A cod fish albumin peptide with fatigue-relieving activity

Abstract

The present invention provides a codfish white peptide with fatigue-relieving activity, which is prepared by enzymatic hydrolysis of codfish meat using alkaline protease and flavor protease as a composite enzyme; one of its amino acid sequences is LPFQR or VPYPR. The codfish white peptide provided by the present invention can be used to prepare anti-fatigue products. The codfish white peptide with fatigue-relieving effect prepared by the present invention using enzymatic hydrolysis technology simplifies the production process, reduces production costs, and does not cause any pollution. The target codfish white peptide prepared by the present invention has a significant fatigue-relieving effect. Animal experiments have shown that the codfish white peptide can significantly prolong the exhaustive swimming time of mice, reduce the blood lactate and blood urea nitrogen levels of mice, increase the glycogen reserve of mice, improve the levels of ATP and ATPase, can improve muscle damage in mice, and has a good fatigue-relieving effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and in particular relates to a cod fish albumin peptide with fatigue relieving activity. Background Art

[0002] According to WHO statistics, more than 35% of people worldwide show symptoms of fatigue, and most people who show fatigue symptoms often have clinically relevant diseases. Studies have shown that fatigue is often caused by the combined action of multiple factors. At present, researchers have formed a variety of theories about the mechanism of fatigue, such as energy depletion, accumulation of metabolites, oxidative stress, homeostasis disorders, protective inhibition of the central nervous system, and mutation theory. Research on bioactive substances with anti-fatigue activity has become a current research hotspot. Therefore, it is imperative to choose safe and effective natural substances to relieve fatigue.

[0003] Natural peptides are considered to be a good source of active substances for relieving fatigue. Oyster peptides have good fatigue-relieving activity; sea cucumber peptides (SCP-1, SCP-2) can inhibit oxidative stress, improve mitochondrial function, and thus play an anti-fatigue role; small molecule oligopeptides FSPPs obtained from soybeans have been shown to prolong the exhaustive swimming time of mice, improve mitochondrial function, and play an anti-fatigue role; after intervention with grass carp protein peptides, the exhaustive time of mice was significantly prolonged, glycogen reserves increased, and the BLA and BUN levels in serum decreased, showing good performance in relieving physical fatigue.

[0004] Yellowtail pollock (Theragra chalcogramma) is an important economic fish species for my country's ocean fishing. In my country, yellowtail pollock will produce scraps accounting for about 40%-50% of the fish weight during processing, of which milt accounts for about 7%. Many studies have shown that milt has a high protein content and good physiological activity. Enzymatic hydrolysis is an effective means to obtain active peptides from milt. However, there are few studies on the anti-fatigue activity pathway of cod milt peptide. Summary of the invention

[0005] The primary purpose of the present invention is to provide a cod fish white peptide with fatigue relieving activity, and a preparation method and application thereof.

[0006] The cod fish white peptide with fatigue relieving activity provided by the present invention is prepared by enzymolysis of cod meat using alkaline protease and flavor protease as composite enzymes;

[0007] The mass ratio of the alkaline protease to the flavor protease is 1:5.

[0008] The enzymatic hydrolysis comprises adding water to the cod milt homogenate to make the solid-liquid ratio 1:8-1:12, then adding protease, adjusting the pH value of the system to 6-9, and hydrolyzing at 50-55°C for 3-6 hours to obtain the cod milt enzymatic hydrolysate; concentrating and drying the enzymatic hydrolysate to obtain the cod milt peptide;

[0009] The pH value of the regulating system is adjusted by using 1 mol / L NaOH solution or HCl solution;

[0010] Preferably, the preparation method is to add a certain mass of water to the cod milt homogenate to make the solid-liquid ratio be 1:10, then add alkaline protease and flavor protease (1:5, mass ratio), the total enzyme amount is 2%, the pH value of the system is adjusted to 8.0, and the hydrolysis is carried out at 55°C for 4 hours to obtain the cod milt enzymatic hydrolyzate.

[0011] The cod fish protein peptide has an amino acid sequence of LPFQR (SEQ ID NO: 1) or VPYPR (SEQ ID NO: 2).

[0012] The cod fish white peptide provided by the present invention can be used to prepare anti-fatigue products;

[0013] The product is a functional food or medicine.

[0014] The present invention also provides an anti-fatigue product, comprising the codfish white peptide in a pharmacologically effective concentration.

[0015] Compared with the prior art, the present invention has the following advantages and effects:

[0016] (1) The codfish white peptide with fatigue-relieving effect prepared by the present invention utilizes enzymatic hydrolysis technology, which simplifies the production process, reduces production costs, and does not cause any pollution.

[0017] (2) The target codfish white peptide prepared by the present invention has a significant fatigue-relieving effect. Animal experiments have shown that the codfish white peptide can significantly prolong the exhaustive swimming time of mice, reduce the blood lactate and blood urea nitrogen levels of mice, increase the glycogen reserve of mice, increase the ATP and ATPase levels, improve the muscle damage of mice, and has a good fatigue-relieving effect.

[0018] (3) The present invention extracts cod fish white peptide with fatigue-relieving effect, which fills the gap in the market and has good application and economic prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The nitrogen recovery rate and hydrolysis degree of different Antarctic krill hydrolysates are shown in Figure 2.

[0020] Figure 2The nitrogen recovery rate and hydrolysis degree of the composite hydrolysate of alkaline protease and flavor protease are shown in Figure 2.

[0021] Figure 3 is the molecular weight distribution diagram of cod fish white peptide, where Figure 3 -A is the liquid chromatogram of the molecular weight of cod white peptide, Figure 3 -B is the molecular weight distribution diagram of cod fish white peptide.

[0022] Figure 4 This is the effect of cod fish protein peptide on the exhaustion time of mice.

[0023] Figure 5 This is the effect of cod fish protein on the serum lactic acid and urea nitrogen content of mice.

[0024] Figure 6 This is the protective effect of cod fish protein peptide on mouse muscle.

[0025] Figure 7 This is the effect of cod fish protein peptide on the levels of lactate dehydrogenase and creatine kinase in mice.

[0026] Figure 8 These are the molecular docking diagrams of codfish white peptide and protein receptors, where Figure A is the molecular docking diagram of LPFQR and AMPK, and Figure B is the molecular docking diagram of VPYPR and Keap1. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0029] Example 1: Preparation of cod fish protein by enzymatic hydrolysis

[0030] The preparation of cod white peptide having fatigue relieving activity comprises the following steps:

[0031] 1) Pretreatment of cod milt: thawing the cod milt in running water, removing impurities and homogenizing.

[0032] 2) Enzymatic hydrolysis reaction: adding water to the cod milt homogenate to make the material-liquid mass ratio 1:10, then adding protease, adjusting the pH value of the system to 8.0, and keeping the hydrolysis at 55°C for 4 hours; boiling and inactivating the enzyme for 10 minutes to obtain the cod milt enzymatic hydrolyzate; concentrating and drying the enzymatic hydrolyzate to obtain the cod milt peptide dry powder;

[0033] Five kinds of proteases (flavor protease, alkaline protease, trypsin, neutral protease, papain) were selected for enzymatic hydrolysis of cod milt. Five enzyme addition gradients (0.5%, 1.0%, 1.5%, 2.0%, 2.5%) were set respectively. Nitrogen recovery rate and hydrolysis degree were used as screening indicators to optimize the enzymatic hydrolysis conditions. Figure 1 The results showed that among the five enzymes, flavor protease and alkaline protease had the highest hydrolysis degree and nitrogen recovery rate. Figure 2 It was shown that when the mass ratio of alkaline protease to flavor protease was 1:5, the degree of hydrolysis and nitrogen recovery rate were the highest. Therefore, the cod white peptide prepared under this condition was used for subsequent research.

[0034] Example 2: Characterization of cod fish protein and enzymatic hydrolysis products

[0035] Water was added to the cod milt homogenate to make the material-liquid mass ratio of 1:10, and then alkaline protease and flavor protease were added in a mass ratio of 1:5, and the total enzyme amount was 2% of the milt mass. The pH value of the system was adjusted to 8.0, and the mixture was hydrolyzed at 55°C for 4 hours; the enzyme was boiled for 10 minutes to inactivate the enzyme to obtain the cod milt hydrolyzate; the hydrolyzate was concentrated and dried to obtain the cod milt peptide dry powder.

[0036] The properties of the prepared cod milt hydrolysate were characterized. Figure 3 The molecular weight distribution of codfish white peptide (TMP). The results show that the molecular weight of the prepared codfish white hydrolysate is basically concentrated in 200-600Da, of which the components with a molecular weight of 200-400Da account for 48%, the components with a molecular weight of 400-600Da account for 22%, and the short peptides below 1000Da account for 91.64%. After calculation, the average molecular weight of codfish white peptide TMP is 292.88Da, indicating a high degree of enzymatic hydrolysis. The amino acid composition results are shown in Table 1. The content of branched-chain amino acids in TMP is 18.33%, of which the content of leucine is 8.65%, the content of isoleucine is 4.14%, and the content of valine is 6.33%. The three amino acids, glutamic acid, arginine and aspartic acid, are relatively high in TMP, at 12.03%, 6.35% and 8.41% respectively.

[0037] Table 1: Amino acid composition of cod milt hydrolysate TMP

[0038]

[0039] Example 2: Detection of the anti-fatigue effect of cod milt hydrolysate

[0040] The present invention established an exhausted swimming mouse model, and intragastric administration of 400 and 800 mg / kg of TMP was performed on the mice. The specific steps are as follows:

[0041] 1. Animal Experiments

[0042] 1. Animal husbandry and experimental design

[0043] The mice were fed adaptively for one week, during which they were subjected to three swimming screening tests, in which the mice were allowed to swim freely in a water tank (25±2°C) with a depth greater than 30 cm. Mice that swam for less than 5 min were considered abnormal and eliminated. Normal mice were randomly divided into groups, with 10 mice in each group. The specific grouping is shown in Table 2.

[0044] Table 2: Experimental design table

[0045]

[0046] On the 24th day after intragastric administration, all groups except the Rest group were subjected to the exhaustion swimming test. 30 minutes after intragastric administration, a certain amount of lead sheet (lead sheet mass = mouse weight × 5%) was tied to the tail of each mouse, and the mouse was placed in a swimming box (water depth ≥ 30 cm, temperature maintained at 25 ± 2°C). The mouse was considered exhausted when its nose tip was submerged in the water for 7 seconds and could not float up. The time from the start of swimming to this time was recorded as the exhaustion time. Each group was subjected to the test at the same time. During this period, if a mouse was suspended and motionless, a glass rod could be used to stir the mouse near it to keep it swimming.

[0047] 2. Animal materials

[0048] On the 30th day after gavage, the mice were sampled. They were fasted for 12 hours before sampling. 30 minutes after the last gavage, all groups except the Rest group performed non-weight-bearing isochronous swimming for 30 minutes. After swimming, blood was collected from the eyeballs, and then the mice were killed by dislocation of the neck, and the liver, gastrocnemius and quadriceps femoris were taken. The serum was separated by centrifugation for later use. The same position of the mouse liver and the left gastrocnemius were taken and fixed in 4% neutral formaldehyde fixative, and three portions of 0.1g of liver and gastrocnemius were taken and placed in liquid nitrogen for later use.

[0049] like Figure 4 As shown, compared with the control group, the exhaustion time of mice in the positive control group and codfish protein hydrolysate TMP group was significantly prolonged (P<0.01), among which the exhaustion time of mice in the low-dose TMP group and the high-dose TMP group was prolonged by 45% and 59%, respectively, indicating that codfish protein peptide can significantly improve the endurance of mice, has good anti-fatigue performance, and shows dose dependence. Lactic acid and urea nitrogen are two metabolites in the body, and their content is closely related to the body's endurance. Figure 5 As shown in (A), compared with the Rest group, the lactic acid content in the serum of the Con group mice showed a significant upward trend, while the lactic acid content in the serum of the mice in the low-dose TMP group and the high-dose TMP group was reduced by 30.7% and 38.5% respectively compared with the Con group (P<0.05). Figure 5As shown in (B), weighted swimming significantly increased the BUN level in the serum of the mice in the exercise control group, while the BUN level in the serum of the mice in the low-dose and high-dose TMP groups showed a significant downward trend and basically returned to normal levels (P<0.01). This shows that TMP can reduce the accumulation of metabolites in mice.

[0050] Excessive exercise may cause muscle cell damage. Figure 6 As shown in the figure, the gastrocnemius muscles of mice were stained with HE. It can be seen that the muscle cells of mice in the Rest group had good morphology, and the muscle fibers were arranged in an orderly and relatively tight manner. After exercise, the muscle cells of mice in the Con group were arranged loosely, and some muscle cells showed shrinkage and cracks. Compared with the Con group, the muscle cell status of mice in the taurine, TMP low-dose group and TMP high-dose group was improved, with less shrinkage and cracks, and the muscle cells were arranged more tightly and orderly, indicating that it has a protective effect on mouse muscles.

[0051] As a key enzyme in the lactate metabolism process, LDH catalyzes the conversion of lactate to pyruvate. Under normal conditions, LDH is mainly distributed in muscle tissue. When exercise causes muscle damage, LDH will flow into the blood, thereby causing an increase in serum LDH levels. Figure 7 As shown in (AB), compared with the Rest group, the LDH level in the muscle of the mice in the Con group was significantly decreased (P<0.01), while the LDH level in the serum was significantly increased (P<0.05), indicating that exercise caused muscle damage in mice. Compared with the Con group, the LDH level in the serum decreased by 22.3% and 25.7%, respectively, and the LDH level in the muscle of the mice in the low-dose and high-dose TMP groups was significantly increased (P<0.01). CK is mainly distributed in various muscle tissues, such as skeletal muscle cells, cardiomyocytes, and smooth muscle cells, and plays an important role in intracellular muscle contraction and ATP regeneration. Under normal physiological conditions, serum CK levels are low, and exercise will cause a large amount of ATP to be consumed, so serum CK levels increase to meet ATP needs. At the same time, part of the reason for the increase in LDH levels is the same as that of exercise causing muscle cell damage and increased cell membrane permeability, causing CK in the muscles to flow into the blood. As Figure 7 As shown in (CD), compared with the Rest group, the CK level in the muscle of the Con group mice was significantly reduced (P<0.05), and the CK level in the serum was increased, indicating that exercise caused muscle damage in mice. Both the low-dose and high-dose TMP groups could alleviate the decrease in the CK level in the muscle of mice and the increase in the CK level in the serum after exercise. This further shows that TMP has a protective effect on muscle.

[0052] Example 3: Purification and Sequencing of Prepared TMP

[0053] The peptide sequence of codfish white peptide was analyzed and identified by LC-MS / MS. The chromatographic column used for UHPLC determination of the peptide sequence was Agilent Advance-Bio Peptide Map C18 column (2.1×150mm, 2.7μm). UHPLC parameters: mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1% formic acid-water, flow rate: 0.25mL / min, column temperature: 40℃, gradient elution program 0-2min (5%A), 2-27min (5%A-20%A), 27-37min (20%A-35%A), 37-39min (35%A-80%A). Scanning range: 50-1500m / z, electrospray mode: electrospray positive ion, electrospray voltage 5500v. 24 peptides with possible biological activity were screened for molecular docking (Table 3).

[0054] Table 3: Docking binding energy table of cod fish white peptide

[0055]

[0056] The results are as follows Figure 8 As shown, it is shown that LPFQR and VPYPR peptides can interact with AMPK and Keap1 receptors, and perform molecular docking with AMPK and Keap1 respectively. The two can form hydrogen bonds and other forces with the receptors and have biological activity.

[0057] LPFQR and VPYPR were tested according to the method of Example 2. The results showed that the two polypeptides in the low-dose group (400 mg / kg / d) reached the exhaustion time of mice in the high-dose group of TMP, showing a more effective anti-fatigue effect. Compared with the Con group, the lactic acid content in the serum of mice fed with LPFQR and VPYPR was reduced by 46.3% and 49.2% respectively (P<0.05).

[0058] In summary, the activity of the codfish white peptide prepared by the present invention was evaluated based on an animal model. TMP can significantly prolong the exhaustive swimming time of mice, reduce the accumulation of metabolites, and has a protective effect on muscles. The H&E staining results also show that TMP has a certain protective effect on muscles. Therefore, the codfish white peptide prepared by the method of the present invention has a good fatigue relief effect and has a good application prospect.

Claims

1. Use of codfish white peptide with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the preparation of anti-fatigue products.

2. The use according to claim 1, characterized in that The product is a functional food or medicine.

3. An anti-fatigue product, characterized in that: The product contains codfish white peptide with an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at a pharmacologically effective concentration.

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