Low-cost large-molecular-weight fish scale collagen extraction method

Abstract

The invention discloses a low-cost large-molecular-weight fish scale collagen extraction method, and belongs to the technical field of fish scale collagen. The method comprises the following steps: (1) pretreatment: repeatedly freezing and storing fish scales and unfreezing and redissolving the fish scales; 2) impurity protein removal: taking out the fish scales, and removing fish silver; soaking in a sodium chloride solution, washing and drying; 3) extracting fish scale collagen, namely extracting the dried fish scales for 1-4 hours at 40-100 DEG C according to a solid-liquid ratio of 1: (5-20) by adopting a hydrothermal extraction mode, carrying out solid-liquid separation, and collecting fish scale collagen extract liquids in different molecular weight intervals; and 4) freeze-drying the collagen extract liquid to obtain a fish scale collagen solid product. According to the method, acid or alkali solution pretreatment is not needed, and a sodium chloride salt solution is used for mildly removing impure protein. And the structure of the fish scale collagen is relatively completely maintained while the relatively high yield is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal collagen, and particularly relates to a method for extracting large-molecular-weight fish scale collagen with low cost. Background Art

[0002] Collagen, as the main component of the extracellular matrix, is a very important protein in the human body. It can supplement the nutrients required by each layer of the skin, and has the effects of moisturizing the skin, delaying aging, and beautifying. Traditionally, collagen was mainly extracted from the skin and bones of terrestrial animals such as cows and pigs; however, with the outbreak of diseases such as mad cow disease and foot-and-mouth disease, people have questioned the safety of traditional collagen products; and with the increasing demand for collagen products in the fields of medicine, cosmetics, etc., it is urgent to find safe and reliable collagen raw materials.

[0003] Research has found that fish scales are rich in protein and fat, and the collagen content is as high as 45%. Therefore, fish scale collagen has become one of the popular candidates for mammalian collagen substitutes. In recent years, there have been more and more studies on extracting collagen from fish scales, but most of them use strong acids and alkalis to pretreat fish scales before extraction. Excessive use will not only cause environmental pollution, but also damage the complete structure of collagen, which is not conducive to the extraction of collagen. In addition, although various new methods for extracting fish scale collagen have emerged one after another, each has its own advantages and disadvantages. Although the acid-enzyme method can retain the complete molecular weight and structure of collagen, the process is complex in large-scale production, resulting in higher costs. The existing hydrothermal method has a simple operation, but the extraction efficiency at low temperature is low; to improve the yield, high temperatures such as 100°C are often selected. Although this method can improve the yield, because collagen is sensitive to heat, high temperatures will destroy its triple helix structure. Through research, it has been found that only collagen with a complete protein structure and appropriate molecular weight can better play its role. In order to balance the extraction efficiency, molecular weight, and integrity of the protein structure, a gradient hydrothermal method for extracting fish scale collagen has been invented.

[0004] The existing technology has the following technical problems:

[0005] 1) The extraction process of fish scale collagen in the existing technology is complex and the cost is high: Although high-purity and water-soluble fish scale collagen can be obtained by using different enzymes and permeable membranes, the price of enzyme preparations is high and the enzyme method process is complex, resulting in high costs in large-scale production, which limits its application range.

[0006] 2) The pretreatment process of the prior art is cumbersome and not conducive to the extraction of fish scale collagen. Soaking with acid or alkali solution can remove impurities and loosen the dense structure of fibers, which is beneficial to the dissolution of fish scale collagen. However, the waste liquid generated will cause environmental pollution. In addition, the residual solution will cause the loss of fish scale collagen, making it unsuitable for large-scale production.

[0007] 3) The degree of denaturation of fish scale collagen extracted by the existing hydrothermal method is serious. Increasing the temperature and prolonging the time are used to obtain a high yield of fish scale collagen. However, fish scale collagen is sensitive to heat, and this method will cause the triple helix structure of the protein to unwind into free polypeptide chains, resulting in a relatively dispersed molecular weight distribution.

[0008] 4) There are differences in the batch-to-batch molecular weight of fish scale collagen extracted by the existing hydrothermal method, and the protein structure is incomplete. Summary of the Invention

[0009] In view of the problems existing in the prior art, the present invention provides a method for extracting large molecular weight fish scale collagen at low cost.

[0010] The present invention mainly realizes the solution of the above problems through the following technical solutions:

[0011] The present invention discloses a method for extracting large molecular weight fish scale collagen at low cost. The method is divided into the following steps:

[0012] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen, thaw and redissolve them, and repeat the operations of frozen storage and thawing and redissolving in sequence to obtain pretreated fish scales.

[0013] 2) Remove impurity proteins: Remove the fish silver from the fish scales pretreated in step 1), and wash them clean with pure water. Then soak them in a salt solution to remove impurity proteins, rinse the fish scales with purified water until there is no turbid liquid, and dry them.

[0014] 3) Extract fish scale collagen: Mix the dried fish scales in step 2) with purified water at a solid-liquid ratio of 1:5 - 20, and perform hydrothermal extraction in the temperature range of 40 - 100 °C for 1 - 4 h, separate the solid and liquid, and collect the solution to obtain the fish scale collagen extraction solution.

[0015] 4) Pre-cool the fish scale collagen extraction solution collected in step 3) and then freeze-dry it to obtain a solid product of fish scale collagen.

[0016] As a further improvement, in step 1) of the present invention, the temperature for frozen storage of the encapsulated fish scales is -80 °C to -20 °C.

[0017] As a further improvement, in step 1) of the present invention, the temperature for thawing and reconstitution is 25 - 45°C, the time is 0.5 h - 3 h, and the number of repetitions of freeze storage - thawing and reconstitution is 1 - 3 times. In the present invention, the temperature difference between freeze storage and thawing and reconstitution is relatively large, and the temperature fluctuations generated during the implementation process can cause the ice crystals inside the fish scales to be more likely to recrystallize. The accumulation and expansion of ice crystals are more likely to damage the internal tissue structure, which is beneficial to the subsequent extraction of collagen; in the present invention, the operation is repeated multiple times, resulting in the complete destruction of the internal interaction force of the fish scales, reducing the energy consumption for the subsequent extraction of fish scale collagen.

[0018] As a further improvement, in step 2) of the present invention, the salt solution is any one of sodium chloride solution and sodium carbonate solution, the mass fraction of the salt solution is 5% - 20%, and the soaking time is 0.5 - 2 h. The present invention adopts the method of soaking the fish scales in the salt solution, with mild conditions, which can not only remove the miscellaneous proteins, but also avoid the destruction of the structural integrity of collagen. The sodium chloride solution can be prepared on site, with simple operation, less energy consumption and convenient use.

[0019] As a further improvement, in step 2) of the present invention, the drying temperature of the fish scales is 20°C - 65°C, and the time is 3 - 24 h. The fish scale collagen is relatively sensitive to the environmental temperature, and too high temperature will cause the inactivation of the fish scale collagen. The drying method adopted in the present invention can ensure the complete dehydration of the fish scales while not destroying the properties of the fish scale collagen.

[0020] As a further improvement, in step 3) of the present invention, the hydrothermal extraction method of fish scale collagen is a process of gradient hydrothermal extraction, and the gradient temperature range is 40 - 70°C, 70 - 90°C, and 90 - 100°C, and the gradient temperature change is gradient heating or gradient cooling.

[0021] As a further improvement, in step 3) of the present invention, the material - liquid ratio of fish scales to water is 1:5 - 1:10, the extraction time at 40 - 70°C is 0.5 - 2 h, the extraction time at 70 - 90°C is 0.5 - 1 h, and the extraction time at 90 - 100°C is 0.5 - 2 h.

[0022] As a further improvement, in step 3) of the present invention, the solid - liquid separation method is filtration or centrifugation, and the collection method of the fish scale collagen extract is segmented collection or cumulative collection. In the present invention, segmented collection is to collect the fish scale collagen solution extracted in the corresponding gradient temperature range according to the material - liquid ratio multiple times. Cumulative collection means collecting the fish scale collagen leaching solution of gradient hydrothermal extraction only once.

[0023] As a further improvement, in step 4) of the present invention, the drying method of the fish scale collagen extract is any one of vacuum freeze-drying, spray freeze-drying, gel filtration drying, and vacuum drying. The collagen freeze-dried product prepared by the present invention has higher stability than the collagen solution.

[0024] As a further improvement, the fish scale collagen solid product obtained by the above method is in any one of the forms of powder, granule, and sponge. After the preparation process of the present invention, fish scales can obtain large-molecular-weight fish scale collagen solid products in various forms such as powder and sponge, which can be widely used in fields such as medicine and cosmetics.

[0025] In the above technical solution, a low-cost method for extracting large-molecular-weight fish scale collagen provided by the present invention has the following beneficial effects:

[0026] 1. The preparation process provided by the present invention avoids the use of enzymes, etc., and screens the molecular weight by osmotic membrane filtration, with simple operation and reduced extraction cost of collagen.

[0027] 2. The present invention uses a salt solution and adopts a process with temperature ranges of 40 - 70°C, 70 - 90°C, and 90 - 100°C, and the temperature change is gradient heating or gradient cooling, avoiding the destruction of the complete structure of collagen by strong acid and alkali solutions and long-time high-temperature cooking.

[0028] 3. Adopting temperature ranges of 40 - 70°C, 70 - 90°C, and 90 - 100°C, the extraction time at 40 - 70°C is 0.5 - 2h, the extraction time at 70 - 90°C is 0.5 - 1h, the extraction time at 90 - 100°C is 0.5 - 2h, and the temperature change is gradient heating or gradient cooling. While retaining a high extraction efficiency, it makes the molecular weight distribution more concentrated and the collagen structure more complete. The traditional hydrothermal method directly cooks the pre-treated raw materials with a high-temperature aqueous solution to obtain collagen. Although the solubility of collagen increases with the increase in temperature, the long-time high-temperature cooking process makes the triple helix structure unwind into free polypeptide chains more thoroughly, and the molecular weight distribution is more dispersed. The gradient hydrothermal method has a low-temperature buffer zone, taking into account a high extraction rate while slowing down the triple helix unwinding rate, enabling it to renature at low temperature, thereby retaining a more complete collagen structure and making the molecular weight concentratedly distributed.

[0029] 4. Adopting a gradient temperature control process reduces the batch-to-batch difference and obtains relatively uniform and complete collagen. Description of the Drawings

[0030] Figure 1 It is the Fourier-transform infrared spectroscopy result graph of fish scale collagen in the comparative case;

[0031] Figure 2 Fourier-transform infrared spectroscopy results of scale collagen in the implementation case;

[0032] Figure 3 SDS-PAGE gel electrophoresis results of scale collagen. Specific implementation mode

[0033] In order to better understand the purpose, technical solution and advantages of the present invention, the following will describe in detail with specific examples of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0034] Example 1:

[0035] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20°C, thaw and redissolve them at 25°C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales;

[0036] 2) Remove impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse them with purified water until there is no turbid liquid; soak them in a 10% sodium chloride solution and let them stand for 1 h; finally, take out the fish scales, wash them, and dry them at 25°C for later use;

[0037] 3) Extract scale collagen: First, mix the dried fish scales with purified water at a solid-liquid ratio of 1:7.5, and perform hydrothermal extraction at 55°C for 1.5 h; then, supplement the purified water at a solid-liquid ratio of 1:5, and continue to extract at 75°C for 1 h; finally, supplement the purified water at a solid-liquid ratio of 1:7.5, and extract at 95°C for 1.5 h. The total immersion extraction time is 4 h. After solid-liquid separation, cumulatively collect the scale collagen extract;

[0038] 4) After pre-cooling the scale collagen extract at -20°C, use a freeze dryer to obtain a solid product of scale collagen.

[0039] Example 2:

[0040] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20°C, thaw and redissolve them at 25°C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales;

[0041] 2) Remove impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse them with purified water until there is no turbid liquid; soak them in a 10% sodium chloride solution and let them stand for 1 h; finally, take out the fish scales, wash them, and dry them at 25°C for later use;

[0042] 3) Extracting fish scale collagen: First, mix the dried fish scales and purified water at a material-liquid ratio of 1:7.5, and perform hydrothermal extraction at 95 °C for 1.5 h; subsequently, supplement the purified water at a material-liquid ratio of 1:5, and continue extraction at 75 °C for 1 h; finally, supplement the purified water at a material-liquid ratio of 1:7.5, and extract at 55 °C for 1.5 h. The total extraction time is 4 h. After solid-liquid separation, collect the cumulative fish scale collagen extract;

[0043] 4) After pre-cooling the fish scale collagen extract at -20 °C, use a freeze dryer to obtain the solid product of fish scale collagen.

[0044] Example 3:

[0045] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20 °C, thaw and redissolve at 25 °C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales;

[0046] 2) Removing impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse with purified water until there is no turbid liquid; soak them in a 10% sodium chloride solution and let stand for 1 h; finally, take out the fish scales, wash them, and dry them at 25 °C for later use;

[0047] 3) Extracting fish scale collagen: First, mix the dried fish scales and purified water at a material-liquid ratio of 1:7.5, and perform hydrothermal extraction at 55 °C for 1.5 h, then perform solid-liquid separation and collect the first fish scale collagen extract; subsequently, mix the solid and purified water at a material-liquid ratio of 1:5, and perform hydrothermal extraction at 75 °C for 1 h, perform solid-liquid separation and collect the second fish scale collagen extract; finally, mix the solid and purified water at a material-liquid ratio of 1:7.5, and continue to extract at 95 °C for 1.5 h. After solid-liquid separation, collect the third fish scale collagen extract. The total extraction time is 4 h, and the solutions collected in three segments are combined;

[0048] 4) After pre-cooling the combined fish scale collagen extract at -20 °C, use a freeze dryer to obtain the solid product of fish scale collagen.

[0049] Example 4:

[0050] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20 °C, thaw and redissolve at 25 °C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales;

[0051] 2) Removing impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse with purified water until there is no turbid liquid; soak them in a 10% sodium chloride solution and let stand for 1 h; finally, take out the fish scales, wash them, and dry them at 25 °C for later use;

[0052] 3) Extract fish scale collagen: First, mix the dried fish scales and purified water at a material-liquid ratio of 1:7.5, and perform hydrothermal extraction at 95°C for 1.5 h. Then, perform solid-liquid separation and collect the first fish scale collagen extract. Subsequently, mix the solid and purified water at a material-liquid ratio of 1:5, and perform hydrothermal extraction at 75°C for 1 h. After solid-liquid separation, collect the second fish scale collagen extract. Finally, mix the solid and purified water at a material-liquid ratio of 1:7.5, continue to extract at 55°C for 1.5 h, and collect the third fish scale collagen extract after solid-liquid separation. The total extraction time is 4 h, and the solutions collected in three segments are combined.

[0053] 4) Pre-cool the combined fish scale collagen extract at -20°C, and then use a freeze dryer to obtain a solid product of fish scale collagen.

[0054] Example Five:

[0055] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20°C, thaw and re-dissolve them at 25°C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales.

[0056] 2) Remove impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse them with purified water until there is no turbid liquid. Immerse them in a 10% sodium chloride solution and let them stand for 1 h. Finally, take out the fish scales, wash them, and dry them at 25°C for later use.

[0057] 3) Extract fish scale collagen: First, mix the dried fish scales and purified water at a material-liquid ratio of 1:7.5, and perform hydrothermal extraction at 60°C for 0.5 h. Subsequently, supplement the amount of purified water at a material-liquid ratio of 1:5, and continue to extract at 80°C for 1 h. Finally, supplement the amount of purified water at a material-liquid ratio of 1:7.5, and extract at 100°C for 2.5 h. The total extraction time is 4 h. After solid-liquid separation, cumulatively collect the fish scale collagen extract.

[0058] 4) Pre-cool the fish scale collagen extract at -20°C, and then use a freeze dryer to obtain a solid product of fish scale collagen.

[0059] Example Six:

[0060] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20°C, thaw and re-dissolve them at 25°C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales.

[0061] 2) Remove impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse them with purified water until there is no turbid liquid. Immerse them in a 10% sodium chloride solution and let them stand for 1 h. Finally, take out the fish scales, wash them, and dry them at 25°C for later use.

[0062] 3) Extracting fish scale collagen: First, mix the dried fish scales and purified water at a material-liquid ratio of 1:7.5 and perform hydrothermal extraction at 60 °C for 1 h; subsequently, supplement the amount of purified water at a material-liquid ratio of 1:5 and continue extraction at 80 °C for 1 h; finally, supplement the amount of purified water at a material-liquid ratio of 1:7.5 and extract at 100 °C for 2 h. The total immersion extraction is 4 h. After solid-liquid separation, collect the cumulative fish scale collagen extract;

[0063] 4) After pre-cooling the fish scale collagen extract at -20 °C, use a freeze dryer to obtain the solid product of fish scale collagen.

[0064] Example VII:

[0065] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20 °C, thaw and re-dissolve at 25 °C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales;

[0066] 2) Removing impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse with purified water until there is no turbid liquid; soak them in a 10% sodium chloride solution and let stand for 1 h; finally, take out the fish scales, wash them, and dry them at 25 °C for later use;

[0067] 3) Extracting fish scale collagen: First, mix the dried fish scales and purified water at a material-liquid ratio of 1:7.5 and perform hydrothermal extraction at 60 °C for 2 h; subsequently, supplement the amount of purified water at a material-liquid ratio of 1:5 and continue extraction at 80 °C for 1 h; finally, supplement the amount of purified water at a material-liquid ratio of 1:7.5 and extract at 100 °C for 1 h. The total immersion extraction is 4 h. After solid-liquid separation, collect the cumulative fish scale collagen extract;

[0068] 4) After pre-cooling the fish scale collagen extract at -20 °C, use a freeze dryer to obtain the solid product of fish scale collagen.

[0069] Example VIII:

[0070] 1) Pretreatment: Collect and encapsulate fish scales, store them frozen at -20 °C, thaw and re-dissolve at 25 °C for 1 h, and repeat the operation 3 times in sequence to obtain pretreated fish scales;

[0071] 2) Removing impurity proteins: Take out the pretreated fish scales, remove the fish silver, and rinse with purified water until there is no turbid liquid; soak them in a 10% sodium chloride solution and let stand for 1 h; finally, take out the fish scales, wash them, and dry them at 25 °C for later use;

[0072] 3) Extract fish scale collagen: First, mix the dried fish scales with purified water at a material-liquid ratio of 1:7.5, and perform hydrothermal extraction at 60 °C for 2.5 h. Subsequently, supplement the amount of purified water at a material-liquid ratio of 1:5, and continue the extraction at 80 °C for 1 h. Finally, supplement the amount of purified water at a material-liquid ratio of 1:7.5, and extract at 100 °C for 0.5 h. The total extraction time is 4 h. After solid-liquid separation, collect the cumulative fish scale collagen extract;

[0073] 4) After pre-cooling the fish scale collagen extract at -20 °C, use a freeze dryer to obtain a solid product of fish scale collagen.

[0074] Comparative Example 1:

[0075] Only for Example 1, replace step 3) with mixing the dried fish scales with purified water at a material-liquid ratio of 1:20, and perform hydrothermal extraction at 55 °C for 4 h. After solid-liquid separation, collect the fish scale collagen extract. Other steps are the same.

[0076] Comparative Example 2:

[0077] Only for Example 1, replace step 3) with mixing the dried fish scales with purified water at a material-liquid ratio of 1:20, and perform hydrothermal extraction at 75 °C for 4 h. After solid-liquid separation, collect the fish scale collagen extract. Other steps are the same.

[0078] Comparative Example 3:

[0079] Only for Example 1, replace step 3) with mixing the dried fish scales with purified water at a material-liquid ratio of 1:20, and perform hydrothermal extraction at 95 °C for 4 h. After solid-liquid separation, collect the fish scale collagen extract. Other steps are the same.

[0080] In the present invention, the biuret method is used to measure the protein content in the solid product and evaluate the extraction rate of the preparation process.

[0081] Measurement method: 1) Prepare a bovine serum albumin reference solution at a concentration of 10 mg / mL. Accurately measure 0 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the reference solution, and place them in test tubes respectively. Add water to each tube to make the volume up to 1.0 mL. Add 4.0 mL of biuret reagent to the different concentrations of bovine serum albumin reference solutions prepared above, mix well at room temperature, let stand for 30 min, and then measure the absorbance at a wavelength of 540 nm to draw a standard curve. 2) Take an appropriate amount of the sample, add water to prepare a sample solution. Take 1 mL and add 4.0 mL of biuret reagent, mix well at room temperature, let stand for 30 min, and then measure the absorbance at a wavelength of 540 nm. Calculate the protein content in the sample from the standard curve and finally convert it into the extraction rate.

[0082] The solid product obtained in the case of the present invention was tested for collagen content, and the results are shown in Table 1.

[0083] Table 1 Results of the extraction rate of fish scale collagen

[0084] Case Temperature (°C) Time (h) Collection method Extraction rate (%) Example 1 55-75-95 1.5-1-1.5 Cumulative collection 32.76 Example 2 95-75-55 1.5-1-1.5 Cumulative collection 33.14 Example 3 55+75+95 1.5-1-1.5 Segmented collection 25.86 Example 4 95+75+55 1.5-1-1.5 Segmented collection 28.83 Example 5 60-80-100 0.5-1-2.5 Cumulative collection 19.59 Example 6 60-80-100 1-1-2 Cumulative collection 20.56 Example 7 60-80-100 2-1-1 Cumulative collection 21.50 Example 8 60-80-100 2.5-1-0.5 Cumulative collection 19.98 Comparative example 1 55 4 Cumulative collection 4.05 Comparative example 2 75 4 Cumulative collection 19.83 Comparative example 3 95 4 Cumulative collection 30.06

[0085] As can be seen from Table 1, in the present invention, the extraction rate of the gradient hydrothermal method is significantly improved compared with that at a low temperature (55 °C), and the extraction rate of the cumulative collection method is higher. In addition, although there are slight differences in the extraction rate compared with that at 95 °C, the gradient hydrothermal method avoids the damage to the collagen structure caused by direct long-term high-temperature cooking, which is more conducive to the use of collagen. In addition, the extraction rate of fish scale collagen in the present invention fluctuates with the change of extraction time, and this property provides an idea for reducing the extraction cost. In the present invention, Fourier-transform infrared spectroscopy was used to measure the structural stability of the solid product.

[0086] Determination method: The fish scale collagen sample and dry KBr powder were placed in a mortar according to a ratio of 1:100, ground thoroughly, pressed into a tablet, and placed in the sample chamber. The measurement range of the spectrum was set to 4000 - 400 cm -1 , and the resolution was 8 cm -1 .

[0087] The solid product obtained in the case of the present invention was subjected to infrared testing, and the results are as Figure 1 shown, Figure 1 is the Fourier-transform infrared spectroscopy result diagram of fish scale collagen in the comparative case; Figure 2 is the Fourier-transform infrared spectroscopy result diagram of fish scale collagen in the implementation case.

[0088] From Figure 1 and Figure 2 it can be seen that the fish scale collagen extracted in the present invention produces absorption peaks near 3474 cm -1 , 2931 cm -1 , 1662 cm -1 , 1538 cm -1 and 1244 cm -1 , which are the characteristic absorption peaks of collagen. In addition, the infrared spectral information of the amide I band is often used for the analysis of the secondary structure of proteins. As can be seen in Figure 1 , as the extraction temperature increases, the peak intensity of the amide I band gradually weakens, indicating that the secondary structure of collagen is damaged; while in Figure 2 the collagen extracted by the gradient hydrothermal method produces a relatively large absorption intensity in the amide I band, indicating that the gradient hydrothermal method can retain a relatively complete protein structure.

[0089] In the present invention, SDS-PAGE gel electrophoresis was used to evaluate the protein molecular weight size and distribution in the solid product.

[0090] Determination method: 1) Preparation of separating gel: Prepare a 7% separating gel solution, pour it into the mold to a certain height, seal it with water, and polymerize at room temperature. 2) Preparation of stacking gel: Prepare a 5% stacking gel solution. After the separating gel solution polymerizes, use filter paper to absorb the upper water layer, then pour in the stacking gel solution, and insert the sample comb. 3) Preparation of sample or reference solution: Take 1 mg of the sample or reference respectively, add 100 μL of purified water, mix it with an equal volume of 2× sample buffer, place it in a 100 °C water bath for 5 - 10 min, and then centrifuge. 4) Electrophoresis: After the stacking gel polymerizes, carefully pull out the sample comb, fill the electrophoresis tank with the electrode buffer. Add the sample reference solution to the sample loading well. The initial voltage of constant voltage electrophoresis is 80 V, and it needs to be adjusted to 100 V when entering the separating gel. Electrophoresis ends when it migrates to the bottom of the gel. 5) Staining and decolorization: Take out the gel, stain it with an excessive amount of Coomassie Brilliant Blue staining solution for 1 - 2 h. Discard the staining solution, and decolorize it with an excessive amount of decolorizing solution multiple times until the gel background becomes transparent. 6) Data analysis: Take pictures and analyze with a gel scanning system.

[0091] The solid product obtained in the case of the present invention was subjected to SDS-PAGE gel electrophoresis test, and the results are as Figure 3 shown. Figure 3 It is the SDS-PAGE gel electrophoresis result diagram of fish scale collagen. As Figure 3 can be seen, under the same conditions, Comparative Example 1 (low-temperature extraction) failed to show obvious protein bands, which is not conducive to the extraction of collagen; and collagen is sensitive to heat, and long-term high-temperature cooking will damage the collagen structure, resulting in a relatively dispersed molecular weight distribution. However, the fish scale collagen extracted by the gradient hydrothermal method has a uniform and stable molecular weight range distribution within the same temperature range and a relatively concentrated overall distribution whether different heating / cooling methods or different collection methods are used.

[0092] Based on the above information, it shows that the method provided by the present invention not only meets a high extraction rate but also retains a relatively complete collagen structure, which is beneficial to the utilization of collagen.

[0093] The above are only several implementation cases of the present invention, but it should not be construed as a limitation on the patent scope of the present invention. Any modifications and deformations made without departing from the concept of the present invention fall within the protection scope of the present invention.

Claims

1. A low-cost method for extracting high molecular weight fish scale collagen, characterized in that: The method described is divided into the following steps: 1) Pretreatment: collecting and packaging fish scales, freezing and storing, thawing and re-melting, and repeating the freezing and storing and thawing and re-melting operations in sequence to obtain pre-treated fish scales; 2) removing impurity proteins: removing the fish silver from the fish scales pre-treated in step 1) and washing them with pure water; then soaking them in a salt solution to remove impurity proteins, washing the fish scales with purified water until there is no turbid liquid, and drying; 3) Extracting fish scale collagen: extracting the dried fish scales in step 2) with purified water at a solid-liquid ratio of 1:5-20 at 40-100° C. by hydrothermal extraction for 1-4 h, separating the solid and the liquid, and collecting the solution to obtain a fish scale collagen extract; 4) The fish scale collagen extract collected in step 3) is precooled and then freeze-dried to obtain a fish scale collagen solid product.

2. The low-cost high-molecular-weight fish scale collagen extraction method according to claim 1, characterized in that: In the step 1), the frozen storage temperature of the packaged fish scales is -80°C to -20°C.

3. The low-cost high-molecular-weight fish scale collagen extraction method according to claim 1, characterized in that: In the step 1), the thawing and re-thawing temperature is 25-45° C., the time is 0.5 h to 3 h, and the number of repetitions of frozen storage-thawing and re-thawing is 1 to 3 times.

4. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 1, 2 or 3, characterized in that: In the step 2), the salt solution is any one of a sodium chloride solution and a sodium carbonate solution, the mass fraction of the salt solution is 5% to 20%, and the soaking time is 0.5 to 2 hours.

5. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 4, characterized in that: In the step 2), the fish scales are dried at a temperature of 20° C. to 65° C. for 3 to 24 hours.

6. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 5, characterized in that: In the step 3), the hydrothermal extraction method of fish scale collagen is a gradient hydrothermal extraction process, the gradient temperature range is 40-70°C, 70-90°C and 90-100°C, and the gradient temperature change is gradient heating or gradient cooling.

7. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 1, 2, 3, 5 or 6, characterized in that: In the step 3), the solid-liquid ratio of fish scale to water is 1:5-1:10, the extraction time at 40-70°C is 0.5-2h, the extraction time at 70-90°C is 0.5-1h, and the extraction time at 90-100°C is 0.5-2h.

8. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 7, characterized in that: In the step 3), the solid-liquid separation method is filtration or centrifugation, and the fish scale collagen extract is collected by segmented collection or cumulative collection.

9. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 8, characterized in that: In the step 4), the fish scale collagen extract is dried by any one of vacuum freeze drying, spray freeze drying, gel filtration drying and vacuum drying.

10. The low-cost method for extracting high-molecular-weight fish scale collagen according to claim 1 or 2 or 3 or 5 or 6 or 8 or 9, characterized in that: The fish scale collagen solid product obtained by the method is in the form of any one of powder, granules and sponge.

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