CN118186037B

CN118186037B - A small molecule hyaluronic acid and its preparation method and application

Info

Publication number: CN118186037B
Application number: CN202410155295.3A
Authority: CN
Inventors: 张晓东
Original assignee: Beijing Wuguan Peptide Dou Biotechnology Co ltd
Current assignee: Zhejiang Shengyan Biomedical Technology Co ltd
Priority date: 2024-02-02
Filing date: 2024-02-02
Publication date: 2025-02-11
Anticipated expiration: 2044-02-02
Also published as: CN118186037A

Abstract

本发明属于生物材料提取技术领域，具体涉及一种小分子透明质酸，更具体涉及一种以鸡冠为原料利用酶催化生物转化法制备小分子透明质酸的方法。本发明所述小分子透明质酸的制备方法，采用生物催化酶解鸡冠子进行透明质酸的提取，采用脂肪酶、α‑半乳糖苷酶和中性蛋白酶的酶制剂体系进行定向酶切及靶向降解，不但能提高透明质酸产品的收率，有利于后续工艺产品纯化；而且，可有效的控制透明质酸的分子量在1500道尔顿以下，含有小分子活性肽和丰富的葡萄糖醛酸，更易于肌体吸收利用。The present invention belongs to the field of biomaterial extraction technology, and specifically relates to a small molecule hyaluronic acid, and more specifically to a method for preparing small molecule hyaluronic acid using cockscomb as raw material by enzyme-catalyzed biotransformation. The preparation method of small molecule hyaluronic acid of the present invention adopts biocatalytic enzymatic hydrolysis of cockscomb to extract hyaluronic acid, and adopts an enzyme preparation system of lipase, α-galactosidase and neutral protease for directional enzyme cutting and targeted degradation, which can not only improve the yield of hyaluronic acid products, but also facilitate the purification of subsequent process products; moreover, the molecular weight of hyaluronic acid can be effectively controlled to be below 1500 Daltons, and it contains small molecule active peptides and rich glucuronic acid, which is easier for the body to absorb and utilize.

Description

Small-molecule hyaluronic acid and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological material extraction, and particularly relates to a method for preparing small-molecule hyaluronic acid by taking cockscomb as a raw material and utilizing an enzyme catalytic bioconversion method.

Background

Hyaluronic acid is an acidic mucopolysaccharide, which was first isolated from bovine vitreous by the university of columbia in the united states, professor Meyer et al, 1934. HA is easily decomposed by acid, alkali or heat treatment, is degraded by oxygen free radical, X-ray, gamma ray, ultraviolet ray and ultrasonic wave in the coexistence of metal ions such as iron and copper and reducing agents such as ascorbic acid or cysteine, and can be decomposed by hyaluronidase and chondroitin sulfate. The HA with high molecular weight can be reduced in molecular weight under the conditions of enzyme, acid, heating and the like, and moreover, the HA aqueous solution is a non-Newtonian fluid and HAs good viscoelasticity and flow deformation. Commercial hyaluronic acid is currently commercially available in the form of sodium salt, namely sodium hyaluronate (SH for short), is a white fibrous or powdery solid, is soluble in water, is insoluble in organic solvents such as alcohol, ketone, diethyl ether and the like, and has strong hygroscopicity. The aqueous solution of sodium hyaluronate is negatively charged and has high viscoelasticity and osmotic pressure at high concentrations.

Hyaluronic acid is widely present in various tissues of animals and has a molecular weight of 500000-730000 daltons. Hyaluronic acid shows various important physiological functions in the body by virtue of unique molecular structure and physicochemical properties, such as joint lubrication, vascular wall permeability regulation, protein regulation, water electrolyte diffusion and operation regulation, wound healing promotion and the like. Particularly, hyaluronic acid has a special water retention effect, and 2% pure hyaluronic acid aqueous solution can firmly retain 98% of water, is the substance with the best water retention property in the nature which is found at present, and is called ideal natural moisturizing factor (Natural moisturizing factor, NMF). Hyaluronic acid is also a multifunctional matrix, which is widely distributed in various parts of the human body. The skin also contains a large amount of hyaluronic acid, and the maturation and aging processes of human skin also change along with the content and metabolism of the hyaluronic acid, so that the skin nutrition metabolism can be improved, the skin is tender, smooth, wrinkle-removing, elasticity-increasing, aging-preventing, moisture-keeping and good transdermal absorption promoter can be realized, and the skin nutrition absorption promoter can play a more ideal role in promoting nutrition absorption when being used together with other nutrition components. In addition, hyaluronic acid is helpful for improving wound healing regeneration capability, reducing scar, enhancing immunity, etc., and can be used as non-steroidal antiinflammatory drug or adjuvant drug for arthritis treatment, ophthalmology, and cardiac surgery, and has unique effect in treating scald, burn, cold injury, and artificial skin. At present, hyaluronic acid is widely used in the fields of foods, health products, cosmetics, medicines and the like, and in addition, biochemical reagents prepared from the hyaluronic acid are widely popularized and applied in the biology field.

Hyaluronic acid has been studied in the thirty-second European and American countries of the last century and extracted from animal organizations in the seventies, while it is only in the nineties in China and is produced in small quantities by conventional extraction methods. The traditional extraction method adopts a large amount of organic solvents such as acetone for extraction, so that the environmental pollution is large and the production safety hidden trouble exists. At present, the production of hyaluronic acid mostly adopts a biological fermentation method, namely, streptococcus is taken as a primary bacterium, and the streptococcus is extracted through mutagenesis fermentation, so that the production cost is effectively reduced, but the organism absorption and utilization are still to be improved due to poor biological cooperativity. More importantly, neither the conventional solvent extraction nor the bio-fermentation method can control the molecular weight of hyaluronic acid, and the yield of the product is not ideal.

Disclosure of Invention

Therefore, a first object of the present invention is to provide a small molecule hyaluronic acid product which is not only controllable in molecular weight but also superior in absorption properties;

The second aim of the invention is to provide a method for preparing small-molecule hyaluronic acid by using cockscomb as a raw material and using an enzyme catalytic bioconversion method, wherein the method can effectively control the molecular weight of a hyaluronic acid product and has higher extraction efficiency.

In order to solve the technical problems, the preparation method of the small molecule hyaluronic acid provided by the invention comprises the following steps:

(1) Taking the chicken crown as a raw material, adding water and pulping to obtain slurry for later use;

(2) Sequentially adding lipase into the slurry for first enzymolysis, continuously adding alpha-galactosidase for second enzymolysis, continuously adding neutral protease for third enzymolysis, and collecting enzymolysis liquid;

(3) Adding an adsorbent into the enzymolysis liquid for purification treatment, and collecting the purified liquid through solid-liquid separation.

Specifically, in the preparation method of the small molecule hyaluronic acid, in the step (1), the mass ratio of the cockscomb to the water is 1:8-10;

Preferably, the cockscomb comprises a broiler chicken breast.

Specifically, in the preparation method of the small molecule hyaluronic acid, in the step (2):

The addition amount of the lipase accounts for 1.5-2wt% of the weight of the cockscomb raw material, and/or,

The addition amount of the alpha-galactosidase accounts for 1.5-2wt% of the weight of the cockscomb raw material, and/or,

The addition amount of the neutral protease accounts for 1-1.5wt% of the weight of the cockscomb raw material.

The time of the first enzymolysis step is 20-40min, and/or,

The second enzymolysis step is performed for 40-80min, and/or,

The time of the third enzymolysis step is 3.0-3.5h.

the rotational speed of the stirrer in the first enzymolysis step is 32-35r/min;

the rotational speed of the stirrer in the second enzymolysis step is 36-38r/min;

the rotational speed of the stirrer in the third enzymolysis step is 38-40r/min.

Specifically, in the preparation method of the small molecule hyaluronic acid, in the step (2), the temperature of the enzymolysis step is 48-50 ℃;

preferably, the method further comprises the step of heating the enzymolysis liquid to 90-95 ℃ for enzyme deactivation treatment;

preferably, the method further comprises the steps of carrying out solid-liquid separation on the enzymolysis liquid and collecting a liquid part.

Specifically, in the preparation method of the small molecule hyaluronic acid, in the step (3), the adsorbent comprises activated carbon;

Preferably, the addition amount of the adsorbent accounts for 5-7wt% of the weight of the cockscomb raw material;

preferably, the temperature of the purification treatment step is from 35 to 37 ℃;

Preferably, the purification treatment step is carried out for a period of time ranging from 0.5 to 0.8 hours.

Specifically, the preparation method of the small molecule hyaluronic acid further comprises the step of concentrating and/or spray drying the purified solution in the step (3).

The invention also discloses the small molecular hyaluronic acid prepared by the method, and the molecular weight of the small molecular hyaluronic acid is controlled between 600 and 2000 daltons by more than 90 percent.

The invention also discloses application of the small molecule hyaluronic acid in preparing foods, health products, cosmetics or medicines.

The preparation method of the small-molecule hyaluronic acid adopts biocatalysis enzymolysis of chicken crown to extract hyaluronic acid, adopts an enzyme preparation system of lipase, alpha-galactosidase and neutral protease, adopts a process of sectional directional enzyme digestion and targeted degradation, can improve the yield of hyaluronic acid products, is beneficial to the purification of subsequent process products, can effectively control the molecular weight of hyaluronic acid to be less than 1500 daltons, contains small-molecule active peptide and rich glucuronic acid, and is easier for human body absorption and utilization.

The preparation method of the micromolecular hyaluronic acid adopts a segmented enzyme cutting process, realizes a directional enzyme cutting technology of raw materials, firstly uses lipase to degrade fat in the raw materials, is beneficial to degreasing and subsequent enzyme addition for enzymolysis, can control the molecular weight of products, has a production period which is only 1/3 of that of a conventional extraction process, has a short production period, and greatly reduces the production cost.

The preparation method of the small-molecule hyaluronic acid adopts a directional enzyme cutting technology, does not need to adopt organic solvents such as ethanol acetone and the like, is beneficial to production safety and environmental protection, and effectively solves the defect that the molecular weight of the hyaluronic acid cannot be controlled in the traditional solvent extraction and fermentation extraction process, and environmental pollution and potential safety hazard brought by the traditional process.

The small-molecule hyaluronic acid provided by the invention has the advantages of lower molecular weight, controllable molecular weight distribution and easier absorption by organisms, and can be widely applied to the fields of foods, health care products, cosmetics and medicines. Is an important medicine dispersing agent which is used for temporarily reducing the viscosity of a cell matrix of a human body, can promote the diffusion of exudates or blood which are subjected to subcutaneous transfusion and local accumulation and is beneficial to absorption. Clinically used as a drug penetrating agent, promotes the absorption of drugs and promotes the dissipation of local oedema or hematoma after surgery and trauma.

Detailed Description

Example 1

The preparation method of the small molecule hyaluronic acid comprises the following steps:

(1) Weighing 1000g of broiler cockscomb, mincing with a meat mincer, adding 8000ml of purified water, and stirring uniformly to obtain slurry for later use;

(2) Heating the slurry to 48 ℃, firstly adding 15g of lipase, controlling the rotation speed of a stirrer to be 32r/min for enzymolysis for 0.5h, then adding 15g of alpha-galactosidase, controlling the rotation speed of the stirrer to be 36r/min for enzymolysis for 1.0h, continuously adding 10g of neutral protease, controlling the rotation speed of the stirrer to be 38r/min for enzymolysis for 3.0h, collecting enzymolysis products, heating to 90 ℃ and keeping the temperature for 10min for enzyme deactivation, firstly filtering impurities by a horizontal gong centrifuge after enzyme deactivation, and then centrifuging and purifying by a high-speed centrifuge, wherein the rotation speed of the centrifuge is kept at 18000r/min to obtain enzymolysis liquid;

(3) Adding 50g of active carbon into the enzymolysis liquid, stirring and purifying for 0.5h, keeping the temperature at 35 ℃, filtering and decarbonizing by a microporous filter, collecting filtrate, concentrating by a concentrator, and spray-drying the concentrated solution to obtain the required micromolecular hyaluronic acid.

Example 2

(1) Weighing 10kg of broiler cocks, mincing with a meat mincer, adding 90L of purified water, and stirring uniformly to obtain slurry for later use;

(2) Heating the slurry to 49 ℃, firstly adding 180g of lipase, controlling the rotation speed of a stirrer to 34r/min for enzymolysis for 0.5h, then adding 180g of alpha-galactosidase, controlling the rotation speed of the stirrer to 37r/min for enzymolysis for 1.0h, continuously adding 140g of neutral protease, controlling the rotation speed of the stirrer to 39r/min for enzymolysis for 3.4h, collecting an enzymolysis product, heating to 93 ℃ and maintaining for 13min for enzyme deactivation, firstly filtering by a horizontal gong centrifuge after enzyme deactivation, centrifuging filtrate by a high-speed centrifuge, and maintaining the rotation speed of the centrifuge for 18000r/min to obtain an enzymolysis solution;

(3) Adding 600g of active carbon into the enzymolysis liquid, stirring and purifying for 0.6 hour, keeping the temperature at 36 ℃, filtering and decarbonizing by a microporous filter, collecting filtrate, concentrating by a concentrator, and spray-drying the collected concentrate to obtain the required micromolecular hyaluronic acid.

Example 3

(1) Weighing 100kg of broiler cocks, mincing with a meat mincer, adding 1000L of purified water, and stirring uniformly to obtain slurry for later use;

(2) Heating the slurry to 50 ℃, firstly adding 2kg of lipase, controlling the rotation speed of a stirrer to be 35r/min for enzymolysis for 0.5h, then adding 2kg of alpha-galactosidase, controlling the rotation speed of the stirrer to be 38r/min for enzymolysis for 1.0h, continuously adding 1.5kg of neutral protease, controlling the rotation speed of the stirrer to be 40r/min for enzymolysis for 3.5h, collecting enzymolysis products, heating to 95 ℃ and keeping for 15min for enzyme deactivation, filtering the enzyme deactivation products by a horizontal gong centrifuge, centrifuging the filtrate by a high-speed centrifuge, keeping the rotation speed of the centrifuge at 18000r/min, and collecting enzymolysis liquid;

(3) Adding 70kg of active carbon into the enzymolysis liquid, stirring and purifying for 0.8 hour, keeping the temperature at 37 ℃, filtering and decarbonizing by a microporous filter, collecting filtrate, concentrating by a concentrator, and spray-drying the collected concentrate to obtain the required micromolecular hyaluronic acid product.

Comparative example 1

The preparation method of the small molecule hyaluronic acid of the comparative example is the same as in example 2, except that the lipase, alpha-galactosidase and neutral protease are simultaneously added to the slurry for enzymolysis treatment for 3.4 hours.

Comparative example 2

The preparation method of the small molecule hyaluronic acid in this comparative example is the same as in example 2, except that the enzymolysis step adopts hyaluronidase and protease for enzymolysis.

Comparative example 3

The preparation method of the micromolecular hyaluronic acid in the comparative example adopts a conventional solvent extraction method, namely chloroform and acetone are used as organic solvents for extraction, and the hidden trouble of the whole process is extremely large.

Comparative example 4

The preparation method of the micromolecular hyaluronic acid in the comparative example adopts a conventional fermentation extraction method, but the production period is 1-2 weeks, and the production period is longer.

Experimental example

1. Yield and production cycle of hyaluronic acid product

The yields of the hyaluronic acid products prepared by the methods of examples 1 to 3 and comparative examples 1 to 4, respectively, were examined and the production cycles thereof were recorded, and the results are shown in Table 1 below.

TABLE 1 hyaluronic acid yield and production cycle

Numbering device	Hyaluronic acid yield/%	Production cycle
			Example 1	>30%	24-28 Hours
Example 2	>30%	24-28 Hours
			Example 3	>30%	24-28 Hours
Comparative example 1	<5%	For 4-5 days
			Comparative example 2	<5%	For 4-5 days
Comparative example 3	<10%	For 8-10 days
			Comparative example 4	<10%	For 10-12 days

Compared with the traditional solvent extraction method, the hyaluronic acid product prepared by the method has the advantages that the product yield is improved by more than 3 times, and the production period is shortened by about 7 times.

In particular, in the scheme of comparative example 2, the fat in the raw material cockscomb is too high, so that the raw material cockscomb is not easy to degrade and fall off, and the product yield is affected. The hyaluronic acid is a glucuronic acid, but the hyaluronidase is not an enzyme for enzyme cutting the molecular weight of the hyaluronic acid, which is not beneficial to controlling the molecular weight of the product. The scheme of the application adopts lipase, alpha-galactosidase and neutral protease for enzymolysis, which is not only beneficial to controlling the molecular weight, but also beneficial to degrading the fat in the raw materials, improving the yield of the product and shortening the production period. 2. Hyaluronic acid molecular weight

The molecular weight distribution of the hyaluronic acid products prepared by the methods of examples 1 to 3 above were examined respectively, the industry standards of the molecular weight of the hyaluronic acid products are shown in table 2 below, and the examination results of the products of the present invention are shown in table 3 below.

Table 2 line molecular weight

Classification	Molecular weight range
		Large molecular weight	1800000-2200000
Medium molecular weight	1000000-1800000
		Small molecular weight	400000-1000000

TABLE 3 molecular weight distribution of hyaluronic acid products

Classification	Molecular weight range
		1-Level small molecule	600-1000
2-Stage small molecule	1000-2000
		3-Stage small molecule	2000-3000

The detection result shows that the method can effectively control the molecular weight of the hyaluronic acid in the examples 1-3 to be less than 1500 daltons, and the molecular weight is lower and the molecular weight distribution is controllable.

3. Absorption rate of hyaluronic acid product

The absorption efficiency of the hyaluronic acid products prepared by the methods of examples 1 to 3 and comparative examples 1 to 2, respectively, was examined, and the results are shown in Table 4 below.

TABLE 4 absorption rate of hyaluronic acid

Numbering device	Hyaluronic acid absorption rate/%
		Example 1	>99.9%
Example 2	>99.9%
		Example 3	>99.9%
Comparative example 1	<50%
		Comparative example 2	<50%
Comparative example 3	<30%
		Comparative example 4	<40%

Therefore, the hyaluronic acid product prepared by the method disclosed by the invention contains small-molecule active peptide and rich glucuronic acid, is easier to be absorbed and utilized by a human body, and compared with the traditional solvent or fermentation and other processes, the absorption rate of the hyaluronic acid product is improved by more than 5 times.

In conclusion, the preparation method of the micromolecular hyaluronic acid can improve the yield of the hyaluronic acid product, is beneficial to the purification of the subsequent process product, can effectively control the molecular weight of the hyaluronic acid to be less than 1500 daltons, has lower molecular weight and controllable molecular weight distribution, contains micromolecular active peptide and rich glucuronic acid, is easier to be absorbed and utilized by a human body, and has a production period which is only 1/3 of that of the conventional extraction process, thus not only having a short production period, but also greatly reducing the production cost.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. A method for preparing small molecule hyaluronic acid, characterized in that it comprises the following steps:

(1) taking cockscomb as raw material and adding water to prepare pulp to obtain slurry for later use;

(2) sequentially adding lipase to the slurry for a first enzymatic hydrolysis treatment, continuing to add α-galactosidase for a second enzymatic hydrolysis treatment, and continuing to add neutral protease for a third enzymatic hydrolysis treatment, and collecting the enzymatic hydrolysis solution; wherein,

The amount of the lipase added is 1.5-2wt% of the weight of the cockscomb raw material, and the time of the first enzymatic hydrolysis step is 20-40min;

The amount of α-galactosidase added is 1.5-2wt% of the weight of the cockscomb raw material, and the time of the second enzymatic hydrolysis step is 40-80min;

The amount of the neutral protease added is 1-1.5wt% of the weight of the cockscomb raw material, and the time of the third enzymatic hydrolysis step is 3.0-3.5h;

(3) adding an adsorbent to the enzymatic hydrolyzate for purification, and collecting the purified liquid through solid-liquid separation to obtain the product.

2. The method for preparing small molecule hyaluronic acid according to claim 1, characterized in that in the step (1), the mass ratio of the cockscomb to water is 1:8-10.

3. The method for preparing small molecule hyaluronic acid according to claim 2, characterized in that the combs include broiler combs.

4. The method for preparing the small molecule hyaluronic acid according to any one of claims 1 to 3, characterized in that in the step (2):

The agitator speed in the first enzymatic hydrolysis step is 32-35 r/min;

The agitator speed of the second enzymatic hydrolysis step is 36-38 r/min;

The agitator speed of the third enzymatic hydrolysis step is 38-40r/min.

5. The method for preparing small molecule hyaluronic acid according to claim 4, characterized in that, in the step (2), the temperatures of the first enzymatic hydrolysis treatment, the second enzymatic hydrolysis treatment, and the third enzymatic hydrolysis treatment steps are independently 48-50°C.

6. The method for preparing small molecule hyaluronic acid according to claim 5, characterized in that, in the step (2), it also includes the step of heating the enzymatic hydrolyzate to 90-95°C for enzyme inactivation.

7. The method for preparing small molecule hyaluronic acid according to claim 6, characterized in that, in the step (2), it also includes the step of separating the enzymatic hydrolysate into solid and liquid and collecting the liquid portion.

8. The method for preparing small molecule hyaluronic acid according to any one of claims 1 to 3, characterized in that in the step (3), the adsorbent comprises activated carbon.

9. The method for preparing small molecule hyaluronic acid according to claim 8, characterized in that, in the step (3), the amount of the adsorbent added accounts for 5-7wt% of the weight of the cockscomb raw material.

10. The method for preparing small molecule hyaluronic acid according to claim 9, characterized in that in the step (3), the temperature of the purification step is 35-37°C.

11. The method for preparing small molecule hyaluronic acid according to claim 10, characterized in that in the step (3), the time of the purification step is 0.5-0.8h.

12. The method for preparing small molecule hyaluronic acid according to claim 11, characterized in that the step (3) further comprises the step of concentrating and/or spray drying the purified solution.