A method for preparing chondroitin A oligosaccharide iron with high enzymatic hydrolysis liquid phase content

Abstract

This invention discloses a method for preparing chondroitin A oligosaccharide ferrous sulfate with high enzymatic hydrolysis content, belonging to the field of bioengineering technology. The invention includes the following steps: softening animal cartilage by soaking in water; pulverizing the softened cartilage using microwaves; enzymatically hydrolyzing the pulverized cartilage with chondroitin sulfate lyase; separating the solid and liquid phases using a disc centrifuge; adsorbing chondroitin sulfate onto the filtrate using a resin column; eluting the resin column with an aqueous iron salt solution; and desalting, concentrating, sterilizing, and drying the eluent to obtain the finished chondroitin A oligosaccharide ferrous sulfate product, effectively preserving the natural structure, bioactivity, and physicochemical properties of chondroitin sulfate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a preparation method of high-enzymolysis liquid content chondroitin sulfate A oligosaccharide iron. BACKGROUND

[0002] Chondroitin sulfate is a polysaccharide compound obtained from natural animal cartilage, is an important bio-chemical raw material, and has the effects of reducing blood fat, resisting thrombosis, resisting tumor, treating arthritis, arteriosclerosis, cardiovascular and cerebrovascular diseases, hearing impairment, nephritis, hepatitis and neuralgia. However, natural chondroitin sulfate has a large molecular weight, is not easy to penetrate the cell membrane, and has the problem of low bioavailability. Low molecular weight chondroitin sulfate or chondroitin sulfate oligosaccharide has strong pharmacological activity and better curative effect on prevention and treatment of rheumatic inflammation and wound healing.

[0003]

[0004] At present, the methods for industrial production of low molecular chondroitin sulfate and chondroitin sulfate oligosaccharide include physical method, chemical method and enzymolysis method. Although the physical method has simple process, it has the disadvantages of easy discoloration of product, poor stability of product, large molecular weight and the like. The chemical method includes oxidative degradation (sodium hypochlorite and hydrogen peroxide), acid hydrolysis and alkali hydrolysis, and the chemical degradation reaction has complex reaction conditions and the disadvantages of poor product activity and much production wastewater. The enzymatic degradation has become a research hotspot of low molecular chondroitin sulfate and chondroitin sulfate oligosaccharide in recent years due to its characteristics of mild reaction conditions and good product biological activity. SUMMARY

[0005] The technical problem to be solved by the application is to provide a preparation method of high-enzymolysis liquid content chondroitin sulfate A oligosaccharide iron, which discards the traditional protease enzymolysis process, realizes one-step efficient production of chondroitin sulfate A oligosaccharide iron by introducing chondroitin sulfate lyase, simplifies the production process and greatly reduces the production cost.

[0006] TECHNICAL SOLUTION

[0007] A preparation method of high-enzymolysis liquid content chondroitin sulfate A oligosaccharide iron, which comprises the following steps:

[0008] (1) cartilage pretreatment: soaking animal cartilage in distilled water for 3-4 h, the ratio of water to cartilage being 2:1-5:1, and then softening the animal cartilage and performing microwave crushing treatment, so that the cartilage particles reach micron level;

[0009] (2) Enzymatic hydrolysis: Adjust the pH of the cartilage powder solution obtained in step (1) to 6.5–8.0 with 6 mol / L ammonia or 6 mol / L hydrochloric acid; add chondroitin sulfate lyase to make the ratio of total chondroitin sulfate lyase activity to cartilage mass 2 × 10⁻⁶. 4 ~4×10 5 U: 1 kg, enzymatic hydrolysis for 4-12 h, preferably 8 h; finally, inactivate the enzyme to obtain mixed system A.

[0010] The method for determining complete enzymatic hydrolysis is as follows: if the concentration of chondroitin sulfate A oligosaccharide no longer increases, it proves that the enzymatic hydrolysis is complete.

[0011] (3) Solid-liquid separation: The mixed system A obtained in step (2) is separated into solid and liquid components using a disc centrifuge. The light liquid is bone oil, the heavy liquid is chondroitin sulfate oligosaccharide solution, and the solid is bone residue, bone protein and inactivated chondroitin sulfate lysin. The speed of the disc centrifuge is 2000-8000 rpm, preferably 6000 rpm.

[0012] (4) Preparation of chondroitin A oligosaccharide

[0013] (4a) Resin adsorption: The chondroitin sulfate oligosaccharide solution obtained in step (3) is added into a chondroitin sulfate-specific adsorption resin column. After adsorption treatment, a resin column adsorbed with chondroitin sulfate oligosaccharide is obtained.

[0014] Method for determining adsorption completion: If no chondroitin sulfate oligosaccharide is detected in the adsorbed solution, adsorption is complete.

[0015] (4b) Elution of chondroitin sulfate oligosaccharides:

[0016] The resin column adsorbed with chondroitin sulfate oligosaccharide was eluted with an aqueous solution of 50-100 g / L iron salt at a flow rate of 2-5 BV / h to obtain an eluent containing chondroitin sulfate oligosaccharide.

[0017] (4c) Desalination: The eluent obtained in step (4b) is subjected to nanofiltration desalination to obtain nanofiltration retentate. The nanofiltration membrane used has a pore size of 160 to 500 Da, preferably 360 Da.

[0018] (4d) Sterilization: The nanofiltration retentate obtained in step (4c) is filtered and sterilized. The nanomembrane used for sterilization has a pore size of 0.1 to 0.45 μm, preferably 0.1 μm. The sterilized filtrate is then pumped into an alcohol precipitation tank.

[0019] (4e) Precipitation: The sterile filtrate obtained in step (4d) is precipitated with ethanol. The precipitate is chondroitin sulfate A oligosaccharide. The volume ratio of ethanol to sterile filtrate is 0.5:1 to 1.5:1, preferably 0.8:1.

[0020] (4f) Dehydration: The chondroitin A oligosaccharide obtained in step (4e) is soaked in anhydrous ethanol for 10 hours to dehydrate; the mass ratio of chondroitin A oligosaccharide to anhydrous ethanol is 1:3 to 1:8, preferably 1:5;

[0021] (4g) Drying and pulverizing: The dehydrated chondroitin sulfate A oligosaccharide obtained in step (4f) is vacuum dried, and then the dried chondroitin sulfate A oligosaccharide is pulverized.

[0022] Beneficial effects:

[0023] 1. This invention introduces chondroitin sulfate lyase to prepare chondroitin sulfate oligosaccharide A iron from animal cartilage in a one-step process, reducing production steps and energy consumption, and lowering the production cycle and production cost;

[0024] 2. This invention uses a biodegradation method to replace the physical and chemical degradation methods in the preparation of chondroitin sulfate oligosaccharide A iron, effectively ensuring the natural structure, bioactivity, and physicochemical properties of chondroitin sulfate. Detailed Implementation

[0025] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0026] The enzymes used in this invention

[0027] Chondroitin sulfate lyases A, B, and C can catalyze the cleavage of glycosaminoglycans formed from chondroitin sulfate A (4-chondroitin sulfate), chondroitin sulfate B (dermatan sulfate), and chondroitin sulfate C (6-chondroitin sulfate), respectively; chondroitin sulfate lyase AC catalyzes the cleavage of glycosaminoglycans formed from chondroitin sulfate A or chondroitin sulfate C; and chondroitin sulfate lyases ABC catalyze the cleavage of glycosaminoglycans formed from chondroitin sulfate A, chondroitin sulfate B, or chondroitin sulfate C.

[0028] Chondroitin sulfate lyase A, chondroitin sulfate lyase B, chondroitin sulfate lyase C, chondroitin sulfate lyase AC, and chondroitin sulfate lyases ABC were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] Example 1: Optimization of the type and ratio of lysin in the preparation method of chondroitin A oligosaccharide iron with high enzymatic hydrolysis liquid phase content.

[0030] This example illustrates the effect of different lysing enzyme ratios on the lysis efficiency of chondroitin sulfate. The ratio of total enzyme activity to cartilage mass was 2 × 10⁻⁶. 4 ~4×10 5Chondroitin sulfate lyase A, chondroitin sulfate lyase B, chondroitin sulfate lyase C, chondroitin sulfate lyase AC, and chondroitin sulfate lyase ABC were added at a ratio of 1 kg / U, with an initial pH of 6.5–8.0. Enzymatic hydrolysis was performed for 6 hours. The yields of chondroitin sulfate A were measured using high-performance liquid chromatography (HPLC) and compared with the results. When the lyases were chondroitin sulfate lyase A and chondroitin sulfate lyase ABC, the yields of chondroitin sulfate A were relatively high, at 32.64% and 33.21%, respectively. Therefore, a compound enzyme of chondroitin sulfate lyase A and chondroitin sulfate lyase ABC was selected for further optimization. Repeat the above steps, adding compound enzymes with ratios of lyase A: lyase ABC = 1:2, 2:1, 2:3, and 3:2 respectively. The initial pH was 6.5–8.0, and enzymatic hydrolysis was performed for 6 hours. The hydrolysates from the reactions under different conditions were collected, and the yield of chondroitin sulfate A was determined by high-performance liquid chromatography (HPLC) and compared. The highest yield of chondroitin sulfate A (38.74%) was observed when the compound enzyme ratio was lyase A: lyase ABC = 2:3. Therefore, the compound enzyme with a ratio of lyase A: lyase ABC = 2:3 was selected as the optimal lyase.

[0031] Table 1. Lysis effect of chondroitin sulfate lyase with different ratios

[0032]

[0033] Example 2: Optimization of the enzymatic hydrolysis time of the lyase in the preparation method of chondroitin A oligosaccharide iron with high enzymatic hydrolysis liquid phase content.

[0034] This example illustrates the effect of different enzymatic hydrolysis times on the cleavage efficiency of chondroitin sulfate, based on the formula: total enzyme activity: cartilage mass = 2 × 10⁻⁶. 4 ~4×10 5 A complex enzyme with a ratio of lyase A:lyase ABC = 2:3 was added at a ratio of U:1 kg. The initial pH was 6.5–8.0. Enzymatic hydrolysis was performed for 4, 6, 8, 10, and 12 hours. The yield of chondroitin sulfate A in the resulting hydrolysate is shown in Table 2. The highest yield of chondroitin sulfate A (40.57%) was achieved at a hydrolysis time of 8 hours. Therefore, 8 hours was selected as the optimal hydrolysis time.

[0035] Table 2. Effects of different enzymatic hydrolysis times on the cleavage of chondroitin sulfate complex lyase.

[0036]

[0037] Example 3: Optimization of resin types and ratios in the preparation method of high-enzymatic hydrolysate chondroitin A oligosaccharide iron.

[0038] This embodiment illustrates the effect of different resin types and their ratios on the adsorption effect of chondroitin sulfate A oligosaccharides. One or a mixture of several of the resins A830, D101, AB-8, DM301, and HPD100 were used, with the amount being 1 to 2.5 times the amount of cartilage fed. The residual chondroitin sulfate A oligosaccharide content in the effluent was measured by high performance liquid chromatography, and the adsorption rate was calculated and compared.

[0039] The highest adsorption rate (65.28%) was observed with AB-8 resin; the adsorption rate with HPD100 resin was 63.97%, while the adsorption rates of the other three resins were all below 60%. Therefore, AB-8 and HPD100 were selected for further optimization of the resin ratio. The above steps were repeated, using mixed resins with ratios of AB-8:HPD100 of 1:1, 1:2, 2:1, 2:3, and 3:2, at amounts 1 to 2.5 times the amount of cartilage fed. The residual chondroitin A oligosaccharide content in the effluent was determined using high-performance liquid chromatography (HPLC), and the adsorption rates were calculated and compared. The highest adsorption rate (70.32%) was observed for chondroitin A oligosaccharides when the mixed resin ratio was AB-8:HPD100 = 2:1. Therefore, the optimal resin is a mixed resin with a ratio of AB-8:HPD100 = 2:1.

[0040] Table 3 Adsorption effects of different resin types and ratios

[0041]

[0042] Example 4: Optimization of iron salt types and ratios in the preparation method of high-enzymatic hydrolysis chondroitin A oligosaccharide iron.

[0043] This example illustrates the effect of different types and ratios of iron salts on the elution efficiency of chondroitin sulfate A oligosaccharides. An AB-8 resin column adsorbed with chondroitin sulfate oligosaccharides was eluted at a flow rate of 3 BV / h using one or more of the following iron salts: ferric chloride, ferric sulfate, ferric nitrate, ferrous sulfate, and ferrous chloride, at a concentration of 120 g / L. The resulting eluent contained chondroitin sulfate A oligosaccharides. The chondroitin sulfate A oligosaccharide content in the eluent was determined by high-performance liquid chromatography (HPLC), and the elution rate was calculated and compared. The highest elution rate (96.71%) was achieved when ferric chloride was used; the elution rate was 93.87% when ferric sulfate was used; and the elution rates of the other three iron salt solutions were all below 90%. Therefore, ferric chloride and ferric sulfate were selected for further optimization. When the ratio of ferric chloride to ferric sulfate in the mixture is 1:1, the elution rate is 95.45%; when the ratio of ferric chloride to ferric sulfate is 1:2, the elution rate is 92.99%, both lower than the elution rate when using ferric chloride aqueous solution alone. Therefore, ferric chloride aqueous solution is selected as the optimal elution solution for iron salts.

[0044] Table 4 Elution effects of different iron salt types and ratios

[0045]

[0046] Example 5: Optimization of the volume ratio of anhydrous ethanol to sterile filtrate in the preparation method of chondroitin A oligosaccharide iron with high enzymatic hydrolysis liquid phase content

[0047] This embodiment illustrates the effect of different volume ratios of anhydrous ethanol to sterile filtrate on the precipitation effect of chondroitin A oligosaccharide iron. Volume ratios of anhydrous ethanol:sterilized filtrate = 0.5:1, 0.8:1, 1:1, and 1.5:1 were used, with a precipitation time of 30 min. The supernatants obtained at different volume ratios of anhydrous ethanol to sterile filtrate were collected, and the residual rate of chondroitin A oligosaccharide was determined and compared using high-performance liquid chromatography (HPLC). When the volume ratio of anhydrous ethanol to sterile filtrate is 0.5:1, the residual rate of chondroitin A oligosaccharides is 3.87%; when the volume ratio is 0.8:1, the residual rate is 2.13%; when the volume ratio is 1:1, the residual rate is 2.41%; and when the volume ratio is 1.5:1, the residual rate is 2.89%. Therefore, an anhydrous ethanol to sterile filtrate volume ratio of 0.8:1 is selected as the optimal anhydrous ethanol precipitation volume ratio.

[0048] Table 5. Precipitation effects of different volume ratios of anhydrous ethanol to sterile filtrate.

[0049]

[0050] Example 6: Production of Chondroitin A Oligosaccharide Iron from 2000 kg of bovine nasal bone

[0051] This example illustrates the yield of chondroitin A oligosaccharide iron produced from 2000 kg of bovine nasal bone. 2000 kg of bovine nasal bone was soaked in water to soften it. The softened nasal bone was then pulverized using an ultrasonic pulverizer. 6000 kg of distilled water was added, and the temperature was raised to 35°C. The pH was adjusted to 7.5 using ammonia or hydrochloric acid, and then 5 × 10⁻⁶ mg / L of the pulverized bone was added. 8The enzyme was a complex enzyme with a ratio of chondroitin sulfate lyase A:chondroitin sulfate lyase ABC = 2:3, and enzymatic hydrolysis was performed for 8 hours. The temperature was then raised to 65℃ and incubated for 0.5 hours to inactivate the enzyme. After enzyme inactivation, the hydrolysate was separated into solid and liquid components using a disc centrifuge, and the heavy liquid (chondroitin sulfate oligosaccharide solution) was collected. The pH of the heavy liquid was adjusted to 6.0 with hydrochloric acid, and then added to a chondroitin sulfate-specific adsorption resin column for chondroitin sulfate adsorption. During adsorption, high-performance liquid chromatography (HPLC) was used to detect whether the chondroitin sulfate adsorption was complete. After adsorption, the resin column containing the adsorbed chondroitin sulfate was rinsed with distilled water. Then, the resin column was eluted with a 120 g / L FeCl3 aqueous solution at a flow rate of 3 BV / h, and the resulting eluent was obtained. The eluent contained chondroitin sulfate oligosaccharides. The eluent was desalted using a 160 Da nanofiltration unit. The desalted chondroitin sulfate oligosaccharide liquid was then sterilized using a 0.1 μm pore size filter, followed by concentration using a triple-effect concentrator. Anhydrous ethanol and sterilized filtrate at a ratio of 0.8:1 were added to precipitate the sterilized filtrate using an organic solvent. The precipitate was dehydrated with anhydrous ethanol and then dried using a vacuum desiccant to obtain 356 kg of chondroitin A oligosaccharide iron, with a yield of 17.8%.

[0052] Table 6. Detection parameters for chondroitin A oligosaccharide iron.

[0053]

Claims

1. A process for the preparation of high enzymatic hydrolysate liquid content chondroitin sulfate A oligosaccharide iron characterized in that, The method comprises the following steps: (1) Cartilage pretreatment: soak the animal cartilage in distilled water for 3-4 hours, the ratio of water to cartilage is 2:1-5:1, after softening, the animal cartilage is subjected to microwave crushing treatment, and the cartilage particles reach micron level; (2) Enzymolysis: the pH of the soft cartilage powder slurry obtained in step (1) is adjusted to 6.5-8.0 with 6 mol / L ammonia water or 6 mol / L hydrochloric acid; chondroitin sulfate lyase is added to the slurry, so that the ratio of total enzyme activity of chondroitin sulfate lyase to the mass of the cartilage is 2 × 10 4 ~ 4 × 10 5 U: 1 kg, enzymeolysis for 4-12 h, and finally inactivate the enzyme to obtain an enzymeolysis solution; (3) Solid-liquid separation: the enzymatic hydrolysate obtained in step (2) is subjected to solid-liquid separation by using a disc centrifuge, wherein the light liquid is bone oil, the heavy liquid is chondroitin sulfate oligosaccharide liquid, and the solid is bone residue, bone protein and inactivated chondroitin sulfate lyase, and the rotation speed of the disc centrifuge is 2000-8000 rpm; (4) Preparation of chondroitin sulfate A oligosaccharide (4a) Resin adsorption: the chondroitin sulfate oligosaccharide liquid obtained in step (3) is added into a chondroitin sulfate special adsorption resin column, and after adsorption treatment, a resin column adsorbed with chondroitin sulfate oligosaccharide is obtained; (4b) Chondroitin sulfate oligosaccharide elution: The resin column adsorbed with chondroitin sulfate oligosaccharide is subjected to elution treatment with 50-100 g / L iron salt aqueous solution at a flow rate of 2-5 BV / h, to obtain an eluate containing chondroitin sulfate oligosaccharide; (4c) Desalination: the eluate obtained in step (4b) is subjected to nanofiltration desalination, to obtain a nanofiltration retentate, and the nanofiltration membrane pore size used is 160-500 Da; (4d) Sterilization: the nanofiltration retentate obtained in step (4c) is subjected to filtration sterilization, and the sterilized filtrate is obtained, and the nanofiltration membrane pore size used is 0.1-0.45 μm, and the sterilized filtrate is poured into an alcohol precipitation tank; (4e) Precipitation: the sterilized filtrate obtained in step (4d) is subjected to organic solvent precipitation, and the precipitate is chondroitin sulfate A oligosaccharide; (4f) Dehydration: the chondroitin sulfate A oligosaccharide obtained in step (4e) is soaked in anhydrous ethanol for dehydration, and the soaking time is 10 h; the mass ratio of chondroitin sulfate A oligosaccharide to anhydrous ethanol is 1:3-1:8; (4g) Drying and crushing: the dehydrated chondroitin sulfate A oligosaccharide obtained in step (4f) is subjected to vacuum drying, and then the dried chondroitin sulfate A oligosaccharide is crushed; The chondroitin sulfate lyase is a complex enzyme of chondroitin sulfate lyase A and chondroitin sulfate lyase ABC; In step (4a), the resin is one or a mixture of several of A830, D101, AB-8, DM301 and HPD100; The resin feeding mass is 1-2.5 times the cartilage feeding mass; In step (4b), the iron salt aqueous solution is an aqueous solution of ferric chloride, ferrous sulfate, ferric nitrate, ferrous sulfate or ferrous chloride; In step (4b), the concentration of the iron salt aqueous solution is 50-100 g / L, and the usage amount is 2-4 times the column volume.

2. The high enzymatic liquid content chondroitin sulfate A oligosaccharide iron of claim 1, characterized in that, In step (4c), the nanofiltration membrane pore size is one of 100 Da, 360 Da, 500 Da and 1000 Da.

3. The high enzymatic liquid content chondroitin sulfate A oligosaccharide iron of claim 1, wherein, In step (4e), the organic solvent is a mixture of one or both of ethanol and acetone.

4. The high enzymatic liquid content chondroitin sulfate A oligosaccharide iron of claim 1, characterized in that, In step (4e), the volume ratio of the organic solvent to the sterilized filtrate is 0.5:1-1.5:1.

Images