A method for precipitating type II collagen by polymer association phase separation

Abstract

The present invention discloses a method for precipitating type II collagen by polymer association phase separation, the method comprising: obtaining type II collagen enzymatic hydrolysate by enzymatic hydrolysis, preparing anionic polysaccharide solution, and adjusting the pH of polysaccharide solution and enzymatic hydrolysate to acidic; adding polysaccharide solution to enzymatic hydrolysate, stirring evenly; standing, centrifuging to obtain precipitation, freeze drying, crushing, and obtaining type II collagen powder. The anionic polysaccharides of the present invention include pectin, chondroitin sulfate, dextran sulfate, sodium alginate, etc., which have relatively small molecular weight and short chain length, and can associate with type II collagen to form a tight complex under acidic conditions. Compared with the traditional salting-out technology, the product obtained by the present invention has significantly increased collagen content, reduced salt content, lower water content, good stability and high activity. The technical scheme of the present invention is simple and easy to implement, and has high yield.

Description

Technical Field

[0001] The invention belongs to the technical field of health care products, and in particular relates to a method for precipitating type II collagen through polymer association phase separation. Background Art

[0002] Type II collagen is an important component of cartilage matrix and is mainly distributed in tissues such as articular cartilage, vitreous body, nucleus pulposus, embryonic cornea and retinal nerve membrane. It is a macromolecule with a complete triple helix structure composed of 3 identical α1 polypeptide chains. Each α1 polypeptide chain contains about 1000 amino acid residues and has a relative molecular mass of about 130kDa. Due to its special molecular structure, type II collagen is widely used in food, medicine, materials and other fields. More importantly, a large number of studies have shown that oral non-denatured type II collagen can inhibit the occurrence of arthritis or relieve arthritis symptoms in animals and humans.

[0003] At present, type II collagen is mainly extracted by enzymatic method, including impurity removal, enzymatic hydrolysis, salting out and other steps. Among them, the salting out step is very important. Its function is to precipitate type II collagen from the solution to facilitate subsequent freeze drying. Commonly used salts include sodium chloride, potassium chloride, etc. On the one hand, they compete with proteins in the solution for water molecules and destroy the water film on the surface of protein colloid particles; on the other hand, they neutralize the charge on protein particles in large quantities, so that protein particles in the water accumulate and precipitate out. The advantage of salting out method for protein precipitation is that it is simple to operate, can improve the purity of the product, and reduce the water content of the product for subsequent drying. However, the disadvantages of the salting out method are also obvious, that is, a large amount of salt will be introduced, making the salt content of the product too high. However, the high salt intake when taking the product may also bring other harms to the human body, which does not meet the requirements of healthy food. In addition, the type II collagen obtained by the salting out method usually needs to be dialyzed to reduce the salt content, which will greatly increase the workload, time and cost. Therefore, it is of great significance to find a convenient and efficient alternative to salting out.

[0004] In the past few decades, with the increasing demand for new functional ingredients in the pharmaceutical and food industries, people have shown increasing interest in protein-polysaccharide complexes. For example, proteins and polysaccharides are used together in gels, foams, and emulsions to improve the strength of gels and the stability of foams and emulsions; proteins are complexed with polysaccharides to improve the thermal stability or gastrointestinal digestion stability of proteins; and the selective complexation phenomenon caused by protein-polysaccharide interactions is used to separate and purify different components in soy protein.

[0005] A Chinese patent application for a method for extracting non-denatured type II collagen from chicken sternal cartilage reports a method for extracting type II collagen from chicken sternal cartilage, including sterilization, crushing and sugar removal, two enzymatic hydrolysis, dialysis, drying and other steps. The supernatant obtained from the two enzymatic hydrolysis is directly dried after dialysis, but due to the addition of a large amount of pure water during the enzymatic hydrolysis and the dilution of the solution after dialysis, the volume is further increased, which will greatly increase the difficulty and workload of the drying step. Therefore, finding a convenient and efficient method for recovering type II collagen from the enzymatic hydrolyzate is a technical bottleneck that urgently needs to be solved in this field.

[0006] Studies have found that aqueous mixed systems of biopolymers (such as proteins and polysaccharides) can achieve phase separation in two different ways: one is to form thermodynamically compatible intermolecular complex coacervates through attractive interactions, which is called associative phase separation. At this time, the two biopolymers are concentrated in the same phase region, and electrostatic interaction is the most important binding force; the other is to form two thermodynamically incompatible phase regions through repulsive interactions, which is called separation phase separation. At this time, each phase is rich in one biopolymer. In addition, the interaction between proteins and polysaccharides is related to their binding affinity, molecular charge density, molecular conformation (such as contour length, molecular weight and flexibility), environmental conditions (pH, mixing ratio and ionic strength), etc. However, there are no reports on the use of polysaccharides for the preparation of type II collagen. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a method for precipitating type II collagen through polymer association phase separation and a product thereof.

[0008] The present invention recovers type II collagen from a solution or an enzymatic hydrolyzate by separating the anionic polysaccharide from the associated phase, thereby avoiding the problems of high salt content and poor stability of the product caused by the traditional salting-out method.

[0009] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0010] The present invention provides a method for precipitating type II collagen by polymer association phase separation, comprising the following steps:

[0011] (1) obtaining type II collagen enzymatic hydrolysate by enzymatic hydrolysis, and selecting anionic polysaccharides with relatively small molecular weight and short chain length to prepare anionic polysaccharide solutions;

[0012] (2) adjusting the pH values ​​of the anionic polysaccharide solution and the type II collagenase hydrolysate to weak acidity to obtain an acidic anionic polysaccharide solution and an acidic hydrolysate;

[0013] (3) Add the acidic anionic polysaccharide solution to the acidic enzymatic hydrolyzate solution in proportion, stirring evenly while adding; let it stand for a period of time, centrifuge to obtain the precipitate, freeze-dry the precipitate, and crush it to obtain type II collagen powder.

[0014] Furthermore, the extraction steps of the type II collagenase hydrolysate in step (1) include: thawing the raw materials, washing, sterilizing, homogenizing, removing impurities, and enzymolysis.

[0015] Furthermore, the extraction step of the type II collagenase hydrolysate in step (1) includes: thawing and washing the chicken sternal cartilage, adding deionized water and homogenizing at a solid-liquid ratio of 1kg: (3-7) L (w / v) to obtain a chicken sternal cartilage homogenate, adding NaCl (final concentration of 0.5-2.0 mol / L) and stirring for 2-12 hours to remove foreign proteins in the cartilage. Then centrifuging at 5000-8000 rpm for 10-20 minutes to obtain a precipitate, adding 0.1-1.0 mol / L acetic acid aqueous solution at a solid-liquid ratio of 1kg: (3-7) L (w / v), and adding 0.1%-2% (w / w) pepsin by weight of the precipitate. After continuous shaking for 10-20 hours in a constant temperature shaker at 10-30°C, centrifuging at 5000-8000 rpm for 10-20 minutes, collecting the enzymatic supernatant, and obtaining an enzymatic hydrolysate.

[0016] Furthermore, the anionic polysaccharide in step (1) has a relatively small molecular weight (<300 kDa) and a short chain length (<60 nm), and can associate with type II collagen to form a tight complex under acidic conditions.

[0017] Preferably, the anionic polysaccharide includes one or more of pectin, sodium alginate, chondroitin sulfate, and dextran sulfate.

[0018] Furthermore, in step (1), the concentration of the type II collagenase hydrolysate is 6-10 mg / mL, and the concentration of the anionic polysaccharide solution is 1-5 mg / mL.

[0019] Furthermore, in step (1), the concentration of the type II collagenase hydrolysate is 8 mg / mL.

[0020] Furthermore, in step (2), the pH values ​​are all adjusted to weak acidity, that is, the pH values ​​are all adjusted to 2.0-5.0.

[0021] Furthermore, in step (3), the volume ratio of the acidic anionic polysaccharide solution to the acidic enzymatic hydrolysate is (0.1-1):1.

[0022] Furthermore, the standing time in step (3) is 1-8 hours.

[0023] Furthermore, the centrifugation conditions in step (3) are 4000-5000 rpm, 10-20 min.

[0024] Furthermore, the centrifugation time in step (3) is 15-20 min.

[0025] Furthermore, the freeze-drying time in step (3) is 12-24 hours.

[0026] The present invention also provides type II collagen prepared by the preparation method.

[0027] Furthermore, the protein content of the type II collagen is >50.0%, the collagen content is >45.0%, the salt content (ie, ash content) is <10.0%, and the moisture content is <8.0%.

[0028] The anionic polysaccharides used in the present invention are the most widely used anionic polysaccharides in the food industry, such as pectin, sodium alginate, dextran sulfate, etc. In addition, the chondroitin sulfate used in the present invention is an anionic polysaccharide with a relatively high content in human and animal cartilage, which has a certain improvement effect on arthritis, has the effects of relieving pain, and promoting cartilage regeneration.

[0029] Compared with the existing salting-out technology, the present invention has the following advantages and beneficial effects:

[0030] 1. The type II collagen prepared by the present invention has significantly increased collagen content, reduced salt content, lower water content, and good stability.

[0031] 2. The protein recovery rate of the preparation method of the present invention is 45.0%±2.0%, and the collagen recovery rate is 40.0%±1.5%.

[0032] 3. The preparation method of the present invention is simple and easy, with high yield, and the prepared type II collagen has high activity, low salt content and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a bar chart of protein content and collagen content of the products prepared in Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4 after freeze-drying.

[0034] Figure 2 The electrophoresis patterns of the products prepared in Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4 at the same protein loading amount.

[0035] Figure 3 The circular dichroism spectra of the products prepared in Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4 at the same protein concentration are shown.

[0036] Figure 4 It is a bar graph of the protein recovery rate and collagen recovery rate of the preparation methods of Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4.

[0037] Figure 5 The salt content and moisture content of the products prepared in Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4 after freeze-drying;

[0038] Figure 6 The scanning electron microscope images of the products prepared in Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4 after freeze-drying.

[0039] Figure 7 The differential scanning calorimetry analysis diagrams of the products prepared in Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4 are shown. DETAILED DESCRIPTION

[0040] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0041] The weight (mass) parts used in the following embodiments and comparative examples are, for example, weight units may be grams, kilograms, etc., or any other commonly used amount in the art.

[0042] Comparative Example 1

[0043] An existing method for preparing type II collagen comprises the following steps:

[0044] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, and 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added. After continuous shaking for 12 h in a constant temperature shaker at 10 ° C, the supernatant was collected by centrifugation at 6000 rpm for 15 min.

[0045] 2. Adjust the pH value of the enzymatic hydrolysate to 7.0 to obtain a neutral enzymatic hydrolysate.

[0046] 3. Prepare 5 mol / L NaCl aqueous solution, mix them in a volume ratio of NaCl aqueous solution: neutral enzymatic solution = 1:1, and stir evenly.

[0047] 4. Let stand for 10 hours, centrifuge at 5000r / min for 15 minutes to collect the precipitate.

[0048] 5. Freeze-dry the precipitate for 20 hours and grind it in a grinder to obtain a type II collagen powder.

[0049] Comparative Example 2

[0050] An existing method for preparing type II collagen comprises the following steps:

[0051] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a 10°C constant temperature shaker, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0052] 2. Adjust the pH value of the enzymatic hydrolysate to 4.0 to obtain an acidic enzymatic hydrolysate.

[0053] 3. Prepare 5 mol / L NaCl aqueous solution, mix them in a volume ratio of NaCl aqueous solution: acidic enzymatic solution = 0.7:1, and stir evenly.

[0054] 4. Let stand for 10 hours, centrifuge at 5000r / min for 15 minutes to collect the precipitate.

[0055] 5. Freeze-dry the precipitate for 24 hours and grind it in a grinder to obtain a type II collagen powder.

[0056] Comparative Example 3

[0057] A method for precipitating type II collagen by polymer association phase separation (when the anionic polysaccharide used has a large molecular weight, a long chain length and a rigid structure), comprising the following steps:

[0058] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a 10°C constant temperature shaker, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0059] 2. Prepare xanthan gum aqueous solution with a concentration of 3 mg / mL.

[0060] 3. Adjust the pH values ​​of the xanthan gum aqueous solution and the enzymatic hydrolysate to 3.0 respectively to obtain an acidic xanthan gum aqueous solution and an acidic enzymatic hydrolysate.

[0061] 4. Add the acidic xanthan gum aqueous solution into the acidic enzymatic hydrolysate in a volume ratio of acidic xanthan gum aqueous solution: acidic enzymatic hydrolysate = 0.2:1, stirring evenly while adding.

[0062] 5. Let stand for 6 hours, centrifuge at 5000r / min for 15 minutes, the system does not separate and no precipitation occurs.

[0063] Comparative Example 4

[0064] A method for precipitating type II collagen by polymer association phase separation (when the pH conditions of the anionic polysaccharide solution and the enzymatic hydrolyzate are neutral), comprising the following steps:

[0065] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a constant temperature shaker at 10 ° C, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0066] 2. Prepare pectin aqueous solution with a concentration of 4 mg / mL.

[0067] 3. Adjust the pH values ​​of the pectin aqueous solution and the enzymatic hydrolysate to 7.0 respectively to obtain a neutral pectin aqueous solution and a neutral enzymatic hydrolysate.

[0068] 4. Add the neutral pectin solution into the neutral enzymatic solution at a volume ratio of neutral pectin aqueous solution: neutral enzymatic solution = 0.1:1, stirring evenly while adding.

[0069] 5. After standing for 4 hours, centrifuge at 5000r / min for 15 minutes. No precipitation occurs.

[0070] Embodiment 1

[0071] A method for precipitating type II collagen by polymer association phase separation, comprising the following steps:

[0072] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a constant temperature shaker at 10 ° C, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0073] 2. Prepare pectin aqueous solution with a concentration of 4 mg / mL.

[0074] 3. The pH values ​​of the pectin aqueous solution and the enzymatic hydrolysate were adjusted to 5.0 respectively to obtain an acidic pectin aqueous solution and an acidic enzymatic hydrolysate.

[0075] 4. Add the acidic pectin aqueous solution to the acidic enzymatic solution in a volume ratio of acidic pectin aqueous solution: acidic enzymatic solution = 0.1:1, stirring evenly while adding.

[0076] 5. Let stand for 4 hours, centrifuge at 4000r / min for 15 minutes to collect the precipitate.

[0077] 6. Freeze-dry the precipitate for 24 hours and grind it with a grinder to obtain the type II collagen.

[0078] Embodiment 2

[0079] A method for precipitating type II collagen by polymer association phase separation, comprising the following steps:

[0080] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a constant temperature shaker at 10 ° C, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0081] 2. Prepare chondroitin sulfate aqueous solution with a concentration of 5 mg / mL.

[0082] 3. The pH of the chondroitin sulfate aqueous solution and the enzymatic hydrolysate are adjusted to 2.0 respectively to obtain an acidic chondroitin sulfate aqueous solution and an acidic enzymatic hydrolysate.

[0083] 4. Add the acidic chondroitin sulfate aqueous solution to the acidic enzymatic solution at a volume ratio of acidic chondroitin sulfate aqueous solution: acidic enzymatic solution = 0.8:1, and stir evenly while adding.

[0084] 5. Let stand for 2 hours, centrifuge at 5000r / min for 10 minutes to collect the precipitate.

[0085] 6. Freeze-dry the precipitate for 12 hours and grind it with a grinder to obtain the type II collagen.

[0086] Embodiment 3

[0087] A method for precipitating type II collagen by polymer association phase separation, comprising the following steps:

[0088] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a constant temperature shaker at 10 ° C, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0089] 2. Prepare sodium alginate aqueous solution with a concentration of 1 mg / mL.

[0090] 3. The pH of the sodium alginate aqueous solution and the enzymatic hydrolysate were adjusted to 4.0 respectively to obtain an acidic sodium alginate aqueous solution and an acidic enzymatic hydrolysate.

[0091] 4. Add the acidic sodium alginate aqueous solution to the acidic enzymatic solution in a volume ratio of acidic sodium alginate aqueous solution: acidic enzymatic solution = 0.7:1, stirring evenly while adding.

[0092] 5. Let stand for 8 hours, centrifuge at 5000r / min for 15 minutes to collect the precipitate.

[0093] 6. Freeze-dry the precipitate for 18 hours and grind it with a grinder to obtain the type II collagen.

[0094] Embodiment 4

[0095] A method for precipitating type II collagen by polymer association phase separation, comprising the following steps:

[0096] 1. Type II collagenase hydrolysate: 5 kg of chicken sternal cartilage was thawed and washed, and 20 L of deionized water was added and homogenized to obtain a chicken sternal cartilage homogenate. 1800 g of NaCl was added and stirred for 10 h to remove the impurities in the cartilage. Then, the precipitate was centrifuged at 6000 rpm for 15 min, 20 L of 0.5 mol / L acetic acid aqueous solution was added, and 2% pepsin was added to the precipitate. After continuous shaking for 12 h in a constant temperature shaker at 10 ° C, the supernatant was collected and the hydrolysate was obtained by centrifugation at 6000 rpm for 15 min.

[0097] 2. Prepare dextran sulfate aqueous solution with a concentration of 3 mg / mL.

[0098] 3. The pH of the dextran sulfate aqueous solution and the enzymatic hydrolyzate were adjusted to 3.0 respectively to obtain an acidic dextran sulfate aqueous solution and an acidic enzymatic hydrolyzate.

[0099] 4. Add the acidic dextran sulfate aqueous solution to the acidic enzymatic hydrolysate at a volume ratio of acidic dextran sulfate aqueous solution: acidic enzymatic hydrolysate = 0.8:1, stirring evenly while adding.

[0100] 5. Let stand for 1 hour, centrifuge at 4000r / min for 10 minutes to collect the precipitate.

[0101] 6. Freeze-dry the precipitate for 16 hours and grind it with a grinder to obtain the type II collagen.

[0102] The appearance of the products prepared in Comparative Examples 1, 2, 3 and 4, and Examples 1, 2, 3 and 4 after centrifugation. The precipitates of Comparative Examples 1 and 2 after centrifugation gathered at the bottom of the centrifuge tube, and no stratification occurred in Comparative Examples 3 and 4 after centrifugation, and no precipitate could be obtained; while the complex of anionic polysaccharide and type II collagen in Examples 1, 2, 3 and 4 became a thin layer after centrifugation, attached to the side of the centrifuge tube, and had more supernatant.

[0103] After freeze drying, the protein content and collagen content of the product were determined. Figure 1 As shown, the protein content and collagen content of type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3 and Example 4 (56%-61% and 50%-58%, respectively) are significantly higher than the type II collagen obtained by the preparation methods of Comparative Example 1 and Comparative Example 2 (32%-34% and 27%-28%, respectively).

[0104] The purity and structure of the product were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Figure 2 and Figure 3As shown, the α1 chain bands of the products obtained by the preparation methods of Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 and Example 2 are darker in color and have higher purity, and contain almost no other impurity proteins; and the circular dichroism spectra of the type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3, and Example 4 have an ellipticity of 0 at a wavelength of 214nm, a positive absorption peak at 222nm, which is a typical feature of a L-polyproline configuration peptide chain, and a strong negative absorption peak at 202nm, which is a typical feature of irregular curling in the triple helix structure of type II collagen, indicating that the triple helix structure of the type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3, and Example 4 is intact.

[0105] The protein recovery rate and collagen recovery rate of the preparation methods in each comparative example and embodiment are as follows: Figure 4 As shown, the protein recovery rate and collagen recovery rate of the preparation methods of Example 1, Example 2, Example 3 and Example 4 are slightly higher than those of the preparation methods of Comparative Example 1 and Comparative Example 2, indicating that the preparation method of the present invention has high feasibility.

[0106] Further determination of the basic components of the products obtained by the preparation methods of the comparative examples and the examples showed that Figure 5 As shown, the ash content of the type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3 and Example 4 is significantly reduced (less than 6%), while the ash content of the type II collagen obtained by the preparation methods of Comparative Example 1 and Comparative Example 2 is very high (more than 55%); and the moisture content (1%-3%) of the type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3 and Example 4 is also slightly lower than the type II collagen obtained by the preparation methods of Comparative Example 1 and Comparative Example 2 (3%-5%).

[0107] Since the freeze-dried samples can maintain their original chemical composition and physical properties (such as porous structure, colloidal properties, etc.), the differences in the microstructures of the products obtained by the preparation methods of the comparative example and the example were observed by scanning electron microscopy. Figure 6 (magnification is 200 times) as shown, the type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3 and Example 4 has a more uniform, flat and dense structure. It can be seen that before freeze-drying, the type II collagen is more tightly combined with the small molecular weight anionic polysaccharide, while the type II collagen obtained by the preparation methods of Comparative Example 1 and Comparative Example 2 contains a large amount of salt crystals on the surface, and the structure may be looser.

[0108] The thermal stability of the products prepared by the methods of each embodiment and comparative example was analyzed by differential scanning calorimetry. Figure 7 (Since the salt content of type II collagen obtained in Comparative Example 1 and Comparative Example 2 was too high, it interfered with differential scanning calorimetry. Figure 7 The results in the table are the results obtained after dialysis of type II collagen) and Table 1 (Table 1 is the denaturation temperature and enthalpy change of the products prepared by Comparative Example 1, Comparative Example 2, Example 1, Example 2, Example 3 and Example 4. Since the salt content of the type II collagen obtained in Comparative Example 1 and Comparative Example 2 is too high and interferes with the differential scanning calorimetry, the results in Table 1 are the results obtained after dialysis of type II collagen). The denaturation temperature of type II collagen obtained by the preparation methods of Example 1, Example 2, Example 3 and Example 4 (52.80-54.45°C) is higher than that of Comparative Example 1 (50.80°C) and Comparative Example 2 (51.20°C), and the enthalpy change value (45.07-63.45J / g -1 ) is also higher than the comparative example 1 (39.92 J / g -1 ) and Comparative Example 2 (37.96 J / g -1 ), this is because type II collagen and anionic polysaccharides are tightly bound through electrostatic attraction interactions, which increases the enthalpy change of type II collagen.

[0109] Table 1

[0110]

[0111] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for precipitating a type II collagen product by polymer association phase separation, characterized in that: The steps include: (1) obtaining type II collagenase hydrolysate by enzymatic hydrolysis, selecting anionic polysaccharides, and preparing anionic polysaccharide solution; the molecular weight of the anionic polysaccharide is less than 300 kDa, and the chain length of the anionic polysaccharide is less than 60 nm; the anionic polysaccharide comprises one or more of pectin, chondroitin sulfate, dextran sulfate, and sodium alginate; the concentration of the type II collagenase hydrolysate is 6-10 mg / mL, and the concentration of the anionic polysaccharide solution is 1-5 mg / mL; (2) adjusting the pH values ​​of the anionic polysaccharide solution and the type II collagenase hydrolysate to acidic to obtain an acidic anionic polysaccharide solution and an acidic hydrolysate; the pH values ​​are adjusted to acidic to a pH value of 2.0-5.0; (3) adding an acidic anionic polysaccharide solution to an acidic enzymatic hydrolysate, wherein the volume ratio of the acidic anionic polysaccharide solution to the acidic enzymatic hydrolysate is (0.1-1):1; stirring evenly while adding, standing and centrifuging to obtain a precipitate, freeze-drying the precipitate, and crushing it to obtain a type II collagen product; the protein recovery rate is 45.0%±2.0%, and the collagen recovery rate is 40.0%±1.5%; the type II collagen product has a protein content of >50.0%, a collagen content of >45.0%, a salt content of <10.0%, and a moisture content of <8.0%.

2. A method for precipitating type II collagen product by polymer association phase separation according to claim 1, characterized in that: The standing time in step (3) is 1-8 hours.

3. The method for precipitating type II collagen product by polymer association phase separation according to claim 1, characterized in that: The centrifugal conditions in step (3) are 4000-5000 rpm, 10-20 min.

4. The method for precipitating type II collagen product by polymer association phase separation according to claim 1, characterized in that: The freeze-drying time in step (3) is 12-24 hours.

5. A type II collagen product obtained by the method according to any one of claims 1 to 4; the type II collagen product has a protein content of >50.0%, a collagen content of >45.0%, a salt content of <10.0%, and a moisture content of <8.0%.

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