Method for fractionating active protein from jellyfish

Abstract

The invention relates to the technical field of jellyfish active protein preparation, and discloses a method for fractionation of jellyfish mucoprotein and collagen. The method comprises the following specific steps: cleaning jellyfish, homogenizing, soaking the homogenate with a sodium chloride solution by utilizing the characteristics of good stability and high solubility of glycoprotein in a dilute salt solution, centrifuging, purifying the supernate to obtain mucoprotein, and performing enzymolysis, centrifugation and ultrafiltration on the precipitate to obtain collagen. According to the method disclosed by the invention, mutual interference of jellyfish active proteins in the extraction process is effectively avoided by utilizing a fractional separation and recovery mode, the jellyfish is utilized to the greatest extent, an organic reagent is not needed in the whole process, the product is safe and free of solvent residues, and the environment is protected.

Description

Technical Field

[0001] The present invention relates to the technical field of jellyfish active protein preparation, in particular to a method for fractionating and separating jellyfish mucin and collagen. The jellyfish mucin and jellyfish collagen prepared by the present invention have high purity and have high application value in the fields of food, cosmetics, medical treatment, etc. Background Art

[0002] With the continuous development and progress in the field of protein technology, more and more methods of protein extraction have appeared in the public eye. As one of the oldest representatives in the field of marine life, jellyfish has rich nutritional value and active functions. The research on jellyfish active ingredients mainly focuses on substances such as proteins, protein complexes and polysaccharides. Jellyfish has become the preferred raw material for protein extraction because it is rich in mucin and collagen. However, there are many limitations and challenges in the separation technology of mucin and collagen. The current separation technology often cannot meet the requirements of high purity and high recovery rate of mucin and collagen at the same time. Although chromatography can achieve a certain degree of separation, it has the problems of high equipment and consumables cost, insufficient resolution or sample loss. In the past, there were related documents that studied the extraction and structural characteristics of jellyfish collagen, as well as the extraction and physicochemical properties of jellyfish collagen. The patent (application number: CN118255872A) proposed a jellyfish collagen and its preparation process, but none of the above involved the extraction of mucin. In addition, there are related patents (application number: 201510479968.1 and publication number: CN 101309932A) and documents that studied the extraction, process purification, physicochemical properties, and biological activity of jellyfish oral wrist glycoproteins, but the extraction process of collagen was not mentioned. In summary, none of the above related patent documents maximized the resource utilization of jellyfish. In addition, the patent (application number: 201380031849.9) proposed a graded extraction method for mucin and collagen. Although the jellyfish raw materials were fully utilized, the extraction rate was low, and organic solvents such as ethanol were used, resulting in high costs and unsuitability for large-scale production.

[0003] Therefore, in order to solve the existing technical problems, the present invention proposes a method for fractionating and separating jellyfish mucin and collagen, which not only ensures that the jellyfish raw materials are fully reacted, but also maximizes the utilization of jellyfish resources. At the same time, no organic solvents such as ethanol are used in the whole extraction process, which is economical and suitable for large-scale production.

[0004] To this end, we propose a method for fractionating active proteins from jellyfish. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for fractionating and separating active proteins from jellyfish, which can simultaneously extract and obtain mucin and collagen. Not only can the recovery rate and purity of active proteins be improved, maximizing the utilization of jellyfish resources, but also no organic solvent is used in the preparation process, thus avoiding environmental pollution and being suitable for large-scale production.

[0006] In order to achieve the above-mentioned method for fractionating active proteins from jellyfish, the present invention provides the following technical solution: the preferred protein separation process comprises the following steps of pretreatment, soaking, centrifugation, etc.: S1 Pretreatment: Take a certain mass of jellyfish, wash it, cut it into small pieces, and use a colloid mill to homogenize it to obtain a jellyfish homogenate for use.

[0007] S2 soaking: add sodium chloride solution to the jellyfish homogenate in proportion and soak.

[0008] S3 centrifugation: take the above jellyfish soaking liquid and centrifuge it at 10000r / min, 4℃ for 30min, and keep the supernatant and precipitate separately.

[0009] Furthermore, in step S2, the soaking condition is to add 0.1M sodium chloride solution to the jellyfish homogenate at a ratio of W:V=1:1 and soak overnight at 4°C.

[0010] Preferably, protein extraction and purification comprises separate preparation of mucin and collagen.

[0011] S4: taking the supernatant to extract mucin: selecting an ultrafiltration membrane with a specific molecular weight cutoff according to the molecular weight of mucin to ultrafilter the supernatant, and retaining the concentrated solution 1.

[0012] S5: Extract collagen from the precipitate according to the following steps: Select the type and amount of acid, enzyme type and amount, reaction temperature and other conditions for enzymatic hydrolysis according to the quality of the precipitate. After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is first centrifuged at a speed of 12000r / min, and the supernatant is retained. Then, an ultrafiltration membrane with a specific molecular weight cutoff is selected according to the molecular weight of the collagen to ultrafilter the supernatant, and the obtained concentrated solution 2 is retained.

[0013] Furthermore, in step S4, an ultrafiltration membrane with a molecular weight cutoff of 30 kDa is selected to perform ultrafiltration to obtain mucin.

[0014] Furthermore, in step S5, the enzymatic hydrolysis conditions include, but are not limited to, the type and amount of acid used for enzymatic hydrolysis, the type and amount of enzyme, and the reaction temperature conditions: adding 0.5M citric acid according to a precipitate mass volume ratio of 1:0.5 (W:V), and adding 3.0wt%-5.0wt% of pepsin by precipitate mass. The enzymatic hydrolysis is carried out at a temperature of 8°C for 24-36 hours.

[0015] Furthermore, in step S5, an ultrafiltration membrane with a molecular weight cutoff of 80 kDa is used for ultrafiltration to obtain collagen.

[0016] Preferably, the concentrated solution 1 is freeze-dried to obtain a solid product of jellyfish mucin. The concentrated solution 2 is freeze-dried to obtain a solid product of jellyfish collagen.

[0017] Compared with the prior art, the present invention provides a method for fractionating and separating active proteins from jellyfish, which has the following beneficial effects: 1. The present invention provides a method for simultaneously extracting mucin and collagen from jellyfish, which not only protects the structure of the protein active components to the greatest extent, but also improves the extraction rate of jellyfish active proteins.

[0018] 2. Through the scientific and reasonable design of process flow, the maximum utilization of kelp resources is achieved, avoiding the waste of resources caused by a single extraction method.

[0019] 3. This method does not use organic solvents in the whole process, thus avoiding environmental pollution. At the same time, it simplifies the extraction steps, reduces energy consumption and raw material consumption, has a high protein extraction rate, is economical, and can achieve large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The SDS-PAGE patterns of jellyfish collagen and mucin prepared in Examples 2 and 3 of the present invention are shown in Figure 1. A: jellyfish mucin, B: jellyfish collagen.

[0021] Figure 2 The SDS-PAGE pattern of the jellyfish mucin prepared in Comparative Example 1 of the present invention is shown in FIG.

[0022] Figure 3 The SDS-PAGE pattern of the jellyfish mucin prepared in Comparative Example 2 of the present invention is shown in FIG.

[0023] Figure 4 The SDS-PAGE pattern of the jellyfish collagen prepared in comparative example 3 of the present invention is shown in FIG.

[0024] Figure 5 The SDS-PAGE pattern of the jellyfish active protein obtained in comparative example 4 of the present invention is shown in Figure 4. Wherein A: a mixture of jellyfish collagen and mucin. Figure 6 The process flow diagram of the present invention includes a protein separation step and a protein extraction and purification step. DETAILED DESCRIPTION

[0025] The technical scheme in the embodiment of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without the creative labor of adding 0.5M citric acid in a volume ratio of 1:0.5 (W:V) and adding 3.0wt% of the precipitate mass, are within the scope of protection of the present invention.

[0026] Weigh 1000g of jellyfish, wash it, cut it into small pieces, and use a colloid mill to homogenize it to obtain a jellyfish homogenate for later use. Add 0.1M sodium chloride solution to the jellyfish crushed liquid according to the ratio of jellyfish mass W:V=1:1, and soak it at 4℃ overnight. Take the above jellyfish soaking liquid and centrifuge it at 10000r / min and 4℃ for 30min, and keep the supernatant and precipitate respectively to separate jellyfish mucin and collagen. At this time, the supernatant contains mucin and the precipitate contains collagen.

[0027] The supernatant containing mucin was ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa until the conductivity reached 28 μs / cm, and the concentrate 1 was retained. The freeze dryer cold trap temperature was set to -75°C and the chamber vacuum degree was set to 15.7 Pa, and the concentrate 1 was freeze-dried to obtain a jellyfish mucin solid product. The yield, total sugar content, and hydroxyproline content of this product are shown in Table 1. The sensory, physical and chemical index, and hygiene index test results are shown in Tables 2 to 4, and the corresponding SDS-PAGE spectra are shown in Figure 1 .

[0028] According to the precipitate mass volume ratio of 1:0.5 (W:V), 0.5M citric acid was added, and pepsin was added at 3.0wt% of the precipitate mass. Enzymolysis was carried out at a temperature of 8°C for 24 to 36 hours. The hydrolyzate was centrifuged at a speed of 12000r / min, and the supernatant was retained. An ultrafiltration membrane with a molecular weight cutoff of 80kDa was selected for ultrafiltration, and the ultrafiltration was stopped when the conductivity reached 36μs / cm, and the concentrated solution 2 was retained. The cold trap temperature of the freeze dryer was set to -75°C and the vacuum degree of the box was set to 15.7Pa to freeze-dry the concentrated solution 2 to obtain a jellyfish collagen solid product. The yield, total sugar content, and hydroxyproline content of this product are shown in Table 1. The sensory, physical and chemical index test results, and hygiene index test results are shown in Tables 2 to 4. The SDS-PAGE spectrum is shown in Tables 2 to 4. Figure 1 .

[0029] Weigh 1000g of jellyfish, wash it, cut it into small pieces, and use a colloid mill to homogenize it to obtain a jellyfish homogenate for use. Add 0.1M sodium chloride solution to the jellyfish crushed liquid according to the jellyfish mass W:V=1:1 and soak it at 4℃ overnight. Centrifuge at 10000r / min, 4℃ for 30min, and take the supernatant. Add ethanol according to the volume ratio Vsupernatant:Vethanol=1:3, centrifuge at 10000r / min, 4℃ for 30min, and take the precipitate. Place the precipitate in a dialysis bag (10000MW) and dialyze it in pure water for 48h, and replace the pure water every 8h. Use a freeze dryer (cold trap temperature -75℃, box vacuum: 15.7Pa) to dry the precipitate after dialysis to obtain a jellyfish mucin solid product. The yield and total sugar content of this product are shown in Table 1, and the SDS-PAGE spectrum is shown in Figure 2 .

[0030] Weigh 1000g of jellyfish, wash it, cut it into small pieces, and use a colloid mill to homogenize it to obtain a jellyfish homogenate for later use. Take the above jellyfish homogenate and treat it with ultrasound at 0℃ for 30min. Add 0.1M sodium chloride solution to the jellyfish homogenate after ultrasound treatment according to the jellyfish mass W:V=1:1 and soak it at 4℃ overnight. Take the above jellyfish soaking liquid and centrifuge it at 10000r / min and 4℃ for 30min to take the supernatant. Use an ultrafiltration membrane with a molecular weight cutoff of 30kDa to ultrafilter the above supernatant until the conductivity is lower than 20μs / cm, and retain the concentrated solution. Use a freeze dryer (cold trap temperature -75℃, box vacuum: 15.7Pa) to dry the above concentrated solution to obtain a jellyfish mucin solid product. The yield, total sugar content, and hydroxyproline content of this product are shown in Table 1, and the SDS-PAGE spectrum is shown in Figure 3 .

[0031] Weigh 1000g of jellyfish, wash it, cut it into small pieces, and use a colloid mill to homogenize it to obtain a jellyfish homogenate for later use. Add 0.5M citric acid according to the jellyfish mass-to-volume ratio of 1:0.5 (W:V), and add 3.0wt% pepsin of the jellyfish mass. Perform enzymolysis at a temperature of 8°C for 24 to 36 hours. Take the above jellyfish soaking liquid and centrifuge it at 10000r / min and 4°C for 30min, and take the supernatant. Add sodium chloride solid to the supernatant to a sodium chloride concentration of 1mol / L, let it stand at 4°C for 24h, and then centrifuge it at 10000r / min and 4°C for 60min to take the precipitate. Place the precipitate in a dialysis bag (10000MW) and dialyze it in pure water for 48h, and replace the pure water every 24h. The precipitate after dialysis was dried using a freeze dryer (cold trap temperature -75°C, box vacuum degree: 15.7Pa) to obtain a solid product of jellyfish collagen. The yield and hydroxyproline content of this product are shown in Table 1, and the SDS-PAGE spectrum is shown in Figure 4 .

[0032] A jellyfish extract extraction process comprises the following steps: weigh 1000g of jellyfish and put it into clean water for washing to remove impurities. Take the washed jellyfish sample and soak it in 0.5M citric acid at 4°C for 12h according to W:V=1:3. Homogenize the treated sample for 2-5min to obtain a jellyfish homogenate. Take the homogenized sample, add 0.5M citric acid at a ratio of W:V=1:0.5, add 3.0wt% of the raw material enzyme, and perform enzymolysis at a temperature of 8°C for 24h. Centrifuge the above enzymolysis solution at 12000r / min and retain the supernatant. Use an ultrafiltration membrane with a molecular weight cutoff of 30kDa to separate the supernatant, stop ultrafiltration when the conductivity is lower than 20μs / cm, and retain the concentrated solution. Freeze-dry the concentrated solution to obtain a jellyfish extract solid product rich in collagen and mucin. The yield, total sugar content, and hydroxyproline content of this product are shown in Table 1, and the SDS-PAGE spectrum is shown in Figure 5 .

Claims

1. A method for fractionating active proteins from jellyfish, including mucin and collagen, characterized in that: The following steps are implemented to achieve the step-by-step recovery of the jellyfish active protein; (1) Protein separation: including pretreatment, soaking, centrifugation and other steps; ① Pretreatment: Take a certain amount of jellyfish, wash it, cut it into small pieces, and homogenize it using a colloid mill to obtain a jellyfish homogenate for later use; ② Soaking: Add sodium chloride solution to the jellyfish homogenate in proportion and soak; ③ Centrifugation: take the above jellyfish soaking solution and centrifuge it at 10000r / min, 4°C for 30min, and keep the supernatant and precipitate respectively; (2) Protein extraction and purification: including preparation of mucin and collagen separately; ① Take the supernatant to extract mucin: select an ultrafiltration membrane with a specific molecular weight cutoff according to the molecular weight of mucin to ultrafilter the supernatant, and retain the concentrated solution 1; ② Take the precipitate and extract collagen according to the following steps: select the type and amount of acid, enzyme type and amount, reaction temperature conditions, etc. for enzymatic hydrolysis according to the quality of the precipitate. After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is first centrifuged at a speed of 12000r / min, and the supernatant is retained. Then, according to the molecular weight of collagen, an ultrafiltration membrane with a specific molecular weight cutoff is selected to ultrafilter the supernatant, and the concentrated solution 2 is retained; (3) Solid drying: freeze-dry the concentrated solution 1 to obtain a solid product of jellyfish mucin. Freeze-dry the concentrated solution 2 to obtain a solid product of jellyfish collagen.

2. A method for fractionating and separating active proteins from jellyfish according to claim 1, characterized in that: (1) Soaking in the second step: adding 0.1 M sodium chloride solution to the jellyfish homogenate at a ratio of W:V of the jellyfish = 1:1 and soaking overnight at 4°C.

3. A method for fractionating and separating active proteins from jellyfish according to claim 1, characterized in that: (2) The enzymatic hydrolysis conditions described in ② include, but are not limited to, the type and amount of acid, type and amount of enzyme, and reaction temperature used for enzymatic hydrolysis: adding 0.5M citric acid according to a precipitate mass volume ratio of 1:0.5 (W:V), and adding 3.0wt% of pepsin by weight of the precipitate. The enzymatic hydrolysis is carried out at a temperature of 8°C for 24-36 hours.

4. A method for fractionating and separating active proteins from jellyfish according to claim 1, characterized in that: During the protein extraction, according to the different molecular weights of mucin and collagen, an ultrafiltration membrane with a molecular weight cutoff of 30 kDa is selected to extract mucin, and an ultrafiltration membrane with a molecular weight cutoff of 80 kDa is selected to extract collagen.

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