WO2023040084A1

WO2023040084A1 - Method for extracting collagen tripeptide and prepared collagen tripeptide

Info

Publication number: WO2023040084A1
Application number: PCT/CN2021/137047
Authority: WO
Inventors: 赵子方; 郭红星; 周尽学
Original assignee: 海南三元星生物科技股份有限公司
Priority date: 2021-09-16
Filing date: 2021-12-10
Publication date: 2023-03-23
Also published as: CN113789360A

Abstract

A method for extracting collagen tripeptide, a collagen tripeptide prepared using said method, and an application thereof in preparing an antioxidant product. The preparation method comprises: crushing fish skin, then washing the fish skin with a NaOH solution, distilled water, n-hexane, and distilled water; mixing the washed fish skin with water, swelling and homogenizing, adding alkaline protease and papain in a mass ratio of 1:3, the total amount of enzymes being 110 U/g, performing enzymolysis at a pH value of 7.5 and a temperature 75°C for 6 hours, and then performing ultrasonic treatment at 600 W for 45 minutes; performing enzyme deactivation and centrifugation on the solution obtained after ultrasonic treatment, and collecting the supernatant to obtain collagen tripeptide.

Description

Extraction method of collagen tripeptide and prepared collagen tripeptide

This application claims the priority of the Chinese patent application submitted to the China Patent Office on September 16, 2021, with the application number 202111087100.9, and the title of the invention is "Collagen Tripeptide Extraction Method and Prepared Collagen Tripeptide", the entire content of which is incorporated by reference incorporated in this application.

technical field

The invention relates to the technical field of extracting active substances, in particular to a method for extracting collagen tripeptide and the prepared collagen tripeptide.

Background technique

Collagen is an extracellular protein consisting of two or more amino acids - protein peptides. The absorption of the human body is carried out in the form of peptides. The absorption and utilization rate of edible protein peptides can reach 100%. Collagen is the most important part of the extracellular matrix. The sources of collagen peptides mainly include natural existence, proteolysis and artificial synthesis. , natural peptides exist in animals, the extraction process is difficult, and the yield rate is very small. Artificial synthesis methods include chemical synthesis, enzymatic synthesis and DNA recombination technology, but the cost is expensive, the technical operation is difficult, and the practicability is very low. Therefore, the current peptides are mainly made of Collagen peptide obtained by protein hydrolysis is the product of enzymatic hydrolysis of collagen or gelatin, and the disintegration and breakage of molecular chains, which contains all the amino acids of collagen. Peptide is a structural fragment of a protein molecule, formed by dehydration condensation of 2 or more amino acids, with a small molecular weight and easy absorption. Collagen peptides usually have a unique glycine-proline-hydroxyproline repeating sequence structure endowed with Its special biological activity, when the amino acid structure in the peptide changes, the biological function of the peptide will also change, so there are thousands of types of peptides, and the functions are also diverse. Collagen peptides have antioxidant activity, reduce Blood pressure activity, antibacterial activity, bone health promotion activity and good moisture absorption and moisturizing properties can repair and repair elastin fibers and endogenous collagen, and reduce damage caused by ultraviolet radiation. It plays an important role and makes full use of collagen to produce active peptides And improving its added value not only saves resources, reduces environmental pollution, but also increases economic benefits.

As far as the current extraction and preparation process of collagen peptides is actually used, most of them use a single method such as enzymatic hydrolysis, acid hydrolysis, and alkaline hydrolysis to extract collagen. The extraction rate of a single enzymatic hydrolysis is not high, so that the extraction rate of raw materials is low. The utilization rate is low, and the extraction rate of collagen is low, resulting in waste of resources. At the same time, when mechanical energy is used to extract collagen with chemical methods, pollution is easy to occur, the process is complicated, and the cost is high, which is not conducive to the extraction and use of collagen peptides.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a method for extracting collagen tripeptide with high victorious activity and the prepared collagen tripeptide.

The preparation method of collagen tripeptide provided by the invention comprises the following steps:

Step 1: After crushing the fish skin, wash it with NaOH solution, distilled water, n-hexane, and distilled water;

Step 2: Mix the washed fish skin with water, after swelling and homogenizing, add alkaline protease and papain, the pH value is 7.5, enzymatic hydrolysis at 75°C for 6 hours, and then 600W ultrasonic treatment for 45 minutes;

Step 3: The solution after ultrasonication is inactivated, centrifuged, and the supernatant is collected to obtain collagen tripeptide;

The mass ratio of alkaline protease and papain is 1:3, and the total amount of enzyme added is 110U/g.

In step 1, the fish skin is the skin of herring. The fish skin was shredded to a size of 10 mm x 10 mm.

In step 1, the concentration of NaOH in the NaOH solution is 0.1 mol/L; the cleaning condition of the NaOH solution is 4° C. and stirring for 1 h.

In step 1, the mass ratio of the fish skin to n-hexane is 1:9, and the cleaning condition of the n-hexane is soaking at 4° C. for 6 h.

In step 2, the ratio of fish skin to water is 1:20.

In step 2, the alkaline protease is an alkaline protease solution, and the papain is a papain solution; the alkaline protease solution has the same mass concentration as the papain solution.

In step 3, the condition for inactivating the enzyme is 95° C. for 10 minutes. The centrifugation conditions include: 4° C., 12000 r/min, centrifugation for 15 minutes.

The supernatant in step 3 is spray-dried, and the spray-drying conditions are that the air inlet temperature is 180°C, the outlet air temperature is 80°C, and the feed temperature is 35°C.

In step 3, the temperature control instrument of the enzymolysis adopts SYC-6 water bath shaker, the equipment for measuring the pH value in the enzymolysis is STARTE2100 type laboratory pH meter, and the equipment used for the centrifugal treatment is TDL-40B bench-top low-speed Large-capacity centrifuge, the raw material pretreatment freezing equipment is Alpha1-2LDplus freeze dryer.

Collagen tripeptide prepared by the preparation method of the invention.

The application of the collagen tripeptide prepared by the preparation method of the invention in the preparation of anti-oxidation products.

An anti-oxidation product, which includes the collagen tripeptide prepared by the preparation method of the present invention.

The collagen tripeptide extraction method provided by the present invention adopts an enzymatic method to extract fish skin, and extracts collagen peptides from fish skin by using alkaline protease and papain in a ratio of 1:3 to extract and prepare collagen peptides, and simultaneously adds 600W ultrasonic waves Treat for 45 minutes, carry out the auxiliary extraction of enzymatic hydrolysis, effectively increase the thoroughness of enzymatic hydrolysis of fish skin, and increase the extraction rate of collagen peptide in fish skin by 15% to 25%, thereby effectively increasing the collagen peptide of mixed enzymatic hydrolysis With high extraction efficiency, the extraction rate of collagen peptide is as high as 92%, and the extraction time is significantly shortened, the energy consumption is low, and the extraction cost can be reduced.

Description of drawings

Fig. 1 is a process flow diagram of the present invention.

Detailed ways

The present invention provides a method for extracting collagen tripeptide and the prepared collagen tripeptide. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is a relationship between these entities or operations. There is no such actual relationship or order between them. However, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device.

Acid method, alkali method, salt method, enzymatic method, etc. are the main methods for extracting collagen from animal tissues. Among them, acid method extraction can maintain its triple helical structure to the greatest extent, with good biological characteristics and good fibrogenesis performance. Applicable to biological materials, the preparation of collagen by the alkaline method is rarely used due to the long cycle and many steps, and the utilization value of the product is low. Different salt types in salt extraction also affect the stability of collagen, some can improve the stability of collagen, while others will reduce the stability of conformation, which restricts the extraction of collagen. The collagen extracted by the enzymatic method has high purity, stable physical and chemical properties, and good water solubility, which greatly shortens the extraction time and reduces environmental pollution. Therefore, the present invention adopts an enzymatic hydrolysis method to enzymatically hydrolyze the collagen.

Fish skin is rich in collagen. In the present invention, herring skin is selected as the raw material for extracting collagen polypeptide, so that the extraction rate of collagen polypeptide is higher, the extraction amount of collagen is more, and the extraction is more convenient.

The pretreatment of the raw material includes removing the head and viscera of the live herring, splitting the fish skin into two pieces, separating the fish skin from the fish meat, removing the scales and fins from the separated fish skin, rinsing it with clean water, and drying it naturally in a cool place , and then cut the clean fish skin into pieces with scissors, the size of the pieces is 10mm×10mm, put them in a sealed bag and freeze them at -20°C for later use.

Degreasing the fish skin by soaking in n-hexane, preventing the fat content in the fish skin from being too high and reducing the extraction rate of collagen peptides, ensuring that the content of other components other than collagen in the fish skin is the lowest, and avoiding extraction When the error is large, so as to prevent other substances from interfering with the extraction. When degreasing, the material-liquid ratio of fish skin and n-hexane is 1:9, fully stir after mixing, and the equipment used for weighing is PTX-FA201S electronic balance,

The enzymolysis includes adjusting the pH to 7.5 and then soaking and swelling the fish skin. The swelling time is 12 hours, stirring and homogenizing after swelling, and adding a mixed enzyme solution to the homogenized solution. By setting the pH gradient value of the enzymolysis For experimental comparison, other variables remained unchanged, the pH value was a single variable, and the pH values were set to 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 to study the effect of pH on the collagen extraction effect. It can be known through experiments that the extraction rate of collagen is the best when the pH value is 7-8, and the extraction rate of collagen is not ideal when the pH value is lower or higher than this pH value range, which is different from the active pH range of alkaline protease and papain Therefore, the pH of enzymatic hydrolysis was chosen to be 7.5 as the best extraction value.

In the embodiment, by setting the ultrasonic power to 300, 400, 500, 600, 700, and 800W respectively, and keeping other environments unchanged, the influence of ultrasonic power on the extraction effect of collagen is studied. When the ultrasonic power is between 300W-600W, The extraction rate of collagen is positively correlated with the ultrasonic power. With the increase of ultrasonic power, a large amount of solvent penetrates into the cells, thereby increasing the extraction rate. When the ultrasonic power reaches 600W, the extraction rate of collagen reaches the highest value. The increase of the power decreases instead, because the ultrasonic power with too much power destroys the molecular structure of fish skin collagen, which leads to a decrease in the extraction rate of collagen, so the ultrasonic power is 600W as the optimal power.

Ultrasound-assisted enzymatic hydrolysis can effectively improve the extraction rate of collagen, shorten the extraction time significantly, and the power of ultrasound is low, which can reduce the cost of extraction. In the embodiment, the gradient value of ultrasonic ultrasonic time is set in turn for testing. The extraction rate of collagen increases continuously, and when it exceeds 50 minutes, the extraction rate is almost unchanged, indicating that basically all collagen is dissolved. However, if the ultrasonic treatment time is too long, the system temperature may be too high to reduce the activity of the enzyme or destroy the enzyme. The structure of collagen, therefore, choose ultrasonic extraction time as 45min.

In some embodiments, the temperature control instrument for enzymolysis adopts SYC-6 water bath shaker, the equipment for measuring pH value in enzymolysis is STARTE2100 laboratory pH meter, and the equipment for centrifugal treatment is TDL-40B desktop low-speed large-capacity centrifuge. The raw material pretreatment freezing equipment is Alpha1-2LDplus freeze dryer.

The parameters and work of the environment in collagen extraction are controlled by various equipment, which is convenient for the extraction of collagen. The drying and collection adopts a pressure spray dryer, and the inlet air temperature is set at 180°C, and the outlet air temperature is 80°C. The feed temperature was 35°C.

The present invention uses alkaline protease and papain to extract and prepare collagen peptides from herring skin by a mixed enzymolysis method at a ratio of 1:3, and at the same time adds a 600W ultrasonic wave for 45 minutes to perform auxiliary extraction of enzymolysis, effectively increasing the amount of enzymolysis fish skin The degree of thoroughness increases the extraction rate of collagen peptides in fish skin by 15%-25%, thereby effectively increasing the extraction efficiency of mixed enzymatically hydrolyzed collagen peptides, making the extraction rate of collagen peptides as high as 92%.

The test materials used in the present invention are all common commercially available products, which can be purchased in the market.

Below in conjunction with embodiment, further set forth the present invention:

Example 1:

As shown in Figure 1, a process method for extracting and enriching highly active collagen tripeptides comprises the following steps:

Sp1: Raw material pretreatment:

Remove the head and viscera of the live herring, cut it into two pieces, separate the skin from the flesh, remove the scales and fins from the separated fish skin, rinse it with clean water, dry it naturally in the shade, and then use scissors to clean it Cut the fish skin into pieces, the size of which is 10mm×10mm, put them into a sealed bag and freeze them at -20°C for later use.

Sp2: Removal of impurities and decolorization: Take out the refrigerated fish skin, store it at 4°C, soak the fish skin in 0.1mol/L NaOH solution for 1 hour to remove impurities and decolorization, wash it with distilled water and set aside;

Sp3: Degreasing: Soak the decontaminated and decolorized fish skin in n-hexane for 6 hours to remove fat, take it out and wash it with distilled water for later use, the volume of n-hexane is twice the volume of the fish skin, and the degreasing ambient temperature is 4°C ;

Sp4: Enzyme hydrolysis: Weigh the degreased fish skin, add distilled water, the ratio of material to liquid is 1:20, adjust the pH to 7.5, then soak and swell the fish skin, the swelling time is 12 hours, stir and homogenize after swelling, Use hydrochloric acid and sodium hydroxide to adjust the pH to 7.5, add a mixed enzyme solution of alkaline protease and papain for enzymolysis, the mixing ratio is 1:3, the amount of enzyme added is 110U/g, the enzymolysis temperature is 75°C, and the enzymolysis time 6h;

Sp5: Ultrasonic-assisted enzymolysis: Ultrasonic treatment is performed on the enzymolysis container during enzymolysis, the ultrasonic power is 600W, and the treatment time is 45min;

Sp6: Enzyme inactivation: Put the solution after enzymatic hydrolysis at 100°C for 15 minutes to inactivate the enzyme;

Sp7: Centrifugation: Cool the solution after deactivation to 4°C, and then centrifuge the cooled solution at a speed of 12000r/min for 15 minutes;

Sp8: dry collection: take the solution after centrifugation and take the supernatant to spray dry, set the inlet air temperature to 180°C, the outlet air temperature to 80°C, and the feed temperature to 35°C to obtain a white powdery crude peptide with a yield of 92% .

After testing, the product has good scavenging ability for free radicals DPPH, OH and superoxide anion free radicals.

The above are only preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can also be made without departing from the principle of the present invention, and these improvements and modifications should also be considered Be the protection scope of the present invention.

Claims

The preparation method of collagen tripeptide, is characterized in that, comprises the steps:

Step 1: After crushing the fish skin, wash it with NaOH solution, distilled water, n-hexane, and distilled water;

Step 2: Mix the washed fish skin with water, after swelling and homogenizing, add alkaline protease and papain, the pH value is 7.5, enzymatic hydrolysis at 75°C for 6 hours, and then 600W ultrasonic treatment for 45 minutes;

Step 3: The solution after ultrasonication is inactivated, centrifuged, and the supernatant is collected to obtain collagen tripeptide;

The mass ratio of alkaline protease and papain is 1:3, and the total amount of enzyme added is 110U/g.
The preparation method according to claim 1, characterized in that in step 1, the concentration of NaOH in the NaOH solution is 0.1 mol/L; the cleaning condition of the NaOH solution is 4° C. and stirring for 1 h.
The preparation method according to claim 1, characterized in that, in step 1, the mass ratio of the fish skin to n-hexane is 1:9, and the cleaning condition of the n-hexane is soaking at 4°C for 6h.
The preparation method according to claim 1, characterized in that in step 2, the ratio of fish skin to water is 1:20.
preparation method according to claim 1, is characterized in that, in step 2, described alkaline protease is alkaline protease solution, and described papain is papain solution; The quality of described alkaline protease solution and papain solution Concentrations are equal.
The preparation method according to claim 1, characterized in that, in step 3, the centrifugation conditions include: 4°C, 12000r/min, centrifugation for 15min.
The preparation method according to claim 1, characterized in that the supernatant in step 3 is spray-dried, and the spray-dried conditions are that the air inlet temperature is 180°C, the outlet air temperature is 80°C, and the feed temperature is 35°C. ℃.
The collagen tripeptide prepared by the preparation method described in any one of claims 1-7.
The application of the collagen tripeptide prepared by the preparation method described in any one of claims 1 to 7 in the preparation of anti-oxidation products.
An anti-oxidation product comprising the collagen tripeptide prepared by the preparation method described in any one of claims 1-7.