Chondrolamin and application thereof in preservation

Abstract

The invention belongs to the technical field of protein extraction and preservation, and discloses a preparation method of chondrolamin, which comprises the following steps: adding recycled cartilage powder obtained by processing residues of chondroitin sulfate extraction into an alcoholic solution, then adjusting the pH value, then carrying out ultrasonic extraction under the ultrasonic extraction condition of 20-40 minutes at the temperature of 40-60 DEG C, and after the extraction is completed, filtering to obtain a filtrate, namely the chondrolamin. Centrifuging to obtain supernate, and freeze-drying to obtain the chondrolamin. The chondroitin sulfate extraction waste liquid is used as a raw material, the ultrasonic-assisted alcohol extraction method is adopted for extracting the chondrolamin, the extraction time can be effectively shortened, the extraction efficiency can be improved, and meanwhile the purity can be improved. A reliable basis is provided for experimental research and large-scale production of chondrolamin extraction, and the chondrolamin is successfully applied to the field of preservation, so that the application of chondrolamin in the field of food industry is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of protein extraction and preservation, and in particular relates to a cartilage alcohol-soluble protein and application thereof in preservation. Background Art

[0002] Alcohol-soluble proteins are rich in proline and glutamine, lacking charged amino acid residues; they can be dissolved in 50%-90% ethanol solution, and have good biocompatibility, biodegradability, high toughness, elasticity, and formability; therefore, the production and utilization of alcohol-soluble proteins is a development direction with great development potential. Alcohol-soluble proteins are widely used in the fields of food and medicine. In the food industry, alcohol-soluble proteins can be used as fresh-keeping proteins, which can effectively delay the loss of nutrients in fruits, vegetables and meats and extend their shelf life. Therefore, cartilage alcohol-soluble proteins are a protein with important biological activity, and the study of their extraction process is of great significance to the improvement of their application value.

[0003] At present, there are mainly acid hydrolysis, enzymatic hydrolysis, salting-out and alcohol dissolution methods for the extraction of cartilage alcohol-soluble proteins. Acid hydrolysis is one of the earliest methods used for the extraction of cartilage alcohol-soluble proteins. In this method, cartilage samples are added to an acidic buffer, hydrolyzed under appropriate conditions, and then neutralized, precipitated and washed to finally obtain cartilage alcohol-soluble proteins. This method has the advantages of being simple and easy to operate and having high extraction efficiency, but it also leads to environmental problems such as the discharge of acidic waste liquid, and the hydrolysis process may damage the structure of the protein.

[0004] The alcohol dissolution method is a method of adding cartilage samples to alcohol solvents of different types and concentrations and extracting them under appropriate conditions. This method has the advantages of simple operation and low cost, but it also has some disadvantages, such as a large impact on protein structure and properties, being easily affected by alcohol and temperature, and easily causing oxidation and denaturation. Therefore, when using the alcohol dissolution method to extract cartilage alcohol-soluble proteins, it is necessary to pay attention to the selection and optimization of extraction conditions to ensure the extraction rate and quality.

[0005] Enzymatic hydrolysis is a method of extracting alcohol-soluble proteins by hydrolyzing proteins in cartilage using proteases. This method has the advantages of strong selectivity and mild hydrolysis process, but it also has problems such as long enzymatic hydrolysis time and high cost. In recent years, improvements to the enzymatic hydrolysis method have also been disclosed, such as the use of ultrasound-assisted enzymatic hydrolysis to improve enzymatic hydrolysis efficiency and reduce costs.

[0006] Studies have shown that alcohol-soluble proteins have multiple functions, but they are all extracted from grains. There are no reports on the extraction of alcohol-soluble proteins from animal cartilage at home and abroad, and there is no application of animal cartilage alcohol-soluble proteins extracted by different methods in preservation applications. Summary of the invention

[0007] In order to overcome the deficiencies in the prior art, one of the purposes of the present invention is to provide a method for preparing chondroitin. The present invention uses chondroitin sulfate extraction waste liquid as raw material, adopts ultrasonic assisted alcohol extraction to extract chondroitin, and uses BCA method to determine the content of the extracted chondroitin. The process conditions of ultrasonic assisted extraction of chondroitin are optimized by single factor and orthogonal test, providing a reliable theoretical basis for experimental research and large-scale production of chondroitin extraction. By optimizing the extraction process of chondroitin, the cost can be reduced and the yield and purity can be increased.

[0008] Another object of the present invention is to provide the use of the above-mentioned cartilage prolamin in the field of preservation.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for preparing cartilage alcohol-soluble protein comprises adding recovered cartilage powder obtained by processing the residue of chondroitin sulfate extraction into an alcohol solution, and then adjusting the pH to 9 for ultrasonic extraction. The ultrasonic extraction conditions are: at a temperature of 40°C to 60°C, with a power of 150 to 300W for 20 to 40 minutes. After the extraction is completed, the supernatant is obtained by centrifugation, and the cartilage alcohol-soluble protein is obtained after freeze-drying.

[0011] Preferably, the recovered cartilage powder is obtained by the following treatment:

[0012] S1. Weigh 1000 kg of bovine cartilage and put it into an extraction tank. Add 8 times distilled water, heat to 100-105°C, keep warm for 3 hours, then take out the upper bone oil and cool to 50°C;

[0013] S2. Adjust the cooking liquid to 9.0, add 0.9% alkaline protease by weight of cartilage, and stir for enzymolysis for 2 hours; readjust the pH value to 8.7, add 5% activated porcine pancreas by weight of cartilage, and stir for enzymolysis for 2 hours; raise the temperature of the enzymolysis liquid to 80°C, stir, adjust the pH value to 4.4, process for 20 minutes, stop stirring, and let stand until stratification is obvious;

[0014] S3, filtering and separating the supernatant, adsorbing the separated liquid by anion exchange resin, the mass ratio of the separated liquid to the anion exchange resin is 1.4:1, and the adsorption time is 1 hour; the ion exchange resin is eluted by a 2 mol / L NaCl solution; the eluate is oxidized and decolorized, filtered, and the filtrate is pumped into an ultrafiltration device, and desalted and concentrated by the ultrafiltration device to obtain a concentrated solution;

[0015] S4. Add 5 times the volume of 95% ethanol to the concentrated solution, centrifuge to obtain a supernatant and a precipitate; introduce the supernatant into a distillation tower for distillation to obtain a distillate and a kettle residue, wherein the distillate is reused after the ethanol concentration reaches 95%, and the kettle residue is spray-dried to obtain the recovered cartilage powder.

[0016] Preferably, the alcohol solution is one or more of ethanol, isopropanol, and tert-butanol.

[0017] The present invention also claims to protect the alcohol-soluble protein prepared by the preparation method.

[0018] Furthermore, the present invention applies the alcohol-soluble protein in the field of meat preservation, and obtains a better preservation effect.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention uses chondroitin sulfate extraction waste liquid as raw material and adopts ultrasonic-assisted alcohol extraction method to extract cartilage alcohol-soluble protein, which can effectively shorten the extraction time, improve the extraction efficiency, improve the purity, etc. It provides a reliable theoretical basis for experimental research and large-scale production of cartilage alcohol-soluble protein extraction. It is successfully applied in the field of preservation, thereby promoting the wide application of cartilage alcohol-soluble protein in the field of food industry. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention below will provide a detailed and comprehensive description of the technical solutions of the present invention. It should be noted that the embodiments provided represent only a part of the present invention, not all of it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0023] Recycling cartilage powder

[0024] Weigh 1000kg of bovine cartilage and put it into an extraction tank, add 8 times distilled water, heat to 105℃, keep warm for 3h, then take out the upper bone oil and cool to 50℃. Adjust the cooking liquid to 9.0, add 0.9% alkaline protease 2709 of cartilage weight, stir at 40rpm, and enzymolyze for 2h; readjust the pH value to 8.7, add 5% activated pig pancreas of cartilage weight (crush the pig pancreas and activate at 25-35℃ for 40h), stir at 40rpm, and enzymolyze for 2h. Raise the temperature of the enzymolysis solution to 80℃, set the stirring speed to 100rpm, adjust the pH value to 4.4, process for 20min, stop stirring, and let stand until stratification is obvious. Filter the supernatant with 747 filter cloth and further separate it with a disc separator. The separation liquid is adsorbed by anion exchange resin, the mass ratio of the separation liquid to the anion exchange resin is 1.4:1, and the adsorption time is 1 hour; the ion exchange resin is eluted by a NaCl solution with a concentration of 2 mol / L; the eluate is oxidatively decolorized, filtered with a 747 filter cloth, and the filtrate is pumped into an ultrafiltration device, and desalted and concentrated by the ultrafiltration equipment to obtain a concentrated solution. The retentate is added with 5 times the volume of 95% ethanol, centrifuged to obtain a supernatant and a precipitate. The precipitate is vacuum dried to obtain chondroitin sulfate. The supernatant is introduced into a distillation tower for distillation to obtain a distillate and a kettle residue, wherein the distillate is reused after the ethanol concentration reaches 95%, and the kettle residue is spray dried to obtain a recovered cartilage powder.

[0025] Example 1 Ultrasonic-assisted extraction and recovery of alcohol-soluble proteins from cartilage powder

[0026] Add 75% ethanol aqueous solution to a 100mL glass bottle with a lid, preheat to a certain temperature, add 2.0000g of pretreated cartilage protein powder (liquid-to-solid ratio is 20ml:1g), adjust pH to 9, ultrasonicate at 200W power for 30min at 50℃ in an ultrasonic cleaner, centrifuge at 3000rpm for 10min, obtain precipitate and supernatant, and determine the content of alcohol-soluble protein in the supernatant by BCA method. The supernatant is freeze-dried to obtain alcohol-soluble protein.

[0027] Example 2 Ultrasonic-assisted extraction and recovery of alcohol-soluble proteins from cartilage powder

[0028] Add 60% ethanol aqueous solution to a 100mL glass bottle with a lid, preheat to a certain temperature, add 2.0000g of pretreated cartilage protein powder (liquid-to-solid ratio is 20:1), adjust pH to 9, ultrasonicate at 200W power for 30min at 50℃ in an ultrasonic cleaner, centrifuge at 3000rpm for 10min, obtain precipitate and supernatant, and determine the content of alcohol-soluble protein in the supernatant by BCA method. After freeze-drying the supernatant, obtain alcohol-soluble protein.

[0029] Example 3 Ultrasonic-assisted extraction and recovery of alcohol-soluble proteins from cartilage powder

[0030] 75 ml of anhydrous ethanol, 15 ml of isopropanol and 10 ml of tert-butyl alcohol were mixed to obtain a 75% volume fraction ethanol mixed solution, which was added to a 100 ml glass bottle with a lid, preheated to a certain temperature, and 2.0000 g of pretreated cartilage protein powder was added (liquid-to-solid ratio was 20:1), the pH was adjusted to 9, and ultrasonic cleaning was performed at 200 W power for 30 min at 50 ° C in an ultrasonic cleaner, and centrifuged at 3000 rpm for 10 min to obtain a precipitate and a supernatant, and the content of alcohol-soluble protein in the supernatant was determined by the BCA method. The supernatant was freeze-dried to obtain alcohol-soluble protein.

[0031] Example 4 Ultrasonic-assisted extraction and recovery of alcohol-soluble proteins from cartilage powder

[0032] Add 90% ethanol aqueous solution to a 100mL glass bottle with a lid, preheat to a certain temperature, add 2.0000g of pretreated cartilage protein powder (liquid-to-solid ratio is 20:1), adjust pH to 9, ultrasonicate at 200W power for 30min at 60℃ in an ultrasonic cleaner, centrifuge at 3000rpm for 10min, obtain precipitate and supernatant, and determine the content of alcohol-soluble protein in the supernatant by BCA method. After freeze-drying the supernatant, obtain alcohol-soluble protein.

[0033] Comparative Example 1 Microwave-assisted extraction and recovery of alcohol-soluble proteins from cartilage powder

[0034] Add 75% ethanol by volume to a 100 mL glass bottle with a lid, preheat to a certain temperature, add 2.0000 g of pretreated cartilage protein powder (liquid-to-solid ratio is 20:1), adjust pH to 9, microwave at 500 W for 30 min, centrifuge at 3000 rpm for 10 min, obtain precipitate and supernatant, and determine the content of alcohol-soluble protein in the supernatant by BCA method. The supernatant is freeze-dried to obtain alcohol-soluble protein.

[0035] The extraction rate and purity of the alcohol-soluble proteins obtained in the examples and comparative examples are shown in Table 1:

[0036] Table 1

[0037] serial number Extraction rate purity% Precipitation Example 1 20.21 89 A small amount of precipitation Example 2 17.21 90 A small amount of precipitation Example 3 23.13 95 There is trace precipitation Example 4 16.28 88 A small amount of precipitation Comparative Example 1 15.46 80 A small amount of precipitation

[0038] As can be seen from the above table, although microwave and ultrasonic extraction are both used as a means to improve the extraction rate, the effect of microwave extraction is not as good as that of ultrasonic assisted extraction for alcohol-soluble protein. In addition, from the effects of Example 3 and other examples, the extraction effect of the mixed solvent of ethanol, isopropanol and tert-butyl alcohol is better than that of a single alcohol solvent.

[0039] Application Example 1: Application of cartilage alcohol-soluble protein in preservation

[0040] 1. Preparation of preservative: The alcohol-soluble protein obtained in Example and Comparative Example 1 is processed as follows: the alcohol-soluble protein is placed in a container, an ethanol water bath is added to dissolve the alcohol-soluble protein, and then centrifuged again, the supernatant is taken, and the container (triangular flask) is added again, and the preservative (glycerol, etc.) is added while heating and stirring, and the preservative (glycerol, etc.) is added, and the mixture is shaken and stored for later use. Fresh pork is soaked in the preservative, and after 5 minutes, it is taken out and dried to form a film naturally, and then refrigerated and stored at 0-4°C.

[0041] 2. Freshness preservation effect

[0042] After storing the refrigerated meat in step 1 for 5 days, the preservation effect is measured, with volatile basic nitrogen (TVB-N) as the inspection index, and the measurement method is based on the national standard method. Volatile basic nitrogen is used as the main criterion for freshness. Volatile basic nitrogen (TVB-N) is an important hygienic physical and chemical indicator for evaluating the freshness of fish, meat, etc. In simple terms, volatile basic nitrogen is the ammonia and alkaline nitrogen-containing substances such as amines produced by the decomposition of protein in fish meat due to the action of enzymes and bacteria. TVB-N ≤ 15mg / 100g is fresh meat that meets the physical and chemical standards.

[0043] The results of sensory testing and TVB-N testing are as follows:

[0044] Color odor TVB-N(mg / 100g) Example 1 Normal color No odor 11 Example 2 Normal color No odor 14 Example 3 Normal color No odor 10 Example 4 Normal color No odor 13 Comparative Example 1 Normal color No odor 22

[0045] From the above data, the sensory state of Comparative Example 1 is normal, but the volatile basic nitrogen index has exceeded the specified value (15 mg / 100 g) by more than 40%.

[0046] The freshness indicators of the embodiments meet the standards both in terms of sensory perception and volatile basic nitrogen. Therefore, the fresh-keeping coating made from the cartilage alcohol-soluble protein raw material of the embodiment group of the present invention can achieve the effects of anti-oxidation, antibacterial and anti-corruption. Compared with the alcohol-soluble protein extracted by microwave assisted extraction, it not only has higher extraction rate and purity, but also has better refrigeration and preservation effect. It has broad application prospects in the field of room temperature and refrigerated preservation of meat.

[0047] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing cartilage alcohol-soluble protein, characterized in that: The recovered cartilage powder obtained by processing the residue of chondroitin sulfate extraction is added to an alcohol solution, and then the pH is adjusted to 9 for ultrasonic extraction. The ultrasonic extraction conditions are: under a temperature of 40°C to 60°C, 150 to 300W power ultrasound for 20 to 40 minutes. After the extraction is completed, the supernatant is obtained by centrifugation, and the cartilage alcohol-soluble protein is obtained after freeze-drying.

2. The preparation method according to claim 1, characterized in that: The liquid-to-solid ratio is 15-20:

1.

3. The preparation method according to claim 1, characterized in that: The recovered cartilage powder is obtained by the following process: S1. Weigh 1000 kg of bovine cartilage and put it into an extraction tank. Add 8 times distilled water, heat to 100-105°C, keep warm for 3 hours, then take out the upper bone oil and cool to 50°C; S2. Adjust the cooking liquid to 9.0, add 0.9% alkaline protease by weight of cartilage, and stir for enzymolysis for 2 hours; readjust the pH value to 8.7, add 5% activated porcine pancreas by weight of cartilage, and stir for enzymolysis for 2 hours; raise the temperature of the enzymolysis liquid to 80°C, stir, adjust the pH value to 4.4, process for 20 minutes, stop stirring, and let stand until stratification is obvious; S3, filtering and separating the supernatant, adsorbing the separated liquid by anion exchange resin, the mass ratio of the separated liquid to the anion exchange resin is 1.4:1, and the adsorption time is 1 hour; the ion exchange resin is eluted by a 2 mol / L NaCl solution; the eluate is oxidized and decolorized, filtered, and the filtrate is pumped into an ultrafiltration device, and desalted and concentrated by the ultrafiltration device to obtain a concentrated solution; S4, add 5 times volume of 95% ethanol to the concentrate, centrifuge to obtain supernatant and precipitate; The supernatant is introduced into a distillation tower for rectification to obtain a distillate and a kettle residue, wherein the distillate is reused after the ethanol concentration reaches 95%, and the kettle residue is spray-dried to obtain the recovered cartilage powder.

4. The preparation method according to claim 1, characterized in that: The alcohol solution is one or more of ethanol, isopropanol and tert-butanol.

5. The preparation method according to claim 4, characterized in that: The volume fraction of the ethanol is 60% to 80%.

6. The alcohol-soluble protein prepared by the preparation method according to claim 1.

7. Use of the alcohol-soluble protein according to claim 6 in the preparation of meat preservation.