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

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 with liver injury repairing effect prepared from tuna leftovers

technical field

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

Background technique

The ocean area accounts for about 71% of the earth's surface, and there are as many as 200,000 species of organisms living in the ocean. The unique living environment and way of life of marine organisms doom the complexity, diversity and particularity of marine products. It is of great significance to extract and isolate effective biologically active substances from marine plants, marine animals and marine microorganisms. The extraction of effective active substances from marine plants, marine animals and marine microorganisms has attracted more and more attention from experts and scholars. At present, the most studied marine biological toxins, anti-tumor factors, antioxidant factors, cardiovascular active peptides and so on. Human life is deeply affected by marine toxins. Among them, toxic organisms still threaten people's life and production at sea. The marine biological toxins that have been discovered include tetrodotoxin, surgeonfish toxin, jellyfish toxin, saxitoxin and so on. Marine biotoxins can be directly developed as natural medicines or further used as lead compounds for the design of innovative medicines. With the rapid development of my country's tuna fishery, tuna processing has become the top priority of the economic development of pelagic fisheries. Tuna products are mainly canned, and the leftovers produced account for 50%-70% of the total weight. Mainly internal organs, minced meat and fish head, the leftovers not only contain high-quality protein, but also rich in a large number of biologically active substances. However, the existing tuna scraps are simply used and wasteful.

Non-alcoholic fatty liver disease is a pathological syndrome not related to alcohol, which is mainly characterized by fat accumulation and steatosis in liver cells. NAFLD can progress to non-alcoholic steatohepatitis, which may eventually progress to liver cirrhosis and even liver cancer. The incidence of NAFLD is high in developed countries, with an incidence rate of 20%-30%. The prevalence of NAFLD in adults in developed areas in China is about 15%, which seriously threatens people's lives, but the incidence is low in backward areas of my country. In recent years, with the improvement of people's living standards, the aging of the population and the prevalence of obesity, people's intake of fats and oils has gradually increased, and NAFLD patients have gradually increased in my country in recent years, becoming the most common disease affecting people's health. Liver Disease.

Regarding the pathogenesis of NAFLD, the most accepted one is the "second hit" theory proposed by Day. The accumulation of lipids in liver cells constitutes the first hit, and the second hit refers to the oxidation that occurs on this basis. Stress and lipid peroxidation. The occurrence of NAFLD includes insulin and leptin resistance, excessive accumulation of fat in the liver, and inflammation in liver tissue and cells. The main function of insulin is to increase the absorption of glucose by increasing the concentration of glucose transporters, thereby reducing the concentration of blood sugar. When cells are metabolized, the glucose ingested by the body will be converted into hepatic glycogen and stored in the liver. Another function of insulin can store lipids and inhibit lipid decomposition. Intracellular insulin resistance will hydrolyze the stored triglycerides. Increases the level of free fatty acids in plasma, which leads to the accumulation of fat in liver cells.

The harm of NAFLD mainly includes the following aspects: It can promote atherosclerosis, so patients are often accompanied by hyperlipidemia, and its blood viscosity will increase. Because of its small molecular weight, low-density lipoprotein is easy to cause blood vessel wall deposition arteries, resulting in reduced arterial elasticity, reducing its flexibility, and ultimately leading to blood circulation disorders, life-threatening; NAFLD can also induce hypertension and coronary heart disease, which can easily lead to myocardial infarction and lead to death; NAFLD can also damage the body's digestion, the human body The ingested proteins, carbohydrates and lipids must be metabolized by the liver, but NAFLD patients with liver damage will inevitably affect the digestive system. NAFLD can worsen liver damage. A buildup of fat in the liver. It makes liver cells swell and degenerate, squeezes the nucleus to one side, and further increases the burden on mitochondria, thereby affecting the metabolism of other nutrients, vitamins and hormones. Long-term hepatocyte degeneration will accelerate the damage of hepatocytes and further develop liver fibrosis and even liver cancer, and NAFLD can induce or aggravate diabetes.

At present, there is no effective treatment for NAFLD, and non-basic treatments such as drug therapy and surgical treatment are mostly used. Liver therapy, bariatric surgery, etc. Studies have shown that biguanides and thiazolidines can significantly improve collective insulin resistance, but the side effects cannot be underestimated. Treatment of NAFLD-related underlying diseases and metabolic symptoms mainly includes weight loss therapy, lipid-lowering therapy, and angiotensin-converting enzyme inhibitor therapy. Weight loss exercises including dieting and aerobic exercise can reduce the fat content in the body to a certain extent, thereby relieving physical stress and achieving a relieving effect. The principle of lipid-lowering therapy is the same as that of weight-loss therapy, but the effect and safety of lipid-lowering drugs also need to be studied.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an active peptide with liver damage repairing effect prepared by utilizing tuna leftovers, using tuna leftovers, a by-product of aquatic product processing as raw material, from which collagen peptides with hepatocyte repairing effect are extracted, which is simple and easy to implement, The low production cost can significantly increase the value of by-products of aquatic processing, avoid waste while protecting the environment, and provide a preliminary basis for the development of related functional foods.

The technical scheme adopted by the present invention to solve its technical problems is:

An active peptide with liver injury repairing effect prepared by using tuna leftovers, its amino acid sequence is: Met-Thr-His-Asp-Asp-Val-Asp-Glu (SEQ ID No. 1).

As preferably, it is prepared by the following steps:

(1) pretreatment of tuna leftovers: the fish meat part of fresh tuna leftovers is cleaned, and then homogenized to obtain meat slurry;

(2) Enzymatic hydrolysis reaction: mix the meat slurry in step (1) with water according to a material-to-liquid ratio of 1 g:3 mL, and then add alkaline protease for enzymatic hydrolysis reaction;

(3) Ultrafiltration: after the enzymatic hydrolysis reaction of step (2), the enzyme is killed, centrifuged, the supernatant is collected, and the supernatant is ultrafiltered with an ultrafiltration membrane to intercept the molecular segment products of three molecular weights, respectively, and freeze-drying , the molecular segment products of three molecular weights were respectively applied to the model group cells at a drug concentration of 10 mg/mL, and the components that could reduce the intracellular TG content the most were screened out;

(4) Sephadex G-25 chromatographic separation: take 100 mg of the component that can reduce the intracellular TG content the most obtained in step (3), add distilled water to prepare a 2 mL solution, centrifuge to get the supernatant and pass it through a 0.22 μm filter membrane, The filtrate was separated by chromatography on a Sephadex G-25 column, and the gel column was equilibrated with distilled water as the eluent; the flow rate of the constant-current pump was adjusted to 1.1 mL/min, and each tube was collected for 3.5 min, and detected at 280 nm. Absorbance, collect the eluate of each peak, and freeze-dry after rotary evaporation; each peak product was formulated into a drug concentration of 10 mg/mL to act on the cells of the model group, and the peak product with the highest reduction rate of intracellular TG content was selected as the target. peptides.

Preferably, the enzymatic hydrolysis conditions in step (2) are as follows: the dosage of alkaline protease is 1000 U per gram of meat pulp, the enzymatic hydrolysis temperature is 50° C., pH 10, and time 6h.

Preferably, the three molecular weights in step (3) are as follows: molecular weight<5kDa, molecular weight 5kDa-8kDa, and molecular weight>8kDa.

The beneficial effects of the invention are as follows: the by-product tuna scraps of aquatic product processing are used as raw materials, and collagen peptides with hepatocyte repairing effect are extracted therefrom. At the same time, it also protects the environment and provides a preliminary basis for the development of related functional foods.

Description of drawings

Fig. 1 is a graph showing the results of G-25 Sephadex chromatography on the <5kDa enzymatic hydrolysis solution of the present invention.

Detailed ways

The technical solutions of the present invention will be further described in detail below through specific examples.

In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or commonly used in the field. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

Tuna leftovers were purchased from Zhejiang Zhoushan Jiahejia Food Processing Co., Ltd.; normal human hepatocytes: Chang Liver cells were purchased from the Cell Center of Xiangya School of Medicine, Central South University, and were subcultured in our laboratory.

Model group cells: Select Chang Liver cells in good growth state, and induce Changliver cells with 15 μg/mL palmitic acid. After 12h, 24h, 48h, and 72h of induction, the cells were collected and the TG content was determined. The TG detection method was based on the reagents. box manual. An in vitro NAFLD cell model was established by selecting the induction time that significantly increased TG content in Chang liver cells and favored cell growth.

Example:

1. Method

1.1. Pretreatment of tuna scraps

Take fresh tuna leftovers and fish meat part into distilled water, stir gently, wash away impurities, homogenize, and store at -20 ℃ for later use.

1.2. Screening of the best enzymes for enzymatic hydrolysis of tuna scraps

Alkaline protease, pepsin, neutral protease, trypsin and papain were used, the optimum temperature and optimum pH were selected respectively, the enzymatic hydrolysis time was 6 h, and the amount of enzyme added was 1000 U/g. Enzymatic hydrolysis was carried out, and the enzymatic hydrolysis conditions were shown in Table 1. After enzymolysis, the enzyme activity was inactivated at 100 °C for 10 min, and then freeze-dried for later use. The dried product was applied to the cells of the model group at a concentration of 10 mg/mL for 24 h, and the protease with the highest reduction rate of TG content in the cells of the model group was screened out. . The calculation method of TG content reduction rate is: TG content reduction rate (%)=(TG content in model group-TG content in drug group)/TG content in model group×100%.

Table 1 Enzymatic hydrolysis conditions of different proteases

1.3. Separation and purification of enzymatic hydrolyzed oligopeptides from tuna scraps

The enzymolysis products obtained under the optimal enzymolysis conditions were subjected to ultrafiltration, and the molecular segment products <5kDa, 5kDa-8kDa, and >8kDa were intercepted, freeze-dried, and placed at -20°C for later use. The above components were applied to the cells of the model group at a drug concentration of 10 mg/mL, and the components that could reduce the intracellular TG content the most were screened.

1.4. Sephadex G-25 chromatographic separation

Take 100 mg of the component (freeze-dried product) that can reduce the intracellular TG content the most obtained in 1.3 and prepare a 2 mL solution. After centrifugation, the supernatant is collected and passed through a 0.22 μm filter membrane, and the filtrate is passed through a Sephadex G-25 column for layering. The gel column was equilibrated with distilled water as the eluent; the flow rate of the constant current pump was adjusted to 1.1 mL/min, each tube was collected for 3.5 min, the absorbance was detected at 280 nm, and the eluent of each peak was collected. , rotary evaporation followed by freeze drying. The drug concentration of 10 mg/mL was formulated to act on the cells of the model group, and the peak component with the highest reduction rate of intracellular TG content was screened out.

1.5. Determination of liver cell function indicators

The contents of ALT, AST, MDA, GSH-ST, γ-GT, SOD and other indicators in Chang liver cells of normal group, model group and 10 mg/mL and 20 mg/mL drug groups were measured respectively. The detection methods were carried out according to the kit instructions.

2 Results analysis

2.1 Screening results of optimal enzymatic hydrolysis conditions for tuna scraps

The tuna leftovers homogenate was subjected to enzymatic hydrolysis with 5 kinds of proteases to collect the products, and the TG content in the cells was measured after 24 hours by treating the cells of the model group. The results are shown in Table 2. It can be concluded from Table 2 that the alkaline protease hydrolyzate has the greatest reduction rate of TG content in cells (P<0.05), so alkaline protease is selected as the best enzyme.

n＝6)Table 2 Changes of TG content in NAFLD cell model by different protease hydrolysis products (

n=6)

Note: ^# Compared with the model group, P<0.05.

2.2 Preliminary separation results of oligopeptides from tuna scraps

The tuna leftover enzymatic hydrolysis solution obtained by alkaline protease under the optimal enzymatic hydrolysis conditions was subjected to ultrafiltration to obtain 3 different molecular segment products of <5kDa, 5kDa-8kDa, and >8kDa, which acted on the cells of the model group for 24 hours, and the TG content The test results are shown in Table 3. As can be seen from Table 3, the molecular segment <5kDa makes the cell TG content decrease the most in the model group, from 36.12 μmol/g in the model group to 14.29 μmol/g (P<0.05). Therefore, the enzyme with the molecular segment <5kDa was selected. The solution was analyzed by G-25 Sephadex chromatography.

n＝6)Table 3 Changes of TG content in NAFLD model cells by protease hydrolysis products of different molecular segments (

n=6)

Note: *P<0.05 compared with the model group.

2.3. Sephadex G-25 chromatographic separation results

The <5kDa enzymatic hydrolysis solution was subjected to G-25 Sephadex chromatography, and the eluate of each tube was collected to measure its absorbance at a wavelength of 280nm. The results were shown in Figure 1 to obtain 4 peaks, namely peak I, peak II, Peak III and Peak IV. The products of the four peaks were freeze-dried respectively, and the dried products acted on the cells of the model group at a concentration of 10 mg/mL. The results are shown in Table 4. The peak I product decreased the TG content of the model group the most, and the reduction rate was as high as 64.89%.

n＝6)Table 4 Changes of TG content in NAFLD model cells with different peak products (

n=6)

Note: *P<0.05 compared with the model group.

2.4. Determination results of oligopeptide sequence of tuna scraps

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

2.5 Determination results of liver cell function indexes

The test results of hepatocyte function index are shown in Table 5. It can be seen that the content of MDA, GSH-ST, ALT, AST, γ-GT in the model group is significantly higher than that in the normal group, and the level in the drug group after the action of the active peptide of the present invention is significantly decreased. (P<0.05). The content of SOD was higher in the cells of the normal group, decreased in the model group, and increased in the drug group (P<0.05).

n＝6)Table 5 Determination results of hepatocyte function indexes (

n=6)

Note: *Compared with the normal group, P<0.05;^#Compared with the model group, P<0.05.

The above-mentioned embodiment is only a preferred solution of the present invention, and does not limit the present invention in any form, and there are other variations and modifications under the premise of not exceeding the technical solution recorded in the claims.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> Active peptide with liver injury repairing effect prepared from tuna waste

<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)

1. an active peptide with liver injury repairing effect prepared by utilizing tuna leftovers is characterized in that, its amino acid sequence is: Met-Thr-His-Asp-Asp-Val-Asp-Glu.

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