CN116790701B - A tilapia skin polypeptide and its preparation method and application in preparing anti-muscle atrophy products - Google Patents

Abstract

The invention discloses a tilapia skin polypeptide, its preparation method and its application in preparing anti-muscle atrophy products, and relates to the field of biotechnology. The preparation method of the tilapia skin polypeptide includes the following steps: remove the scales of the tilapia skin and crush it, and then dry it after degreasing to obtain tilapia skin dry powder; the tilapia skin dry powder is enzymatically processed After hydrolysis and enzyme-killing treatment, an enzymatic hydrolyzate is obtained; the enzymatic hydrolyzate is subjected to ultrafiltration, and a filtrate with a molecular weight of less than 10 kDa is collected, and then concentrated and freeze-dried to obtain the tilapia skin polypeptide. The tilapia skin polypeptide provided by the invention has the effect of improving muscle atrophy, and has guiding significance for the development of products for preventing and treating muscle atrophy.

Description

A tilapia skin polypeptide and its preparation method and its use in preparing anti-muscle atrophy products Applications in

Technical field

The invention relates to the field of biotechnology, and in particular to a tilapia skin polypeptide, its preparation method and its application in preparing anti-muscle atrophy products.

Background technique

Muscle atrophy, also known as sarcopenia, refers to a syndrome caused by continued loss of skeletal muscle mass, strength and function. Skeletal muscle is the driving force of the human movement system. Muscle aging and atrophy are important signs of human aging and can easily cause fractures, joint injuries and other problems. Muscle atrophy is a degenerative disease of muscles that causes muscle fibers to become thinner or even disappear, resulting in a decrease in muscle mass and muscle volume. Among people aged 65 to 70 years, the prevalence of sarcopenia is as high as 24%, and elderly people between the ages of 70 and 80 lose 15% of their total muscle mass every 10 years. Sarcopenia also affects organ function and may lead to heart and lung failure and even death. Muscle atrophy affects the mobility and quality of life of the elderly. The high distribution of glucocorticoids in the body is one of the causes of muscle atrophy. Glucocorticoids represented by dexamethasone (DEX) can successfully induce muscle atrophy models in vitro and in vivo.

There are currently no effective treatments for muscle atrophy. Tilapia is a common species in my country, with large output and low utilization rate. Currently, there are no research reports on the extraction of active peptides from tilapia skin for anti-muscle atrophy.

Contents of the invention

The purpose of the present invention is to provide a tilapia skin polypeptide and its preparation method and application in preparing anti-muscle atrophy products, so as to solve the problems existing in the above-mentioned prior art. The tilapia skin polypeptide has better Antimuscle atrophy activity.

In order to achieve the above objects, the present invention provides the following solutions:

The invention provides a method for preparing tilapia skin polypeptide, which is characterized in that it includes the following steps:

(1) Remove the scales from the tilapia skin, crush it, and then dry it to obtain tilapia skin dry powder;

(2) After the tilapia skin dry powder is subjected to enzymatic hydrolysis and enzyme-killing treatment, an enzymatic hydrolysis liquid is obtained;

(3) Perform ultrafiltration treatment on the enzymatic hydrolyzate, collect the filtrate with a molecular weight below 10 kDa, and then concentrate and freeze-dry to obtain the tilapia skin polypeptide.

Further, in step (1), the reagent used in the degreasing treatment includes isopropyl alcohol.

Further, in step (2), the enzymes used in the enzymatic hydrolysis include neutral protease and alkaline protease.

Further, in step (2), the enzymatic hydrolysis includes the step of sequentially adding the neutral protease and the alkaline protease to enzymatically hydrolyze the dry tilapia skin powder.

Further, the added mass of the neutral protease is 3% of the mass of the tilapia skin dry powder; the added mass of the alkaline protease is 0.3% of the mass of the tilapia skin dry powder.

Further, the conditions for adding the neutral protease for enzymatic hydrolysis include: pH is 7.0±0.2, the temperature is 50±5°C, and the enzymatic hydrolysis time is 2 h; the conditions for adding the alkaline protease for enzymatic hydrolysis include: the pH is 9.0±0.2, the temperature is 50±5℃, and the enzymatic hydrolysis time is 2h.

The invention also provides a tilapia skin polypeptide prepared according to the above preparation method.

The present invention also provides the use of the above-mentioned tilapia skin polypeptide in preparing products for preventing and treating muscle atrophy.

Further, the product is a medicine.

The invention also provides a product for preventing and treating muscle atrophy, including the above-mentioned tilapia skin polypeptide.

The invention discloses the following technical effects:

The invention provides an anti-muscle atrophy tilapia skin polypeptide, which can significantly improve the weight loss, strength loss, muscle weight loss, muscle cross-sectional area reduction, and human dystrophin protein in DEX-induced muscle atrophy mice. The up-regulation of Fbox-1 (Atrogin-1) and ubiquitin protein ligase 1 (MURF1) expression, as well as the decrease in C2C12 fibroblast viability, can be used to improve muscle atrophy and have guiding significance for the development of products to prevent and treat muscle atrophy. .

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the cytotoxicity MTT results of tilapia skin peptides against C2C12 Among them, compared with the blank group, #p<0.05;

Figure 2 shows the MTT results of the anti-muscle atrophy activity of tilapia skin peptides Among them, compared with the blank group, ^## p<0.01; compared with the model group, ^* p<0.05, ^** p<0.01;

Figure 3 shows the Western blot expression results of muscle atrophy-related proteins in cells in each group; (A) is a representative picture of protein expression of muscle atrophy-related proteins; (B) is the relative expression of Atrogin-1; (C) is MURF1 Relative expression amount; compared with the blank group, ^## p＜0.01; compared with the model group, ^* p＜0.05, ^** p＜0.01;

Figure 4 shows the grip strength test results of mice in each group. Among them, compared with the blank group, ^## p<0.01; compared with the model group, ^** p<0.01;

Figure 5 shows the running endurance test results of mice in each group. Among them, compared with the blank group, ^## p<0.01; compared with the model group, ^** p<0.01;

Figure 6 shows the weight results of muscle tissues such as quadriceps femoris, gastrocnemius, tibialis anterior and soleus in each group of mice (X±SEM); among them, compared with the blank group, ^# p<0.05, ^## p<0.01; Compared with the model group, ^* p<0.05, ^** p<0.01;

Figure 7 shows the H&E staining (A) and cross-sectional area statistical results (B) of paraffin sections of gastrocnemius tissue; among them, compared with the blank group, ^## p<0.01; compared with the model group, ^** p<0.01; in A The scales are all 100μm;

Figure 8 shows the H&E staining (A) and cross-sectional area statistical results (B) of paraffin sections of tibialis anterior muscle tissue; among them, compared with the blank group, ^## p<0.01; compared with the model group, ^** p<0.01; The scale bars in A are all 100 μm;

Figure 9 shows the results of lean meat content and fat content of mice in each group: (A) is lean meat content; (B) is fat content; compared with the blank group, ^## p<0.01; compared with the model group, ^* p<0.05, ^** p<0.01;

Figure 10 shows the Western blot expression results of muscle atrophy-related proteins in the tibialis anterior muscle of each group; (A) is a representative picture of the protein expression of muscle atrophy-related proteins; (B) is the relative expression of Atrogin-1; (C) is Relative expression of MURF1; compared with the blank group, ^## p＜0.01; compared with the model group, ^* p＜0.05.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range, and any other stated value or value intermediate within a stated range, is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples of the present invention are exemplary only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

Example 1

Preparation method of tilapia skin polypeptide:

(I) Sample pretreatment. First, remove the scales from the tilapia skin, rinse it under running water and drain it. Use a tissue homogenizer to mince the tilapia skin and homogenize for 3 minutes each time for 4 times. Add 4 times the volume of isopropyl alcohol to the homogenate for defatting for 4 hours, during which the medium was changed 4 times. The entire degreasing process was carried out on a magnetic stirrer, with the stirring speed set to 100 rpm and the stirring temperature set to 55°C. After degreasing, centrifuge with a high-speed centrifuge (centrifugation parameter: 12000 rpm, 10 min), collect the filter residue, and dry it in an electric constant temperature oven at 50°C to obtain dry tilapia skin powder.

(II) Enzymatic hydrolysis. Add 5 times the volume of ultrapure water to soak the dry tilapia skin powder, add 3% neutral protease based on the mass of the dry skin powder, maintain the pH of the solution system at 7.0±0.2, and perform enzymatic hydrolysis at 50±5°C for 2 hours. After 2 hours, add 0.3% alkaline protease by mass of dry fish skin powder, maintain the pH of the solution system at 9.0±0.2, and perform enzymatic hydrolysis at 50±5°C for 2 hours. All enzymatic hydrolysis processes were stirred for 1 min on a magnetic stirrer every 15 min. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis solution is heated to 100°C for 10 minutes to interrupt the enzymatic hydrolysis process, and then centrifuged to collect the supernatant and store it at -80°C for later use.

(III) Ultrafiltration. After thawing the aforementioned supernatant, add 5 times the weight of ultrapure water, mix evenly and pour it into the inlet liquid cylinder of the pellicon tangential flow ultrafiltration system. Adjust the inlet pressure to 30psi and the reflux pressure to 10psi. Use 10kDa ultrapure water at 4°C. Perform ultrafiltration treatment on the filter membrane, collect the ultrafiltrate, use a rotary evaporator, and set the water bath temperature to 55°C for concentration. The concentrated solution was freeze-dried using a Labconco freeze dryer under operating parameters of -50°C and 0.20mBar to obtain Tiliapia skin peptides (TSP).

Example 2

The tilapia skin polypeptide prepared in Example 1 was subjected to an in vitro anti-muscle atrophy activity test:

(1) Culture and differentiation of C2C12 mouse fibroblasts

Cell culture C2C12 mouse fibroblasts are cells commonly used internationally to study muscle pathology, physiology and pharmacological effects. After recovery, the mouse C2C12 fibroblast cell line (purchased from Nanjing Kebai Biotechnology Co., Ltd.) was cultured in DMEM medium (C11995500BT, GIBCO, USA): C2C12 cells were inoculated into cells containing 10% (v/v) fetal bovine Serum and 1wt% penicillin sulfate/streptomycin sulfate in DMEM medium were placed in a 37°C constant-temperature incubator containing 5% _CO2 , and the medium was replaced every two days.

Cell differentiation: C2C12 cell density grows to 70-80%. C2C12 cells are cultured in DMEM medium containing 2% (v/v) horse serum for 5 days. The cells differentiate from round to long strips of myotubes to indicate successful differentiation. .

(2) MTT method to detect the cytotoxicity of tilapia skin peptides

The differentiated C2C12 cells in the logarithmic stage were digested with trypsin, adjusted to an appropriate density with DMEM complete medium containing 10% bovine serum, and evenly seeded into a 96-well plate. When the cell density in the well reaches 80%, the cells are randomly divided into blank group, TSP experimental group 1 (containing 1.0 mg/mL TSP), TSP experimental group 2 (containing 1.5 mg/mL TSP) and TSP experimental group 3. (Contains 2.0mg/mL TSP). Add 100 μL of MTT (thiazolyl blue) working solution (0.5 mg/mL MTT in PBS) to each well and incubate at 37°C for 3 h. After removing the MTT solution, dissolve the purple crystals formed within the cells by adding 200 μL DMSO (dimethyl sulfoxide) to each well. After shaking, detect the absorbance value of each well at 490 nm. Calculate the relative viability of cells in each group according to the following formula:

Relative cell viability = (OD experimental group/OD blank group) × 100%

(3) The improving effect of tilapia skin peptide on muscle atrophy cell model

Inoculate the differentiated C2C12 cells into a 96-well plate according to the method in step (2), and randomly divide the cells into blank group, model group, and TSP intervention group 1, 2, and 3. After the cell density in the well reached 70%, the liquid in the well was sucked off, and 200 μL/well of DMEM maintenance solution containing 1% bovine serum was added to the blank group, and 200 μL/well of DMEM maintenance solution containing 10 μM DEX was added to the model group. TSP intervention Groups 1, 2, and 3 added 200 μL/well of maintenance solution containing 10 μM DEX+1.0 mg/mL TSP, 10 μM DEX+1.5 mg/mL TSP, and 10 μM DEX+2.0 mg/mL TSP respectively. After 24 hours, the relative viability of cells in each group was detected according to the MTT method in step (2), and the relative viability of cells reflected the in vitro anti-muscle atrophy activity of TSP.

Western blot measured the expression levels of proteins Atrogin-1 and MURF1 in cells of the blank group, model group and intervention group. Add protein lysis buffer to lyse the cells, and centrifuge (12000 rpm, 12 min) to obtain the supernatant. Use BCA kit to detect the protein concentration of cell lysis supernatant. Mix the cell lysis supernatant to be tested and the loading buffer (4×) at a ratio of 1:3, cook in boiling water for 5 minutes, and store in a -20°C refrigerator. After the sample proteins were separated by SDS-polyacrylamide gel electrophoresis, they were electrotransferred to a PVDF membrane. The PVDF membrane was blocked with TBST containing 5% skimmed milk powder for 1 hour. The PVDF membrane was coated with primary antibodies Atrogin-1 (1:1000) and MuRF1. (1:1000) overnight (4°C). The next day, the PVDF membrane was washed with TBS containing 0.1% Tween-20, incubated with HRP-labeled secondary antibody at room temperature for 2 hours, and washed again with TBS containing 0.1% Tween-20 for 15 minutes. Soak the PVDF membrane in chemiluminescent solution for 1 minute, use a gel imaging system for exposure imaging, and use Image Pro Plus 6.0 for optical density analysis.

(4)Data processing

All experimental data in this example were analyzed by One-way ANOVA using the software GraphPad Prism 8.0. One-way ANOVA further performed Dunnett's method for multiple comparisons. All data are expressed as mean±standard error Indicates; compared with the blank group, ^# p＜0.05, ^## p＜0.01; compared with the model group, ^* p＜0.05, ^** p＜0.01.

(5)Experimental results

The evaluation results of the in vitro anti-muscle atrophy activity of tilapia skin peptides are shown in Figures 1 and 2. Tilapia skin peptide has no cytotoxicity to C2C12 (Figure 1). 1.0~2.0 mg/mL tilapia skin peptide can significantly restore the relative viability of C2C12 cells induced by 10 μM DEX (Figure 2). Tilapia skin peptide significantly inhibited the up-regulation of Atrogin-1 and MURF1 protein expression levels in C2C12 cells caused by dexamethasone (Figure 3), indicating that tilapia skin peptide has the effect of promoting proliferation and inhibiting muscle atrophy of C2C12 cells. .

Example 3

The tilapia skin polypeptide prepared in Example 1 was tested for in vivo anti-muscle atrophy activity:

(1) Animal feeding

Four-week-old C57BL/6 male mice purchased from the Guangdong Provincial Medical Experimental Animal Center were raised in a standard animal room. Maintain the temperature of the animal room at 25±1°C and the air humidity between 50% and 60%. Set the lighting system to turn on at 8:00 in the morning and turn off at 20:00 in the evening. The animal experiment protocol was approved by the Animal Ethics Committee of Guangdong Ocean University and was conducted in compliance with the requirements of the "Guidelines for the Care and Use of Laboratory Animals of Guangdong Ocean University". The mice were not restricted in food and water during the animal experiments.

(2) Animal grouping and drug intervention

An animal model of muscle wasting disease was constructed using dexamethasone subcutaneous injection. 45 C57BL/6 male mice (7-8 weeks) were randomly divided into 5 groups. Control group: Oral administration of pure water + subcutaneous injection of physiological saline. Model group: subcutaneous injection of 20 mg/kg DEX + pure water orally. Intervention group: subcutaneous injection of 20mg/kg DEX+400mg/kg TSP intervention. Except for the control group, mice in other groups were injected subcutaneously with DEX at a dose of 20 mg/kg per day for 8 days, followed by continuous TSP intervention or oral administration of pure water for 8 days. Mice were placed at controlled room temperature with a dark-light cycle (12 hours: 12 hours). Measure relevant behavioral indicators.

(3) Recording of mouse body weight and muscle weight

All mice were weighed every two days for determination and changes in body weight were recorded. Mice were anesthetized with barbiturate reagent, and muscle tissues, including quadriceps femoris (Quad), gastrocnemius (Gast), tibialis anterior (TA), and soleus (Sol), were collected and weighed for relevant examinations.

(4) Grip strength measurement

The mouse grip strength was tested using a rat and mouse dynamometer (XR501, Shanghai Xinruan Company, China). Place the mouse on the tension net, pull the tail evenly and horizontally, and record the pulling force value. This procedure was repeated three times.

(5)Running endurance measurement

The running distance of mice was measured using a treadmill (ZH-PT, Anhui Zhenghua Biological Instrument Equipment Co., Ltd., China). Based on previous experiments, the treadmill test protocol was slightly modified. Mice ran on a treadmill for 10 min at an initial speed of 12 m/min and increased the speed by 2 m/min every 2 min until the speed reached 16 m/min. Additional electrical stimulation was applied at the end of each track, and the distance run by the mouse when it fell from the track was recorded until the mouse was exhausted, and the entire running distance of the mouse was recorded.

(6)Body composition analysis

Magnetic resonance imaging (MRI, Neumed Analytical Instruments Co., Ltd., China) was used to determine the body composition and fat distribution (i.e., lean body mass and fat mass) of each mouse. Each mouse was weighed before the test and then placed on the cylindrical stand and scanned for 1 to 2 min.

(7)H&E staining and cross-sectional area statistics

4 μm paraffin sections of tibialis anterior muscle and gastrocnemius muscle were placed at room temperature for 30 min, fixed in PBS containing 4% paraformaldehyde for 30 min, and rinsed with PBS for 10 min to remove the fixative. Tissue sections were sequentially deparaffinized with xylene and dehydrated in an ascending ethanol series. The sections were stained with hematoxylin for 5 min and then rinsed with tap water. Then, use hydrochloric acid ethanol for 30 seconds to identify, rinse with tap water, turn blue with 0.6% ammonia water, and rinse with tap water. The sections were stained with eosin for 2 min. The shooting records were evaluated under a light microscope, and Image Pro Plus 6.0 software was used for muscle cross-sectional area statistics.

(8) Western blot determination of related protein expression in mouse muscles

Place the tibialis anterior muscle tissue in a test tube, add protein lysate and steel beads, homogenize using a tissue grinder and centrifuge (12000 rpm, 12 min) to obtain the supernatant. According to the instruction manual, use the BCA kit to detect the protein concentration of the supernatant. Mix the supernatant and loading buffer (4×) at a ratio of 1:3, cook in boiling water for 5 minutes, and store in a -20°C refrigerator. After the sample proteins were separated by SDS-polyacrylamide gel electrophoresis, they were electrotransferred to a PVDF membrane. The PVDF membrane was blocked with TBST containing 5% skimmed milk powder for 1 hour. The PVDF membrane was coated with primary antibodies Atrogin-1 (1:1000) and MuRF1. (1:1000) overnight (4°C). The next day, the PVDF membrane was washed with TBS containing 0.1% Tween-20, incubated with HRP-labeled secondary antibody at room temperature for 2 hours, and washed again with TBS containing 0.1% Tween-20 for 15 minutes. Soak the PVDF membrane in chemiluminescent solution for 1 minute, use a gel imaging system for exposure imaging, and use Image Pro Plus 6.0 for optical density analysis.

(7)Data processing

All experimental data in this example were analyzed by One-way ANOVA and Two-way ANOVA using the software GraphPad Prism 8.0. One-way ANOVA and Two-way ANOVA further performed Dunnett's method for multiple comparisons. All data are expressed as mean±standard error Indicates; compared with the blank group, ^# p＜0.05, ^## p＜0.01; compared with the model group, ^* p＜0.05, ^** p＜0.01.

(8)Experimental results

In vivo experimental results of tilapia skin peptides show that tilapia skin peptides can significantly improve mouse grip strength (Figure 4), running distance (Figure 5), and muscle tissue (including quadriceps, gastrocnemius, and tibialis anterior muscles). ) mass (Figure 6) and tibialis anterior muscle cross-sectional area (Figures 7 and 8). The tilapia skin polypeptide can significantly increase lean meat content, reduce fat content in mice (Figure 9), and inhibit the up-regulation of the expression levels of muscle atrophy-related proteins Atrogin-1 and MURF1 induced by DEX (Figure 10).

The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

Claims (3)

1. The application of a tilapia skin polypeptide in the preparation of products for preventing and treating dexamethasone-induced muscle atrophy, characterized in that the preparation method of the tilapia skin polypeptide includes the following steps: (1) Remove the scales from the tilapia skin, crush it, and then dry it to obtain tilapia skin dry powder; (2) After the tilapia skin dry powder is subjected to enzymatic hydrolysis and enzyme-killing treatment, an enzymatic hydrolysis liquid is obtained; (3) Perform ultrafiltration treatment on the enzymatic hydrolyzate, collect the filtrate with a molecular weight below 10 kDa, and then concentrate and freeze-dry to obtain the tilapia skin polypeptide; In step (2), the enzymatic hydrolysis includes the step of first adding neutral protease for enzymatic hydrolysis, and then adding alkaline protease to continue enzymatic hydrolysis; the added mass of the neutral protease is the mass of the tilapia skin dry powder 3%; the added mass of the alkaline protease is 0.3% of the mass of the tilapia skin dry powder; The conditions for adding the neutral protease for enzymatic hydrolysis include: pH is 7.0±0.2, temperature is 50±5°C, and enzymatic hydrolysis time is 2 hours; The conditions for adding the alkaline protease for enzymatic hydrolysis include: pH is 9.0±0.2, temperature is 50±5°C, and enzymatic hydrolysis time is 2 hours. 2. The application according to claim 1, characterized in that in step (1), the reagent used in the degreasing treatment includes isopropyl alcohol. 3. The application according to claim 1, characterized in that the product is a medicine.