阅读说明：本技术 胶原三肽的提取方法及制备的胶原三肽 (Method for extracting collagen tripeptide and collagen tripeptide prepared by same ) 是由 赵子方 郭红星 周尽学 于 2021-09-16 设计创作，主要内容包括：本发明涉及活性物质提取技术领域,尤其涉及胶原三肽的提取方法及制备的胶原三肽。本发明提供的胶原三肽提取方法采用酶法对鱼皮进行提取,通过采用碱性蛋白酶和木瓜蛋白酶以1:3比例混合酶解法对鱼皮进行胶原蛋白肽的提取制备,同时加以600W的超声波进行处理45min,进行酶解的辅助提取,有效增加酶解鱼皮的彻底程度,使鱼皮中对胶原蛋白肽的提取率提高了15％～25％,从而有效增加混合酶解的胶原蛋白肽的提取效率,使胶原蛋白肽提取率高达92％,且提取时间明显缩短,能耗低,能够降低提取成本。(The invention relates to the technical field of active substance extraction, in particular to a method for extracting collagen tripeptide and the prepared collagen tripeptide. The method for extracting the collagen tripeptide provided by the invention adopts an enzyme method to extract the fish skin, the alkali protease and the papain are adopted to carry out extraction and preparation of the collagen peptide by a mixed enzymolysis method in a ratio of 1:3, and meanwhile, 600W of ultrasonic wave is added for treatment for 45min to carry out auxiliary extraction of enzymolysis, so that the thorough degree of enzymolysis of the fish skin is effectively increased, the extraction rate of the collagen peptide in the fish skin is improved by 15-25%, the extraction efficiency of the collagen peptide subjected to mixed enzymolysis is effectively increased, the extraction rate of the collagen peptide is up to 92%, the extraction time is obviously shortened, the energy consumption is low, and the extraction cost can be reduced.)

1. The preparation method of the collagen tripeptide is characterized by comprising the following steps:

step 1: crushing fish skin, and then cleaning with NaOH solution, distilled water, normal hexane and distilled water;

step 2: mixing the cleaned fish skin with water, swelling, homogenizing, adding alkaline protease and papain, performing enzymolysis at 75 deg.C and pH of 7.5 for 6 hr, and performing ultrasonic treatment at 600W for 45 min;

and step 3: inactivating enzyme and centrifuging the solution after ultrasonic treatment, and collecting supernatant to obtain collagen tripeptide;

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

2. The preparation method according to claim 1, wherein in step 1, the concentration of NaOH in the NaOH solution is 0.1 mol/L; the NaOH solution is stirred for 1h at 4 ℃.

3. The preparation method according to claim 1, wherein in the step 1, the mass ratio of the fish skin to the n-hexane is 1:9, and the n-hexane is washed under 4 ℃ for soaking for 6 hours.

4. The preparation method according to claim 1, wherein in the step 2, the ratio of the fish skin to the water is 1: 20.

5. the method according to claim 1, wherein in step 2, the alkaline protease is an alkaline protease solution, and the papain is a papain solution; the mass concentration of the alkaline protease solution is equal to that of the papain solution.

6. The method according to claim 1, wherein in step 3, the centrifugation conditions include: centrifuging at 12000r/min at 4 deg.C for 15 min.

7. The method according to claim 1, wherein the supernatant in step 3 is dried by spraying, and the conditions of the drying are that the inlet air temperature is 180 ℃, the outlet air temperature is 80 ℃ and the inlet air temperature is 35 ℃.

8. A tripeptide of collagen obtainable by the process according to any one of claims 1 to 7.

9. Use of a collagen tripeptide prepared by the preparation method according to any one of claims 1 to 7 in the preparation of an antioxidant product.

10. An antioxidant product comprising a collagen tripeptide prepared by the preparation method of any one of claims 1 to 7.

Technical Field

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

Background

Collagen is an extracellular protein, which is composed of two or more amino acids, namely protein peptide. The absorption of human body is carried out in a peptide mode, the absorption utilization rate of edible protein peptide can reach 100%, collagen is the most important component in extracellular matrix, the source of the collagen peptide mainly comprises natural existence, proteolysis and artificial synthesis, the natural peptide exists in the animal body, the extraction process is difficult, the yield is low, the artificial synthesis method comprises chemical synthesis, enzymatic synthesis and DNA recombination technology, but the cost is high, the technical operation is difficult, and the practicability is low, so the collagen peptide mainly obtained by proteolysis of the existing peptide is a product obtained by enzymolysis of collagen or gelatin and molecular chain disintegration and breakage, and contains all amino acids of the collagen. The peptide is a structural fragment forming protein molecules, is formed by dehydration condensation of 2 or more than 2 amino acids, has small molecular weight and easy absorption, and the collagen peptide generally has a unique repeated sequence structure of glycine-proline-hydroxyproline to endow the collagen peptide with special biological activity, when the amino acid structure of the peptide is changed, the biological function of the peptide is changed, so that the variety of the peptide is thousands, the function is diversified, the collagen peptide has antioxidant activity, antihypertensive activity, antibacterial activity, bone health promotion activity and good moisture absorption and retention, can repair and repair elastin fiber and endogenous collagen, reduce the damage caused by ultraviolet radiation, have important function, fully utilize collagen to produce active peptide and improve the added value thereof, not only save resources, reduce environmental pollution, but also increase economic benefit.

At present, when the extraction preparation process of collagen peptide is actually used, single methods such as enzymolysis, acidolysis and alkaline hydrolysis are mostly adopted for extracting collagen, the single enzymolysis extraction rate is low, the extraction rate of raw materials is low, the utilization rate is low, the extraction rate of collagen is low, the waste of resources is caused, and meanwhile, when the collagen peptide is extracted by matching with a chemical method and machinery, the pollution is easy to generate, the process is complex, the cost is high, and the extraction and the use of collagen peptide are not facilitated.

Disclosure of Invention

In view of the above, the present invention provides a method for extracting a collagen tripeptide with high activity and a collagen tripeptide prepared by the method.

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

step 1: crushing fish skin, and then cleaning with NaOH solution, distilled water, normal hexane and distilled water;

step 2: mixing the cleaned fish skin with water, swelling, homogenizing, adding alkaline protease and papain, performing enzymolysis at 75 deg.C and pH of 7.5 for 6 hr, and performing ultrasonic treatment at 600W for 45 min;

and step 3: inactivating enzyme and centrifuging the solution after ultrasonic treatment, and collecting supernatant to obtain collagen tripeptide;

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

In the step 1, the fish skin is the skin of black carp. The fish skin is minced to a size of 10mm x 10 mm.

In the step 1, the concentration of NaOH in the NaOH solution is 0.1 mol/L; the NaOH solution is stirred for 1h at 4 ℃.

In the step 1, the mass ratio of the fish skin to the n-hexane is 1:9, and the n-hexane is cleaned under the condition of soaking for 6 hours at 4 ℃.

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

in the step 2, the alkaline protease is an alkaline protease solution, and the papain is a papain solution; the mass concentration of the alkaline protease solution is equal to that of the papain solution.

In step 3, the enzyme deactivation condition is that the temperature is kept at 95 ℃ for 10 min. The conditions of the centrifugation include: centrifuging at 12000r/min at 4 deg.C for 15 min.

And (3) spraying the supernatant in the step 3, wherein the air inlet temperature is 180 ℃, the air outlet temperature is 80 ℃, and the feeding temperature is 35 ℃.

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

The collagen tripeptide prepared by the preparation method of the invention.

The collagen tripeptide prepared by the preparation method is applied to preparing an antioxidant product.

An antioxidant product comprises the collagen tripeptide prepared by the preparation method.

The method for extracting the collagen tripeptide provided by the invention adopts an enzyme method to extract the fish skin, the alkali protease and the papain are adopted to carry out extraction and preparation of the collagen peptide by a mixed enzymolysis method in a ratio of 1:3, and meanwhile, 600W of ultrasonic wave is added for treatment for 45min to carry out auxiliary extraction of enzymolysis, so that the thorough degree of enzymolysis of the fish skin is effectively increased, the extraction rate of the collagen peptide in the fish skin is improved by 15-25%, the extraction efficiency of the collagen peptide subjected to mixed enzymolysis is effectively increased, the extraction rate of the collagen peptide is up to 92%, the extraction time is obviously shortened, the energy consumption is low, and the extraction cost can be reduced.

Drawings

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

Detailed Description

The invention provides an extraction method of collagen tripeptide and the prepared collagen tripeptide, and the technical personnel can use the contents for reference and appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. However, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

The acid method, the alkaline method, the salt method, the enzyme method and the like are main methods for extracting collagen from animal tissues, wherein the acid method can maintain the three-strand helical structure of the collagen to the maximum extent, has good biological characteristics and good fiber forming performance, is suitable for biological materials, and the alkaline method for preparing the collagen has long period, more steps are rarely adopted, and the utilization value of the product is low. Different salt types in the salt extraction process also influence the stability of the collagen, some salts can improve the stability of the collagen, and some salts can reduce the conformational stability, thereby restricting the extraction of the collagen. The collagen extracted by the enzyme method has high purity, stable physicochemical property and good water solubility, greatly shortens the extraction time and reduces the environmental pollution, so the invention adopts the enzymolysis method to carry out the enzymolysis of the collagen.

The fish skin contains rich collagen, and the black carp skin is selected as the raw material for extracting the collagen polypeptide, so that the extraction rate of the collagen polypeptide is higher, the extraction amount of the collagen is more, and the extraction is more convenient

The raw material pretreatment comprises the steps of removing heads and internal organs of live black carps, separating skin from fish meat after cutting the live black carps into two pieces, removing scales and fins of the separated skin, washing the skin with clear water, naturally drying the skin in the shade, cutting the clean skin into fragments with the size of 10mm multiplied by 10mm by using scissors, putting the fragments into a sealing bag, sealing the sealing bag, and freezing and storing the fragments at the temperature of-20 ℃ for later use.

The fish skin is degreased by soaking in n-hexane, so that the reduction of the extraction rate of collagen polypeptide due to the overhigh fat content in the fish skin is prevented, the content of other components except collagen in the fish skin is ensured to be the lowest, the large error in extraction is avoided, and the interference of other substances in extraction is prevented. The ratio of the fish skin to the normal hexane is 1:9 during degreasing, the fish skin and the normal hexane are fully stirred after mixing, and the device used for weighing is a PTX-FA201S electronic balance,

the enzymolysis comprises the steps of adjusting the pH value to 7.5, soaking and swelling the fish skin, wherein the swelling time is 12 hours, stirring and homogenizing the fish skin after swelling, adding mixed enzyme liquid into the homogenized solution, carrying out experimental comparison by setting the pH gradient value of the enzymolysis, keeping other variables unchanged, setting the pH value to be a single variable and setting the pH values to be 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 respectively, and researching the influence of the pH value on the extraction effect of the collagen. Experiments show that the collagen extraction rate is optimal when the pH value is 7-8, and is lower than or higher than the pH value range, the collagen extraction rate is not ideal and is consistent with the activity pH value range of alkaline protease and papain, so that the pH value of enzymolysis is selected to be 7.5 as the optimal extraction value.

In the embodiment, the ultrasonic power is set to be 300, 400, 500, 600, 700 and 800W respectively, other environments are kept unchanged, the influence of the ultrasonic power on the collagen extraction effect is researched, when the ultrasonic power is 300W-600W, the collagen extraction rate and the ultrasonic power are in positive correlation, a large amount of solvent permeates into cells along with the increase of the ultrasonic power, so that the extraction rate is improved, when the ultrasonic power reaches 600W, the collagen extraction rate reaches the highest value, and then the extraction rate is reduced along with the increase of the ultrasonic power, and the collagen extraction rate is reduced because the molecular structure of the fish skin collagen is damaged by the ultrasonic with overlarge power, so that the ultrasonic power is 600W, so that the optimal power is obtained.

The ultrasonic-assisted enzymolysis method can effectively improve the extraction rate of collagen, obviously shorten the extraction time, reduce the ultrasonic power and reduce the extraction cost, in the embodiment, the gradient value of the ultrasonic time is sequentially set for testing, the extraction rate of the collagen is continuously increased along with the extension of the ultrasonic treatment time, and when the extraction rate exceeds 50min, the extraction rate is almost unchanged, which shows that all the collagen is basically dissolved out, but the overhigh system temperature caused by overlong ultrasonic treatment time can reduce the activity of enzyme or destroy the structure of the collagen, so the ultrasonic extraction time is selected to be 45 min.

In some embodiments, the temperature control apparatus for enzymolysis is a SYC-6 water bath shaker, the apparatus for measuring PH during enzymolysis is a star 2100 laboratory PH meter, the apparatus for centrifugation is a TDL-40B desk-top low-speed large-capacity centrifuge, and the raw material pretreatment freezing apparatus is an Alpha1-2LDplus freeze dryer.

Each parameter and the work of environment are controlled during collagen extraction through each device, collagen extraction is facilitated, drying and collection are achieved by adopting a pressure type spray dryer, the air inlet temperature is set to be 180 ℃, the air outlet temperature is set to be 80 ℃, and the feeding temperature is set to be 35 ℃.

The method adopts alkaline protease and papain to carry out extraction and preparation of the collagen peptide from the black carp skin by a mixed enzymolysis method in a ratio of 1:3, simultaneously carries out treatment for 45min by ultrasonic waves of 600W, carries out auxiliary extraction of enzymolysis, effectively increases the thoroughness of enzymolysis of the fish skin, improves the extraction rate of the collagen peptide from the fish skin by 15 to 25 percent, thereby effectively increasing the extraction efficiency of the collagen peptide by mixed enzymolysis and ensuring that the extraction rate of the collagen peptide reaches 92 percent,

the test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1:

as shown in figure 1, a process for extracting and enriching high-activity collagen tripeptide comprises the following steps:

sp 1: pretreatment of raw materials:

removing head and viscera of live black carp, cutting into two pieces, separating skin from fish, removing scales and fins from the separated skin, washing with clear water, naturally drying in the shade, cutting into pieces with size of 10mm × 10mm, sealing in a sealed bag, and freezing at-20 deg.C.

Sp 2: removing hybrid protein and decoloring: taking out the refrigerated fish skin, storing the fish skin at the temperature of 4 ℃, soaking the fish skin in 0.1mol/L NaOH solution for 1 hour for removing foreign proteins and decoloring, and washing the fish skin with distilled water for later use;

sp 3: degreasing: soaking the fish skin subjected to impurity removal and decolorization in n-hexane for 6h to remove fat, taking out, washing with distilled water for later use, wherein the volume of the n-hexane is twice that of the fish skin, and the degreasing environment temperature is 4 ℃;

sp 4: enzymolysis: weighing the degreased fish skin, adding distilled water, wherein the material-liquid ratio is 1: 20, adjusting the pH to 7.5, soaking and swelling the fish skin for 12 hours, stirring and homogenizing after swelling, adjusting the pH to 7.5 by using sodium hydroxide hydrochloride, adding a mixed enzyme solution of alkaline protease and papain for enzymolysis, wherein the mixing ratio is 1:3, the enzyme adding amount is 110U/g, the enzymolysis temperature is 75 ℃, and the enzymolysis time is 6 hours;

sp 5: ultrasonic-assisted enzymolysis: carrying out ultrasonic treatment on an enzymolysis container during enzymolysis, wherein the ultrasonic power is 600W, and the treatment time is 45 min;

sp 6: enzyme deactivation: putting the solution into 100 deg.C to inactivate enzyme for 15 min;

sp 7: centrifuging: cooling the solution after enzyme deactivation to 4 ℃, and centrifuging the cooled solution at the speed of 12000r/min for 15 min;

sp 8: drying and collecting: and (3) taking the centrifuged solution and taking the supernatant for spray drying, setting the air inlet temperature to be 180 ℃, the air outlet temperature to be 80 ℃ and the feeding temperature to be 35 ℃ to obtain white powdery crude peptide, wherein the yield is 92%.

The detection shows that the product has good cleaning capability on DPPH, OH and superoxide anion free radicals.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.