High-efficiency extraction method of animal skin enzymatic gelatin

Abstract

The application provides a high-efficiency extraction method of animal skin enzymatic gelatin. The method is characterized in that the expansion degree and thermal denaturation law of collagen fibers of animal skin are controlled through the synergistic effect of acid, alkali, salt and heat, the exposure degree of the specific site of protease in the collagen fibers is controlled, the endo-type hydrolysis of the protease on the collagen fibers is promoted, the gelatin extraction rate and gel strength are improved, and the performance of the gelatin can meet the quality standard of industrial gelatin without desalting treatment. The method can realize one-time extraction of animal skin gelatin, the gelatin yield is more than 90%, the gel strength is more than 200 Bloom g, the production efficiency of the gelatin can be obviously improved, and good energy-saving and emission-reducing effects are achieved. The method has important significance for developing the high-efficiency preparation technology of high-performance animal skin gelatin.

Description

Technical Field

[0001] This invention relates to a highly efficient enzymatic extraction method for gelatin from animal skin and its application, belonging to the fields of biochemistry and animal skin resource processing. Background Technology

[0002] Bovine hide contains a high amount of collagen, making it a crucial raw material for gelatin production. Extracting gelatin from animal tissues such as bovine hide involves moderately disrupting the natural structure of insoluble collagen fibers, hydrolyzing some peptide bonds, and releasing soluble α-chains and oligomers. The peptide bond hydrolysis rate directly affects the molecular weight, viscosity, gel strength, and other properties of gelatin, as well as its application performance in various fields. Common methods for extracting gelatin from animal hides include hot water extraction, acid extraction, alkali extraction, and enzymatic extraction. Different preparation methods result in significant differences in the quality and performance characteristics of the obtained gelatin. Hot water extraction involves high-temperature boiling to denature collagen and hydrolyze some peptide bonds, yielding gelatin with high purity and strength. Acid and alkali extraction methods use acid or alkali at appropriate temperatures to accelerate the denaturation and partial hydrolysis of collagen, producing gelatin of relatively high quality. However, these methods generally suffer from long extraction times, low yields, high water requirements, and the generation of neutral salts, making them increasingly difficult to meet the demands of green development. Various methods, including ultrasound-assisted extraction, microwave treatment, high-pressure treatment, ionic liquid extraction, and steam explosion, can improve the extraction efficiency and strength of gelatin to varying degrees and have been applied to some extent. However, these methods also increase energy consumption, initial investment, and production costs. Furthermore, the degree of hydrolysis of collagen peptide bonds under different acid and alkali conditions is difficult to control, easily leading to excessive hydrolysis of peptide chains and excessively low molecular weight, thus affecting the gel strength, viscosity, and film-forming properties of gelatin. To obtain high-quality gelatin, multiple extraction processes are generally employed, resulting in water consumption of 300-500 tons per ton of gelatin during production. This places significant pressure on subsequent concentration, drying, and wastewater management processes, representing a key issue currently faced in the production of gelatin from animal hides such as bovine hides.

[0003] Enzymatic gelatinization is a process in which collagen undergoes partial hydrolysis to form gelatin under the action of proteases. It generally has advantages such as mild processing conditions, low water consumption, and low pollution, and has long attracted considerable attention. Attempts have been made to apply pepsin, alkaline protease, papain, bromelain, and irisin to gelatin preparation. However, in practical applications, it has been found that the extraction yield of enzymatic gelatin is generally below 30%, and there are widespread problems with controlling gel strength, viscosity, and molecular weight, making it difficult to simultaneously achieve high extraction rates and high gel strength. While protease optimization, acid-base swelling to loosen collagen fibers, and microwave or ultrasound-assisted enzyme action can improve enzyme efficiency, the technical defects of low gelatin yield and low strength remain difficult to effectively solve.

[0004] Studies have shown that using alkaline protease and lipase, combined with enzyme activators such as urea, calcium chloride, and hydrochloric acid, as well as hydrogen bond breakers and cross-linking breakers, can improve gelatin yield, but it puts significant pressure on subsequent desalination processes. Su Minghui, in his paper "Green Preparation of Gelatin," used a combination of heat treatment and a single enzyme to prepare gelatin, reducing the use of chemical reagents, lowering water and energy consumption, and making it more environmentally friendly; however, the final gelatin yield was low (16.5%). Gao Peiru et al.'s technology showed that treating bovine hide in a 50-80 °C water bath, followed by treatment with alkaline protease and lipase, can improve collagen extraction rate, but in this technology, the enzymes only play a role in promoting the efficiency of the alkaline-heat extraction method (CN 109354624 A). The reasons for these problems are complex, mainly because proteases are generally bond-specific enzymes, primarily catalyzing the hydrolysis of peptide bonds formed by specific amino acid residues. However, the collagen in animal hide has a relatively tight triple helix structure, providing a certain degree of resistance to degradation by acids, alkalis, and enzymes. In the enzymatic gelatin preparation process, because most of the enzyme's action sites in collagen are encapsulated within a triple-helix structure, proteases cannot preferentially act on their specific sites. Instead, they generally and preferentially act on the non-helix terminal peptide regions where the cross-links between collagen fibers are relatively weak. This results in proteases hydrolyzing animal skin collagen primarily in an exolytic manner, which is the fundamental reason for the low efficiency of enzymatic gelatin preparation, the difficulty in producing high molecular weight gelatin, and the fact that only oligopeptides, amino acids, and other collagen hydrolysates are obtained. Therefore, improving the efficiency and quality of enzymatic gelatin preparation requires appropriate denaturation treatment of animal skin collagen to disrupt its natural structure, expose more enzyme action sites, promote the preferential action of proteases on their specific sites, and increase the probability of endolytic hydrolysis of collagen fibers. However, it is crucial to simultaneously avoid the hydrolysis of collagen peptide bonds to prevent negative impacts on gelatin extraction rate and quality.

[0005] This study shows that animal skin collagen fibers swell to varying degrees with changes in pH and salt concentration. The degree of damage to the natural structure of collagen fibers varies significantly under different temperature conditions due to different degrees of swelling. Consequently, the exposure of specific protease sites in collagen differs, effectively controlling the degree of enzyme hydrolysis of collagen. This improves gelatin extraction rate while ensuring that the gelatin molecular weight is sufficiently large. The gelatin viscosity, gel strength, ash content, and insoluble content all meet the basic requirements for industrial gelatin (QB / T 1995-2024). This lays the foundation for the efficient and clean preparation of animal skin gelatin, effectively improving gelatin production efficiency and achieving good energy-saving and emission-reduction effects. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing research methods by providing a highly efficient enzymatic method for preparing animal skin gelatin. This method enables a one-time extraction rate of over 90% for animal skin gelatin, while achieving a gel strength of over 200 Bloom g. It also avoids the excessive use and discharge of large amounts of chemicals such as acids, alkalis, and salts, thus realizing the efficient and green extraction and preparation of animal skin gelatin.

[0007] The present invention is characterized in that the method controls the pretreatment conditions and degree of animal skin to destroy the natural structure of collagen fibers in the animal skin, promotes the endo-hydrolysis of collagen fibers by proteases, and the denaturation pretreatment and enzymatic hydrolysis of animal skin can be completed in one pot, thereby improving the one-time extraction rate and gel strength of gelatin, reducing gelatin ash content, and simplifying the gelatin preparation process.

[0008] In the above technical solution, a highly efficient enzymatic extraction method for gelatin from animal skin is characterized by controlling the pretreatment conditions and degree of animal skin. This refers to controlling the degree of swelling and thermal denaturation of collagen fibers in animal skin through the synergistic effect of acid, alkali, salt, and heat, controlling the degree of exposure of specific sites of enzyme proteins within the collagen fibers, promoting the endo-hydrolysis of collagen fibers by proteases, improving the gelatin extraction rate and gel strength, and enabling the gelatin to meet the quality standards of industrial gelatin without desalting treatment.

[0009] In the above technical solution, a method for efficient extraction of gelatin from animal skin using enzymatic methods is characterized by controlling the pretreatment conditions and degree of animal skin, with the synergistic effects of acid, alkali, salt, and heat as follows: water volume is 0.1-10 times the skin weight, salt concentration is 0-100 g / L, pH value is 0-5.0 or 9-14.0, temperature is 20-100 °C, and treatment time is 1-120 min.

[0010] In the above technical solution, a highly efficient method for extracting gelatin from animal skin using enzymatic methods is characterized in that the protease can be at least one of acidic protease, neutral protease, alkaline protease, keratinase, papain, and trypsin.

[0011] In the above technical solution, a highly efficient method for extracting gelatin from animal skin using enzymatic hydrolysis is characterized in that, after enzymatic hydrolysis, animal skin collagen fibers no longer require secondary treatment with acid, alkali, salt, or heat. The primary dissolution rate of animal skin collagen fibers reaches over 90%, and the gel strength of the gelatin can reach over 200 Bloom g. Even without ultrafiltration and desalination, the ash content of the obtained gelatin can meet the quality requirements of industrial gelatin.

[0012] In the above technical solution, a method for efficient extraction of gelatin from animal skin using enzymatic methods is characterized by comprising the following steps, wherein the materials used in each step are by weight unless otherwise specified: (1) Animal skin pretreatment: Take 100 parts of animal skin, after hair removal, alkali swelling, deashing, and thorough washing, adjust the pH to 7.0-9.0, pulverize, or dry and then pulverize; (2) Animal skin denaturation treatment: Take the animal skin treated in step (1), add 0-20 parts water and 0-2 parts salt, then adjust the pH to 0-5.0 or 9.0-14.0, treat at 55-100 °C for 10-60 min, and cool to 25-65 °C; (3) Enzymatic hydrolysis reaction: Adjust the pH of the animal skin treated in step (2) to the range of protease activity, add 0.1-1000 U of protease per gram of animal skin collagen fiber, hydrolyze at 25-65 °C for 0.5-10 h, and then adjust the pH to 6.0-8.0 to inactivate the enzyme; (4) Filtration, concentration and drying.

[0013] In the above technical solution, a method for efficient extraction of gelatin from animal skin using enzymatic methods is characterized in that the animal skin pulverization can be achieved by cutting the animal skin into small pieces of any size from 1×1cm to 3×3cm, dispersing it into fibrous form using a homogenizer, or pulverizing it into fibrous form after drying.

[0014] The advantages of this invention are as follows.

[0015] This method proposes a scheme for the controllable hydrolysis of animal skin collagen fibers during the preparation of animal skin enzymatic gelatin. In particular, the technical design schemes for enzyme selection and process parameter control have achieved a very significant energy-saving and emission-reduction effect.

[0016] Based on further optimization of enzyme action conditions, the preparation process of bovine gelatin was comprehensively optimized. The steps of acid washing, deacidification, gelatin recycling and desalting in the traditional acid hydrolysis gelatin preparation technology were integrated into one step, forming a "one-pot" gelatin preparation technology based on acid-heat pretreatment and protease synergistic effect.

[0017] This study examines the changes in the thermal stability and denaturation degree of hide collagen with acidic swelling, using acid-heat denaturation to partially expose specific protease action sites that were previously shielded within the triple helix structure of collagen. Subsequently, a suitable protease was selected to promote the endo-hydrolysis of bovine hide collagen fibers while controlling the degree of collagen hydrolysis. This research addresses the issue that in the development of enzymatic gelatin preparation technologies, most studies have focused on enzyme specificity selection, neglecting the effective exposure of specific peptide bonds within collagen fibers.

[0018] The technology demonstrates significant advantages. By scientifically controlling conditions and designing the process route, a one-pot extraction method for enzymatic gelatin has been achieved. This means that after enzymatic hydrolysis, secondary treatments involving acid, alkali, salt, and heat are no longer required, simplifying the gelatin production process, improving production efficiency, and achieving an extraction rate of 92.7%. When the gel strength reaches at least 210.0 Bloom g, this extraction rate is significantly higher than previously reported. Furthermore, all product indicators meet industry quality requirements (QB / T 1995-2024). Attached Figure Description

[0019] Figure 1 Yield and gel strength of enzymatic extraction of gelatin from bovine hide under different pH (A) and acid types (B) Figure 2 Extraction rate and gel strength of enzymatically hydrolyzed gelatin from bovine hide at different heat treatment temperatures Figure 3 Effect of heat treatment time on the extraction rate and gel strength of enzymatic gelatin from bovine hide Figure 4 Enzymatic gelatin preparation process flow chart Figure 5 SDS-PAGE electrophoresis image of enzymatic gelatin Detailed Implementation The present invention will be specifically described below through embodiments. It should be noted that these embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above content of the present invention.

[0020] Experimental Example 1: Performance of Enzymatic Gelatin Preparation under Different Pretreatment pH Select high-quality fresh cowhide, wash, rehydrate, remove hair, lime, and remove flesh. Use a splitting machine to remove the grain layer (approximately 2.2 mm thick), then slice off two layers of lime (approximately 2.5 mm thick). Wash, delim, and adjust the pH to 7.0-8.0. Then wash thoroughly to remove neutral salts from the hide. Cut the split hide into small pieces and freeze-dry. Grind into hide powder using a hide powdering machine. Take a certain mass of cowhide powder and add a certain volume of water at a material-to-liquid ratio of 1:20 (m:v). Rehydrate thoroughly at room temperature for 2 hours. Adjust the pH to different levels (2.0, 3.0, 4.0, 5.0, 6.0) with 1 mol / L HCl, boil for 10 minutes, cool to room temperature, adjust the pH, add protease, and stir at a certain temperature for a period of time to stop the enzymatic hydrolysis. After the reaction, adjust the pH to neutral to terminate the hydrolysis. Centrifuge the hydrolysate at 7500 r / min for 10 minutes, collect the supernatant, and freeze-dry to obtain gelatin.

[0021] from Figure 1(A) It can be seen that under the same conditions (pH around 3.0), compared with organic weak acids such as formic acid and acetic acid, inorganic strong acids such as hydrochloric acid and sulfuric acid have a stronger swelling effect on collagen fibers, resulting in lower heat resistance. After heat treatment, the triple helix structure is more severely damaged, which is more conducive to the action of enzymes and to increasing the yield of enzymatically hydrolyzed gelatin. At the same time, strong acids have a stronger ability to hydrolyze peptide bonds, which will lead to a slight decrease in gel strength, but it still reaches more than 200 Bloom g, which can effectively meet the basic requirements of the industry (QB / T 1995-2024, ≥200 Bloom g). Hydrochloric acid has a stronger loosening effect on collagen fibers, which is more conducive to the endo-hydrolysis of collagen fibers by proteases. Therefore, the gelatin yield and gel strength are better than those of other acids. Moreover, as the pH value decreases from 7.0 to 2.0, the gelatin yield gradually increases from 20.0% to about 60.0%, while the gel strength decreases from 401.4 Bloom g to 245.4 Bloom g. Figure 1 (B) This is because, as the pH decreases, the absorption and swelling of collagen intensifies, its heat resistance gradually decreases, and the degree of collagen structure destruction intensifies during heat treatment, which is conducive to exposing more enzyme action sites and improving the efficiency of enzyme catalytic hydrolysis, thereby significantly increasing gelatin yield; however, when the pH value is too low, it is easy to cause excessive hydrolysis of peptide chains, which reduces the gel strength of gelatin and may lead to a high neutral salt content in the obtained gelatin. Therefore, the pH value is controlled at around 3.0.

[0022] Experimental Example 2: Performance of Enzymatic Gelatin Preparation at Different Pretreatment Temperatures A certain mass of bovine secondary leather powder was taken and a certain volume of water was added at a material-to-liquid ratio of 1:20 (m:v). The mixture was allowed to fully rehydrate at room temperature for 2 hours. The pH was adjusted to 3.0 with 1 mol / L HCl, and the mixture was heat-treated at different temperatures for 10 minutes. After cooling to room temperature, the pH was adjusted, and protease was added. The enzymatic hydrolysis reaction was carried out by stirring at a certain temperature for a period of time. After the reaction was completed, the pH was adjusted to neutral to terminate the enzymatic hydrolysis. The hydrolysate was centrifuged at 7500 r / min for 10 minutes, and the supernatant was collected and freeze-dried to obtain gelatin.

[0023] from Figure 2As can be seen, with the increase of heat treatment temperature, the gelatin extraction yield gradually increased from 11.5% to 56.3%. Although the gel strength of the obtained gelatin also gradually decreased, even at a pretreatment temperature of around 100 ℃, the gel strength still reached about 220 Bloom g. Acid hydrolysis alone, while increasing the gelatin yield to around 66.7%, only yielded a gel strength of 158.9 Bloom g, which is insufficient to meet the basic requirements of the industry. This is because, under acidic conditions (around pH 3.0), with the increase of heating temperature, the probability of breakage of secondary bonds such as hydrogen bonds, hydrophobic bonds, and van der Waals forces between and within the bovine collagen protein fibers increases. The degree of damage to the higher-order structure is enhanced, further causing the collagen triple helix conformation to unwind. This increases the degree to which the peptide chain changes from a folded conformation to an unfolded conformation, exposing more enzyme action sites. This facilitates the endo-hydrolysis of the collagen peptide chain by the enzyme, thereby improving the efficiency of enzymatic gelatin extraction and the gel strength of the gelatin. It is evident that the synergistic effect of acid, heat, and enzyme helps to balance the contradiction between gelatin yield and gel strength, thereby improving gelatin production efficiency.

[0024] Experimental Example 3: Performance of Enzymatic Gelatin Preparation under Different Pretreatment Times A certain mass of bovine secondary leather powder was taken and a certain volume of water (salt concentration of 0.1 g / mL) was added at a material-to-liquid ratio of 1:20 (m:v). The mixture was allowed to fully rehydrate at room temperature for 2 hours. The pH was adjusted to 3.0 with 1 mol / L HCl, and the mixture was heat-treated for different times (5, 15, 30, 45, 60 min). After cooling to room temperature, the pH was adjusted, and 1000 U / g of protease was added. The enzymatic hydrolysis reaction was carried out at 35 °C with stirring for 6 hours. After the reaction, the pH was adjusted to neutral to terminate the enzymatic hydrolysis. The hydrolysate was centrifuged at 7500 r / min for 10 min, and the supernatant was collected and freeze-dried to obtain gelatin.

[0025] The result is from Figure 3 It can be seen that while extending the heat treatment time of the cowhide fiber effectively increased the yield of enzymatically hydrolyzed gelatin from 41.81% to 87.81%, the gel strength of the resulting gelatin also decreased rapidly. When the heat treatment time reached 30 minutes, the gel strength of the enzymatically hydrolyzed gelatin dropped below 139.7 Bloom g. This is because, during the heat treatment process, extending the heat treatment time not only breaks the secondary bonds of the protein but also causes partial hydrolysis of the peptide bonds, generating some gelatin. After the addition of protease, the protease preferentially acts on the dissolved gelatin, increasing the content of low molecular weight components in the resulting gelatin and leading to a decrease in gel strength. Therefore, to obtain gelatin that meets industry standards, the heat treatment time needs to be controlled within 10 minutes.

[0026] Experimental Example 4: Enzymatic Gelatin Preparation Technology from Bovine Hide like Figure 4 As shown, 200 g of crushed cowhide was weighed, 15 parts water and 0.5 parts salt were added, and the pH was adjusted to 3.5. The mixture was treated at 95 °C for 10 min and then cooled to 40 °C. The pH was adjusted to the range of protease activity, and 300 U of protease was added per gram of animal skin collagen fiber. The mixture was hydrolyzed at 40 °C for 4 h, and then the pH was adjusted to 6.0-8.0 to inactivate the enzyme. The mixture was filtered, concentrated, and dried to obtain acidic enzymatic gelatin.

[0027] Table 1. Quality stability of enzymatic bovine hide gelatin preparation technology

[0028] *: Enzyme action conditions: pH approximately 4.0, temperature approximately 40 ℃, dosage approximately 350 U / g; **: rotational viscosity; ***: Industry standard "Industrial Gelatin" (QB / T 1995-2024).

[0029] In five batches of production, the average gelatin yield reached approximately 93% (Table 1). This was mainly due to the enhanced suspension of the hide powder through mechanical stirring during the scale-up experiment, which allowed for more thorough contact between the enzyme and the animal hide powder. This further promoted the gradual hydrolysis and dissolution of collagen fibers by the enzyme, enabling efficient gelatin preparation. Moreover, from... Figure 5 It can be seen that the SDS-PAGE band characteristics of the five consecutive batches of enzymatically prepared gelatin are similar, indicating that the molecular weight distribution, structure, and subunit composition of the gelatin in each batch are relatively consistent. This further demonstrates that heat denaturation treatment facilitates more efficient hydrolysis of specific peptide bonds on collagen by proteases, promotes "endo-glucanization" hydrolysis of collagen, and better controls the degree and pattern of collagen hydrolysis, ensuring the basic gel strength of gelatin and improving the quality stability of the product. Therefore, as can be further seen from Table 1, the various performance indicators of the bovine gelatin prepared from five consecutive batches are relatively stable. The gelatin gel strength is approximately 210.0 ± 6.1 Bloom g, viscosity is approximately 6.9 ± 0.3 mPa·s, ash content is approximately 2.1% ± 0.3%, water-insoluble matter content is approximately 0.15 ± 0.03%, moisture content is approximately 12.3% ± 0.8%, and pH value is approximately 6.4 ± 0.2. All indicators meet the industry quality standards (QB / T 1995-2024). Furthermore, the water retention capacity (WHC%), oil retention capacity (OHC%), and emulsification performance (EAI%) of each batch of gelatin are also relatively stable. 2Functional properties such as gelatin extract yield (g), ESI (min), foaming capacity (FBC%), and foam stability (FS%) are also relatively stable (Table 2). This also indicates that the amino acid composition, molecular weight distribution, structure, and subunit composition of different batches of gelatin are quite similar. In summary, the newly constructed novel enzymatic gelatin preparation technology effectively solves the technical problem of balancing gelatin extraction rate and gel strength in the preparation of bovine hide enzymatic gelatin, and ensures product quality stability, demonstrating good technical acceptability.

[0030] Table 2 Functional properties of novel enzymatically processed bovine gelatin

[0031] This study aims to develop a bovine gelatin preparation technology based on acidic protease catalysis to address the bottleneck issue of achieving both high gelatin extraction rate and high gel strength in common gelatin preparation techniques. It also seeks to improve preparation efficiency, reduce chemical usage at the source, and develop an energy-saving, water-saving, and highly efficient enzymatic gelatin preparation technology. Compared to common acid-based gelatin preparation techniques, the novel enzymatic gelatin preparation technology integrates the acid washing-deacidification-gelatin boiling process into a single step, avoiding multiple boiling-filtration, compounding, and desalination operations, resulting in significant process simplification. Furthermore, as shown in Table 3, the novel enzymatic gelatin preparation technology reduces the total water consumption, wastewater discharge, acid usage, and energy consumption during gelatin boiling by approximately 78.9%, 100.0%, 90.0%, and 28.6%, respectively, compared to common acid-based gelatin preparation techniques. It also reduces energy consumption during the gelatin drying process by 63.5%, demonstrating excellent energy-saving and emission-reduction effects, and shortens the production cycle by 3-6 days, effectively improving the daily production efficiency of gelatin.

[0032] In summary, the "one-pot" preparation technology based on heat-denatured bovine hide enzymatic gelatin has good acceptability in terms of technical stability, production efficiency, energy saving and emission reduction. It is expected to provide new technical support for improving the resource utilization efficiency of animal hides such as bovine hides in the field of gelatin preparation and promote the green development of the animal hide resource utilization industry.

[0033] Table 3. Energy saving and emission reduction effects of enzymatic gelatin preparation technology*

[0034] *Calculations are based on the production of 1 ton of gelatin; the indicators of the acid-process gelatin preparation technology are based on survey data from bovine gelatin production enterprises, and the indicators of the new enzymatic gelatin preparation technology are extrapolated based on the results of scale-up experiments. **The specific heat capacity of water is used for calculation, without considering heat conversion: Q = cmΔt; c - specific heat capacity of water (4.2 × 10⁻⁶ cmΔt).** 3J / (kg ℃)); m - hot water volume (kg); Δt - water temperature rise (℃); ***The drying temperature is controlled at 55 ℃. The energy consumption calculation formula for water evaporation is used, without considering heat conversion: Q=m(h+c△t); m - mass of evaporated water (kg), h - latent heat of vaporization of water (55 ℃), 2371 kJ / g, c - specific heat capacity of water (4.2×10⁻⁶ kJ / g). 3 J / (kg ℃)); △t - water temperature rise (℃).

Claims

1. A highly efficient enzymatic extraction method for gelatin from animal skin, characterized in that, The method involves controlling the pretreatment conditions and degree of animal hides to disrupt the natural structure of collagen fibers, promote the endo-hydrolysis of collagen fibers by proteases, and complete the denaturation pretreatment and enzymatic hydrolysis of animal hides in one batch. This improves the one-time extraction rate and gel strength of gelatin, reduces gelatin ash content, and simplifies the gelatin preparation process.

2. The method for efficient enzymatic extraction of gelatin from animal skin according to claim 1, characterized in that, Controlling the pretreatment conditions and degree of animal skin refers to controlling the degree of swelling and thermal denaturation of collagen fibers in animal skin through the synergistic effects of acids, alkalis, salts, and heat. It also controls the degree of exposure of specific sites of enzyme proteins within collagen fibers, promotes the endo-hydrolysis of collagen fibers by proteases, and improves the gelatin extraction rate and gel strength. This allows the gelatin to meet the quality standards of industrial gelatin without desalting.

3. The method for efficient enzymatic extraction of gelatin from animal skin according to claim 1, characterized in that, The conditions and extent of animal skin pretreatment were controlled. The synergistic effects of acid, alkali, salt, and heat were as follows: water volume was 0.1-10 times the skin weight, salt concentration was 0-100 g / L, pH value was 0-5.0 or 9-14.0, temperature was 20-100 °C, and treatment time was 1-120 min.

4. The method for efficient enzymatic extraction of gelatin from animal skin according to claim 1, characterized in that, The protease may be at least one of acidic protease, neutral protease, alkaline protease, keratinase, papain, and trypsin.

5. The method for efficient extraction of gelatin from animal skin using enzymatic extraction according to claim 1, characterized in that, Animal skin collagen fibers, after enzymatic hydrolysis, no longer require secondary treatment with acids, alkalis, salts, or heat. The primary solubility of animal skin collagen fibers reaches over 90%, and the gel strength of the gelatin can reach over 200 Bloom g. Even without ultrafiltration and desalination, the ash content of the resulting gelatin can meet the quality requirements of industrial gelatin.

6. The method for efficient enzymatic extraction of gelatin from animal skin according to claim 1, characterized in that... The following steps are included, and unless otherwise specified, all materials used in each step are by weight: (1) Animal skin pretreatment: Take 100 parts of animal skin, after hair removal, alkali swelling, deashing, and thorough washing, adjust the pH to 7.0-9.0, pulverize, or dry and then pulverize; (2) Animal skin denaturation treatment: Take the animal skin treated in step (1), add 0-20 parts water and 0-2 parts salt, then adjust the pH to 0-5.0 or 9.0-14.0, treat at 55-100 °C for 10-60 min, and cool to 25-65 °C; (3) Enzymatic hydrolysis reaction: Adjust the pH of the animal skin treated in step (2) to the range of protease activity, add 0.1-1000 U of protease per gram of animal skin collagen fiber, hydrolyze at 25-65 °C for 0.5-10 h, and then adjust the pH to 6.0-8.0 to inactivate the enzyme; (4) Filtration, concentration and drying.

7. The method for efficient enzymatic extraction of gelatin from animal skin according to claim 6, characterized in that, The animal skin pulverization can be achieved by cutting the animal skin into small pieces of any size from 1×1cm to 3×3cm, dispersing it into fibrous form using a homogenizer, or pulverizing it into fibrous form after drying.

8. A method for efficient enzymatic extraction of gelatin from animal skin according to claims 1 and 6, characterized in that, The traditional acid hydrolysis gelatin preparation technology integrates the processes of acid washing, deacidification, gelatin recycling and desalting into a single operation, forming the "one-pot" gelatin preparation technology.

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