Processing technology of low-gi protein powder of donkey-hide gelatin

Abstract

The application belongs to the technical field of food processing, and particularly relates to a low GI donkey-hide gelatin protein powder processing technology. The low GI donkey-hide gelatin protein powder processing technology provided by the application comprises steps of degreasing, extraction, enzymolysis, fermentation, drying and the like. The application adopts whole-process low-temperature treatment, maximally retains heat-sensitive active ingredients in the donkey-hide gelatin, and realizes maximization of the nutritional value. Ultrasonic waves and microwaves are used for synergistic extraction under stirring, so that the collagen protein is accelerated to dissolve under low-temperature conditions, high-temperature extraction of the donkey-hide gelatin is avoided, and deformation and degradation of the collagen protein and active peptides are significantly reduced. A composite enzymolysis process is used to hydrolyze the macromolecular collagen protein into small molecular peptides, so as to improve the bioavailability, solubility and dispersibility of the donkey-hide gelatin protein. Composite strains are used for fermentation of the collagen peptide liquid, so that the glucose and maltose existing in the collagen peptide liquid are consumed, and the glycemic index of the donkey-hide gelatin protein powder is effectively reduced. Auxiliary materials are added into the donkey-hide gelatin protein powder, so as to improve the palatability and nutritional value of the donkey-hide gelatin protein powder.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a processing technology for low-GI donkey-hide gelatin protein powder. Background Technology

[0002] With the development of the times and the improvement of economic level, more and more people are paying attention to improving their quality of life. Donkey-hide gelatin (Ejiao) has the effects of nourishing blood, moisturizing dryness, beautifying the skin, calming the mind, and improving intelligence. As a traditional Chinese tonic with thousands of years of history, it holds an important position in the fields of food health care and traditional Chinese medicine.

[0003] Traditional donkey-hide gelatin is mostly in block or paste form, which suffers from slow dissolution and inconvenience in carrying. Furthermore, traditional donkey-hide gelatin products often contain added sucrose and maltose to improve taste, resulting in a high glycemic index (GI), making them unsuitable for the consumption needs of special populations such as diabetics and obese individuals. Protein powder, on the other hand, is a nutritional food supplement that provides essential nutrients for children, the elderly, patients, athletes, and those trying to lose weight, supplementing the body's protein intake. With increasing health awareness and diversified consumption scenarios, the functions and forms of traditional donkey-hide gelatin can no longer meet market demands, providing a clear direction for the technological development of donkey-hide gelatin protein powder.

[0004] The high-temperature boiling process in traditional donkey-hide gelatin extraction can easily denature and degrade collagen, active peptides, and other components, affecting the product's nutritional value. Furthermore, the processing of donkey-hide gelatin protein powder presents a unique challenge: on the one hand, collagen in donkey-hide gelatin needs to be moderately hydrolyzed to form small-molecule peptides to improve absorption; on the other hand, the hydrolysis process may increase the product's viscosity, affecting its taste and solubility. In addition, commonly used dietary fibers such as inulin and resistant dextrin added to traditional low-GI protein powders may interact with donkey-hide gelatin proteins, leading to gelation and affecting the product's appearance and taste.

[0005] Chinese patent application CN106473138A discloses a donkey-hide gelatin and ginseng protein powder and its preparation method. The formula includes donkey-hide gelatin, ginseng, coix seed, polygonatum extract, selenium-enriched yeast, wolfberry extract, jujube extract, soy protein powder, and whey protein powder. The specific preparation method includes enzymatic hydrolysis of soy protein powder, ultrafiltration, jujube pretreatment, pulverization, donkey-hide gelatin powder preparation, and mixing. The protein components in this donkey-hide gelatin protein powder are mainly soy protein powder and whey protein powder. Furthermore, the purity of the wolfberry extract, polygonatum extract, and jujube extract added to the formula is low, making them prone to interaction with the protein powder, leading to changes in product color and a sour or astringent taste. The formula also contains a large variety of raw materials with significant density differences, resulting in uneven mixing and the problem of locally excessively high donkey-hide gelatin content. Chinese patent application CN115152999A discloses a preparation process for low-sugar donkey-hide gelatin blocks, which includes steps such as acid removal, concentration and boiling, cooling and forming into blocks, and cutting. During the concentration and boiling process, the effective components such as collagen and active peptides in the donkey-hide gelatin blocks produced by this method will denature and degrade, resulting in the loss of nutrients in the low-sugar donkey-hide gelatin blocks. In addition, the bone collagen added to the formula will cross-link with the collagen in the donkey-hide gelatin, reducing the digestibility and absorption rate of the product. Summary of the Invention

[0006] In order to solve the technical problems of active ingredient denaturation, poor solubility, and high glycemic index in the above-mentioned related technologies, the purpose of this invention is to provide a low-GI donkey-hide gelatin protein powder processing technology.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A low-GI donkey-hide gelatin protein powder processing technology includes the following steps:

[0009] S1: After washing the dehaired donkey hide, cut it into pieces, immerse it in a degreasing solution for ultrasonic treatment, wash it clean, and obtain degreased donkey hide;

[0010] S2: Mix the defatted donkey skin obtained in step S1 with purified water, and extract it using ultrasound and microwave under stirring. After filtration, a crude extract of donkey-hide gelatin is obtained.

[0011] S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution, stir and hydrolyze at a constant temperature of 45-55℃, raise the temperature to 70-75℃ after hydrolysis, keep warm for 15-25 minutes, and filter with a nanofiltration membrane to obtain collagen peptide solution.

[0012] S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 10%-12%, adjust the pH value to 6.0-7.0, sterilize by steam, inoculate with compound bacterial culture, stir evenly, ferment at a constant temperature of 30-35℃ for 12-16 hours, and then continue fermentation for 4-6 hours under stirring. After sterilization, filter to obtain donkey-hide gelatin peptide fermentation liquid.

[0013] S5: Freeze-dry the fermented broth of donkey-hide gelatin peptides obtained in step S4, pulverize it at low temperature, add excipients, sterilize, and fill to obtain low-GI donkey-hide gelatin protein powder.

[0014] This invention employs a fully low-temperature processing method to maximize the preservation of heat-sensitive active ingredients in donkey-hide gelatin, thus maximizing its nutritional value. It utilizes a synergistic extraction process combining ultrasound and microwaves under stirring. The cavitation effect of ultrasound and the internal heating characteristics of microwaves rapidly disrupt the dense structure of donkey hide under mild low-temperature conditions, accelerating collagen dissolution and significantly reducing the deformation and degradation of collagen and active peptides. The crude extract of donkey-hide gelatin undergoes complex enzymatic hydrolysis at low temperatures, directionally hydrolyzing large, fibrous, and aggregated collagen molecules into smaller peptides, improving bioavailability as well as solubility and dispersibility. During fermentation, the complex microbial strain preferentially utilizes small sugars such as glucose and maltose as carbon sources for growth and metabolism, converting them into metabolites such as lactic acid, ethanol, and carbon dioxide, effectively reducing the glycemic index of the donkey-hide gelatin protein powder.

[0015] Furthermore, the method for preparing the defatting solution in step S1 is as follows: sodium carbonate, lipase, neutral protease, and alkaline protease are added to purified water at 20-30℃ and stirred evenly to obtain the defatting solution.

[0016] This invention employs a degreasing agent composed of lipase, neutral protease, and alkaline protease, which effectively removes fat from donkey hide while protecting its collagen fibers. The lipase specifically hydrolyzes triglycerides in donkey hide fat, acting directly on the adipose tissue surrounding hair follicles and between collagen fibers. The neutral and alkaline proteases assist in removing fat-protein complexes, breaking down the binding between fat and protein, and effectively improving the fat removal rate. The addition of sodium carbonate to the degreasing solution makes it weakly alkaline, enhancing the stability of the lipase and slightly swelling the donkey hide fibers, promoting enzyme penetration into the hide and further increasing the degreasing rate.

[0017] Furthermore, the defatting solution contains 3%-7% sodium carbonate, 2%-5% lipase, 0.02%-0.06% neutral protease, and 0.01%-0.03% alkaline protease by mass.

[0018] Furthermore, the ultrasonic treatment in step S1 has a frequency of 30-40kHz, a power of 100-300W, a temperature of 30-45℃, and a time of 40-60min.

[0019] Furthermore, in step S2, the frequency of the ultrasound is 20-30kHz and the power is 300-400W, the power of the microwave is 200-300W, the temperature of the synergistic extraction is 55-60℃, and the time of the synergistic extraction is 3-5h.

[0020] Furthermore, the complex enzyme solution in step S3 is composed of collagenase, papain and purified water, with the collagenase having a mass percentage of 10%-15% and the papain having a mass percentage of 20%-30%.

[0021] This invention employs a synergistic enzymatic hydrolysis system composed of collagenase and papain. Through their combined action, it solves the technical problems of poor solubility and low bioavailability in traditional donkey-hide gelatin products, achieving the targeted conversion of donkey-hide gelatin collagen into highly active small molecule peptides. Papain efficiently breaks the glycine-proline peptide bonds in collagen, while collagenase hydrolyzes the three-dimensional helical structure of collagen, forming polypeptides and amino acids, thus improving the extraction and utilization rate of collagen. The enzymatic hydrolysis process significantly reduces the molecular weight of collagen in the donkey-hide gelatin extract, effectively improving the solubility and bioavailability of the donkey-hide gelatin protein powder.

[0022] Furthermore, the amount of the compound enzyme solution used in step S3 is 2%-5% of the mass of the crude extract of donkey-hide gelatin.

[0023] Furthermore, the nanofiltration membrane described in step S3 has a molecular weight cutoff of 300 Da.

[0024] Furthermore, the preparation method of the compound bacterial culture in step S4 is as follows: freeze-dried *Lactobacillus plantarum* is inoculated into MRS liquid culture medium and cultured at 37-40℃ for 18-24 hours until the viable count is ≥10⁻⁶. 9 CFU / mL was used to obtain *Lactobacillus plantarum* solution; freeze-dried *Saccharomyces cerevisiae* was inoculated into YPD liquid medium and cultured in a shaker at 30-35℃ for 12-16 hours until the viable count was ≥10⁻⁶. 9 CFU / mL was used to obtain the Saccharomyces cerevisiae culture; the Lactobacillus plantarum culture and the Saccharomyces cerevisiae culture were mixed evenly at a volume ratio of 3:1 to obtain the compound culture culture.

[0025] This invention utilizes the synergistic effect of Lactobacillus plantarum and Saccharomyces cerevisiae. This not only decomposes the residual monosaccharides and disaccharides in the collagen peptide solution, but Lactobacillus plantarum can also convert some absorbable sugars into oligosaccharides such as fructooligosaccharides and galactooligosaccharides, which are not easily absorbed, thus reducing the glycemic index from the source. No artificial sweeteners are added. At the same time, after fermentation, Lactobacillus plantarum and Saccharomyces cerevisiae not only retain the antioxidant and immune-regulating functions of donkey-hide gelatin peptides, but also produce prebiotics to synergize with donkey-hide gelatin peptides to exert a dual effect of intestinal protection and nutritional supplementation. The lactic acid produced by Lactobacillus plantarum fermentation and the small amount of alcohol produced by Saccharomyces cerevisiae fermentation can also neutralize the fishy smell of donkey-hide gelatin peptides, giving the product a rich taste.

[0026] Furthermore, the inoculation amount of the compound bacterial culture in step S4 is 2%-5% of the mass of the concentrated collagen peptide solution.

[0027] Furthermore, the excipients in step S5 consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 4-8:3-5:6-9, and the amount of the excipients is 4%-7% of the mass of the donkey-hide gelatin peptide fermentation broth.

[0028] In this invention, whey protein isolate, casein, and soy protein isolate are added to the donkey-hide gelatin protein powder as excipients. Whey protein isolate and soy protein isolate contain essential amino acids required by the human body, which can make up for the deficiency of essential amino acids in donkey-hide gelatin protein. They also have high bioavailability and excellent solubility, which helps to improve the solubility of donkey-hide gelatin protein powder in water. Casein has a dense molecular structure and forms a gel-like substance in the stomach. Its slow digestion and absorption can prolong the feeling of fullness of donkey-hide gelatin protein powder. In addition, casein has a milky aroma, which can enrich the taste of donkey-hide gelatin protein powder and improve the palatability of the product.

[0029] Compared with existing technologies, the low-GI donkey-hide gelatin protein powder processing technology provided by this invention has the following technical advantages:

[0030] (1) The present invention adopts low-temperature processing throughout the entire process, which preserves the heat-sensitive active ingredients in donkey-hide gelatin to the greatest extent and maximizes its nutritional value;

[0031] (2) The present invention uses ultrasound and microwave in a stirring state for synergistic extraction, which accelerates the dissolution of collagen under low temperature conditions, avoids the high temperature extraction of donkey-hide gelatin, and significantly reduces the deformation and degradation of collagen and active peptides.

[0032] (3) The present invention uses a compound enzymatic hydrolysis process to hydrolyze macromolecular collagen into small molecule peptides, thereby improving the bioavailability, solubility and dispersibility of donkey-hide gelatin protein;

[0033] (4) The present invention uses a compound strain to ferment collagen peptide liquid, which consumes glucose and maltose present in collagen peptide liquid, effectively reducing the glycemic index of donkey-hide gelatin protein powder.

[0034] (5) The present invention adds excipients to donkey-hide gelatin protein powder to improve the palatability and nutritional value of donkey-hide gelatin protein powder. Detailed Implementation

[0035] The following description, in conjunction with specific embodiments, provides further details, but the present invention is not limited to these embodiments. Those skilled in the art can make various modifications based on the fundamental principles of the present invention, but all modifications that do not depart from the fundamental principles of the present invention are within its scope.

[0036] The formulation of the MRS liquid culture medium in this specific embodiment is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diammonium citrate, 1mL Tween-80, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, with water added to 1000mL and the pH adjusted to 6.2-6.4; the formulation of the YPD liquid culture medium is as follows: 10g yeast extract, 20g peptone, 20g glucose, with water added to 1000mL and the pH set to natural.

[0037] Example 1

[0038] S1: After the dehaired donkey hide is washed by high-pressure (pressure 0.3MPa) spraying, it is cut into small pieces of 2cm×2cm, immersed in degreasing solution (material-liquid ratio of 1:8) and ultrasonically treated at 30℃ for 40min (frequency 30kHz, power 100W). After removing the donkey hide pieces, it is rinsed twice with 0.5% sodium citrate aqueous solution, and then rinsed three more times with clean water to obtain degreased donkey hide.

[0039] The defatting solution was prepared as follows: sodium carbonate, lipase, neutral protease, and alkaline protease were added to purified water at 20°C and stirred until homogeneous to obtain the defatting solution; wherein the mass percentage of sodium carbonate was 3%, the mass percentage of lipase was 2%, the mass percentage of neutral protease was 0.02%, and the mass percentage of alkaline protease was 0.01%.

[0040] S2: Mix the defatted donkey hide obtained in step S1 with purified water (material-liquid ratio of 1:8), add it to an ultrasonic-microwave extraction tank, add 5% sodium citrate solution to adjust the pH of the system to 5.5, heat to 55℃ at 150 rpm, and simultaneously turn on ultrasonic (frequency 20kHz, power 300W) and microwave (power 200W) for 3 hours of synergistic extraction, filter with a filter cloth with a pore size of 0.2μm to obtain crude extract of donkey-hide gelatin;

[0041] S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution (the amount of compound enzyme solution is 2% of the mass of the crude extract of donkey-hide gelatin), stir and hydrolyze at a constant temperature of 45°C, and after the enzymatic hydrolysis is completed, raise the temperature to 70°C and keep it at that temperature for 15 minutes. Filter the solution using a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain collagen peptide solution.

[0042] The complex enzyme solution consists of collagenase, papain, and purified water, with collagenase comprising 10% by mass and papain comprising 20% ​​by mass.

[0043] S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 10%, adjust the pH value to 6.0, autoclave at 115℃ for 15 min, inoculate with compound bacterial culture (the inoculation amount of compound bacterial culture is 2% of the mass of the concentrated collagen peptide solution), stir evenly, ferment at 30℃ for 12 h, and then continue fermentation for 4 h with stirring (speed is 80 rpm). After sterilization at 60℃ for 30 min, filter with a 0.45 μm microporous nanofiltration membrane to obtain the donkey-hide gelatin peptide fermentation broth.

[0044] The preparation method of the compound bacterial culture solution is as follows: Freeze-dried *Lactobacillus plantarum* is inoculated into MRS liquid medium and incubated at 37℃ for 18 hours until the viable count is ≥10⁻⁶. 9 CFU / mL was used to obtain Lactobacillus plantarum solution; freeze-dried Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 30℃ in a shaker for 12 h until the viable count was ≥10. 9 CFU / mL was used to obtain the Saccharomyces cerevisiae culture; the Lactobacillus plantarum culture and the Saccharomyces cerevisiae culture were mixed evenly at a volume ratio of 3:1 to obtain the compound culture culture.

[0045] S5: The donkey-hide gelatin peptide fermentation broth obtained in step S4 was atomized in a freezing tower at -50℃ using a two-fluid nozzle (atomization pressure 0.2MPa). It was then dried for 10 hours under vacuum of 10Pa and sublimation temperature of -20℃, followed by a desorption drying at 30℃ for 4 hours. The frozen protein block was then placed in an ultrafine pulverizer and pulverized to 120 mesh at 0℃ using pulsed high-intensity light (energy density 25J / cm²). 2 The mixture is sterilized for 30 seconds with a pulse frequency of 3 Hz, then filled to obtain low-GI donkey-hide gelatin protein powder. The excipients consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 4:3:6, and the amount of the excipients is 4% of the mass of the donkey-hide gelatin peptide fermentation broth.

[0046] Example 2

[0047] S1: After the dehaired donkey hide is washed by high-pressure (pressure is 0.5MPa) spraying, it is cut into small pieces of 2cm×2cm, immersed in degreasing solution (material-liquid ratio is 1:10) and ultrasonically treated at 45℃ for 60min (frequency is 40kHz, power is 300W). After removing the donkey hide pieces, it is rinsed 3 times with 1% sodium citrate aqueous solution, and then rinsed 5 times with clean water to obtain degreased donkey hide.

[0048] The defatting solution is prepared as follows: sodium carbonate, lipase, neutral protease, and alkaline protease are added to purified water at 30°C and stirred until homogeneous to obtain the defatting solution; wherein the mass percentage of sodium carbonate is 7%, the mass percentage of lipase is 5%, the mass percentage of neutral protease is 0.06%, and the mass percentage of alkaline protease is 0.03%.

[0049] S2: Mix the defatted donkey hide obtained in step S1 with purified water (material-liquid ratio of 1:10), add it to an ultrasonic-microwave extraction tank, add 7% sodium citrate solution to adjust the pH of the system to 5.5, heat to 60℃ at 200 rpm, and simultaneously turn on ultrasonic (frequency 30kHz, power 400W) and microwave (power 300W) for 5 hours of synergistic extraction, filter with a filter cloth with a pore size of 0.2μm to obtain crude extract of donkey-hide gelatin;

[0050] S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution (the amount of compound enzyme solution is 5% of the mass of the crude extract of donkey-hide gelatin), stir and hydrolyze at 55℃, raise the temperature to 75℃ after hydrolysis, keep warm for 25 min, and filter with a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain collagen peptide solution.

[0051] The complex enzyme solution consists of collagenase, papain, and purified water, with collagenase comprising 15% by mass and papain comprising 30% by mass.

[0052] S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 12%, adjust the pH value to 7.0, autoclave at 115℃ for 15 min, inoculate with compound bacterial culture (the inoculation amount of compound bacterial culture is 5% of the mass of the concentrated collagen peptide solution), stir evenly, ferment at 35℃ for 16 h, and then continue fermentation for 6 h with stirring (speed is 100 rpm). After sterilization at 60℃ for 30 min, filter with a 0.45 μm microporous nanofiltration membrane to obtain the donkey-hide gelatin peptide fermentation broth.

[0053] The preparation method of the compound bacterial culture solution is as follows: Freeze-dried *Lactobacillus plantarum* is inoculated into MRS liquid medium and cultured at 40℃ for 24 hours until the viable count is ≥10⁻⁶. 9CFU / mL was used to obtain Lactobacillus plantarum solution; freeze-dried Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 35℃ in a shaker for 16 h until the viable count was ≥10. 9 CFU / mL was used to obtain the Saccharomyces cerevisiae culture; the Lactobacillus plantarum culture and the Saccharomyces cerevisiae culture were mixed evenly at a volume ratio of 3:1 to obtain the compound culture culture.

[0054] S5: The donkey-hide gelatin peptide fermentation broth obtained in step S4 was atomized in a freezing tower at -50℃ using a two-fluid nozzle (atomization pressure 0.2MPa). It was then dried for 10 hours under vacuum of 10Pa and sublimation temperature of -20℃, followed by a desorption drying at 30℃ for 4 hours. The frozen protein block was then placed in an ultrafine pulverizer and pulverized to 120 mesh at 0℃ using pulsed high-intensity light (energy density 25J / cm²). 2 The mixture is sterilized for 30 seconds with a pulse frequency of 3Hz, then filled to obtain low-GI donkey-hide gelatin protein powder. The excipients consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 8:5:9, and the amount of the excipients is 7% of the mass of the donkey-hide gelatin peptide fermentation broth.

[0055] Example 3

[0056] S1: After the dehaired donkey hide is washed by high-pressure (pressure 0.4MPa) spraying, it is cut into small pieces of 2cm×2cm, immersed in degreasing solution (material-liquid ratio of 1:9) and ultrasonically treated at 35℃ for 50min (frequency 35kHz, power 200W). After removing the donkey hide pieces, it is rinsed 3 times with 0.7% sodium citrate aqueous solution, and then rinsed 4 times with clean water to obtain degreased donkey hide.

[0057] The defatting solution is prepared as follows: sodium carbonate, lipase, neutral protease, and alkaline protease are added to purified water at 25°C and stirred until homogeneous to obtain the defatting solution; wherein the mass percentage of sodium carbonate is 5%, the mass percentage of lipase is 4%, the mass percentage of neutral protease is 0.05%, and the mass percentage of alkaline protease is 0.02%.

[0058] S2: Mix the defatted donkey hide obtained in step S1 with purified water (material-liquid ratio of 1:9), add it to an ultrasonic-microwave extraction tank, add 6% sodium citrate solution to adjust the pH of the system to 5.5, heat to 58°C at 180 rpm, and simultaneously turn on ultrasonic (frequency 25 kHz, power 350 W) and microwave (power 250 W) for 4 hours of synergistic extraction, filter with a filter cloth with a pore size of 0.2 μm to obtain crude extract of donkey-hide gelatin;

[0059] S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution (the amount of the compound enzyme solution is 3% of the mass of the crude extract of donkey-hide gelatin), stir and hydrolyze at 50°C, raise the temperature to 72°C after hydrolysis, keep warm for 20 min, and filter with a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain collagen peptide solution.

[0060] The complex enzyme solution consists of collagenase, papain, and purified water, with collagenase comprising 13% by mass and papain comprising 25% by mass.

[0061] S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 11%, adjust the pH value to 6.5, autoclave at 115℃ for 15 min, inoculate with compound bacterial culture (the inoculation amount of compound bacterial culture is 4% of the mass of the concentrated collagen peptide solution), stir evenly, ferment at 33℃ for 15 h, and then continue fermentation for 5 h with stirring (speed is 90 rpm). After sterilization at 60℃ for 30 min, filter with a 0.45 μm microporous nanofiltration membrane to obtain the donkey-hide gelatin peptide fermentation broth.

[0062] The preparation method of the compound bacterial culture solution is as follows: Freeze-dried *Lactobacillus plantarum* is inoculated into MRS liquid medium and cultured at 38℃ for 20 hours until the viable count is ≥10⁻⁶. 9 CFU / mL was used to obtain Lactobacillus plantarum solution; freeze-dried Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 33℃ in a shaker for 14 h until the viable count was ≥10. 9 CFU / mL was used to obtain the Saccharomyces cerevisiae culture; the Lactobacillus plantarum culture and the Saccharomyces cerevisiae culture were mixed evenly at a volume ratio of 3:1 to obtain the compound culture culture.

[0063] S5: The donkey-hide gelatin peptide fermentation broth obtained in step S4 was atomized in a freezing tower at -50℃ using a two-fluid nozzle (atomization pressure 0.2MPa). It was then dried for 10 hours under vacuum of 10Pa and sublimation temperature of -20℃, followed by a desorption drying at 30℃ for 4 hours. The frozen protein block was then placed in an ultrafine pulverizer and pulverized to 120 mesh at 0℃ using pulsed high-intensity light (energy density 25J / cm²). 2 The mixture is sterilized for 30 seconds with a pulse frequency of 3 Hz, then filled to obtain low-GI donkey-hide gelatin protein powder. The excipients consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 7:4:8, and the amount of the excipients is 5% of the mass of the donkey-hide gelatin peptide fermentation broth.

[0064] Example 4

[0065] S1: After the dehaired donkey hide is washed by high-pressure (pressure 0.4MPa) spraying, it is cut into small pieces of 2cm×2cm, immersed in degreasing solution (material-liquid ratio of 1:9) and ultrasonically treated at 35℃ for 50min (frequency 35kHz, power 200W). After removing the donkey hide pieces, it is rinsed 3 times with 0.7% sodium citrate aqueous solution, and then rinsed 4 times with clean water to obtain degreased donkey hide.

[0066] The defatting solution was prepared as follows: sodium carbonate, lipase, neutral protease, and alkaline protease were added to purified water at 25°C and stirred until homogeneous to obtain the defatting solution; wherein the mass percentage of sodium carbonate was 6%, the mass percentage of lipase was 3%, the mass percentage of neutral protease was 0.04%, and the mass percentage of alkaline protease was 0.03%.

[0067] S2: Mix the defatted donkey hide obtained in step S1 with purified water (material-liquid ratio of 1:9), add it to an ultrasonic-microwave extraction tank, add 6% sodium citrate solution to adjust the pH of the system to 5.5, heat to 58°C at 180 rpm, and simultaneously turn on ultrasonic (frequency 25 kHz, power 380 W) and microwave (power 270 W) for 4.2 h of synergistic extraction, filter with a filter cloth with a pore size of 0.2 μm to obtain crude extract of donkey-hide gelatin;

[0068] S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution (the amount of compound enzyme solution is 3% of the mass of the crude extract of donkey-hide gelatin), stir and hydrolyze at 50°C, raise the temperature to 73°C after hydrolysis, keep warm for 22 min, and filter with a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain collagen peptide solution.

[0069] The complex enzyme solution consists of collagenase, papain, and purified water, with collagenase comprising 13% by mass and papain comprising 27% by mass.

[0070] S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 11%, adjust the pH value to 6.5, autoclave at 115℃ for 15 min, inoculate with compound bacterial culture (the inoculation amount of compound bacterial culture is 4% of the mass of the concentrated collagen peptide solution), stir evenly, ferment at 33℃ for 15 h, and then continue fermentation for 5 h with stirring (speed is 90 rpm). After sterilization at 60℃ for 30 min, filter with a 0.45 μm microporous nanofiltration membrane to obtain the donkey-hide gelatin peptide fermentation broth.

[0071] The preparation method of the compound bacterial culture solution is as follows: Freeze-dried *Lactobacillus plantarum* is inoculated into MRS liquid medium and incubated at 39℃ for 22 hours until the viable count is ≥10⁻⁶. 9CFU / mL was used to obtain *Lactobacillus plantarum* solution; freeze-dried *Saccharomyces cerevisiae* was inoculated into YPD liquid medium and cultured at 33℃ in a shaker for 15 h until the viable count was ≥10⁻⁶. 9 CFU / mL was used to obtain the Saccharomyces cerevisiae culture; the Lactobacillus plantarum culture and the Saccharomyces cerevisiae culture were mixed evenly at a volume ratio of 3:1 to obtain the compound culture culture.

[0072] S5: The donkey-hide gelatin peptide fermentation broth obtained in step S4 was atomized in a freezing tower at -50℃ using a two-fluid nozzle (atomization pressure 0.2MPa). It was then dried for 10 hours under vacuum of 10Pa and sublimation temperature of -20℃, followed by a desorption drying at 30℃ for 4 hours. The frozen protein block was then placed in an ultrafine pulverizer and pulverized to 120 mesh at 0℃ using pulsed high-intensity light (energy density 25J / cm²). 2 The mixture is sterilized for 30 seconds with a pulse frequency of 3 Hz, then filled to obtain low-GI donkey-hide gelatin protein powder. The excipients consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 6:4:7, and the amount of the excipients is 6% of the mass of the donkey-hide gelatin peptide fermentation broth.

[0073] Example 5

[0074] S1: After the dehaired donkey hide is washed by high-pressure (pressure 0.4MPa) spraying, it is cut into small pieces of 2cm×2cm, immersed in degreasing solution (material-liquid ratio of 1:9) and ultrasonically treated at 35℃ for 50min (frequency 35kHz, power 200W). After removing the donkey hide pieces, it is rinsed 3 times with 0.7% sodium citrate aqueous solution, and then rinsed 4 times with clean water to obtain degreased donkey hide.

[0075] The defatting solution is prepared as follows: sodium carbonate, lipase, neutral protease, and alkaline protease are added to purified water at 25°C and stirred until homogeneous to obtain the defatting solution; wherein the mass percentage of sodium carbonate is 5%, the mass percentage of lipase is 3%, the mass percentage of neutral protease is 0.03%, and the mass percentage of alkaline protease is 0.02%.

[0076] S2: Mix the defatted donkey hide obtained in step S1 with purified water (material-liquid ratio of 1:9), add it to an ultrasonic-microwave extraction tank, add 6% sodium citrate solution to adjust the pH of the system to 5.5, heat to 58°C at 180 rpm, and simultaneously turn on ultrasonic (frequency 28 kHz, power 360 W) and microwave (power 270 W) for 4.5 h of synergistic extraction, filter with a filter cloth with a pore size of 0.2 μm to obtain crude extract of donkey-hide gelatin;

[0077] S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution (the amount of the compound enzyme solution is 4% of the mass of the crude extract of donkey-hide gelatin), stir and hydrolyze at 52℃, raise the temperature to 73℃ after hydrolysis, keep warm for 22 min, and filter with a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain collagen peptide solution.

[0078] The complex enzyme solution consists of collagenase, papain, and purified water, with collagenase comprising 12% by mass and papain comprising 26% by mass.

[0079] S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 11%, adjust the pH value to 6.3, autoclave at 115℃ for 15 min, inoculate with compound bacterial culture (the inoculation amount of compound bacterial culture is 4% of the mass of the concentrated collagen peptide solution), stir evenly, ferment at 32℃ for 15 h, and then continue fermentation for 5.2 h with stirring (speed is 90 rpm). After sterilization at 60℃ for 30 min, filter with a 0.45 μm microporous nanofiltration membrane to obtain the donkey-hide gelatin peptide fermentation broth.

[0080] The preparation method of the compound bacterial culture solution is as follows: Freeze-dried *Lactobacillus plantarum* is inoculated into MRS liquid medium and cultured at 38℃ for 20 hours until the viable count is ≥10⁻⁶. 9 CFU / mL was used to obtain Lactobacillus plantarum solution; freeze-dried Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 34℃ in a shaker for 15 h until the viable count was ≥10. 9 CFU / mL was used to obtain the Saccharomyces cerevisiae culture; the Lactobacillus plantarum culture and the Saccharomyces cerevisiae culture were mixed evenly at a volume ratio of 3:1 to obtain the compound culture culture.

[0081] S5: The donkey-hide gelatin peptide fermentation broth obtained in step S4 was atomized in a freezing tower at -50℃ using a two-fluid nozzle (atomization pressure 0.2MPa). It was then dried for 10 hours under vacuum of 10Pa and sublimation temperature of -20℃, followed by a desorption drying at 30℃ for 4 hours. The frozen protein block was then placed in an ultrafine pulverizer and pulverized to 120 mesh at 0℃ using pulsed high-intensity light (energy density 25J / cm²). 2 The mixture is sterilized for 30 seconds with a pulse frequency of 3Hz, then filled to obtain low-GI donkey-hide gelatin protein powder. The excipients consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 5:4:8, and the amount of the excipients is 6% of the mass of the donkey-hide gelatin peptide fermentation broth.

[0082] Comparative Example 1

[0083] The processing technology of the donkey-hide gelatin protein powder in this comparative example is similar to that in Example 4. The difference between this comparative example and Example 4 is that step S3 was not performed in this comparative example, that is, the crude extract of donkey-hide gelatin was directly fermented.

[0084] Comparative Example 2

[0085] The processing technology of the donkey-hide gelatin protein powder in this comparative example is similar to that in Example 4. The difference between this comparative example and Example 4 is that an equal amount of papain is used instead of collagenase in step S3 of this comparative example.

[0086] Comparative Example 3

[0087] The processing technology of the donkey-hide gelatin protein powder in this comparative example is similar to that in Example 4. The difference between this comparative example and Example 4 is that step S4 was not performed in this comparative example, that is, collagen peptide liquid was directly freeze-dried.

[0088] Comparative Example 4

[0089] The processing technology of the donkey-hide gelatin protein powder in this comparative example is similar to that in Example 4. The difference between this comparative example and Example 4 is that an equal amount of Saccharomyces cerevisiae is used instead of Lactobacillus plantarum in this comparative example.

[0090] Comparative Example 5

[0091] The processing technology of the donkey-hide gelatin protein powder in this comparative example is similar to that in Example 4. The difference between this comparative example and Example 4 is that the excipients in this comparative example use an equal amount of casein instead of whey protein isolate.

[0092] Test case

[0093] Solubility and sensory test: The donkey-hide gelatin protein powders prepared in Examples 1-5 and Comparative Examples 1-5 were mixed with 60°C warm water at a mass ratio of 1:6 and then their solubility and taste were evaluated.

[0094] Glycemic Index (GI) Test: The donkey-hide gelatin protein powders prepared in Examples 1-5 and Comparative Examples 1-5 were placed in test tubes. 10 mL of 0.2 mol / L CH3COONa buffer (pH = 5.2) was added, and the mixture was stirred and heated in a boiling water bath for 30 min to gelatinize. Then, 0 mL (300 U / mL) of freshly prepared porcine pancreatic α-amylase and 40 μL (10000 U / mL) of saccharifying enzyme were added and mixed thoroughly. The mixture was then reacted with shaking in a water bath at 37°C and 170 rpm. Samples were taken at 0, 30, 60, 90, and 120 min, respectively. The enzymes were inactivated in a boiling water bath for 10 min, and the mixture was centrifuged at 2000 rpm for 5 min. 1 mL of the supernatant was collected, and the glucose content was determined using a glucose kit. The GI calculation formula is: HI = ((initial time point glucose content - sampling time point glucose content) × 0.9) / test sample mass × 100%, GI = 39.21 + 0.803HI 90 .

[0095] The test results are shown in Table 1.

[0096] Table 1 Performance Test Results

[0097]

[0098] As shown in Table 1, the low-GI donkey-hide gelatin protein powder prepared by this invention has a harmonious aroma, a rich milky fragrance, a smooth mouthfeel, and good palatability. It also shows no sedimentation when mixed with warm water, and the GI value is below 55 within 2 hours. This indicates that the low-GI donkey-hide gelatin protein powder prepared by this invention has good palatability and good solubility, which meets the requirements for "low-GI foods" in "T / CNLIC0145-2024 General Technical Requirements for Low Glycemic Index (GI) Foods".

[0099] The above embodiments are merely illustrative of the present invention and are not intended to limit the invention. Those skilled in the art should not modify the above embodiments without departing from the spirit and scope of the present invention. All equivalent modifications or alterations made by those skilled in the art without departing from the technical concept of the present invention are still within the protection scope of the present invention.

Claims

1. A processing technology for low-GI donkey-hide gelatin protein powder, characterized in that, Includes the following steps: S1: After washing the dehaired donkey hide, cut it into pieces, immerse it in a defatting solution for ultrasonic treatment, and wash it to obtain defatted donkey hide; wherein, the defatting solution is prepared by adding sodium carbonate, lipase, neutral protease and alkaline protease to purified water at 20-30℃, stirring evenly to obtain the defatting solution. S2: Mix the defatted donkey hide obtained in step S1 with purified water, and extract it using ultrasound and microwave under stirring. After filtration, obtain crude extract of donkey-hide gelatin. The frequency of ultrasound is 20-30kHz and the power is 300-400W. The power of microwave is 200-300W. The temperature of co-extraction is 55-60℃ and the time of co-extraction is 3-5h. S3: Mix the crude extract of donkey-hide gelatin obtained in step S2 with the compound enzyme solution, stir and hydrolyze at a constant temperature of 45-55℃, raise the temperature to 70-75℃ after hydrolysis, keep warm for 15-25 minutes, and filter with a nanofiltration membrane to obtain collagen peptide solution. S4: Concentrate the collagen peptide solution obtained in step S3 to a protein concentration of 10%-12%, adjust the pH to 6.0-7.0, sterilize by steam, inoculate with the compound inoculum solution, stir evenly, and ferment at a constant temperature of 30-35℃ for 12-16 hours. Then continue fermentation for 4-6 hours under stirring. After sterilization, filter to obtain the donkey-hide gelatin peptide fermentation broth. The preparation method of the compound inoculum solution is as follows: inoculate freeze-dried *Lactobacillus plantarum* in MRS liquid medium and culture at a constant temperature of 37-40℃ for 18-24 hours until the viable cell count is ≥10⁹ CFU / mL to obtain *Lactobacillus plantarum* solution; inoculate freeze-dried *Saccharomyces cerevisiae* in YPD liquid medium and culture at a constant temperature of 30-35℃ in a shaker for 12-16 hours until the viable cell count is ≥10⁹ CFU / mL to obtain *Saccharomyces cerevisiae* solution; mix the *Lactobacillus plantarum* solution and *Saccharomyces cerevisiae* solution at a volume ratio of 3:1 evenly to obtain the compound inoculum solution. S5: Freeze-dry the fermented broth of donkey-hide gelatin peptides obtained in step S4, pulverize it at low temperature, add excipients, sterilize, and fill to obtain low-GI donkey-hide gelatin protein powder.

2. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, In step S1, the defatting solution contains 3%-7% sodium carbonate, 2%-5% lipase, 0.02%-0.06% neutral protease, and 0.01%-0.03% alkaline protease by mass.

3. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, The ultrasonic treatment in step S1 has a frequency of 30-40kHz, a power of 100-300W, a temperature of 30-45℃, and a time of 40-60min.

4. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, The complex enzyme solution described in step S3 consists of collagenase, papain and purified water, with collagenase comprising 10%-15% by mass and papain comprising 20%-30% by mass.

5. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, The amount of the compound enzyme solution used in step S3 is 2%-5% of the mass of the crude extract of donkey-hide gelatin.

6. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, The nanofiltration membrane described in step S3 has a molecular weight cutoff of 300 Da.

7. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, The inoculation amount of the compound bacterial culture in step S4 is 2%-5% of the mass of the concentrated collagen peptide solution.

8. The low-GI donkey-hide gelatin protein powder processing technology according to claim 1, characterized in that, The excipients in step S5 consist of whey protein isolate, casein and soy protein isolate in a mass ratio of 4-8:3-5:6-9, and the amount of the excipients is 4%-7% of the mass of the donkey-hide gelatin peptide fermentation broth.

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