WO2023000370A1 - 非变性ii型胶原蛋白的制备方法 - Google Patents

非变性ii型胶原蛋白的制备方法 Download PDF

Info

Publication number
WO2023000370A1
WO2023000370A1 PCT/CN2021/109251 CN2021109251W WO2023000370A1 WO 2023000370 A1 WO2023000370 A1 WO 2023000370A1 CN 2021109251 W CN2021109251 W CN 2021109251W WO 2023000370 A1 WO2023000370 A1 WO 2023000370A1
Authority
WO
WIPO (PCT)
Prior art keywords
cartilage
collagen
denatured type
preparing non
collagen according
Prior art date
Application number
PCT/CN2021/109251
Other languages
English (en)
French (fr)
Inventor
朱美红
罗程
王伙根
Original Assignee
嘉兴恒杰生物制药股份有限公司
福建恒杰生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 嘉兴恒杰生物制药股份有限公司, 福建恒杰生物科技有限公司 filed Critical 嘉兴恒杰生物制药股份有限公司
Publication of WO2023000370A1 publication Critical patent/WO2023000370A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/10—Process efficiency

Definitions

  • the invention discloses a method for extracting collagen from animal cartilage, relates to the field of food biotechnology, and in particular relates to a preparation method of non-denatured type II collagen.
  • Collagen is a structural protein widely distributed in mammals. It is an important part of tissues such as skin, bones and joints, and is also one of the main components of the extracellular matrix of multicellular animals. At present, there are more than 20 types of collagen known, which can be divided into three categories according to tissue classification: interstitial collagen, basement membrane collagen and cartilage collagen. Among them, type II collagen is mainly distributed in articular cartilage, and a small amount exists. In the vitreous body, embryonic cornea and other parts.
  • Type II collagen is composed of three intertwined polypeptide chains. Because it has a stable triple helix structure, it mainly plays a role in supporting organs and protecting the body in the body. Recent studies have shown that type II collagen has been widely concerned by consumers because it can be used as a structural and functional nutrient for joints, has low immunogenicity and good biocompatibility [1] .
  • type II collagen Most of the currently commercially available type II collagen products are fully enzymatically hydrolyzed collagen products. Due to the limitations of the extraction process and preparation process, under the action of enzymes, high temperature and other factors, type II collagen loses its original triple helix structure, collagen is decomposed into polypeptides and amino acids below 20,000 Daltons, and loses its original pair of peptides and amino acids. Preventive and auxiliary therapeutic functions of rheumatoid arthritis and arthritis [2] .
  • CN106916870A discloses a method for preparing cartilage extracts containing non-denatured type II collagen. Filtration, drying and other steps; wherein, in the enzymatic hydrolysis step: adjust the pH of the slurry obtained in the homogenization step to 2.5 to 8.5, add 0.001% to 2% of the cartilage weight enzyme, add cartilage weight 1/20-1/500 of the papaya and/or pineapple juice obtained by filtering at low temperature, enzymatic hydrolysis for 12-48 hours.
  • Li Jie, Zhu Dakai and Gong Hui Li Jie, Zhu Dakai and Gong Hui (Li Jie, Zhu Dakai, Gong Hui.
  • non-denatured type II collagen used pepsin to remove impurities from cartilage for 24 hours, and then carried out enzymatic extraction for 24 hours to obtain soluble non-denatured type II collagen protein.
  • US7083820 discloses a production method of biologically active products. Chicken breast bones are cleaned, sterilized, chopped, mixed (inorganic salts), dried, etc., wherein the above-mentioned steps are operated below 110°F to obtain biologically active products. Type II collagen in its original structure.
  • the present invention provides a method for preparing non-denatured type II collagen, aiming to solve one or more of the above problems.
  • the invention provides a method for preparing non-denatured type II collagen, which is characterized in that it comprises the following steps in sequence:
  • the selected raw material is fresh chicken breast cartilage. After manually removing fat and muscle, cut the part within 3 cm from the tip of the chicken breast cartilage for later use;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • the raw material is selected within 1.5 cm from the tip of chicken breast cartilage.
  • the pepsin is added through a loading carrier, and the loading carrier is food-grade silicon dioxide.
  • the loading carrier is food-grade silicon dioxide.
  • pepsin is added in the form of a load carrier, which can effectively control the degree of enzymatic hydrolysis, combined with the concentration of pepsin and the time of enzymatic hydrolysis, the obtained non-denatured II collagen has a high content.
  • the addition ratio of the pepsin to the loading carrier is 0.3-0.6:15.
  • beating is performed first, and then acid or lye is used to adjust the pH value of the feed solution between 2.0 and 4.0.
  • the beating temperature is controlled below 25°C.
  • the number of cleanings in step (2) is 1 to 3 times, and each time is 10 to 15 minutes.
  • the enzymolysis temperature is 30°C to 32°C.
  • the non-denatured type II collagen is obtained by the extraction method of the present invention.
  • the present invention has the advantages of:
  • the extraction process is fast and simple: the total extraction time does not exceed 4 hours, and the preparation time is significantly shortened compared with the long-term extraction of more than 24 hours in the prior art;
  • the content of non-denatured type II collagen is high, and the product performance is good: the non-denatured type II collagen prepared by the method of the present invention has a content of more than 15% and the water absorption ratio in aqueous solution is 10 ⁇ 12 times, the volume expansion rate is 5 ⁇ 7 times;
  • Fig. 1 is the non-denatured type II collagen extracted from animal cartilage according to the present invention, a picture of the characteristic and regular striated tissue of natural collagen under a transmission electron microscope.
  • Material selection select fresh chicken breast bones, remove fat and muscle by hand, and cut 1.0 kg of cartilage within 1 cm from the tip;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 89 edition, and the content of glycosaminoglycans was 22.3% (calculated as chondroitin sulfate, and the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted According to the test, the total protein content was 79.1% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect the hydroxyproline content of 6.8%; the ELISA kit was used to detect the non-denatured type II collagen content was 21.88%.
  • Water absorption multiple detection Weigh 20 g of the above sieve into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume expands rapidly from 42 mL to 260 mL, and the volume expands by 6.2 times. Quantitative filter paper was filtered to remove free water. After wetting, the product had a mass of 276.1 g and a water absorption of 256.1 g. g, 12.8 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above sieve material, make it into a 5% (w/v) liquid with purified water, and let it stand for 2 hours without stratification.
  • Material selection select fresh chicken breast bones, remove fat and muscle by hand, and cut them 2 ⁇ 3 inches away from the tip. 1.0 kg of cartilage within 1 cm;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 89 edition, and the glycosaminoglycan content was 28.9% (calculated as chondroitin sulfate, the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted According to the test, the total protein content was 70.3% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect the content of hydroxyproline, and the content of hydroxyproline was 5.2%; the content of non-denatured type II collagen detected by ELISA kit was 16.77%.
  • Water absorption multiple detection Weigh 20 g of the above sieve into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume expands rapidly from 39 mL to 200 mL, and the volume expands by 5.1 times. Quantitative filter paper was filtered to remove free water. After wetting, the product had a mass of 252.2 g and a water absorption of 232.2 g, 11.6 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above sieve material, make it into a 5% (w/v) liquid with purified water, and let it stand for 2 hours without stratification.
  • Material selection select fresh chicken breast bones, remove fat and muscle by hand, and cut 1.0 kg of cartilage within 3 cm from the tip;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 89 edition, and the content of glycosaminoglycans was 25.2% (calculated as chondroitin sulfate, and the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted According to the test, the total protein content was 74.5% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect the content of hydroxyproline, and the content of hydroxyproline was 5.9%; the content of non-denatured type II collagen detected by ELISA kit was 18.81%.
  • Water absorption multiple detection Weigh 20 g of the above product into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume rapidly expands from 40 mL to 212 mL, and the volume expansion is 5.3 times, using quantitative filter paper Filter to remove free water, the product quality after wetting is 266.1 g, water absorption 246.1 g, 12.3 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above product, make it into a 5% (w/v) liquid with purified water, and let it stand for 2 hours without separation.
  • Material selection select fresh chicken breast bones, remove fat and muscle by hand, and cut 1.0 kg of cartilage within 3 cm from the tip;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 1989 edition, and the glycosaminoglycan content was 24.8% (calculated as chondroitin sulfate, the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted According to the test, the total protein content was 75.1% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect the content of hydroxyproline, and the content of hydroxyproline was 5.9%; the content of non-denatured type II collagen detected by ELISA kit was 23.2%.
  • Water absorption multiple detection Weigh 20 g of the above product into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume rapidly expands from 40 mL to 256 mL, and the volume expansion is 6.4 times, using quantitative filter paper Filter to remove free water, the product quality after wetting is 278.2 g, water absorption 258.2 g, 12.9 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above product, make it into a 5% (w/v) liquid with purified water, and let it stand for 2 hours without separation.
  • Material selection select fresh chicken breast bones, remove fat and muscle by hand, and cut 1.0 kg of cartilage within 3 cm from the tip;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 89 edition, and the content of glycosaminoglycans was 25.0% (calculated as chondroitin sulfate, and the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted According to the test, the total protein content was 75.1% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect the content of hydroxyproline, and the content of hydroxyproline was 5.8%; the content of non-denatured type II collagen detected by ELISA kit was 23.5%.
  • Water absorption multiple detection Weigh 20 g of the above product into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume rapidly expands from 40 mL to 260 mL, and the volume expands by 6.5 times, and is filtered with quantitative filter paper After removing the free water, the product has a mass of 282 g after wetting, absorbs 262 g of water, and is 13.1 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above product, make it into a 5% (w/v) liquid with purified water, and let it stand for 2 hours without separation.
  • Material selection select fresh chicken breast bones, remove fat and muscle by hand, and cut 1.0 kg of cartilage away from the tip 3 cm away;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 1989 edition.
  • the content of glycosaminoglycans was 33.1% (calculated as chondroitin sulfate, and the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted Tested, the total protein content was 64.2% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect, and the content of hydroxyproline was 4.1%; the content of non-denatured type II collagen was detected by ELISA kit to be 6.71%.
  • Water absorption multiple detection Weigh 20 g of the above product into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume rapidly expands from 38 mL to 156 mL, and the volume expansion is 4.1 times, using quantitative filter paper Filter to remove free water, the product quality after wetting is 208.1 g, water absorption 188.1 g, 9.4 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above product, prepare 5% (w/v) liquid with purified water, let it stand for 0.5 h, the suspension starts to separate, and stand for 1.25 h, the suspension is 1:1.
  • Material selection select 1.0 kg of fresh chicken breast bone, remove fat and muscle by hand;
  • step (3) Pulverization: After draining the cartilage cleaned in step (2), use a pulverizer to pulverize the cartilage into cartilage particles smaller than 2 mm;
  • Glycosaminoglycans were detected by the hexosaminoglycan method in the first volume of the "Ministry of Health Drug Standard Biochemical Drugs" 89 edition, and the content of glycosaminoglycans was 30.4% (calculated as chondroitin sulfate, the coefficient was calculated as 2.82); the method of GB 5009.5 was adopted Tested, the total protein content was 66.2% (the protein conversion coefficient was calculated as 5.79); the method of GB/T 9695.23 was used to detect, and the content of hydroxyproline was 4.7%; the content of non-denatured type II collagen was detected by ELISA kit to be 6.9%.
  • Water absorption multiple detection Weigh 20 g of the above product into a graduated measuring cylinder, add purified water until the sample is completely wet and gelatinous, the product volume rapidly expands from 38 mL to 170 mL, and the volume expands by 4.5 times, using quantitative filter paper Filter to remove free water, the product quality after wetting is 212.0 g, water absorption 192.0 g, 9.6 times the weight of the product itself.
  • Suspension ability test Take 20 g of the above product, prepare 5% (w/v) liquid with purified water, let it stand for 47 minutes, the suspension starts to separate, and stand for 65 minutes, the suspension is 1:1.
  • the non-denatured type II collagen was prepared by the method of Example 4 for different parts of the samples, and the corresponding product properties are shown in Table 1.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

非变性II型胶原蛋白的制备方法和产品，以鸡胸软骨为原料，经选材、清洗、粉碎、酶解、干燥、粉碎、过筛等步骤，得到吸水倍数为10-13倍，体积膨胀倍数为5-7倍，保留了非变性II型胶原蛋白天然长链结构且含量不低于15％。通过制备的含非变性II型胶原蛋白的产品在提高了非变性II型胶原蛋白含量、吸水性和体积膨胀系数的前提下，具备提取过程快速、产品溶液悬浮性能好的优点，具有良好的市场应用前景。

Description

[根据细则37.2由ISA制定的发明名称]　非变性II型胶原蛋白的制备方法 技术领域
本发明公开的是一种从动物软骨中提取胶原蛋白的方法，涉及食品生物技术领域，尤其涉及一种非变性II型胶原蛋白的制备方法。
 背景技术
胶原蛋白是一种广泛分布于哺乳动物体内的结构性蛋白，是构成皮肤、骨骼和关节等组织的重要组成部分，同时也是多细胞动物细胞外基质的主要成分之一。目前已知的胶原蛋白种类已有二十余种，根据组织分类可分为间隙胶原蛋白、基膜胶原蛋白和软骨胶原蛋白三大类，其中II型胶原蛋白主要分布于关节软骨中，少量存在于玻璃体、胚胎角膜等部位。
II型胶原蛋白是由三股缠绕的多肽链组成，由于其具备稳定的三螺旋结构，因此，在体内主要起到支撑器官、保护机体的作用。近年来的研究表明，II型胶原蛋白因其可作为关节的结构和功能性营养素，具有低免疫原性和生物相容性好等特点 [1]，受到消费者的广泛关注。
目前市售的II型胶原蛋白产品，多为完全酶解的胶原蛋白产品。由于提取工艺和制剂工艺的局限，在酶、高温等因素作用下，II型胶原蛋白丧失原有的三螺旋结构，胶原蛋白被分解为20,000道尔顿以下的多肽和氨基酸，失去原有对类风湿关节炎和关节炎的预防及辅助治疗功能 [2]。
目前，国内外已公开多种非变性II型胶原蛋白的制备工艺，例如CN106916870A公开的一种含非变性II型胶原蛋白的软骨提取物的制备方法，通过脱脂、消毒、匀浆、酶解、过滤、干燥等步骤；其中，在所述酶解步骤中：将在匀浆步骤中获得的浆状物的pH调节为2.5～8.5，加入软骨重量的0.001％～2％的酶，加入软骨重量的1/20～1/500的木瓜和/或菠萝低温榨汁后过滤获得的清液，酶解12～48h。李洁，朱大开和龚辉（李洁, 朱大开, 龚辉. 非变性Ⅱ型胶原蛋白生产方法.），采用胃蛋白酶先对软骨除杂24h，再进行酶解提取24h得到可溶性非变性II型胶原蛋白。US7083820公开的一种生物活性产品的生产方法，鸡胸骨通过清洗、消毒、切碎、混合（无机盐）、干燥等步骤，其中上述所述步骤是在110℉以下操作，得到不改变生物活性产品原始结构的II型胶原蛋白。
 技术问题
但已公开的技术方法及制备的非变性II型胶原蛋白依然存在问题：一产品在溶液中悬浮性能差，易分层；二是产品制备提取经过蛋白酶或酶提取原料进行长时间的充分水解，非变性II胶原蛋白含量较低，且提取时间长，不利于工业生产推广。
 技术解决方案
本发明提供了一种非变性II型胶原蛋白的制备方法，旨在解决上述问题中的一个或多个问题。
具体技术方案如下：
本发明提供了一种非变性II型胶原蛋白的制备方法，它的特征是依次包括以下步骤：
（1）选材：所选原料为新鲜鸡胸软骨，手工剔除脂肪和肌肉后，切取距离鸡胸软骨尖端3厘米以内部分备用；
（2）清洗：使用温度不高于37℃的清水清洗步骤（1）中切取备用的软骨；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：向步骤（3）所得软骨颗粒中加水，加水量为软骨颗粒质量的0.5~3倍（m/V），使用酸液或碱液调节料液pH值在2.0~4.0之间，加入软骨颗粒重量1.0~5.0%的胃蛋白酶，酶解温度不超过37℃，搅拌并酶解0.5~2.0 h；酶解结束后，使用碱液调节pH值至8.5~9.5，静置15~60 min；
（5）过滤：使用20 ~ 60目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行真空干燥或冷冻干燥，干燥后获得干燥品；
（7）粉碎：将干燥品进行粉碎，经60~200目筛网过筛，取筛下物，得非变性II型胶原蛋白粉末。
本发明的上述方案中，首先通过对原料进行选择，选取鸡胸软骨，并选择距离骨尖端3厘米以内，结合对工艺的改进，尤其是酶解步骤的改进，可以获得蛋白含量大于15%的非变性II型胶原蛋白。而且提取过程快速、简便：总提取时间不超过4 h。
更加优选，原料选用鸡胸软骨尖端1.5厘米以内。
作为优选，所述胃蛋白酶通过负载载体加入，所述负载载体为食品级二氧化硅。本方案中，胃蛋白酶通过负载载体的形式加入，能够有效控制酶解程度，结合胃蛋白酶的浓度以及酶解时间，得到的非变性II胶原蛋白含量高。
作为优选，所述胃蛋白酶与所述负载载体的添加比例为0.3~0.6：15。
作为优选，步骤（4）中在软骨颗粒中加水后，先进行打浆，再使用酸液或碱液调节料液pH值在2.0~4.0之间。
作为优选，打浆温度控制在25℃以下。
作为优选，步骤（2）中清洗次数为1~3次，每次10 ~15min。
作为优选，酶解温度为30℃~32℃。
通过本发明的提取方法获得非变性II型胶原蛋白。
 有益效果
与现有技术相比，本发明的优点在于：
（1）提取过程快速、简便：总提取时间不超过4 h，与现有技术24 h以上的长时间提取相比，制备时间明显缩短；
（2）非变性II型胶原蛋白含量高，产品性能好：通过本发明所述方法制备的非变性II型胶原蛋白，非变性II型胶原蛋白含量大于15%，在水溶液中吸水倍数为10~12倍，体积膨胀率为5~7倍；
（3）在水溶液中悬浮性能好，不易分层，除可应用于硬胶囊、片剂外，还适合软胶囊、固体饮料、凝胶糖果等剂型。
 附图说明
图1为本发明从动物软骨中提取的非变性II型胶原蛋白，在透射电镜下的天然胶原蛋白特征性、规律性横纹组织图片。
 本发明的实施方式
下面结合实施例进一步说明本发明的实质性内容，但并不以此限定本发明保护范围。尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
实施例1
（1）选材：选择新鲜鸡胸骨，手工剔去脂肪和肌肉后，切取距离尖端1 cm以内部位的软骨1.0kg；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒投入反应罐，加入1 L清水，温度控制在35℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入20 g胃蛋白酶，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品S1，共计91.6 g。；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为22.3%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为79.1%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量6.8%；采用ELISA试剂盒检测非变性II型胶原蛋白含量为21.88%。
吸水倍数检测：称取上述筛下物20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由42 mL迅速膨胀至260 mL，体积膨胀6.2倍，使用定量滤纸过滤除去自由水，湿润后产品质量为276.1 g，吸水256.1 g，为产品自身重的12.8倍。
悬浮能力测试：取上述筛下物20 g，用纯化水配制成5%（w/v）液体，静置2 h，未分层。 
实施例2
（1）选材：选择新鲜鸡胸骨，手工剔去脂肪和肌肉后，切取距离尖端2~3 cm以内部位的软骨1.0 kg；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒投入反应罐，加入1 L清水，温度控制在35 ℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入30 g胃蛋白酶，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品S2，共计81.5 g；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为28.9%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为70.3%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量5.2 %；采用ELISA试剂盒检测非变性II型胶原蛋白含量为16.77%。
吸水倍数检测：称取上述筛下物20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由39 mL迅速膨胀至200 mL，体积膨胀5.1倍，使用定量滤纸过滤除去自由水，湿润后产品质量为252.2 g，吸水232.2 g，为产品自身重的11.6倍。
悬浮能力测试：取上述筛下物20 g，用纯化水配制成5%（w/v）液体，静置2 h，未分层。 
实施例3
（1）选材：选择新鲜鸡胸骨，手工剔去脂肪和肌肉后，切取距离尖端3 cm以内部位的软骨1.0 kg；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒投入反应罐，加入1 L清水，温度控制在35 ℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入50 g胃蛋白酶，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品S3，共计90.1 g；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为25.2%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为74.5%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量5.9 %；采用ELISA试剂盒检测非变性II型胶原蛋白含量为18.81%。
吸水倍数检测：称取上述产品20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由40 mL迅速膨胀至212 mL，体积膨胀5.3倍，使用定量滤纸过滤除去自由水，湿润后产品质量为266.1 g，吸水246.1 g，为产品自身重的12.3倍。
悬浮能力测试：取上述产品20 g，用纯化水配制成5%（w/v）液体，静置2 h，未分层。
实施例4
（1）选材：选择新鲜鸡胸骨，手工剔去脂肪和肌肉后，切取距离尖端3 cm以内部位的软骨1.0 kg；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒加入1 L清水，25℃以下打浆，浆液投入反应罐，温度控制在35 ℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入负载胃蛋白酶的食品级二氧化硅，胃蛋白酶10 g，食品级二氧化硅500g，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品S3，共计86.2g；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为24.8%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为75.1%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量5.9 %；采用ELISA试剂盒检测非变性II型胶原蛋白含量为23.2%。
吸水倍数检测：称取上述产品20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由40 mL迅速膨胀至256 mL，体积膨胀6.4倍，使用定量滤纸过滤除去自由水，湿润后产品质量为278.2 g，吸水258.2 g，为产品自身重的12.9倍。
悬浮能力测试：取上述产品20 g，用纯化水配制成5%（w/v）液体，静置2 h，未分层。
实施例5
（1）选材：选择新鲜鸡胸骨，手工剔去脂肪和肌肉后，切取距离尖端3 cm以内部位的软骨1.0 kg；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒加入1 L清水，25℃以下打浆，浆液投入反应罐，温度控制在35 ℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入负载胃蛋白酶的食品级二氧化硅，胃蛋白酶20 g，食品级二氧化硅500g，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品S3，共计86.2g；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为25.0%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为75.1%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量5.8 %；采用ELISA试剂盒检测非变性II型胶原蛋白含量为23.5%。
吸水倍数检测：称取上述产品20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由40 mL迅速膨胀至260mL，体积膨胀6.5倍，使用定量滤纸过滤除去自由水，湿润后产品质量为282g，吸水262 g，为产品自身重的13.1倍。
悬浮能力测试：取上述产品20 g，用纯化水配制成5%（w/v）液体，静置2 h，未分层。
比较例1
（1）选材：选择新鲜鸡胸骨，手工剔去脂肪和肌肉后，切取距离尖端3 cm以外部位的软骨1.0 kg；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒投入反应罐，加入1 L清水，温度控制在35 ℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入20 g胃蛋白酶，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品D1，共计90.1 g；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为33.1%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为64.2%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量4.1 %；采用ELISA试剂盒检测非变性II型胶原蛋白含量为6.71%。
吸水倍数检测：称取上述产品20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由38 mL迅速膨胀至156 mL，体积膨胀4.1倍，使用定量滤纸过滤除去自由水，湿润后产品质量为208.1 g，吸水188.1 g，为产品自身重的9.4倍。
悬浮能力测试：取上述产品20 g，用纯化水配制成5%（w/v）液体，静置0.5 h，悬浮液开始分层，静置1.25 h，悬浮液呈1:1分层。 
比较例2
（1）选材：选择1.0 kg新鲜鸡胸骨，手工剔去脂肪和肌肉；
（2）清洗：使用不高于37℃的清水清洗1~3次，每次10 min；沥干；
（3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
（4）酶解：将步骤（3）所得软骨颗粒投入反应罐，加入1 L清水，温度控制在35 ℃，使用酸液或碱液调节料液pH值在3.0~4.0之间，加入20 g胃蛋白酶，搅拌并酶解2.0 h；酶解结束后，使用碱液调节pH值至9.0，静置30 min；
（5）过滤：使用40目筛过滤步骤（4）中的酶解液，收集筛上物；
（6）干燥：将筛上物进行冷冻干燥，干燥后获得干燥品D2，共计93.6 g；
（7）粉碎：将干燥品进行粉碎，经100目标准筛过筛，取筛下物。
采用《卫生部药品标准生化药品》89年版第一册氨基己糖法检测糖胺聚糖，糖胺聚糖含量为30.4%（以硫酸软骨素计，系数按2.82计）；采用GB 5009.5的方法检测，总蛋白质含量为66.2%（蛋白质折算系数按5.79计）；采用GB/T 9695.23的方法检测，羟脯氨酸含量4.7 %；采用ELISA试剂盒检测非变性II型胶原蛋白含量为6.9%。
吸水倍数检测：称取上述产品20 g至带刻度的量入式量筒中，加入纯化水至样品全部湿润呈胶状，产品体积由38 mL迅速膨胀至170 mL，体积膨胀4.5倍，使用定量滤纸过滤除去自由水，湿润后产品质量为212.0 g，吸水192.0 g，为产品自身重的9.6倍。
悬浮能力测试：取上述产品20 g，用纯化水配制成5%（w/v）液体，静置47 min，悬浮液开始分层，静置65 min，悬浮液呈1:1分层。
针对不同取材部位，通过实施例4的方法制备非变性II型胶原蛋白，其对应的产品性能如表1。
Figure dest_path_image001
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化，均仍属于本发明技术方案的保护范围内。
参考文献
[1] 刘斌. 巴氏毕赤酵母基因工程菌高密度发酵表达重组人源胶原蛋白[D]. 南京理工大学, 2012.
[2] Trentham D E . Autoimmunity to type II collagen an experimental model of arthritis.[J]. Journal of Experimental Medicine, 1977, 146(3):857-868.

Claims (10)

  1. 一种非变性II型胶原蛋白的制备方法，其特征在于，依次包括以下步骤：
    （1）选材：所选原料为新鲜鸡胸软骨，手工剔除脂肪和肌肉后，切取距离鸡胸软骨尖端3厘米以内部分备用；
    （2）清洗：使用温度不高于37℃的清水清洗步骤（1）中切取备用的软骨；
    （3）粉碎：将步骤（2）中经清洗的软骨沥干后，使用粉碎机将软骨粉碎为2 mm以下的软骨颗粒；
    （4）酶解：向步骤（3）所得软骨颗粒中加水，加水量为软骨颗粒质量的0.5~3倍（m/V），使用酸液或碱液调节料液pH值在2.0~4.0之间，加入软骨颗粒重量1.0~5.0%的胃蛋白酶，酶解温度不超过37℃，搅拌并酶解0.5~2.0 h；
    （5）过滤：使用20 ~ 60目筛过滤步骤（4）中的酶解液，收集筛上物；
    （6）干燥：将筛上物进行真空干燥或冷冻干燥，干燥后获得干燥品；
    （7）粉碎：将干燥品进行粉碎，经60~200目筛网过筛，取筛下物，得非变性II型胶原蛋白粉末。
  2. 根据权利要求1所述的一种非变性II型胶原蛋白的制备方法，其特征在于，原料选用鸡胸软骨尖端1.5厘米以内。
  3. 根据权利要求1所述的一种非变性II型胶原蛋白的制备方法，其特征在于，酶解结束后，使用碱液调节pH值至8.5~9.5，静置15~60 min后，再进行步骤（5）的过滤。
  4. 根据权利要求1所述的一种非变性II型胶原蛋白的制备方法，其特征在于，所述胃蛋白酶通过负载载体加入，所述负载载体为食品级二氧化硅。
  5. 根据权利要求4所述的一种非变性II型胶原蛋白的制备方法，其特征在于，所述胃蛋白酶与所述负载载体的添加比例为0.3~0.6：15。
  6. 根据权利要求1所述的一种非变性II型胶原蛋白的制备方法，其特征在于，步骤（4）中在软骨颗粒中加水后，先进行打浆，再使用酸液或碱液调节料液pH值在2.0~4.0之间。
  7. 根据权利要求6所述的一种非变性II型胶原蛋白的制备方法，其特征在于，打浆温度控制在25℃以下。
  8. 根据权利要求1所述的一种非变性II型胶原蛋白的制备方法，其特征在于，步骤（2）中清洗次数为1~3次，每次10 ~15min。
  9. 根据权利要求1所述的一种非变性II型胶原蛋白的制备方法，其特征在于，酶解温度为30℃~32℃。
  10. 如权利要求1-9任一项所述的非变性II型胶原蛋白的制备方法制得的非变性II型胶原蛋白。
PCT/CN2021/109251 2021-07-19 2021-07-29 非变性ii型胶原蛋白的制备方法 WO2023000370A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110812193.0A CN113563458B (zh) 2021-07-19 2021-07-19 一种非变性ii型胶原蛋白的制备方法
CN202110812193.0 2021-07-19

Publications (1)

Publication Number Publication Date
WO2023000370A1 true WO2023000370A1 (zh) 2023-01-26

Family

ID=78165464

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/109251 WO2023000370A1 (zh) 2021-07-19 2021-07-29 非变性ii型胶原蛋白的制备方法

Country Status (2)

Country Link
CN (1) CN113563458B (zh)
WO (1) WO2023000370A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116217707A (zh) * 2023-02-21 2023-06-06 华南理工大学 一种通过聚合物缔合相分离作用沉淀ii型胶原蛋白的方法
CN116253791A (zh) * 2023-02-10 2023-06-13 完美（广东）日用品有限公司 一种具有改善骨关节健康作用的非变性ii型胶原蛋白的制备方法与应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5840848A (en) * 1996-01-05 1998-11-24 Autoimmune, Inc. Method for preparation of type II collagen
CN102154425A (zh) * 2011-03-18 2011-08-17 北京华达杰瑞生物技术有限公司 非变性ⅱ型胶原蛋白生产方法
US20130123468A1 (en) * 2010-09-13 2013-05-16 Hiroyoshi Moriyama Method for extracting undenatured type ii collagen having active epitope
CN105132502A (zh) * 2015-09-08 2015-12-09 四川大学 一种从鸡软骨中提取纯净ⅱ型胶原的方法
CN106916870A (zh) * 2017-04-26 2017-07-04 北京盛美诺生物技术有限公司 一种含非变性ii型胶原蛋白的软骨提取物的制备方法
CN109851669A (zh) * 2017-11-30 2019-06-07 扬州中福生物技术有限公司 一种鸡胸软骨提取ⅱ型胶原蛋白的方法
CN111004320A (zh) * 2019-12-30 2020-04-14 中国农业科学院农产品加工研究所 Ⅱ型胶原蛋白的提取方法及用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2248319B2 (zh) * 1973-10-19 1978-06-02 Tepral
AUPR311601A0 (en) * 2001-02-15 2001-03-08 Adp Pharmaceutical Pty Limited Matrix gene expression in chondrogenesis
JP6881708B2 (ja) * 2017-03-02 2021-06-02 新田ゼラチン株式会社 多孔質シリカおよびその製造方法
CN109384842A (zh) * 2018-12-25 2019-02-26 美泰科技（青岛）股份有限公司 一种产业化非变性ⅱ胶原蛋白的制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5840848A (en) * 1996-01-05 1998-11-24 Autoimmune, Inc. Method for preparation of type II collagen
US20130123468A1 (en) * 2010-09-13 2013-05-16 Hiroyoshi Moriyama Method for extracting undenatured type ii collagen having active epitope
CN102154425A (zh) * 2011-03-18 2011-08-17 北京华达杰瑞生物技术有限公司 非变性ⅱ型胶原蛋白生产方法
CN105132502A (zh) * 2015-09-08 2015-12-09 四川大学 一种从鸡软骨中提取纯净ⅱ型胶原的方法
CN106916870A (zh) * 2017-04-26 2017-07-04 北京盛美诺生物技术有限公司 一种含非变性ii型胶原蛋白的软骨提取物的制备方法
CN109851669A (zh) * 2017-11-30 2019-06-07 扬州中福生物技术有限公司 一种鸡胸软骨提取ⅱ型胶原蛋白的方法
CN111004320A (zh) * 2019-12-30 2020-04-14 中国农业科学院农产品加工研究所 Ⅱ型胶原蛋白的提取方法及用途

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116253791A (zh) * 2023-02-10 2023-06-13 完美（广东）日用品有限公司 一种具有改善骨关节健康作用的非变性ii型胶原蛋白的制备方法与应用
CN116253791B (zh) * 2023-02-10 2024-02-23 完美（广东）日用品有限公司 一种具有改善骨关节健康作用的非变性ii型胶原蛋白的制备方法与应用
CN116217707A (zh) * 2023-02-21 2023-06-06 华南理工大学 一种通过聚合物缔合相分离作用沉淀ii型胶原蛋白的方法

Also Published As

Publication number Publication date
CN113563458B (zh) 2023-07-21
CN113563458A (zh) 2021-10-29

Similar Documents

Publication Publication Date Title
US11884953B2 (en) Method for preparing protein peptide based on connective tissue and prepared protein peptide and use thereof
AU783184B2 (en) Freeze-dried composite materials and processes for the production thereof
WO2023000370A1 (zh) 非变性ii型胶原蛋白的制备方法
US10918675B2 (en) Water resistant enhanced wound healing film and preparation method thereof
CN108796017B (zh) 牛骨肽及其酶解提取方法
JPS58170795A (ja) コラ−ゲン質材料の製造方法
CN101805775B (zh) 一种鹿筋胶原蛋白的制备方法
JP2000506367A (ja) ▲ｉｉ▼型コラーゲンの調製法
CN109349419A (zh) 一种修复人体细胞的复合牦牛骨胶原蛋白肽粉
CN110922475A (zh) 一种酶解提取猪皮胶原蛋白的方法
CN109796529A (zh) 一种胶原蛋白及其提取方法
CN107630061A (zh) 牦牛骨ⅰ型胶原蛋白的制备方法
CN111635920A (zh) 一种二级仿生酶解技术制备驴骨胶原蛋白肽粉的方法
CN108785751B (zh) 鱼鳞胶原蛋白/海藻酸钠复合多孔骨组织工程支架及其制备方法与应用
WO2007131424A1 (fr) Procédé de préparation de compositions de protéoglycane et de collagène à faible poids moléculaire, produits résultants et utilisations de ceux-ci
CN112501229B (zh) 一种牛骨胶原肽的生产工艺
CN109258916A (zh) 一种鱼鳞胶原肽钙及其制备方法、用途
CN106039403B (zh) 具有生物活性的角膜修复植片的制备方法和角膜修复植片
CN111718825A (zh) 一种具有补肾益阳作用的鲟鱼骨肽酒、其制备方法及应用
CN107574217A (zh) 一种纳米级胶原蛋白的提纯方法
US20210393501A1 (en) Preparation method and application of recombinant mutant collagenase
CN104448038B (zh) 一种硫酸软骨素钙盐的制备工艺
CN111296755A (zh) 一种含海洋硫酸软骨素的营养强化麦片食品及其制备方法
CN106173853B (zh) 一种降血脂的营养组合物及其应用
RU2304441C1 (ru) Способ получения сульфатированных гликозаминогликанов из биологических тканей

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21950616

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE