A small molecule hyaluronic acid and its preparation method and application

Abstract

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

Technical Field

[0001] The invention belongs to the technical field of biomaterial extraction, and specifically relates to a small molecule hyaluronic acid, and more specifically to a method for preparing the small molecule hyaluronic acid by using chicken comb as a raw material and utilizing an enzyme-catalyzed biotransformation method. Background Art

[0002] Hyaluronic acid is an acidic mucopolysaccharide. It was first isolated from the vitreous body of bovine eyes by Meyer and others, professor of ophthalmology at Columbia University in the United States, in 1934. HA is easily decomposed by acid, alkali or heat treatment; it is degraded by oxygen free radicals, x-rays, gamma-rays, ultraviolet rays and ultrasound in the presence of metal ions such as iron and copper and reducing agents such as ascorbic acid or cysteine; it can also be decomposed by hyaluronidase and chondroitin sulfate enzymes. High molecular weight HA can be reduced to low molecular weight under the conditions of enzymes, acids, heating, etc. In addition, HA aqueous solution is a non-Newtonian fluid with good viscoelasticity and flow deformation. The commercial hyaluronic acid currently on the market is in the form of its sodium salt, namely sodium hyaluronate (abbreviated as SH), which is a white fibrous or powdery solid, soluble in water, insoluble in organic solvents such as alcohol, ketones, ether, etc., and has strong hygroscopicity. The aqueous solution of sodium hyaluronate is negatively charged and has high viscoelasticity and osmotic pressure at high concentrations.

[0003] Hyaluronic acid is widely present in various tissues of animals, with a molecular weight of 500,000-730,000 Daltons. With its unique molecular structure and physical and chemical properties, hyaluronic acid shows a variety of important physiological functions in the body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating the diffusion and operation of proteins, water and electrolytes, and promoting wound healing. More importantly, hyaluronic acid has a special water-retaining effect. A 2% pure hyaluronic acid aqueous solution can firmly retain 98% of water. It is the best moisturizing substance found in nature and is called the ideal natural moisturizing factor (NMF). Hyaluronic acid is also a multifunctional matrix, widely distributed in various parts of the human body. The skin also contains a large amount of hyaluronic acid, and the maturation and aging process of human skin also changes with the content and metabolism of hyaluronic acid. It can improve the skin's nutritional metabolism, make the skin soft, smooth, wrinkle-free, increase elasticity, and prevent aging. While moisturizing, it is also a good transdermal absorption promoter. When used in combination with other nutrients, it can achieve a more ideal effect of promoting nutrient absorption. In addition, hyaluronic acid helps improve wound healing and regeneration, reduce scars, enhance immunity, etc. It can be used in non-steroidal anti-inflammatory drugs, or auxiliary drugs for arthritis treatment, ophthalmology, and cardiac surgery; it has a unique effect in treating scalds, burns, frostbite, and artificial skin. At present, hyaluronic acid is widely used in food, health products, cosmetics, medicines and other fields. In addition, biochemical reagents prepared by hyaluronic acid are widely promoted and applied in the biological community.

[0004] Hyaluronic acid has been studied in Europe and the United States in the 1930s, and began to be extracted from animal tissues in the 1970s. However, it was not until the 1990s that China began to study and produce it in small batches using traditional extraction methods. Traditional extraction methods use a large amount of organic solvents such as acetone for extraction, which not only pollutes the environment but also poses a potential safety hazard in production. At present, the production of hyaluronic acid mostly adopts biological fermentation, that is, streptococci are used as the original bacteria, and extraction is carried out through mutagenesis fermentation. Although it effectively reduces the production cost, due to poor biological synergy, the body's absorption and utilization still needs to be improved. More importantly, neither the traditional solvent extraction method nor the biological fermentation method can control the molecular weight of hyaluronic acid, and the yield of the product is not ideal. Summary of the invention

[0005] To this end, the first object of the present invention is to provide a small molecule hyaluronic acid product, wherein the hyaluronic acid product has a controllable molecular weight and better product absorption performance;

[0006] The second object of the present invention is to provide a method for preparing small molecule hyaluronic acid using chicken comb as raw material by enzyme-catalyzed biotransformation method, wherein the method can effectively control the molecular weight of the hyaluronic acid product and has higher extraction efficiency.

[0007] In order to solve the above technical problems, the present invention provides a method for preparing small molecule hyaluronic acid, comprising the following steps:

[0008] (1) taking cockscomb as raw material and adding water to prepare pulp to obtain slurry for later use;

[0009] (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 hydrolyzate;

[0010] (3) adding an adsorbent to the enzymatic hydrolyzate for purification, and collecting the purified liquid through solid-liquid separation to obtain the product.

[0011] Specifically, in the method for preparing small molecule hyaluronic acid, in the step (1), the mass ratio of the cockscomb to water is 1:8-10;

[0012] Preferably, the comb comprises a broiler comb.

[0013] Specifically, in the method for preparing the small molecule hyaluronic acid, in the step (2):

[0014] The amount of lipase added is 1.5-2wt% of the weight of the cockscomb raw material; and / or,

[0015] The amount of α-galactosidase added is 1.5-2wt% of the weight of the cockscomb raw material; and / or,

[0016] The amount of the neutral protease added is 1-1.5 wt % of the weight of the cockscomb raw material.

[0017] Specifically, in the method for preparing the small molecule hyaluronic acid, in the step (2):

[0018] The duration of the first enzymatic hydrolysis step is 20-40 min; and / or,

[0019] The duration of the second enzymatic hydrolysis step is 40-80 min; and / or,

[0020] The duration of the third enzymatic hydrolysis step is 3.0-3.5 h.

[0021] Specifically, in the method for preparing the small molecule hyaluronic acid, in the step (2):

[0022] The agitator speed in the first enzymatic hydrolysis step is 32-35 r / min;

[0023] The agitator speed of the second enzymatic hydrolysis step is 36-38 r / min;

[0024] The agitator speed of the third enzymatic hydrolysis step is 38-40r / min.

[0025] Specifically, in the method for preparing small molecule hyaluronic acid, in the step (2), the temperature of the enzymatic hydrolysis step is 48-50°C;

[0026] Preferably, the method further comprises heating the enzymatic hydrolysate to 90-95° C. to inactivate the enzyme;

[0027] Preferably, the method further comprises the step of performing solid-liquid separation on the enzymatic hydrolysate and collecting the liquid portion.

[0028] Specifically, in the method for preparing small molecule hyaluronic acid, in the step (3), the adsorbent includes activated carbon;

[0029] Preferably, the adsorbent is added in an amount of 5-7 wt % of the weight of the cockscomb raw material;

[0030] Preferably, the temperature of the purification step is 35-37°C;

[0031] Preferably, the purification step takes 0.5-0.8 h.

[0032] Specifically, in the method for preparing small molecule hyaluronic acid, the step (3) further comprises the step of concentrating and / or spray drying the purified solution.

[0033] The invention also discloses the small molecule hyaluronic acid prepared by the method, and more than 90% of the molecular weight of the small molecule hyaluronic acid is controlled between 600 and 2000 Daltons.

[0034] The invention also discloses the use of the small molecule hyaluronic acid in preparing food, health products, cosmetics or medicines.

[0035] The preparation method of small molecule hyaluronic acid of the present invention adopts biocatalytic enzymatic hydrolysis of cockscomb to extract hyaluronic acid, adopts an enzyme preparation system of lipase, α-galactosidase and neutral protease, and adopts a process of segmented directional enzyme cutting and targeted degradation, which can not only improve the yield of hyaluronic acid products and facilitate the purification of subsequent process products; but also effectively control the molecular weight of hyaluronic acid to be below 1500 Daltons, contains small molecule active peptides and rich glucuronic acid, and is easier for the body to absorb and utilize.

[0036] The preparation method of small molecule hyaluronic acid described in the present invention adopts a segmented enzymatic cutting process to realize a directional enzymatic cutting technology for raw materials. The fat in the raw materials is first degraded with lipase, which is not only beneficial to degreasing, but also beneficial to the subsequent addition of enzymes for enzymatic hydrolysis. It can not only control the molecular weight of the product, but also the production cycle is only 1 / 3 of the conventional extraction process. Not only is the production cycle short, but the production cost is also greatly reduced.

[0037] The preparation method of small molecule hyaluronic acid of the present invention adopts directional enzyme cutting technology, does not need to use organic solvents such as ethanol acetone, is beneficial to production safety and environmental protection, and effectively solves the defects of traditional solvent extraction and fermentation extraction processes that the molecular weight of hyaluronic acid cannot be controlled, as well as the environmental pollution and safety hazards caused by traditional processes.

[0038] The small molecule hyaluronic acid of the present invention has a lower molecular weight and a controllable molecular weight distribution, and is easier to be absorbed by the body, and can be widely used in the fields of food, health products, cosmetics and medicine. It is a kind of drug that can temporarily reduce the viscosity of the intercellular matrix in the human body, promote the diffusion of subcutaneous infusion, local accumulation of exudate or blood, and facilitate absorption, and is an important drug diffusion agent. It is used clinically as a drug permeation agent to promote the absorption of drugs and promote the dissipation of local edema or hematoma after surgery and trauma. DETAILED DESCRIPTION

[0039] Example 1

[0040] The preparation method of the small molecule hyaluronic acid described in this embodiment comprises the following steps:

[0041] (1) Weigh 1000 g of broiler combs, mince with a meat grinder, add 8000 ml of purified water and stir evenly to obtain a slurry for later use;

[0042] (2) The slurry is heated to 48° C., 15 g of lipase is first added, the stirrer speed is controlled at 32 r / min, and the enzymolysis is carried out for 0.5 h; then 15 g of α-galactosidase is added, the stirrer speed is controlled to be adjusted to 36 r / min, and the enzymolysis is carried out for 1.0 h; 10 g of neutral protease is continued to be added, the stirrer speed is controlled to be increased to 38 r / min, and the enzymolysis is carried out for 3.0 h; the enzymolysis product is collected and heated to 90° C., and maintained for 10 min to inactivate the enzyme; after the enzyme is inactivated, impurities are first filtered out using a horizontal centrifuge, and then centrifuged and purified using a high-speed centrifuge, and the centrifuge speed is maintained at 18000 r / min to obtain an enzymolysis solution;

[0043] (3) Add 50 g of activated carbon to the enzymatic hydrolyzate, stir and purify for 0.5 h, maintain the temperature at 35° C., and then filter and decarbonize using a microporous filter; collect the filtrate and concentrate it using a concentrator; collect the concentrated solution and spray dry it to obtain the desired small molecule hyaluronic acid.

[0044] Example 2

[0045] The preparation method of the small molecule hyaluronic acid described in this embodiment comprises the following steps:

[0046] (1) Weigh 10 kg of broiler combs, mince with a meat grinder, add 90 L of purified water, stir evenly, and obtain a slurry for later use;

[0047] (2) The slurry is heated to 49° C., 180 g of lipase is first added, the stirrer speed is controlled at 34 r / min, and the enzymolysis is carried out for 0.5 h; then 180 g of α-galactosidase is added, the stirrer speed is controlled to increase to 37 r / min, and the enzymolysis is carried out for 1.0 h; 140 g of neutral protease is continued to be added, the stirrer speed is controlled to increase to 39 r / min, and the enzymolysis is carried out for 3.4 h; the enzymolysis product is collected and heated to 93° C., and maintained for 13 min to inactivate the enzyme; after the enzyme is inactivated, it is first filtered using a horizontal centrifuge, and the filtrate is then centrifuged using a high-speed centrifuge, and the centrifuge speed is maintained at 18000 r / min to obtain an enzymolysis solution;

[0048] (3) Add 600 g of activated carbon to the enzymatic hydrolyzate, stir and purify for 0.6 hours, maintain the temperature at 36° C., and then filter and decarbonize using a microporous filter; collect the filtrate and concentrate it using a concentrator; collect the concentrated solution and spray dry it to obtain the desired small molecule hyaluronic acid.

[0049] Example 3

[0050] The preparation method of the small molecule hyaluronic acid described in this embodiment comprises the following steps:

[0051] (1) Weigh 100 kg of broiler combs, mince with a meat grinder, add 1000 L of purified water, stir evenly to obtain a slurry, and set aside;

[0052] (2) The slurry is heated to 50° C., 2 kg of lipase is first added, the stirrer speed is controlled at 35 r / min, and the enzymolysis is carried out for 0.5 h; then 2 kg of α-galactosidase is added, the stirrer speed is controlled to increase to 38 r / min, and the enzymolysis is carried out for 1.0 h; 1.5 kg of neutral protease is continued to be added, the stirrer speed is controlled to increase to 40 r / min, and the enzymolysis is carried out for 3.5 h; the enzymolysis product is collected and heated to 95° C., and maintained for 15 min to inactivate the enzyme; after the enzyme is inactivated, it is first filtered using a horizontal centrifuge; the filtrate is then centrifuged using a high-speed centrifuge, the centrifuge speed is maintained at 18000 r / min, and the enzymolysis liquid is collected;

[0053] (3) adding 70 kg of activated carbon to the enzymatic hydrolyzate, stirring and purifying for 0.8 hours, maintaining the temperature at 37° C., and then filtering and decarbonizing with a microporous filter; collecting the filtrate and concentrating it with a concentrator; collecting the concentrated liquid and spray drying it to obtain the desired small molecule hyaluronic acid product.

[0054] Comparative Example 1

[0055] The preparation method of the small molecule hyaluronic acid in this comparative example is the same as that in Example 2, the only difference is that the lipase, α-galactosidase and neutral protease are added to the slurry at the same time for enzymatic hydrolysis for 3.4 hours.

[0056] Comparative Example 2

[0057] The preparation method of the small molecule hyaluronic acid in this comparative example is the same as that in Example 2, the only difference is that the enzymatic hydrolysis step uses hyaluronidase and protease for enzymatic hydrolysis.

[0058] Comparative Example 3

[0059] The preparation method of the small molecule hyaluronic acid described in this comparative example adopts a conventional solvent extraction method, that is, chloroform and acetone are used as organic solvents for extraction, and the production hidden dangers of the whole process are extremely great.

[0060] Comparative Example 4

[0061] The preparation method of the small molecule hyaluronic acid described in this comparative example adopts a conventional fermentation extraction method, but its production cycle is 1-2 weeks, which is relatively long.

[0062] Experimental example

[0063] 1. Yield and production cycle of hyaluronic acid products

[0064] The yields of the hyaluronic acid products prepared by the methods in the above Examples 1-3 and Comparative Examples 1-4 were respectively tested, and their production cycles were recorded. The results are shown in Table 1 below.

[0065] Table 1 Hyaluronic acid yield and production cycle

[0066] serial number 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％ 4-5 days Comparative Example 2 ＜5％ 4-5 days Comparative Example 3 ＜10％ 8-10 days Comparative Example 4 ＜10％ 10-12 days

[0067] It can be seen that the hyaluronic acid product prepared by the method of the present invention has a product yield increased by more than 3 times and a production cycle shortened by about 7 times compared with the traditional solvent extraction method.

[0068] Especially in the scheme of comparative example 2, since the fat content in the raw chicken comb is too high, it is not easy to degrade and remove, and the product yield is affected. And because hyaluronic acid itself is a kind of glucuronic acid, but hyaluronidase is not an enzyme that cleaves the molecular weight of hyaluronic acid, it is not conducive to controlling the molecular weight of the product. The present application scheme uses lipase, α-galactosidase and neutral protease for enzymatic hydrolysis, which is not only conducive to controlling the molecular weight, but also helps to degrade the fat in the raw material, improve the yield of the product, and shorten the production cycle. 2. Molecular weight of hyaluronic acid

[0069] The molecular weight distribution of the hyaluronic acid products prepared by the methods in the above Examples 1-3 was tested respectively. The industry standard of the molecular weight of the hyaluronic acid product is shown in Table 2 below. The test results of the product of the present invention are shown in Table 3 below.

[0070] Table 2 Molecular weight

[0071] Classification Molecular weight range Large molecular weight 1800000-2200000 Medium molecular weight 1000000-1800000 Small molecular weight 400000-1000000

[0072] Table 3 Molecular weight distribution of hyaluronic acid products

[0073] Classification Molecular weight range Tier 1 small molecules 600-1000 Second level small molecules 1000-2000 Level 3 small molecules 2000-3000

[0074] The test results show that the method of the present invention can effectively control the molecular weight of hyaluronic acid in Examples 1-3 to be below 1500 Daltons, which not only makes the molecular weight lower but also makes the molecular weight distribution controllable.

[0075] 3. Absorption rate of hyaluronic acid products

[0076] The absorption efficiency of the hyaluronic acid products prepared by the methods in the above Examples 1-3 and Comparative Examples 1-2 was tested respectively, and the results are shown in Table 4 below.

[0077] Table 4 Hyaluronic acid absorption rate

[0078] serial number 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％

[0079] It can be seen that the hyaluronic acid product prepared by the method of the present invention contains small molecule active peptides and rich glucuronic acid, which is easier for the body to absorb and utilize. Compared with traditional solvent or fermentation processes, its absorption rate is increased by more than 5 times.

[0080] In summary, the preparation method of small molecule hyaluronic acid described in the present invention can not only improve the yield of hyaluronic acid products, but also be beneficial to the purification of subsequent process products; moreover, the molecular weight of hyaluronic acid can be effectively controlled below 1500 Daltons, which not only has a lower molecular weight but also a controllable molecular weight distribution; it contains small molecule active peptides and rich glucuronic acid, which is easier for the body to absorb and utilize; and the production cycle is only 1 / 3 of that of the conventional extraction process, which not only has a short production cycle but also greatly reduces the production cost.

[0081] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing small molecule hyaluronic acid, characterized in that: The steps include: (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: The step (2) also includes the step of heating the enzymatic hydrolysate to 90-95° C. to inactivate the enzyme.

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

8. The method for preparing the small molecule hyaluronic acid according to any one of claims 1 to 3, characterized in that: In the step (3), the adsorbent includes 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 is 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 purification step is performed for 0.5-0.8 h.

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