers which are aquatic product processing byproducts as raw materials and extracting the collagen peptide with the liver cell repairing function from the raw materials, is simple and easy to implement, has low production cost, can obviously improve the value of the aquatic product processing byproducts, avoids waste, protects the environment and also provides an early basis for developing related functional foods.

The technical scheme adopted by the invention for solving the technical problems is as follows:

an active peptide with liver injury repairing effect prepared from tuna leftovers has an amino acid sequence as follows: Met-Thr-His-Asp-Asp-Val-Asp-Glu (SEQ ID No. 1).

Preferably, the preparation method comprises the following steps:

(1) pretreatment of tuna leftovers: cleaning the fish meat part of the leftovers of the fresh tuna, and then homogenizing to obtain meat pulp;

(2) and (3) carrying out enzymolysis reaction: mixing the meat pulp obtained in the step (1) with water according to a feed-liquid ratio of 1g:3mL, and then adding alkaline protease for enzymolysis reaction;

(3) and (3) ultrafiltration: after the enzymolysis reaction in the step (2) is finished, inactivating enzyme, centrifuging, collecting supernatant, ultrafiltering the supernatant by using an ultrafiltration membrane, respectively intercepting molecular segment products with three molecular weights, freeze-drying, respectively acting the molecular segment products with the three molecular weights on model group cells at the drug concentration of 10mg/mL, and screening out components capable of reducing the content of TG in the cells to the maximum;

(4) sephadex G-25 chromatographic separation: taking 100mg of the component which is obtained in the step (3) and can reduce the content of TG in cells to the maximum, adding distilled water to prepare 2mL of solution, centrifuging, taking supernatant, filtering through a 0.22 mu m filter membrane, passing filtrate through a Sephadex G-25 column for chromatographic separation, taking distilled water as eluent, and carrying out balanced elution on the gel column; regulating the flow rate of a constant flow pump to be 1.1mL/min, collecting each tube for 3.5min, detecting the absorbance of each tube at 280nm, collecting the eluent of each peak, and freeze-drying after rotary evaporation; each peak product is respectively prepared into 10mg/mL medicine concentration to act on model group cells, and the peak product which enables the content reduction rate of TG in the cells to be the maximum is selected as the target peptide.

Preferably, the enzymolysis conditions in step (2) are as follows: the dosage of the alkaline protease is 1000U per g of meat pulp, the enzymolysis temperature is 50 ℃, the pH value is 10, and the time is 6 h.

Preferably, the three molecular weights in step (3) are specified below: the molecular weight is less than 5kDa, the molecular weight is 5kDa-8kDa, and the molecular weight is more than 8 kDa.

The invention has the beneficial effects that: the method has the advantages of simple and easy operation and low production cost, can obviously improve the value of aquatic product processing byproducts, avoids waste, protects the environment and also provides early basis for developing related functional foods.

Drawings

FIG. 1 is a graph showing the results of G-25 Sephadex chromatography performed on < 5kDa enzymatic hydrolysate of the present invention.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

Tuna offal was purchased from Zhongshan Jia Zhejiang and Jia food processing, Inc.; normal human hepatocytes: zhang's hepatocytes (Chang Liver cells) were purchased from the cell center of Xiangya medical college, school of Hunan university, and subcultured in this laboratory.

Model group cells: selecting Chang Liver cells in good growth state, inducing the Chang Liver cells by using 15 mu g/mL palmitic acid, collecting the cells after inducing for 12h, 24h, 48h and 72h respectively, and determining the content of TG, wherein the TG detection method is carried out according to the kit instructions. And selecting the induction time which obviously increases the TG content in the Chang lever cell and ensures that the cell grows well, and establishing an in vitro NAFLD cell model.

Example (b):

1. method of producing a composite material

1.1 pretreatment of tuna offcuts

Taking the fish meat part of the fresh tuna leftovers, slightly stirring the fish meat part in distilled water, washing off impurities, homogenizing the mixture, and storing the homogenate at the temperature of minus 20 ℃ for later use.

1.2 screening of the optimal enzyme species for tuna leftover enzymolysis

Alkaline protease, pepsin, neutral protease, trypsin and papain are adopted, the optimum temperature and the optimum pH are respectively selected, the enzymolysis time is 6 hours, the enzyme adding amount is 1000U/g, the homogenate of the tuna leftovers is subjected to enzymolysis, and the enzymolysis conditions are shown in table 1. Inactivating enzyme at 100 deg.C for 10min after enzymolysis, freeze drying, allowing the dried product to act on model group cells at concentration of 10mg/mL for 24h, and screening protease with maximum TG content reduction rate. The calculation method of the TG content reduction rate is as follows: the TG content reduction rate (%) (model group TG content-drug group TG content)/model group TG content × 100%.

TABLE 1 enzymatic conditions for different proteases

1.3, separation and purification of tuna leftover enzymatic hydrolysis oligopeptide

Ultrafiltering the enzymolysis product obtained under the optimal enzymolysis condition, respectively intercepting molecular segment products less than 5kDa, 5kDa-8kDa and more than 8kDa, freeze-drying, and standing at-20 deg.C for use. The product of the above components was allowed to act on the cells of the model group at a drug concentration of 10mg/mL, and the components capable of reducing the intracellular TG content most were selected.

1.4 Sephadex G-25 chromatography separation

Preparing 100mg of the components (freeze-dried products) which are screened out in the step 1.3 and can reduce the content of TG in cells to the maximum into 2mL of solution, centrifuging, taking supernatant, filtering the supernatant through a 0.22 mu m filter membrane, carrying out chromatographic separation on the filtrate through a Sephadex G-25 column, taking distilled water as eluent, and carrying out balanced elution on the gel column; regulating the flow rate of a constant flow pump to be 1.1mL/min, collecting each tube for 3.5min, detecting the absorbance of each tube at 280nm, collecting the eluent of each peak, performing rotary evaporation, and performing freeze drying. The drug concentration of 10mg/mL is prepared to act on model group cells, and peak components which enable the content reduction rate of TG in the cells to be the maximum are screened.

1.5 determination of hepatocyte function index

Respectively measuring the contents of indexes such as ALT, AST, MDA, GSH-ST, gamma-GT, SOD and the like in cells of the normal group, the model group and the 10mg/mL and 20mg/mL drug group Chang lever, and carrying out the detection method according to the kit instructions.

2 analysis of results

2.1 screening results of the optimal conditions for the enzymatic hydrolysis of tuna offcuts

The product was collected by subjecting tuna leftover homogenate to enzymatic hydrolysis using 5 proteases, and the intracellular TG content was measured 24h after treatment of model group cells, the results are shown in Table 2. From Table 2, it can be concluded that the alkaline protease substrate maximizes the reduction of TG content in the cells (P <0.05), and thus the alkaline protease is selected as the optimum enzyme species.

Table 2 variation of TG content in different protease cleavage products versus NAFLD cell model: (

n＝6)

Note:^#compared with the model group, P is less than 0.05.

2.2 preliminary separation result of oligopeptide from tuna offcuts

Ultrafiltering tuna leftover enzymolysis liquid obtained by alkaline protease under the optimal enzymolysis condition to obtain 3 different molecular segment products of less than 5kDa, 5kDa-8kDa and more than 8kDa, and acting on model group cells for 24h, wherein the TG content detection result is shown in Table 3. As can be seen from Table 3, the < 5kDa molecular fragment maximizes the reduction in TG content in the cells of the model group from 36.12. mu. mol/G to 14.29. mu. mol/G (P <0.05) of the model group, and therefore the enzymatic hydrolysate of the < 5kDa molecular fragment was selected for G-25 Sephadex chromatography.

TABLE 3 variation of protease enzymatic products of different molecular weight segments on TG content in NAFLD model cells: (

n＝6)

Note: p <0.05 compared to model group.

2.3 Sephadex G-25 chromatographic separation result

G-25 sephadex chromatography is carried out on the < 5kDa enzymolysis solution, and the eluates of all tubes are collected to measure the absorbance of the solution at the wavelength of 280nm, so that 4 peaks, namely a peak I, a peak II, a peak III and a peak IV, are obtained as shown in figure 1. The four peak products were lyophilized and the dried products were applied to the model group cells at a concentration of 10mg/mL, and as a result, as shown in Table 4, the peak I product resulted in the greatest decrease in TG content of the model group, with a decrease rate of 64.89%.

Table 4 different peak products on NAFLD model intracellular TG content change: (

n＝6)

Note: p <0.05 compared to model group.

2.4 determination result of oligopeptide sequence of tuna offal

The amino acid sequence of the target peptide of the peak I product is detected as follows: Met-Thr-His-Asp-Asp-Val-Asp-Glu, ESI/MS molecular weight 960.10 Da.

2.5 measurement results of hepatocyte function index

The result of detecting the liver cell function index is shown in Table 5, and it can be seen that the content model group of MDA, GSH-ST, ALT, AST and gamma-GT is obviously higher than the normal group, and the level of the drug group after the action of the active peptide of the invention is obviously reduced (P is less than 0.05). The SOD content is higher in normal group cells, is reduced in a model group, and is increased in a drug group level (P < 0.05).

TABLE 5 measurement results of hepatocyte function index: (

n＝6)

Note: comparison with normal group, P<0.05；^#Comparison with model groups, P<0.05。

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

SEQUENCE LISTING

<110> Zhejiang ocean university

<120> active peptide with liver injury repair effect prepared by tuna leftovers

<130>2016.12

<160>1

<170>PatentIn version 3.3

<210>1

<211>8

<212>PRT

<213> tuna

<400>1

Met Thr His Asp Asp Val Asp Glu

1 5

Claims

1. A bioactive peptide with liver injury repairing effect prepared from tuna leftovers is characterized in that the amino acid sequence is as follows: Met-Thr-His-Asp-Asp-Val-Asp-Glu.