A method for removing the fishy smell of cow hooves based on the Maillard reaction

Abstract

The present invention discloses a preparation method for removing fishy smell and enhancing fragrance of bovine by-products of bovine hoof. The method comprises the following steps: (1) dividing and rinsing fresh bovine hoof samples to obtain clean bovine hoof samples; (2) enzymatically hydrolyzing the bovine hoof samples obtained in step (2) to obtain an enzymatic hydrolysate; (3) adding reducing sugar to the enzymatic hydrolysate for saccharification treatment; (4) cooking the bovine hoof obtained in step (3) to obtain the bovine hoof with removed fishy smell. This method can effectively improve the generation of fishy smell of bovine hoof caused by heat treatment, thereby achieving the removal of fishy smell and enhancement of fragrance, improving the comprehensive utilization rate of bovine by-products, and can be applied to the development and application of high-value products of bovine by-products.

Description

Technical Field

[0001] The present invention relates to the field of improving the flavor of meat products, and particularly to a method for removing the fishy smell of cow hooves by Maillard reaction. Background Art

[0002] In recent years, the cattle industry has developed into the leading industry of animal husbandry, and the per capita consumption of beef in China has also been increasing year by year. According to the data of the beef supply and demand balance sheet monitored by the United States Department of Agriculture (USDA), the beef production in China in 2022 was 6.92 million tons, a year-on-year increase of 1.32%; the domestic consumption of beef was 10.149 million tons, a year-on-year increase of 3.95%. Relevant research shows that cattle by-products account for about 56% of the live weight of cattle. Then, with the growth of beef consumption, a large amount of cattle by-products need to be solved and processed. In daily life, because cattle by-products are rich in a large amount of protein, amino acids, minerals and bioactive peptides, they are also often used as edible foods, such as blood sausage, beef tripe, beef bone soup and other related products. However, most of them are directly listed in the form of raw materials, and related high-value products are relatively rare. The food processing industry pays insufficient attention to such by-products, and the by-products themselves have a strong fishy smell. Except for the vast majority of by-products sold directly on the market, they are directly discarded, which is also a major problem in the processing and utilization of cattle by-products.

[0003] At present, the global demand for high-quality protein is increasing day by day. As a typical high-protein food, the demand for meat is also increasing. At the same time, on the one hand, more breeding land is needed to raise livestock and poultry, and on the other hand, the large amount of discarded by-products will cause serious environmental pollution, which is undoubtedly a huge challenge to the earth's environment. Plant alternative proteins and cell-cultured meat are also excellent countermeasures to solve this problem. In addition, attention should be paid to the processing and utilization of by-products, improve the comprehensive utilization rate, and improve their flavor characteristics, so that they can be more acceptable to consumers, laying a solid foundation for the high-value utilization and product development of cattle by-products.

[0004] The Maillard reaction is one of the chemical methods for removing fishy smell. The principle of removing fishy smell is that on the one hand, the reaction products react chemically with fishy smell substances to generate non-fishy components or volatile substances with higher thresholds, so as to remove fishy smell; on the other hand, the generated odor will cover the fishy smell. At present, the Maillard method is often used for removing fishy smell and enhancing fragrance in food. Existing research includes CN102726697B "A Preparation Method of a Water-Soluble Maillard Reaction-Type Food Flavor", which uses Maillard reaction products as the base material and prepares a Maillard reaction-type food flavor through relevant process treatments, expanding the application range of Maillard reaction-type food flavors in the food field. CN103815222A "A Method for Preparing Durian Jam by Maillard Reaction" uses durian, coconut powder, etc. as raw materials, conducts Maillard reaction, and prepares durian jam with toffee flavor, covering the smell of durian itself and realizing the popular consumption of durian.

[0005] As one of the by-products of cattle, cattle hooves are composed of cowhide and tendons. They are rich in nutrients, containing a large amount of collagen and elastin. Collagen can improve the elasticity and luster of the skin, delay skin aging. In addition, for the elderly, it has a certain effect of alleviating osteoporosis. However, it has an unacceptable fishy smell problem for consumers. Improving and studying its flavor can not only realize the economic value of cattle hooves, develop corresponding high-value products, but also solve the environmental pollution and resource waste caused by related enterprises disposing of them. At present, however, there is no reported research method for removing the fishy smell of cattle hooves by enzymatic hydrolysis combined with saccharification. The present invention studies this problem and hopes to solve the problem of its poor flavor through the combined treatment of enzymatic hydrolysis and saccharification. Summary of the Invention

[0006] In view of the problems raised in the above background technology, the object of the present invention is to provide a research method for removing the fishy smell of cattle hooves based on the combined treatment of enzymatic hydrolysis and Maillard reaction.

[0007] To achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0008] A method for removing the fishy smell of cattle hooves based on Maillard reaction, the method comprising the following steps:

[0009] (1) Divide and rinse the fresh cattle hoof samples to obtain clean cattle hoof samples;

[0010] (2) Enzymatically hydrolyze the cattle hoof samples obtained in step (2) to obtain an enzymatic hydrolysate;

[0011] (3) Add reducing sugar to the enzymatic hydrolysate for saccharification treatment;

[0012] (4) Cook the cattle hooves obtained in step (3) (i.e., carry out Maillard reaction) to obtain the cattle hooves with the fishy smell removed.

[0013] Further, the rinsing in step (1) is rinsing with running water for 15 minutes to ensure the cleaning effect.

[0014] Further, the enzyme used in step (2) is selected from one or more of neutral protease, bromelain, papain, and flavor protease.

[0015] Further, the addition amount of the enzyme in step (2) does not exceed 0.6% of the mass of the cattle hoof samples.

[0016] Further, the enzymatic hydrolysis in step (2) is to dissolve the enzyme in pure water to obtain an enzyme solution, and then enzymatically hydrolyze the cattle hoof samples.

[0017] Furthermore, there is no special limitation on the material-liquid ratio of the beef hoof sample to the enzyme solution in step (2). In a particular embodiment, the material-liquid ratio is 1:2 to fully immerse the beef hoof sample.

[0018] Furthermore, in step (2), the enzymatic hydrolysis temperature is 40-60°C and the enzymatic hydrolysis time is 10-40 min. Preferably, the enzymatic hydrolysis time is 10-30 min. More preferably, the enzymatic hydrolysis time is 40 min, 30 min, 20 min or 10 min.

[0019] The above enzymatic hydrolysis treatment conditions can achieve excellent enzymatic hydrolysis effects and are beneficial to the deodorization effect of beef hoof.

[0020] Furthermore, the reducing sugar in step (3) is selected from one or more of glucose, galactose, fructose, and maltose.

[0021] Furthermore, the addition amount of the reducing sugar in step (3) does not exceed 9% of the mass of the beef hoof sample.

[0022] The above saccharification treatment conditions can achieve excellent Maillard reaction effects and are beneficial to the deodorization effect of beef hoof.

[0023] Furthermore, the cooking in step (4) is boiling. Preferably, the boiling temperature is 90°C and the boiling time is 90 min.

[0024] Advantages of the present invention:

[0025] The preparation method of the present invention uses enzymatic hydrolysis combined with Maillard reaction to improve the fishy smell of beef hoof, with obvious flavor improvement effect, and can effectively reduce the contents of fishy smell components such as nonanal, undecane, and dodecane. The edible materials are all food-grade, and the preparation process is simple and the process is mild.

[0026] The beef hoof product obtained by the present invention can be used for the research and development of high-value products and applied in the food industry. The enzymatic hydrolysate can also be applied to the research and development of essence and flavor.

[0027] The preparation process of the present invention is simple to operate, low in cost, and suitable for industrial production. Description of the Drawings

[0028] Figure 1 Influence of protease types on the deodorization effect of beef hoof;

[0029] Figure 2 Influence of enzymatic hydrolysis time on the deodorization effect of beef hoof;

[0030] Figure 3 Influence of enzyme addition amount on the deodorization effect of beef hoof;

[0031] Figure 4 Influence of sugar types on the deodorization effect of beef hoof;

[0032] Figure 5 Effect of sugar addition amount on the removal effect of the fishy smell of ox hooves;

[0033] Figure 6 Comparison results of sensory evaluation between Example 1 and Comparative Examples 1-3, where Figure 6 A is the score of fishy smell after treatment, Figure 6 B is the score of acceptance degree after treatment;

[0034] Figure 7 Comparison results of electronic nose detection between Example 1 and Comparative Examples 1-3, where Figure 7 A is the result of electronic nose detection of tendon, Figure 7 B is the result of electronic nose detection of hide;

[0035] Figure 8 Comparison results of GC-MS detection of tendon between Example 1 and Comparative Examples 1-3;

[0036] Figure 9 Comparison results of GC-MS detection of hide between Example 1 and Comparative Examples 1-3. Detailed implementation mode

[0037] The present invention will be further explained below in conjunction with examples, but the examples do not limit the present invention in any form.

[0038] Since the ox hoof includes tendon, hide and a very small amount of muscle, and the muscle has no fishy smell, therefore, the following examples conduct experiments on the tendon and hide in the ox hoof.

[0039] Example 1

[0040] Take 500 g each of tendon and hide, place them in a cooking bag at a material-liquid ratio of 1:2, and carry out enzymatic hydrolysis at 50 °C. The tendon is hydrolyzed with 0.6% papain for 30 min, and the hide is hydrolyzed with 0.4% neutral protease for 10 min. After the enzymatic hydrolysis is completed, 9% maltose is added to the cooking bags of tendon and hide, and then transferred to a constant temperature water bath at 90 °C and boiled for 90 min to inactivate the enzyme in the enzymatic hydrolysis process. The obtained product is a product for removing fishy smell by combined enzymatic hydrolysis and saccharification treatment.

[0041] Comparative Example 1

[0042] Take 500 g each of tendon and hide, place them in a cooking bag at a material-liquid ratio of 1:2, and carry out water bath at 50 °C in a constant temperature water bath without adding protease. After the tendon is bathed for 30 min and the hide is bathed for 10 min, the cooking bag is transferred to a constant temperature water bath at 90 °C and boiled for 90 min. The obtained product is an untreated product for removing fishy smell.

[0043] Comparative Example 2

[0044] Take 500 g of each of beef tendon and cowhide, place them in a cooking bag at a material-liquid ratio of 1:2, and carry out enzymatic hydrolysis at 50 °C. For beef tendon, use 0.6% papain for enzymatic hydrolysis for 30 min, and for cowhide, use 0.4% neutral protease for enzymatic hydrolysis for 10 min. After the enzymatic hydrolysis is completed, transfer it to a constant temperature water bath at 90 °C and boil for 90 min to inactivate the enzyme in the enzymatic hydrolysis process. The resulting product is a product for removing fishy smell only by enzymatic hydrolysis.

[0045] Comparative Example 3

[0046] Take 500 g of each of beef tendon and cowhide, place them in a cooking bag at a material-liquid ratio of 1:2, and carry out a water bath at 50 °C in a constant temperature water bath without adding protease. After the beef tendon is water-bathed for 30 min and the cowhide is water-bathed for 10 min, add 9% maltose to the cooking bags of beef tendon and cowhide, and then transfer the cooking bags to a constant temperature water bath at 90 °C and boil for 90 min. The resulting product is a product for removing fishy smell only by saccharification treatment.

[0047] Performance test of experimental examples

[0048] Index detection method

[0049] 1. Sensory evaluation

[0050] Select a number of postgraduate students in the laboratory of Nanjing Agricultural Food Science and Technology College for sensory evaluation training. Weigh 2.0 g of the obtained sample and place it in a headspace vial, and score the fishy smell degree on a ten-point scale. 0 points represent no smell, and 10 points represent the strongest smell. The specific smell ratings are as follows:

[0051]

[0052] 2. Electronic nose test

[0053] Place 2 g of samples with different cooking times in a 20 mL headspace vial, and carry out incubation treatment in a water bath at 45 °C for 20 min for each sample. The parameters of the electronic nose are set as follows: the sample interval time is 1 s, the cleaning time is 120 s, the zeroing time is 10 s, the sample preparation time is 5 s, the detection time is 120 s, the carrier gas flow rate is 300 mL / min, and the injection volume is 300 mL / min.

[0054] 3. GC-MS detection

[0055] To extract volatile compounds, 2 g of the sample was taken and placed at the bottom of a headspace vial, and 1.5 μL of 0.11 μg / μL ortho-dichlorobenzene was added. The sample was incubated in a 20-ml headspace vial, and the SPME fiber (50 / 30 μm DVB / CAR / PDMS) was exposed to the headspace of the vial containing the sample extract, allowing the volatile compounds to be absorbed in the SPME fiber for 5 min. Then the volatile compounds were desorbed in the GC injection port at 240 °C for 5 minutes.

[0056] GC conditions: HP-5MS column (30 m × 0.25 mm, 0.25 μm); injection port temperature 240 °C; temperature program: initial column temperature 38 °C, held for 13 min, increased to 100 °C at 3 °C / min, held for 5 min, then increased to 150 °C at 4 °C / min, and then increased to 240 °C at 10 °C / min; carrier gas (He) purity not less than 99.999%, flow rate 1.0 mL / min.

[0057] MS conditions: electron ionization source: transfer line temperature 240 °C; ion source temperature 240 °C; mass scan range m / z 35 - 450.

[0058] Results of single-factor, orthogonal experiments and index detections

[0059] (1) Screening of proteases for enzymatic hydrolysis

[0060] Four proteases, namely neutral protease, bromelain, papain, and flavor protease, were selected as pre-screening enzymes. 500 g of beef tendon and 500 g of cowhide were taken, with a material-liquid ratio of 1:2 (m / v) as the substrate, 0.2% of the enzyme was added, and the enzymatic hydrolysis was carried out at 50 °C for 30 min. 3% of glucose was added, and it was heated in a water bath at 90 °C for 90 min.

[0061] The control group was to take 500 g of beef tendon and 500 g of cowhide, with a material-liquid ratio of 1:2 (m / v) as the substrate, no enzyme was added, it was water-bathed at 50 °C for 30 min, 3% of glucose was added, and it was heated in a water bath at 90 °C for 90 min.

[0062] Taking the fishy smell score and acceptance score of sensory evaluation as indicators, the fishy smell removal effects of the four proteases are as Figure 1 shown. The treatment results of the four proteases on beef tendon and cowhide are significantly better than those of the control group. For the beef tendon sample, papain has the best fishy smell removal effect, and for cowhide, neutral protease has the best effect.

[0063] (2) Single-factor experiments

[0064] Taking the fishy smell score and acceptance score of sensory evaluation as indicators, for the enzymolysis time (10, 20, 30, 40 min), enzyme addition amount (0%, 0.2%, 0.4%, 0.6%), sugar type (glucose, galactose, fructose, maltose), and sugar addition amount (0%, 3%, 6%, 9%), single-factor experiments were carried out to explore the influence of this factor on the flavor of ox hooves and determine the optimal single-factor conditions for preparing ox hoof products with the best flavor.

[0065] ① Influence of enzymolysis time on the fishy smell removal effect of ox hooves

[0066] Under the test conditions, papain was used for tendons and neutral protease was used for cowhide. The enzyme addition amount was 0.2%, the enzymolysis was carried out at 50 °C for 30 min, the maltose addition amount was 3%, and then the sample was heated in a water bath at 90 °C for 90 min. The influence of different enzymolysis times on the fishy smell removal effect of ox hooves was studied. As Figure 2 shown, for cowhide and tendons, with the increase of enzymolysis time, the fishy smell score showed a decreasing trend and the acceptance score showed an increasing trend; for cowhide, when the enzymolysis time was 20 min, the fishy smell removal effect and acceptance were the highest; for tendons, the fishy smell scores at different enzymolysis times were relatively low and there was no significant difference. The enzymolysis time of 30 min was selected as the best fishy smell removal time for tendons; because the enzymolysis time was short, the hardness might be high and it might be difficult to chew, and too long enzymolysis time would lead to the loss of collagen and might cause problems such as the product not taking shape.

[0067] ② Influence of enzyme addition amount on the fishy smell removal effect of ox hooves

[0068] Under the test conditions, the enzymolysis was carried out at 50 °C. Papain was used for tendons and the enzymolysis time was 30 min, and neutral protease was used for cowhide and the enzymolysis time was 20 min. The maltose addition amount was 3% for both. Then the sample was heated in a water bath at 90 °C for 90 min. The influence of different enzyme addition amounts on the fishy smell of ox hooves was studied. As Figure 3 shown, with the increase of enzyme addition amount, the fishy smell score first decreased and then tended to be flat. The effect was the best when the enzyme addition amount for cowhide was 0.6%, and the best fishy smell removal effect for tendons was at 0.4%; considering the comprehensive acceptance score, the optimal enzyme addition amount for fishy smell removal of tendons was 0.4%, and the optimal enzyme addition amount for fishy smell removal of cowhide was 0.6%.

[0069] ③ Influence of sugar type on the fishy smell removal effect of ox hooves

[0070] The enzymatic hydrolysis was carried out at a test temperature of 50 °C. For beef tendon, papain was used with an enzyme addition amount of 0.4% and an enzymatic hydrolysis time of 30 min. For cowhide, neutral protease was used with an enzyme addition amount of 0.6% and an enzymatic hydrolysis time of 20 min. Then, the samples were heated in a water bath at 90 °C for 90 min. The effects of different sugar types on removing fishy smell from cow hooves were studied. The control group was as follows: for beef tendon, papain was used with an enzyme addition amount of 0.4%, and it was enzymatically hydrolyzed at 50 °C for 30 min, then cooked in a water bath at 90 °C for 90 min without adding sugar; for cowhide, neutral protease was used with an enzyme addition amount of 0.6%, and it was enzymatically hydrolyzed at 50 °C for 20 min, then cooked in a water bath at 90 °C for 90 min without adding sugar; the results of fishy smell removal are as Figure 4 shown. Glucose, galactose, fructose, and maltose all have effects on removing fishy smell from cow hooves. For cowhide, the best sugar type is fructose; for beef tendon, the best sugar type is maltose. Different sugars have different sensitivities to the Maillard reaction, which may result in different effects on removing fishy smell.

[0071] ④ Effect of sugar addition amount on removing fishy smell from cow hooves

[0072] The enzymatic hydrolysis was carried out at a test temperature of 50 °C. For beef tendon, papain was used with an enzyme addition amount of 0.4%, an enzymatic hydrolysis time of 30 min, and the sugar type was maltose; for cowhide, neutral protease was used with an enzyme addition amount of 0.6%, an enzymatic hydrolysis time of 20 min, and the sugar type was fructose. Then, the samples were heated in a water bath at 90 °C for 90 min to obtain the samples. The effects of different sugar addition amounts on removing fishy smell from cow hooves were studied. As Figure 5 shown, for both cowhide and beef tendon, a sugar addition amount of 6% is the best. Different sugar addition amounts may cause different degrees of Maillard reaction, resulting in different effects on flavor improvement.

[0073] According to the results of single-factor experiments, the factors and levels of the orthogonal experiment for enzymatic hydrolysis combined with saccharification to remove fishy smell from beef tendon and cowhide were designed as shown in Tables 1 and 2.

[0074] Table 1 Orthogonal experiment design table for beef tendon

[0075]

[0076]

[0077] Table 2 Orthogonal experiment design table for cowhide

[0078]

[0079] Note: The numbers 1, 2, and 3 in the table are abbreviations, representing the specific experimental quantities of different factors, corresponding to the orthogonal experiment in the following table. (3) Optimize the best fishy smell removal process conditions by orthogonal experiment

[0080] On the basis of single-factor optimization experiments, experiments were designed using the Orthogonal Design Assistant II V3.1. With the enzyme addition amount, enzymolysis time, sugar type, and sugar addition amount as the four variables and the sensory evaluation fishy smell score as the measurement index, a four-factor and three-level experiment was carried out to further optimize the best process for removing fishy smell from tendon and cowhide. The experimental results are shown in Tables 3 and 4.

[0081] Table 3 Results of Orthogonal Experiments on Tendon

[0082]

[0083] Table 4 Results of Orthogonal Experiments on Cowhide

[0084]

[0085]

[0086] Through the analysis of the orthogonal experimental results, the best process for removing fishy smell from tendon was obtained as A 3 B 2 C 3 D 3 , that is, the enzyme addition amount was 0.6%, enzymolysis was carried out for 30 min, and 9% maltose was added; the best process for removing fishy smell from cowhide was: the enzyme addition amount was 0.4%, the enzymolysis time was 10 min, the sugar type was maltose, and the sugar addition amount was 9%. Under these conditions, a sensory evaluation was carried out to confirm that there was no difference from the theoretical results.

[0087] (4) Sensory Evaluation

[0088] Sensory evaluation is an intuitive and reliable technique that can convert the color, smell, and taste of food into measurable data that can directly reflect the characteristics and quality of food. As Figure 6 shown, compared with Comparative Example 1, Comparative Example 2, and Comparative Example 3, the fishy smell of the product in Example 1 was significantly reduced, and both tendon and cowhide were 3 points (the lowest score); the acceptance degree was the highest, and both tendon and cowhide reached 6 points.

[0089] (5) Electronic Nose Detection

[0090] The radar chart of the electronic nose shows that obvious changes have occurred in the corresponding sensor response values of Example 1 of tendon and cowhide compared with Comparative Example 1, Comparative Example 2, and Comparative Example 3; as Figure 7As shown, the first and second principal components of tendon are 72.7% (PC1) and 19.4% (PC2) respectively, and those of cowhide are 58.6% (PC1) and 29.9% (PC2) respectively. The total contribution rate is greater than 80%, which is credible and can reflect the main characteristics of the volatile flavor of the samples. Through principal component analysis, it is found that Example 1 is significantly separated from Comparative Example 1, Comparative Example 2, and Comparative Example 3 on PC1, indicating that its odor characteristics have changed, which is consistent with the sensory results. For the samples after treatment, the fishy smell has decreased accordingly; relevant studies have shown that selecting a suitable enzyme for enzymatic hydrolysis can not only be used to extract proteins and bioactive compounds, but also be used to enhance the flavor of the final product.

[0091] (6) GC-MS detection

[0092] GC-MS is a detection technology that combines gas chromatography and mass spectrometry. The role of GC is to separate, and the role of MS is to detect. The working principle is to vaporize the sample through GC, separate the vaporized compounds through a capillary column, enter the ion source of MS, and ionize them after being bombarded by an electron beam to obtain a chromatogram. Matching the obtained unknown volatile components in the mass spectrometry library can obtain the detection results. As Figure 8 、 Figure 9 shown, the mixed treatment in Example 1 can effectively reduce the concentrations of fishy smell components such as nonanal, undecane, and dodecane. For tendon, the mixed treatment reduces the nonanal content from 30.84 μg / kg to 12.58 μg / kg, the undecane content from 33.44 μg / kg to 5.79 μg / kg, and the dodecane content from 7.78 μg / kg to 2.78 μg / kg; for cowhide, the mixed treatment reduces the nonanal content from 29.90 μg / kg to 3.34 μg / kg, the undecane content from 29.69 μg / kg to 0.91 μg / kg, and the dodecane content from 18.50 μg / kg to 2.50 μg / kg; compared with Comparative Example 1 where the fishy smell volatile components are the highest, resulting in the strongest fishy smell; the content of fishy smell volatile components in Comparative Example 2 and Comparative Example 3 is higher than that in Example 1, and the content of fishy smell components is lower than that in Comparative Example 1. Therefore, the fishy smell removal effect of Comparative Example 2 and Comparative Example 3 is higher than that of Comparative Example 1 but lower than that of Example 1.

[0093] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for removing the odor of cattle hooves based on the Maillard reaction, characterized in that: The method comprises the following steps: (1) Split and rinse the fresh cow hoof sample to obtain a clean cow hoof sample; (2) enzymatically hydrolyzing the cattle hoof sample obtained in step (2) to obtain an enzymatic hydrolyzate; (3) Adding reducing sugar to the enzymatic hydrolysate for saccharification; (4) cooking the ox hoof obtained in step (3) to obtain an ox hoof with the fishy smell removed; the contents of the fishy components nonanal, undecane, and dodecane are reduced; The enzyme used in the enzymatic hydrolysis in step (2) is selected from one or more of neutral protease, bromelain, papain, and flavor protease; The amount of enzyme added in step (2) does not exceed 0.6% of the mass of the cattle hoof sample; In step (2), the enzymatic hydrolysis temperature is 40-60°C and the enzymatic hydrolysis time is 10-40 min; The reducing sugar in step (3) is selected from one or more of glucose, galactose, fructose and maltose; The amount of reducing sugar added in step (3) does not exceed 9% of the mass of the cattle hoof sample.

2. The method according to claim 1, characterized in that: The flushing in step (1) is performed by running water flushing, and the flushing time is 15 minutes.

3. The method according to claim 1, characterized in that The enzymatic hydrolysis in step (2) is to dissolve the enzyme in pure water to obtain enzyme solution, and then perform enzymatic hydrolysis on the cattle hoof sample.

4. The method according to claim 1, characterized in that The cooking in step (4) is boiling.

5. The method according to claim 4, characterized in that The cooking temperature in step (4) is 90° C. and the cooking time is 90 min.

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