阅读说明：本技术 一种利用鱿鱼软骨制备硫酸软骨素的方法 (Method for preparing chondroitin sulfate from squid cartilage ) 是由 吴文惠 杨劲峰 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种利用鱿鱼软骨制备硫酸软骨素的方法,其解决提取硫酸软骨素的传统方法存在提取工艺复杂、产品得率低、蛋白含量高于5%的问题。其经过以下工艺步骤：1)硫酸软骨素裂解酶制备；2)鱿鱼软骨酶解；3)纯化；4)干燥。其是以鱿鱼软骨为原料,通过鸭小肠提取硫酸软骨素裂解酶,并应用于鱿鱼软骨酶解制备硫酸软骨素,发酵产物经醇-盐第一次纯化,联合膜-电场第二次纯化,干燥获得本发明产品。本发明采用生物法和联合纯化法,提取率高、生物活性强,制备的硫酸软骨素主要是硫酸软骨素C。本发明工艺流程简单高效,易控制,利于大规模推广生产。(The invention discloses a method for preparing chondroitin sulfate by utilizing squid cartilage, which solves the problems of complex extraction process, low product yield and protein content higher than 5 percent of the traditional method for extracting the chondroitin sulfate. The method comprises the following process steps: 1) preparing chondroitin sulfate lyase; 2) carrying out enzymolysis on squid cartilage; 3) purifying; 4) and (5) drying. The method comprises the steps of taking squid cartilage as a raw material, extracting chondroitin sulfate lyase through duck small intestines, applying the chondroitin sulfate lyase to the squid cartilage for enzymolysis to prepare chondroitin sulfate, purifying a fermentation product for the first time through alcohol-salt, purifying the fermentation product for the second time through a combined membrane-electric field, and drying the fermentation product to obtain the product. The invention adopts a biological method and a combined purification method, has high extraction rate and strong biological activity, and the prepared chondroitin sulfate is mainly chondroitin sulfate C. The method has the advantages of simple and efficient process flow, easy control and contribution to large-scale popularization and production.)

1. A method for preparing chondroitin sulfate by utilizing squid cartilage is characterized by comprising the following process steps:

1) chondroitin sulfate lyase preparation

a. Crushing fresh duck small intestines by using a wall breaking machine, adding sterile water according to the weight part of 1:5-6 to prepare duck small enteric solution, stirring for 10-20min, filtering by using a 160-sand 180-mesh sieve, coating the filtrate on a culture medium, performing plate culture separation, selecting single colonies, and purifying by using a slant streaking method; preparing a culture medium containing squid cartilage powder, dibbling the purified single colony on a culture medium flat plate, culturing at constant temperature of 30-37 ℃ for 1-3 days, pouring sterile water into the flat plate, and soaking for 3-5 min; selecting a strain generating a transparent ring, and performing slant streak culture by controlling the temperature to be 30-37 ℃ and performing shaking aerobic culture at the speed of 120-; after the culture is finished, inoculating the strain into a liquid culture medium for culturing production bacteria, and culturing the strain for 1-3 days at the temperature of 30-37 ℃ and the shaking aeration culture at the speed of 120-⁸ - 5×10⁸When the strain per ml is detected, obtaining the seed bacterial liquid of the chondroitin sulfate lyase producing strain;

b. inoculating the chondroitin sulfate lyase producing strain seed bacterial liquid into a fermentation tank according to the inoculation amount of 4-6% v/v, fermenting the culture medium at the temperature of 30-37 ℃, fermenting for 48-60h, centrifuging for 20-30min at 5 ℃ and 4000 + 5500r/min by using a low-temperature centrifuge, taking the supernatant, slowly adding a sodium chloride, ammonium sulfate and ethanol compound into the supernatant in an ice bath, standing for 18-20h at 5 ℃, centrifuging for 25-38min at 5 ℃ and 8000 + 10000r/min by using a low-temperature centrifuge, taking the precipitate, dissolving with deionized water, transferring into a dialysis bag, dialyzing to remove impurities to obtain the chondroitin sulfate lyase;

2) enzymolysis of squid cartilage

a. Washing cartilage of squid with clear water to remove impurities, air drying, pulverizing with a pulverizer, soaking in anhydrous ethanol for 2-5h, centrifuging to obtain precipitate, washing with anhydrous ethanol for several times, and air drying;

b. mixing the squid cartilage for standby in the step a with water according to the weight percentage of 20-30 percent, adding the chondroitin sulfate lyase extracted in the step 1) b according to the weight percentage of 2-5 percent of the mixture, and carrying out enzymolysis for 4-6h at the temperature of 45-51 ℃ and the pH value of 8.5-10 at the speed of 100-120 r/min;

c. after enzymolysis, centrifuging to remove precipitate, collecting supernatant, and vacuum concentrating to obtain solid substance with weight percentage of 30-35%;

3) purification of

a. Adding 65-80% V/V of absolute ethanol into the fermentation concentrated solution obtained in the step 2), adjusting the pH to 5.2-7.0, stirring for 30-60min, standing for 4-5h, and centrifuging to remove the supernatant;

b. dissolving the centrifugal precipitate with deionized water, transferring the solution into a dialysis bag for dialysis and purification, simultaneously adding a direct current electric field of 0.5-0.8V, collecting trapped fluid, adding absolute ethanol into the trapped fluid to enable the ethanol concentration to reach 74-81% V/V, simultaneously adjusting the pH value to 5.5-6.5, standing for 8-10h, centrifuging, and repeatedly crystallizing for 2-3 times;

4) drying

Freeze-drying the crystal obtained in the step 3) by cold air to obtain the chondroitin sulfate glycoprotein.

2. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 1, wherein in the step 1), the culture medium is one of LB culture medium, nutrient agar culture medium or tryptone soy broth culture medium.

3. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 1, wherein, in the step 1), the duck small intestine is obtained from a duck which is fed with shrimp and crab shells as feed ingredients for 2-3 years.

4. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 1, wherein in step 2) b, CaCl2 with the weight of 2-4% and ethanol with the weight of 3-5% are added before adding chondroitin sulfate lyase.

5. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 4, wherein in the step 2) b, 3.5% CaCl2 and 4% ethanol by weight of the mixture are added before adding the chondroitin sulfate lyase.

6. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 1, wherein, in the step 3) b, the dialysis bag has a size of 4-10kD and is ready-to-use.

7. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 1, wherein in the step 1) b, the sodium chloride, ammonium sulfate and ethanol compound are added in an amount of 85-92% by weight.

8. The method for preparing chondroitin sulfate from squid cartilage as claimed in claim 1 or 7, wherein the composition ratio of the sodium chloride, ammonium sulfate and ethanol in step 1) b is W_{Sodium chloride}：W_{Ammonium sulfate}：V_EthanolIs 1:6-8:3, wherein W_{Sodium chloride}For sodium chloride addition by weight, W_{Ammonium sulfate}Adding weight V to ammonium sulfate_EthanolVolume is added for ethanol.

Technical Field

The invention relates to preparation of chondroitin sulfate, in particular to a method for preparing chondroitin sulfate by utilizing squid cartilage.

Background

The squid processing is often accompanied with the generation of a large amount of byproducts, including cartilage, inner shells and the like, the leftovers are rarely utilized, and except the part of the leftovers which are processed into fish meal or feed, the leftovers are easily rotten and deteriorated to be treated as garbage, so that the waste of resources is caused. Researches show that the main components of the squid cartilage comprise protein and chondroitin sulfate, wherein the chondroitin is a biological medicine product and has various activities of reducing blood fat, preventing angiosclerosis and the like. Therefore, the comprehensive utilization is enhanced, waste is changed into valuable, the added value of the sleeve-fish processing method can be effectively improved, the cost of waste treatment is reduced, and the development of sleeve-fish processing is promoted. The squid cartilage has wide sources, so the method is an ideal new source of chondroitin sulfate.

Chondroitin sulfate is glycosaminoglycan, and the prior chondroitin sulfate extraction methods such as the traditional alkaline method, the enzyme method, the neutral salt method, the alkali-salt method, the acetic acid extraction method, the dilute alkali-enzyme method and the like have the defects of complex extraction process, low product yield, high protein content higher than 5 percent and the like. At present, reports of preparing chondroitin sulfate by utilizing enzymolysis and electrical purification are not found.

Disclosure of Invention

In order to overcome the defects of complex extraction process, low product yield and more than 5% of protein content in the traditional method for extracting chondroitin sulfate, the invention aims to provide a method for preparing the chondroitin sulfate by utilizing squid cartilage. The invention adopts a biological method and a combined purification method, has high extraction rate and strong biological activity, the prepared chondroitin sulfate is mainly chondroitin sulfate C, the process flow is simple and efficient, and is easy to control and beneficial to large-scale popularization and production.

The technical scheme adopted by the invention for solving the technical problems is as follows: a method for preparing chondroitin sulfate by utilizing squid cartilage is characterized by comprising the following process steps:

1) chondroitin sulfate lyase preparation

a. Crushing fresh duck small intestines by using a wall breaking machine, adding sterile water according to the weight part of 1:5-6 to prepare duck small enteric solution, stirring for 10-20min, filtering by using a 160-sand 180-mesh sieve, coating the filtrate on a culture medium, performing plate culture separation, selecting single colonies, and purifying by using a slant streaking method; preparing a culture medium containing squid cartilage powder, dibbling the purified single colony on a culture medium flat plate, culturing at constant temperature of 30-37 ℃ for 1-3 days, pouring sterile water into the flat plate, and soaking for 3-5 min; selecting a strain generating a transparent ring, and performing slant streak culture by controlling the temperature to be 30-37 ℃ and performing shaking aerobic culture at the speed of 120-; after the culture is finished, inoculating the strain into a liquid culture medium for culturing production bacteria, and culturing the strain for 1-3 days at the temperature of 30-37 ℃ and the shaking aeration culture at the speed of 120-⁸ - 5×10⁸When the strain per ml is detected, obtaining the seed bacterial liquid of the chondroitin sulfate lyase producing strain;

b. inoculating the chondroitin sulfate lyase producing strain seed bacterial liquid into a fermentation tank according to the inoculation amount of 4-6% v/v, fermenting the culture medium at the temperature of 30-37 ℃, fermenting for 48-60h, centrifuging for 20-30min at 5 ℃ and 4000 + 5500r/min by using a low-temperature centrifuge, taking the supernatant, slowly adding a sodium chloride, ammonium sulfate and ethanol compound into the supernatant in an ice bath, standing for 18-20h at 5 ℃, centrifuging for 25-38min at 5 ℃ and 8000 + 10000r/min by using a low-temperature centrifuge, taking the precipitate, dissolving with deionized water, transferring into a dialysis bag, dialyzing to remove impurities to obtain the chondroitin sulfate lyase;

2) enzymolysis of squid cartilage

a. Washing cartilage of squid with clear water to remove impurities, air drying, pulverizing with a pulverizer, soaking in anhydrous ethanol for 2-5h, centrifuging to obtain precipitate, washing with anhydrous ethanol for several times, and air drying;

b. mixing the squid cartilage for standby in the step a with water according to the weight percentage of 20-30 percent, adding the chondroitin sulfate lyase extracted in the step 1) b according to the weight percentage of 2-5 percent of the mixture, and carrying out enzymolysis for 4-6h at the temperature of 45-51 ℃ and the pH value of 8.5-10 at the speed of 100-120 r/min;

c. after enzymolysis, centrifuging to remove precipitate, collecting supernatant, and vacuum concentrating to obtain solid substance with weight percentage of 30-35%;

3) purification of

a. Adding 65-80% V/V of absolute ethanol into the fermentation concentrated solution obtained in the step 2), adjusting the pH to 5.2-7.0, stirring for 30-60min, standing for 4-5h, and centrifuging to remove the supernatant;

b. dissolving the centrifugal precipitate with deionized water, transferring the solution into a dialysis bag for dialysis and purification, simultaneously adding a direct current electric field of 0.5-0.8V, collecting trapped fluid, adding absolute ethanol into the trapped fluid to enable the ethanol concentration to reach 74-81% V/V, simultaneously adjusting the pH value to 5.5-6.5, standing for 8-10h, centrifuging, and repeatedly crystallizing for 2-3 times;

4) drying

Freeze-drying the crystal obtained in the step 3) by cold air to obtain the chondroitin sulfate glycoprotein.

Preferably, in step 1), the culture medium is one of LB culture medium, nutrient agar culture medium or tryptone soy broth culture medium.

Preferably, in the step 1), the small duck intestines are taken from ducks fed with the shrimp and crab shells as feed ingredients for 2-3 years.

Preferably, in step 2) b, CaCl2 2-4 wt% and ethanol 3-5 wt% of the mixture are added before adding chondroitin sulfate lyase.

Preferably, in step 2) b, CaCl2 of 3.5% and ethanol of 4% by weight of the mixture are added before adding the chondroitin sulfate lyase.

Preferably, in step 3) b, the dialysis bag has a size of 4-10kD and is ready-to-use.

Preferably, in step 1) b, the sodium chloride, ammonium sulfate and ethanol compound is added in an amount of 85-92% by weight.

Preferably, in step 1) b, the composition ratio of the sodium chloride, the ammonium sulfate and the ethanol is W_{Sodium chloride}：W_{Ammonium sulfate}：V_EthanolIs 1:6-8:3, wherein W_{Sodium chloride}For sodium chloride addition by weight, W_{Ammonium sulfate}Adding weight V to ammonium sulfate_EthanolVolume is added for ethanol.

The invention has the beneficial effects that:

(1) the method adopts an enzymolysis coupling dilute salt method to cooperatively extract the chondroitin sulfate, has high extraction rate compared with the existing chondroitin sulfate extraction process, has simple, mild and efficient process flow and easy control, and is beneficial to large-scale popularization and production;

(2) the dialysis coupling electric field purification method adopted by the invention can effectively remove impurity salts while removing macromolecular impurities, and the chondroitin sulfate is gathered in the direction of a negative electrode due to negative electricity, so that the chondroitin sulfate is separated from impurity proteins, and the purity of the product is high;

(3) the chondroitin sulfate enzyme is separated and purified by adopting a salting-out precipitation method coupled with an alcohol precipitation method and an ammonium sulfate method, and is combined with a dialysis method. Compared with a single purification method, the method has the advantages of high enzyme activity and high purity;

(4) the product prepared by the invention is mainly chondroitin sulfate C, wherein the protein content is less than 2.5%.

Drawings

FIG. 1 is a Fourier infrared spectrum of a sample of the product of example 1 with a C-S2 standard.

Detailed Description

The invention is described in detail below with reference to specific embodiments, which are intended to facilitate the understanding and implementation of the invention and are not intended to limit the invention. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

Unless otherwise specified, the culture media in the following examples are all LB media.

Example 1

A method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:

1) chondroitin sulfate lyase preparation

a. Taking fresh duck small intestine from duck fed with shrimp and crab shell as feed ingredients for 2 years, crushing by a wall breaking machine, adding sterile water according to the weight part of 1:5.5 to prepare duck small intestine solution, stirring for 15min, filtering by a 180-mesh sieve, coating the filtrate on a culture medium, performing plate culture separation, selecting single bacterial colony, and purifying by an inclined plane scribing method; preparing a culture medium containing squid cartilage powder, dibbling the purified single colony on a culture medium flat plate, culturing at the constant temperature of 32 ℃ for 2 days, pouring sterile water into the flat plate, and soaking for 4 min; selecting strains which generate transparent circles, carrying out slant streak culture, controlling the temperature to be kept at 32 ℃, and carrying out shaking aerobic culture at 130r/min for 2 days; after the culture, inoculating into liquid culture medium for culturing production bacteria, and performing shake aeration culture at 32 deg.C and 130r/min for 1 day with thallus density of 3 × 10⁸When the strain per ml is detected, obtaining the seed bacterial liquid of the chondroitin sulfate lyase producing strain;

b. inoculating the chondroitin sulfate lyase producing strain seed bacterial liquid into a fermentation tank according to the inoculation amount of 5% v/v, fermenting a culture medium at 32 ℃, fermenting for 48 hours, centrifuging for 25 minutes at 5 ℃ and 5000r/min by using a low-temperature centrifuge, taking supernatant, slowly adding 90% of sodium chloride, ammonium sulfate and ethanol compound into the supernatant in ice bath according to the weight percentage, standing for 18.5 hours at 5 ℃, centrifuging for 30 minutes at 5 ℃ and 9000r/min by using a low-temperature centrifuge, taking precipitate, dissolving with deionized water, transferring into a dialysis bag, and dialyzing to remove impurities to obtain the chondroitin sulfate lyase; the composition ratio of the sodium chloride, the ammonium sulfate and the ethanol compound is W_{Sodium chloride}：W_{Ammonium sulfate}：V_EthanolIs 1:7: 3;

2) enzymolysis of squid cartilage

a. Washing cartilage of squid with clear water to remove impurities, air drying, pulverizing with a pulverizer, soaking in anhydrous ethanol for 4h, centrifuging to obtain precipitate, washing with anhydrous ethanol for several times, and air drying;

b. mixing 28 wt% of squid cartilage for later use in the step a with water, and adding CaCl 3 wt% of the mixture₂4 percent of ethanol and 3.5 percent of chondroitin sulfate lyase, and the temperature is 50 ℃, the pH value is 9, and the enzymolysis is carried out for 5 hours at 120 r/min;

c. after the enzymolysis is finished, centrifuging to remove precipitates, collecting supernate and concentrating in vacuum until the weight percentage content of solid matters is 32%;

3) purification of

a. Adding 70% V/V absolute ethanol into the fermentation concentrated solution obtained in the step 2), adjusting the pH to 6.3, stirring for 40min, standing for 4.5h, and centrifuging to remove the supernatant;

b. dissolving the centrifugal precipitate with deionized water, transferring the solution into a dialysis bag with the specification of 8kD for use, dialyzing and purifying, simultaneously adding a direct current electric field of 0.6V, collecting trapped fluid, adding absolute ethyl alcohol into the trapped fluid to enable the concentration of the ethyl alcohol to reach 78% V/V, simultaneously adjusting the pH value to 6.3, standing for 9h, centrifuging, and repeatedly crystallizing for 3 times;

4) drying

Freeze-drying the crystal obtained in the step 3) by cold air to obtain the chondroitin sulfate glycoprotein.

Example 2

A method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:

1) chondroitin sulfate lyase preparation

a. Taking fresh duck small intestine from duck fed with shrimp and crab shell as feed ingredients for 2 years, crushing by a wall breaking machine, adding sterile water according to the weight part of 1:5 to prepare duck small intestine solution, stirring for 10min, filtering by a 160-mesh sieve, taking filtrate, coating a culture medium on the filtrate, performing plate culture separation, selecting single bacterial colony, and purifying by a slant surface scribing method; preparing culture medium containing squid cartilage powder, inoculating purified single colony on culture medium plate, culturing at 30 deg.C for 1 day, and pouring sterile liquidSoaking in water for 3 min; selecting strains which generate transparent rings, carrying out slant streak culture, controlling the temperature to be kept at 30 ℃, and carrying out shaking aerobic culture at 120r/min for 1 day; after the culture, inoculating into liquid culture medium for culturing producing bacteria, and culturing at 30 deg.C and 120r/min under shaking and aeration conditions for 1 day with cell density of 1 × 10⁸When the strain per ml is detected, obtaining the seed bacterial liquid of the chondroitin sulfate lyase producing strain;

b. inoculating the chondroitin sulfate lyase producing strain seed bacterial liquid into a fermentation tank according to the inoculation amount of 4% v/v, fermenting a culture medium at 30 ℃, fermenting for 48 hours, centrifuging for 2 minutes at 5 ℃ and 4000r/min by using a low-temperature centrifuge, taking supernatant, slowly adding 85% of sodium chloride, ammonium sulfate and ethanol compound into the supernatant according to the weight percentage in an ice bath, standing for 18 hours at 5 ℃, centrifuging for 25 minutes at 5 ℃ and 8000r/min by using a low-temperature centrifuge, taking precipitate, dissolving with deionized water, transferring into a dialysis bag, and dialyzing to remove impurities to obtain the chondroitin sulfate lyase; the composition ratio of the sodium chloride, the ammonium sulfate and the ethanol compound is W_{Sodium chloride}：W_{Ammonium sulfate}：V_EthanolIs 1:6: 3;

2) enzymolysis of squid cartilage

a. Washing cartilage of squid with clear water to remove impurities, air drying, pulverizing with a pulverizer, soaking in anhydrous ethanol for 2h, centrifuging to obtain precipitate, washing with anhydrous ethanol for several times, and air drying;

b. mixing the squid cartilage reserved in the step a with water according to the weight percentage of 20%, adding CaCl2 with the weight of 2%, ethanol with the weight of 3% and chondroitin sulfate lyase with the weight of 2%, performing enzymolysis for 4 hours at the temperature of 45 ℃ and the pH value of 8.5 at the speed of 100 r/min;

c. after the enzymolysis is finished, centrifuging to remove the precipitate, collecting supernatant, and vacuum concentrating until the weight percentage content of solid matters is 30%;

3) purification of

a. Adding 65% V/V absolute ethanol into the fermentation concentrated solution obtained in the step 2), adjusting the pH to 5.2, stirring for 30min, standing for 4h, and centrifuging to remove the supernatant;

b. dissolving the centrifugal precipitate with deionized water, transferring the solution into a dialysis bag with a specification of 4kD for use, dialyzing and purifying, simultaneously adding a direct current electric field of 0.5V, collecting trapped fluid, adding absolute ethyl alcohol into the trapped fluid to enable the concentration of the ethyl alcohol to reach 74% V/V, simultaneously adjusting the pH value to 5.5, standing for 8h, centrifuging, and repeatedly crystallizing for 2 times;

4) drying

Freeze-drying the crystal obtained in the step 3) by cold air to obtain the chondroitin sulfate glycoprotein.

Example 3

A method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:

1) chondroitin sulfate lyase preparation

a. Taking fresh duck small intestine from duck fed with shrimp and crab shell as feed ingredients for 3 years, crushing by a wall breaking machine, adding sterile water according to the weight part of 1:6 to prepare duck small intestine solution, stirring for 20min, filtering by a 180-mesh sieve, taking filtrate, coating a culture medium on the filtrate, performing plate culture separation, selecting single bacterial colony, and purifying by a slant surface scribing method; preparing a culture medium containing squid cartilage powder, dibbling the purified single colony on a culture medium flat plate, culturing at the constant temperature of 37 ℃ for 3 days, pouring sterile water into the flat plate, and soaking for 5 min; selecting strains which produce transparent circles, carrying out slant streak culture, controlling the temperature to be 37 ℃, and carrying out shaking aerobic culture at 150r/min for 3 days; after the culture, the strain was inoculated into a liquid medium to culture a producer, and the strain was cultured at 37 ℃ and 150r/min with shaking aeration for 3 days at a cell density of 5X 10⁸When the strain per ml is detected, obtaining the seed bacterial liquid of the chondroitin sulfate lyase producing strain;

b. inoculating the chondroitin sulfate lyase producing strain seed bacterial liquid into a fermentation tank according to the inoculation amount of 6% v/v, fermenting a culture medium at 37 ℃, fermenting for 60 hours, centrifuging for 30 minutes at 5 ℃ and 5500r/min by using a low-temperature centrifuge, taking supernatant, slowly adding 92% of sodium chloride, ammonium sulfate and ethanol compound into the supernatant in ice bath according to the weight percentage, standing for 20 hours at 5 ℃, centrifuging for 38 minutes at 5 ℃ and 10000r/min by using the low-temperature centrifuge, taking precipitate, dissolving with deionized water, transferring into a dialysis bag, and dialyzing to remove impurities to obtain the chondroitin sulfate lyase; the composition ratio of the sodium chloride, the ammonium sulfate and the ethanol compound is W_{Sodium chloride}：W_{Ammonium sulfate}：V_EthanolIs 1:8: 3;

2) enzymolysis of squid cartilage

a. Washing cartilage of squid with clear water to remove impurities, air drying, pulverizing with a pulverizer, soaking in anhydrous ethanol for 5h, centrifuging to obtain precipitate, washing with anhydrous ethanol for several times, and air drying;

b. mixing the squid cartilage prepared in the step a with water according to the weight percentage of 20-30%, adding CaCl2, 5% ethanol and 5% chondroitin sulfate lyase which are based on the weight of the mixture, and carrying out enzymolysis for 6 hours at the temperature of 51 ℃, the pH value of 10 and the speed of 120 r/min;

c. after enzymolysis, centrifuging to remove precipitate, collecting supernatant, and vacuum concentrating to obtain solid substance with weight percentage of 35%;

3) purification of

a. Adding 80% V/V absolute ethanol into the fermentation concentrated solution obtained in the step 2), adjusting the pH to 7.0, stirring for 60min, standing for 5h, and centrifuging to remove the supernatant;

b. dissolving the centrifugal precipitate with deionized water, transferring the solution into a dialysis bag with the specification of 10kD, dialyzing and purifying the solution in a ready-to-use dialysis bag, simultaneously adding a direct current electric field of 0.8V, collecting trapped fluid, adding absolute ethyl alcohol into the trapped fluid to enable the concentration of the ethyl alcohol to reach 81% V/V, simultaneously adjusting the pH value to 6.5, standing for 10h, centrifuging, and repeatedly crystallizing for 3 times;

4) drying

Freeze-drying the crystal obtained in the step 3) by cold air to obtain the chondroitin sulfate glycoprotein.

Comparative example 1

In this comparative example, the preparation was essentially the same as in example 1, except that: in the step 1), the chondroitin sulfate lyase producing strain is not extracted, and the crushed small duck intestine is directly used for fermentation in the step 2) b.

Comparative example 2

In this comparative example, the preparation was essentially the same as in example 1, except that: no CaCl is added before fermentation in the step 2) b₂And ethanol as an adjuvant without adjusting the pH.

Comparative example 3

In this comparative example, the preparation was essentially the same as in example 1, except that: no ultrasonic auxiliary treatment is carried out in step 2) b.

Comparative example 4

In this comparative example, the preparation method thereofThe process is essentially the same as in example 1, with the following differences: in the step 2) b, CaCl is not added₂And ethanol as an auxiliary agent without adjusting pH and without assisting ultrasonic treatment.

Comparative example 5

In this comparative example, the preparation was essentially the same as in example 1, except that: in step 2) b, no microorganisms are inoculated.

Comparative example 6

In this comparative example, the preparation was essentially the same as in example 1, except that: in step 3) no dialysis bag dialysis operation is performed.

Comparative example 7

In this comparative example, the preparation was essentially the same as in example 1, except that: no electric field treatment is applied in step 3).

Comparative example 8

In this comparative example, the preparation was essentially the same as in example 1, except that: the operation of step 3) is not performed.

Comparative example 9

In this comparative example, the preparation was essentially the same as in example 1, except that: in step 1) b, sodium chloride and ammonium sulfate are not added.

Comparative example 10

In this comparative example, the preparation was essentially the same as in example 1, except that: in step 1) b, no ammonium sulfate and no ethanol are added.

Comparative example 11

In this comparative example, the preparation was essentially the same as in example 1, except that: no sodium chloride and no ethanol were added in step 1) b.

The above examples and comparative examples were compared in extraction yield, which is the ratio of the finally obtained product to the weight of the raw material, and purity, and the results are shown in Table 1.

TABLE 1 analysis of chondroitin sulfate extraction yield and purity under different treatment conditions

Table 1 shows that the examples have ideal extraction rate and purity, and the combination of the extraction method and the purification method constitutes an advanced process for preparing chondroitin sulfate. The extraction of chondroitin sulfate in comparative examples 1-5 is significantly influenced by microbial purification and fermentation aids, wherein the microbial extraction and purification are determining factors influencing the extraction rate, but the extraction rate of chondroitin sulfate by a single microorganism is low, and the extraction rate of chondroitin sulfate is significantly improved by adopting a composite extraction method. Comparative examples 6-8 chondroitin sulfate purification was affected by both the dialysis procedure and the electric field, but the dialysis bag was more pronounced. The chondroitin sulfate prepared in comparative examples 9-11 is significantly affected by the precipitant, and compared with example 1, the chondroitin sulfate prepared by using only one precipitant has low activity, resulting in poor enzymolysis effect and reduced product purity.

The above examples and comparative examples were compared for protein and chondroitin sulfate contents, the protein/chondroitin sulfate content being the ratio of the amount of protein/chondroitin sulfate in the finally obtained product to the weight of the product, and the results are shown in Table 2.

TABLE 2 analysis of the product protein with chondroitin sulfate

Table 2 shows that the protein contents of the examples are less than 2.5%, and the protein contents of the other operations are higher, the chondroitin sulfate contents of the finished chondroitin sulfate obtained from the respective groups of examples using the preparation conditions provided by the present invention are higher than those of the comparative examples, and the protein contents of the finished chondroitin sulfate are lower than those of the comparative examples, and then, the effects of dialysis and electric field on the extraction of chondroitin sulfate are significant.

The chemical structure of a sample of the product of example 1 was analyzed by fourier infrared spectroscopy (KBr tabletting). Wherein the Fourier infrared spectrum of the product sample of example 1 and the C-S2 standard is shown in FIG. 1.

As can be seen from FIG. 1, the infrared spectrum of the product sample of example 1 is substantially consistent with that of the standard product, namely, the cartilage C sulfate at 918cm^-1There is a characteristic absorption of typical chondroitin sulfate a in the vicinity. The weak characteristic absorption of the product sample shows that a small amount of Chs-A exists in the sample, which is consistent with the report at home and abroad. That is, the method adopted by the application is reliable and effective, and the prepared chondroitin sulfate is relatively good in uniformity and basically is chondroitin sulfate C.

The effectiveness of the process product was tested for in vitro anti-tumor activity using the product sample of example 1.

Culturing the breast cancer cell line 4T1 cells under the following culture conditions: adding 10% fetal calf serum, 100U/mL penicillin and 100 μ g/mL streptomycin double antibody into DMEM culture solution containing 2mM non-essential amino acids and 2mM L-glutamine, adding appropriate concentration of cells, 37 deg.C, and 5% CO₂Culturing in an incubator. The specific steps of the detection are described in patent CN 202010552297.8.

TABLE 3 analysis of antitumor Activity

Group of	0μg/mL	25μg/mL	50μg/mL	100μg/mL	200μg/mL
						Control group/%)	100	100	100	100	100
Administration group/%	100	73	65	54	48

The detection result shows that the number of 4T1 cells added into the product of example 1 is reduced; and the ability of anti-tumor cell proliferation is gradually enhanced along with the increase of polysaccharide concentration, under the action of 200 mug/mL of the product, the cell number only reaches 45-50% of that of the product under the sugar-free action, and the product has the activity of inhibiting tumor cell proliferation, and the result shows that the product and the extraction method are feasible and effective.

The chondroitinase activities were measured spectrophotometrically using the chondroitinase prepared in example 1 and comparative examples 9 to 11 as the study subjects.

TABLE 4 results of enzyme Activity measurement

Group of	Comparative example 9	Comparative example 10	Comparative example 11	Example 1
					Activity value	0.5726	0.6324	0.7943	0.9817

The results in Table 4 show that the chondroitin sulfate prepared in example 1 has a significantly affected enzyme activity compared to comparative examples 9-11, the enzyme precipitant composition is poor, and the enzyme activity prepared from the composite precipitant is higher.