Cattlehide preservation method for improving extraction efficiency of collagen in cattlehide

Abstract

The invention discloses a cow leather preservation method for improving the extraction efficiency of collagen in cow leather, and belongs to the technical field of collagen preservation. The cattle hide with fat and muscle removed is placed in a mixed aqueous solution containing an enzymolysis regulator and a depilation enzyme preparation for enzymolysis depilation treatment, the cattle hide with depilation is placed in an antiseptic solution to be soaked, and cryopreservation is conducted at the cryopreservation temperature ranging from-5 DEG C to-20 DEG C after soaking; the enzymolysis regulator is prepared from mercaptoethanol, cysteine, calcium citrate and sodium dodecyl sulfate; the unhairing enzyme preparation consists of keratinase, heparinase, lipase and neutral amylase; the anti-corrosion solution is composed of glutaraldehyde, hydrochloric acid, zinc sulfate and water. When the method is adopted to cryopreserve the cow leather, the cryopreservation temperature can be reduced in the presence of the preservative solution, the loss of the cryopreserved collagen at the temperature is small, the cow leather can be preserved for about 6 months, and the application effect is good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of collagen preservation, and particularly relates to a method for preserving cowhide to improve the extraction efficiency of collagen in the cowhide. BACKGROUND

[0002] In the production of beef, a large amount of by-products is generated. Cowhide accounts for about 12% of the live body weight before slaughter, and is used to prepare food and leather. In the leather production process, about 70% is low-value by-products or discarded as waste, and the comprehensive utilization rate is low. The protein content in cowhide accounts for 30-35%, of which about 85% is collagen, and the content is much higher than the sum of other proteins. At present, the utilization of protein and collagen in cowhide is still very limited, which causes great waste. Cowhide is mostly used as raw material for food industry and leather industry, and has the advantages of low environmental pollution, high resource utilization rate and wide commodity coverage. In order to make cowhide better play its commodity value, timely and good processing is necessary. After the hide is peeled off, if it is placed in an environment with high temperature, the hide will rot and deteriorate due to the action of bacteria, which will cause protein decomposition and reduce the content of collagen. The commonly used preservation methods at present include salt curing method, drying preservation method, acid immersion preservation method and freezing preservation method. The salt curing method uses a large amount of salt, which will destroy the activity of collagen and affect the subsequent separation and extraction; the acid immersion preservation method will cause abnormal breakage of collagen molecular peptide bond, affecting the activity of collagen. The drying preservation method is generally natural air drying, which is time-consuming and may cause collagen gelation. The water in the skin layer is difficult to evaporate, and bacteria may grow in the inner layer.

[0003] Low-temperature preservation can reduce cell oxygen consumption, maintain cell viability, and thus prolong the preservation time. Low-temperature preservation can be generally divided into the following temperature ranges: cold storage (2-8℃), freezing storage (-20-0℃), deep low-temperature freezing storage (-80--20℃) and liquid nitrogen storage (-196℃), and deep low-temperature freezing storage is mostly used. However, through research, it is found that the lower the freezing temperature and the longer the freezing time, the greater the loss of collagen in the cowhide during enzymolysis.

[0004] Unhairing is an essential step in the processing of bovine hide. Traditional hot water unhairing is widely used due to its simple operation and low cost, but it also has problems such as environmental pollution, residual hair roots, and cross-contamination, which affect the subsequent processing of bovine hide and the quality of the products. In recent years, enzyme unhairing has made significant progress as a green and environmentally friendly technology. It can effectively reduce environmental pollution and improve unhairing efficiency, so it has been applied to some extent in the unhairing of edible bovine hide. Enzyme unhairing uses enzymes to hydrolyze the mucin around the hair roots, weakening the connection between the hair and the skin, thereby promoting the shedding of the hair. Although enzyme unhairing has good unhairing effects, it still has problems such as long processing time and limited enzyme activity, which limit its further promotion. In addition, the hair is not completely removed, and in order to achieve complete unhairing and loosen the collagen fibers, a certain amount of sulfide is often used after enzyme unhairing to supplement the unhairing. SUMMARY

[0005] In view of the problems of large collagen protein loss, short storage time, and low extraction rate of collagen protein in the later stage in the existing technology during the storage of bovine hide, the present application provides a bovine hide storage method for improving the extraction efficiency of collagen protein in bovine hide. The enzyme hydrolysis process is improved, and the improved preservative is used for freezing preservation, thereby prolonging the storage period of bovine hide. The extraction rate of collagen protein remains at a high level after six months of low-temperature storage.

[0006] The present application is realized by the following technical solutions: The present application provides a bovine hide storage method for improving the extraction efficiency of collagen protein in bovine hide. The bovine hide after removing fat and muscle is placed in a mixed aqueous solution containing enzyme hydrolysis regulator and unhairing enzyme preparation for enzyme hydrolysis unhairing treatment. The unhairing bovine hide is soaked in a preservative solution, and then frozen for storage. The freezing storage temperature is-5~-20℃. The enzyme hydrolysis regulator is composed of the following raw materials by weight: mercaptoethanol 3~10 parts, cysteine 5~10 parts, calcium citrate 6~8 parts, and sodium dodecyl sulfonate 3~5 parts. The unhairing enzyme preparation is composed of keratinase, heparinase, lipase, and neutral protease. The preservative solution is composed of the following raw materials by weight: glutaraldehyde 5~10 parts, hydrochloric acid 3~6 parts, zinc sulfate 5~10 parts, and water 60~100 parts.

[0007] Further, the specific enzyme hydrolysis unhairing method is as follows: the bovine hide after removing fat and muscle is placed in a rotating drum containing circulating liquid for enzyme hydrolysis unhairing treatment. The circulating liquid is a mixed aqueous solution containing enzyme hydrolysis regulator and unhairing enzyme preparation.

[0008] Further, the temperature of the enzyme hydrolysis unhairing treatment is 30~40℃, and the time is 2~4h.

[0009] Further, the mass content of the enzyme hydrolysis regulator in the mixed aqueous solution is 2.0-5.0%, and the mass content of the depilating enzyme preparation is 1.0-2.0%.

[0010] Further, the mass ratio of keratinase, heparinase, lipase and neutral amylase in the depilating enzyme preparation is 3-5:1-3:1-2:1.

[0011] Further, the soaking time of the depilated cowhide in the preservative solution is 10-50 min, and the soaking temperature is 5-15℃.

[0012] Further, the soaking time of the depilated cowhide in the preservative solution is 20-30 min, and the soaking temperature is 5-10℃.

[0013] Further, the freezing preservation temperature is-10--15℃.

[0014] Further, the mass concentration of hydrochloric acid in the preservative solution is 20-30%.

[0015] Further, when the collagen is extracted, the frozen preserved cowhide is directly immersed in water after thawing, and the soaking is performed for 30-60 min and 2-3 times.

[0016] Compared with the prior art, the present application has the following beneficial effects: (1) The depilating enzyme preparation in the present application is composed of keratinase, heparinase, lipase and neutral protease, the keratinase and the neutral protease decompose keratin and mucin (protein polysaccharide) between the cowhide and the cowhair, the lipase can decompose the fat around the hair follicle, so that the keratinase and the neutral protease can penetrate into the hair follicle and decompose the residual hair in the hair follicle, and the heparinase is an alpha exo-enzyme, which is used in combination with the keratinase to achieve better depilation effect.

[0017] (2) The enzyme hydrolysis regulator is composed of mercaptoethanol, cysteine, calcium citrate and sodium dodecyl sulfonate, the mercaptoethanol can shorten the enzyme hydrolysis time, and replace the use of sulfide (sodium sulfide, which can damage collagen and cause collagen loss during the depilation process) in the traditional depilation method, the sodium dodecyl sulfonate can be used as a penetration enhancer to accelerate the enzyme hydrolysis sequence, and the cysteine and the calcium citrate form a complex, which can act as an enzyme activator and combine with the collagen on the surface of the cowhide to reduce the loss of the surface collagen caused by enzyme hydrolysis.

[0018] (3) After the hair is removed, the cowhide is soaked in the preservative solution, the preservative solution is composed of glutaraldehyde, hydrochloric acid, zinc sulfate and water, the glutaraldehyde has strong acid resistance, can produce weak cross-linking with the collagen in the cowhide, the hydrochloric acid can acidify and expand the surface of the cowhide, the glutaraldehyde can cross-link with more collagen, the zinc sulfate plays an antibacterial role, the glutaraldehyde, the hydrochloric acid, the zinc sulfate and the surface collagen form a protective film on the surface of the cowhide, the film will dissolve into water in the thawing and soaking process of the cowhide, which can slow down the damage of the frozen environment to the collagen, and the film itself will not cause the loss of the collagen.

[0019] (4) The enzyme solution adjusting agent and the depilation enzyme preparation can quickly depilate the cowhide, and the collagen is less damaged in the depilation process; in the presence of the preservative solution, the freezing temperature can be reduced, the collagen is less damaged in the freezing process at the temperature, the cowhide can be preserved for about 6 months, and the application effect is good. DETAILED DESCRIPTION

[0020] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples are not specified, and the methods are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. The reagents or raw materials used in the application can be purchased through a conventional route, and the reagents or raw materials used in the application are used according to the conventional manner in the art or according to the product instructions, unless otherwise specified.

[0022] In the following examples and comparative examples, the alkaline protease and the neutral protease are conventional commercially available products.

[0023] The above only describes the preferred embodiments of the application and is not used to limit the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

[0024] The keratinase, heparinase, lipase and neutral amylase in the application are commercially available products; the "parts" in the following examples are weight parts.

[0025] Example 1 The composition of the enzyme solution adjusting agent: mercaptoethanol 5 parts, cysteine 8 parts, calcium citrate 6 parts, sodium dodecyl sulfonate 3 parts; The depilation enzyme preparation is composed of keratinase, heparinase, lipase and neutral protease, and the mass ratio of the keratinase, heparinase, lipase and neutral protease is 3:2:1:1; The composition of the preservative solution: glutaraldehyde 8 parts, hydrochloric acid (25%) 4 parts, zinc sulfate 5 parts, water 83 parts; (1) The enzymatic regulation agent and the depilation enzyme preparation are added to water to obtain a mixed aqueous solution, and the mass content of the enzymatic regulation agent in the mixed solution is 3.0%, and the mass content of the depilation enzyme preparation is 1.5%; (2) The fat and muscle removed cowhide is placed in a rotating drum containing circulating liquid for enzymatic depilation treatment, and the circulating liquid is the mixed aqueous solution in step (1), the temperature of the enzymatic depilation treatment is 35℃, the time is 3h, and after the enzymatic depilation treatment, the cowhide is repeatedly washed with clean water; (3) The cowhide after the depilation treatment in step (2) is soaked in the preservative solution, the soaking time is 25min, the soaking temperature is 10℃, and after the soaking, the cowhide is stored in a freezer, the freezing storage temperature is set to-10℃, and after 15 days, 30 days, two months, three months and six months of storage, part of the cowhide is taken out, and collagen is extracted.

[0026] Example 2 The composition of the enzymatic regulation agent: mercaptoethanol 8 parts, cysteine 5 parts, calcium citrate 6 parts, and sodium dodecyl sulfonate 5 parts; The depilation enzyme preparation is composed of keratinase, heparinase, lipase and neutral protease, and the mass ratio of the keratinase, heparinase, lipase and neutral protease is 3:2:1:1; The composition of the preservative solution: glutaraldehyde 8 parts, hydrochloric acid (25%) 4 parts, zinc sulfate 5 parts, water 83 parts; (1) The enzymatic regulation agent and the depilation enzyme preparation are added to water to obtain a mixed aqueous solution, and the mass content of the enzymatic regulation agent in the mixed solution is 3.0%, and the mass content of the depilation enzyme preparation is 1.5%; (2) The fat and muscle removed cowhide is placed in a rotating drum containing circulating liquid for enzymatic depilation treatment, and the circulating liquid is the mixed aqueous solution in step (1), the temperature of the enzymatic depilation treatment is 35℃, the time is 3h, and after the enzymatic depilation treatment, the cowhide is repeatedly washed with clean water; (3) The cowhide after the depilation treatment in step (2) is soaked in the preservative solution, the soaking time is 25min, the soaking temperature is 10℃, and after the soaking, the cowhide is stored in a freezer, the freezing storage temperature is set to-15℃, and after 15 days, 30 days, two months, three months and six months of storage, part of the cowhide is taken out, and collagen is extracted.

[0027] Example 3 The composition of the enzymatic regulation agent: mercaptoethanol 5 parts, cysteine 8 parts, calcium citrate 6 parts, and sodium dodecyl sulfonate 3 parts; Enzyme preparation for depilation: composed of keratinase, heparinase, lipase and neutral protease, the mass ratio of keratinase, heparinase, lipase and neutral protease is 3:2:1:1; Composition of preservative solution: glutaraldehyde 8 parts, hydrochloric acid (25%) 4 parts, zinc sulfate 5 parts, water 83 parts; (1) The enzyme regulation agent and the enzyme preparation for depilation are added to water to obtain a mixed aqueous solution, the mass content of the enzyme regulation agent in the mixed solution is 3.0%, and the mass content of the enzyme preparation for depilation is 1.5%; (2) The fat and muscle removed cowhide is placed in a rotating drum containing circulating liquid for enzymatic depilation treatment, the circulating liquid is the mixed aqueous solution in step (1), the temperature of the enzymatic depilation treatment is 35℃, the time is 3h, and after the enzymatic depilation, the cowhide is repeatedly washed with clean water; (3) The cowhide after the depilation treatment in step (2) is soaked in a preservative solution, the soaking time is 25min, the soaking temperature is 10℃, after soaking, the cowhide is stored in a frozen state, the freezing storage temperature is -5℃, after 15 days, 30 days, two months, three months and six months of storage, part of the cowhide is taken out, and collagen is extracted.

[0028] Example 4 Composition of enzyme regulation agent: mercaptoethanol 8 parts, cysteine 5 parts, calcium citrate 6 parts, sodium dodecyl sulfonate 5 parts; Enzyme preparation for depilation: composed of keratinase, heparinase, lipase and neutral protease, the mass ratio of keratinase, heparinase, lipase and neutral protease is 3:2:1:1; Composition of preservative solution: glutaraldehyde 8 parts, hydrochloric acid (25%) 4 parts, zinc sulfate 5 parts, water 83 parts; (1) The enzyme regulation agent and the enzyme preparation for depilation are added to water to obtain a mixed aqueous solution, the mass content of the enzyme regulation agent in the mixed solution is 3.0%, and the mass content of the enzyme preparation for depilation is 1.5%; (2) The fat and muscle removed cowhide is placed in a rotating drum containing circulating liquid for enzymatic depilation treatment, the circulating liquid is the mixed aqueous solution in step (1), the temperature of the enzymatic depilation treatment is 35℃, the time is 3h, and after the enzymatic depilation, the cowhide is repeatedly washed with clean water; (3) The cowhide after the depilation treatment in step (2) is soaked in a preservative solution, the soaking time is 25min, the soaking temperature is 15℃, after soaking, the cowhide is stored in a frozen state, the freezing storage temperature is -20℃, after 15 days, 30 days, two months, three months and six months of storage, part of the cowhide is taken out, and collagen is extracted.

[0029] Comparative Example 1 Compared with Example 1, the hide in Comparative Example 1 was directly stored at -10℃ after step (2) of unhairing, and the remaining steps were the same as Example 1.

[0030] Comparative Example 2 Compared with Example 1, the hide in Comparative Example 2 was directly stored at -40℃ after step (2) of unhairing.

[0031] Comparative Example 3 Compared with Example 1, the hide in Comparative Example 3 was stored after step (3) of soaking, and the freezing temperature was set to -40℃.

[0032] Comparative Example 4 Compared with Example 1, no cysteine and calcium citrate were added in the enzyme regulation agent in Comparative Example 4, and the remaining steps were the same as Example 1; in this comparative example, the hair follicle pores were larger and the surface was rougher after unhairing, which was caused by partial collagen enzymolysis.

[0033] Comparative Example 5 Compared with Example 1, no glutaraldehyde was added in the preservative solution in Comparative Example 5, and the remaining steps were the same as Example 1.

[0034] Comparative Example 6 Compared with Example 1, no hydrochloric acid was added in the preservative solution in Comparative Example 6, and the remaining steps were the same as Example 1.

[0035] Comparative Example 7 Compared with Example 1, the soaking temperature of the hide in the preservative solution in step (3) in Comparative Example 7 was 25℃, and the remaining steps were the same as Example 1.

[0036] Performance test 1. Extraction of collagen (1) The frozen hide of the examples and comparative examples was thawed at room temperature, and the frozen hide was directly immersed in water after thawing, and soaked for 3 times, each time for 30 min; (2) The hide was cut into pieces, and immersed in a 5% Na2CO3 solution and a 5% NaCl solution for 24 h respectively to obtain a hide with defatted and deproteinized, and then washed with water; (3) The defatted and deproteinized hide was added to 3 times the volume of water, homogenized, and 240 U / g (hide mass) of pepsin was added, and ultrasonic (150 W) assisted enzymolysis was carried out for 1 h, and after stopping the ultrasonic, the enzymolysis was continued for 6 h, the enzymolysis temperature was 37±2℃, and after the enzymolysis was completed, centrifugation (10000 r / min, 15 min) was performed for separation, and the supernatant was taken; (4) The supernatant of step (3) is added with sodium chloride solution for salting-out, and after salting-out, centrifugation (10000 r / min, 15 min) is performed for separation, and the precipitate is reserved; (5) The precipitate of step (4) is dissolved in a citric acid solution (0.5 mol / L), and the solution is dialyzed in the citric acid solution and distilled water for 24 h respectively, and the dialysis external solution is replaced every 8 h; (6) Freeze-drying is performed to obtain collagen, and the extraction rate of collagen is calculated by using the following formula The extraction rate of bovine collagen = (the mass of collagen after freeze-drying / the dry weight of bovine skin) * 100%; 2. Extraction rate of bovine collagen with different storage times The bovine skin stored by freezing in examples 1-4 and comparative examples 1-7 is taken out at the 15th day, 30th day, two months, three months and six months of storage, and the collagen is extracted by using the above method, and the extraction rate is calculated, and the results are shown in Table 1. Table 1: Results of extraction rate of bovine collagen with different storage times As shown in Table 1, the technical effects of example 1 and example 2 are equivalent, which indicates that when stored at-10℃ and-15℃, the collagen will not be greatly affected, and with the prolongation of the storage time, the extraction rate of collagen will not decrease obviously within six months. The storage temperature of example 3 is-5℃, and the extraction rate of collagen will decrease obviously with the prolongation of time, which is caused by the lower storage temperature, and it is indicated that after two months, the bovine skin will be obviously corrupted, and the collagen will be greatly lost, and therefore, the storage time is preferably within two months. The storage temperature of example 4 is-20℃, and at this temperature, the collagen will be lost to a certain extent (caused by low temperature), but with the prolongation of the storage time, the attenuation trend is not obvious.

[0037] The collagen extracted by the preservation method of Comparative Example 4 was lower than that of Example 1, indicating that cysteine and calcium citrate in the enzyme regulation agent can protect the collagen of the bovine skin epidermis from being enzymatically degraded, and reduce the loss of collagen during the depilation process. The collagen extracted by the preservation method of Comparative Example 1 was lower than that of Example 1, glutaraldehyde, hydrochloric acid, zinc sulfate and the surface collagen formed a protective film on the surface of the bovine skin, and zinc sulfate had antibacterial activity, which could prevent the loss of collagen in the bovine skin at a relatively low preservation temperature. The extraction rates of collagen in Comparative Example 2 and Comparative Example 3 were roughly equivalent, indicating that the loss of collagen in the bovine skin would be relatively large and the extraction would be difficult under the condition of freezing preservation at -40℃, and the protective film formed by glutaraldehyde, hydrochloric acid, zinc sulfate and the surface collagen on the surface of the bovine skin could not play its role at this temperature. In Comparative Example 5, glutaraldehyde was absent in the preservative solution, and in Comparative Example 6, glutaraldehyde was absent in the preservative solution. Hydrochloric acid can acidify and swell the surface of the bovine skin, and glutaraldehyde can crosslink more collagen. The absence of any one component will reduce the protective effect of the preservative solution. Comparative Example 7 confirmed that the soaking temperature of the preservative solution also had a certain influence on its protective effect.

Claims

1. A method for preserving bovine hide to improve the efficiency of collagen extraction from the bovine hide, the method comprising: The fat and muscle removed cowhide is placed in a mixed aqueous solution containing an enzymatic regulation agent and a depilation enzyme preparation for enzymatic depilation treatment, the depilated cowhide is soaked in a preservative solution, and the soaked cowhide is stored in a frozen state at a temperature of-5~-20℃. ​ The enzymatic regulation agent is composed of the following raw materials by weight: mercaptoethanol 3~10 parts, cysteine 5~10 parts, calcium citrate 6~8 parts, and sodium dodecyl sulfonate 3~5 parts. The depilation enzyme preparation is composed of keratinase, heparinase, lipase, and neutral amylase. The preservative solution is composed of the following raw materials by weight: glutaraldehyde 5~10 parts, hydrochloric acid 3~6 parts, zinc sulfate 5~10 parts, and water 60~100 parts.

2. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized by, The specific enzymatic depilation method is as follows: the fat and muscle removed cowhide is placed in a rotating drum containing a circulating liquid for enzymatic depilation treatment, and the circulating liquid is a mixed aqueous solution containing an enzymatic regulation agent and a depilation enzyme preparation.

3. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized by, The temperature of the enzymatic depilation treatment is 30~40℃, and the time is 2~4h.

4. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized by, The mass content of the enzymatic regulation agent in the mixed aqueous solution is 2.0~5.0%, and the mass content of the depilation enzyme preparation is 1.0~2.0%.

5. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized in that, The mass ratio of keratinase, heparinase, lipase, and neutral amylase in the depilation enzyme preparation is 3~5:1~3:1~2:

1.

6. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized by, The depilated cowhide is soaked in the preservative solution for 10~50min at a temperature of 5~15℃.

7. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 6, characterized by, The depilated cowhide is soaked in the preservative solution for 20~30min at a temperature of 5~10℃.

8. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized by, The freezing storage temperature is-10~-15℃.

9. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized by, The mass concentration of hydrochloric acid in the preservative solution is 20~30%.

10. The hide preserving method for improving the efficiency of collagen extraction from bovine hide according to claim 1, characterized in that, When extracting collagen, the frozen and stored cowhide is thawed and directly immersed in water, soaked for 30~60min, and soaked for 2~3 times.

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