Donkey-hide gelatin purification method and application thereof

Abstract

The invention provides a donkey-hide gelatin purification method and application thereof, and belongs to the technical field of donkey-hide gelatin. The colla corii asini purification method comprises the following steps: crushing colla corii asini slices, mixing the crushed colla corii asini slices with an extracting solution, performing ultrasonic extraction and filtration, adding a compound enzyme into filtrate for enzymolysis, performing centrifugation, filtering supernate through an ultrafiltration membrane, collecting permeate liquid, and performing drying to obtain a colla corii asini extract, the extracting solution comprises L-cysteine, acetic acid and sodium chloride. According to the extracting solution disclosed by the invention, efficient dissolution of insoluble protein in donkey-hide gelatin is realized through synergistic effects of destroying hydrogen bonds in an acidic environment, collapsing hydrophobic cross-linking by L-cysteine and enhancing dissolution stability by salt ions, and active small molecule components are obtained through enzymolysis by compound enzyme. The invention further provides application of the colla corii asini extract and the radix rehmanniae preparata extract in preparation of products for nourishing yin, enriching blood and promoting lactation. Animal experiments show that by compounding the colla corii asini extract and the radix rehmanniae preparata extract, the functions of nourishing yin and enriching blood can be remarkably improved, and lactation is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of donkey-hide gelatin, and particularly relates to a donkey-hide gelatin purification method and application thereof. BACKGROUND

[0002] Donkey-hide gelatin is a solid gelatin made of donkey skin as the main raw material through decoction and concentration, and is a traditional precious Chinese medicinal material and tonifying product. According to traditional Chinese medicine, donkey-hide gelatin has the effects of tonifying blood and yin, moistening dryness and stopping bleeding, and can be used for treating blood deficiency and emaciation, dizziness and palpitation, restlessness and insomnia, and dry cough.

[0003] However, the main component collagen has a large molecular weight and poor solubility, resulting in low bioavailability. The existing extraction methods (such as water boiling and alcohol precipitation) have the following problems: low extraction efficiency, the cross-linked collagen in donkey-hide gelatin is difficult to dissolve in water, and the extraction rate is less than 50% by using conventional methods; activity loss, long-time high-temperature extraction easily destroys small-molecule active peptides (such as hydroxyproline oligopeptides).

[0004] In addition, the traditional donkey-hide gelatin products are mostly used in a single form, and the comprehensive conditioning effect on complex syndromes such as postpartum deficiency of both qi and blood and insufficient lactation is limited. Therefore, it is of great significance to develop a new technology that can improve the extraction effect of active components of donkey-hide gelatin to promote the modernization of traditional Chinese medicine. SUMMARY

[0005] The present application aims to provide a donkey-hide gelatin purification method and application thereof, and the donkey-hide gelatin extract obtained by using the purification method has the effects of nourishing yin and promoting lactation.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a donkey-hide gelatin purification method, which comprises the following steps: crushing donkey-hide gelatin tablets, mixing with an extraction liquid, ultrasonic extraction, adding a compound enzyme into the filtrate for enzymolysis after filtration, centrifugation, filtering the supernatant through an ultrafiltration membrane, collecting the permeate and drying to obtain a donkey-hide gelatin extract; the concentration of L-cysteine in the extraction liquid is 0.1-0.2 mol / L, the volume fraction of acetic acid is 1%-2%, and the concentration of sodium chloride is 0.1-0.2 mol / L.

[0007] Preferably, the crushing is to less than 600 mesh.

[0008] Preferably, the mass-volume ratio of the donkey-hide gelatin to the extraction liquid is 1:(30-40) g / mL.

[0009] Preferably, the ultrasonic extraction is performed at a temperature of 25-30 DEG C, a power of 300-400 W, a frequency of 25-30 kHz, and for 20-30 min.

[0010] Preferably, the complex enzyme is composed of collagen hydrolase and ficin at a weight ratio of (3-7):1, and the complex enzyme is added in an amount of 0.5%-1.5% of the weight of the donkey-hide gelatin.

[0011] More preferably, the enzymolysis of the complex enzyme is performed at a temperature of 35-40 DEG C, a pH value of 6-7, and for 3-5 h.

[0012] Preferably, the ultrafiltration membrane has a molecular weight cut-off of 3-5 kDa and is a hollow fiber ultrafiltration membrane.

[0013] The application further provides a donkey-hide gelatin extract obtained by the above-mentioned donkey-hide gelatin purification method.

[0014] The application further provides an application of the above-mentioned donkey-hide gelatin extract in the preparation of a product for nourishing yin and blood and promoting lactation.

[0015] Preferably, the product comprises the donkey-hide gelatin extract and a rehmannia extract.

[0016] More preferably, the rehmannia extract is obtained by microbial fermentation of rehmannia.

[0017] The application further provides an application of the above-mentioned donkey-hide gelatin extract in the preparation of a product for promoting lactation.

[0018] Compared with the prior art, the application has the following beneficial effects: The application provides a donkey-hide gelatin purification method, which comprises: crushing donkey-hide gelatin tablets, mixing with an extraction liquid, performing ultrasonic extraction, adding a complex enzyme to the filtrate for enzymolysis after filtration, centrifuging, filtering the supernatant through an ultrafiltration membrane, collecting the permeate and drying to obtain a donkey-hide gelatin extract; the extraction liquid has a L-cysteine concentration of 0.1-0.2 mol / L, an acetic acid volume fraction of 1%-2%, and a sodium chloride concentration of 0.1-0.2 mol / L. The extraction liquid of the application realizes efficient dissolution of the insoluble protein in donkey-hide gelatin through the synergistic effect of the destruction of hydrogen bonds in an acidic environment, the chemical cleavage of disulfide bonds by L-cysteine through a reduction reaction, and the enhancement of dissolution stability by salt ions, and the active small molecule components are obtained through the enzymolysis of the complex enzyme.

[0019] The application also provides application of the donkey-hide gelatin extract combined with the rehmannia extract in preparation of a product for nourishing yin and blood and promoting lactation. Animal experiment verification shows that the combination of the donkey-hide gelatin extract and the rehmannia extract can significantly improve the levels of red blood cells, hemoglobin and reticulocytes of blood deficiency model rats, improve the function of nourishing yin and blood; meanwhile, the combination can improve the weight gain rate of the young rats and the lactation amount of the mother rats of the bromocriptine inhibited lactation model rats, and the effect of promoting lactation is significant, and is superior to the donkey-hide gelatin or the rehmannia extract and other process control groups, indicating that the selection of the donkey-hide gelatin extract and the rehmannia extract, and the specific extract, the complex enzyme and the fermentation strain plays a key role in the effect. DETAILED DESCRIPTION

[0020] The application provides a donkey-hide gelatin purification method, which comprises the following steps: crushing donkey-hide gelatin tablets, mixing with an extract, ultrasonic extraction, adding a complex enzyme for enzymolysis after filtration, centrifugation, filtering the supernatant through an ultrafiltration membrane, collecting the permeate and drying to obtain a donkey-hide gelatin extract; the concentration of L-cysteine in the extract is 0.1-0.2 mol / L, the volume fraction of acetic acid is 1%-2%, and the concentration of sodium chloride is 0.1-0.2 mol / L.

[0021] Preferably, the preparation method of the extract comprises the following steps: adding glacial acetic acid into water, stirring at 300-400 rpm for 3-5 min, adding L-cysteine, stirring at 300-400 rpm for 5-10 min, and adding sodium chloride, stirring at 300-400 rpm for 3-5 min.

[0022] The main component of donkey-hide gelatin is collagen, and a large number of disulfide bonds (-S-S-) exist in and between collagen molecules, which form a firm three-dimensional network structure, so that the protein is difficult to be dissolved. In order to improve the solubility of collagen, the L-cysteine in the extract is a high-efficiency reducing agent, which can break the disulfide bonds, so that the tight collagen protein network structure is disintegrated into smaller and more dispersed peptide chain fragments, loses the cross-linking constraint, and is more easily stretched and dissolved in the aqueous solution. The acetic acid reduces the pH of the extract to reach the easy-soluble pH range of collagen, destroys the hydrogen bonds in the collagen molecules, reduces the intermolecular aggregation, and the acidic environment of acetic acid can make the L-cysteine more stable and not easily oxidized by oxygen in the air too early, so that the reducing effect can be more persistent and more effective; the acetic acid increases the polarity of the solution to promote the dissolution of the soluble collagen peptide. The sodium ions and chloride ions dissociated from the sodium chloride in the extract combine with the charged groups on the surface of the donkey-hide gelatin protein to increase the hydrophilicity and the thickness of the solvation layer of the protein molecules, and reduce the precipitation caused by the intermolecular electrostatic attraction; the sodium ions and chloride ions increase the ionic strength of the extract to promote the penetration of the L-cysteine and the acetic acid into the protein and enhance the destruction efficiency of the two on the structure.

[0023] The extract liquid can effectively improve the dissolution rate and solubility of the protein in the donkey-hide gelatin through functional complementation and synergistic effect. The multiple forces are synergistically destroyed: acetic acid destroys hydrogen bonds, L-cysteine chemically breaks disulfide bonds through reduction reaction, and sodium chloride optimizes the ion environment. The three act on different mechanisms of protein stability, forming a comprehensive dissolution promotion system, especially for the cross-linked insoluble protein in the donkey-hide gelatin formed during storage or processing. The single reagent concentration is reduced, and the protein damage is reduced: when the three are used together, the individual concentration of each reagent can be reduced, which can achieve efficient dissolution through synergy, avoid excessive denaturation of protein, peptide bond breakage or inferior flavor and odor caused by high-concentration single reagent (such as high-concentration L-cysteine or acetic acid), and is more conducive to retaining the activity or functionality of the protein in the donkey-hide gelatin. The solution stability is improved: sodium chloride helps to stabilize the charged soluble peptide chains generated after reduction by L-cysteine by adjusting the ionic strength, prevents the soluble peptide chains from combining with each other again to precipitate, and ensures the stability of the extraction system. At the same time, the acidic environment of acetic acid can ensure the uniformity and stability of the extraction system.

[0024] The crushing is to less than 600 mesh, and more preferably to less than 700 mesh. The crushing can significantly increase the specific surface area of the donkey-hide gelatin, reduce the penetration resistance of the extraction liquid, make the L-cysteine, acetic acid and other components in the subsequent extraction liquid more easily contact with the collagen inside the donkey-hide gelatin, lay a foundation for efficient dissolution, and avoid the problem of insufficient extraction caused by coarse particles, and improve the utilization rate of raw materials.

[0025] The mass-to-volume ratio of the donkey-hide gelatin to the extraction liquid is preferably 1: (30-40) g / mL, and more preferably 1:35 g / mL.

[0026] The temperature of the ultrasonic extraction is preferably 25-30℃, more preferably 35℃, the power is preferably 300-400W, more preferably 350W, the frequency is preferably 25-30kHz, more preferably 28kHz, and the time is preferably 20-30min, more preferably 25min. The low temperature (25-30℃) avoids thermal denaturation of collagen, and the optimization of power and frequency balances the dissolution efficiency and protein structure protection (avoiding peptide bond breakage caused by strong ultrasound). Mechanical vibration and micro-flow field are generated through the ultrasonic cavitation effect, the agglomeration structure of the donkey-hide gelatin particles is dispersed, the penetration of the extraction liquid into the protein is accelerated, and the dissolution rate of the soluble protein is improved.

[0027] The composite enzyme of this invention is preferably composed of collagen hydrolase and fig protease in a weight ratio of (3-7):1, more preferably 5:1. The amount of composite enzyme added is preferably 0.5%-1.5% of the weight of the donkey-hide gelatin, more preferably 1%. The enzymatic hydrolysis temperature is preferably 35-40℃, more preferably 37℃, the pH value is preferably 6-7, more preferably 6.5, and the time is preferably 3-5h, more preferably 4h. The collagen hydrolase has an activity of 300,000 U / g and was purchased from Guangzhou Chenyang Biotechnology Co., Ltd.; the fig protease has an activity of 100,000 U / g and was purchased from Guangzhou Longde Biotechnology Co., Ltd. In this invention, the collagen protease in the composite enzyme specifically hydrolyzes the triple helix core structure of collagen (destroying the glycine-proline-hydroxyproline repeat sequence), while the fig protease assists in hydrolyzing the non-helical region and weakly cross-linked regions. Together, they degrade large-molecule collagen into small-molecule active peptides (easier to be purified by subsequent ultrafiltration).

[0028] The ultrafiltration membrane of this invention preferably has a molecular weight cutoff of 3-5 kDa and is made of hollow fiber ultrafiltration membrane. This ultrafiltration membrane retains large molecular impurities larger than 3-5 kDa (such as incompletely hydrolyzed proteins, residual enzyme molecules, and colloidal particles), allowing only small molecule active peptides to permeate through the membrane. This achieves molecular weight fractionation purification, improves the uniformity and activity of the extract, and makes the small molecule active peptides more readily absorbed by the human body.

[0029] The present invention also provides a donkey-hide gelatin extract obtained according to the above-described donkey-hide gelatin purification method.

[0030] The donkey-hide gelatin extract obtained by this invention is rich in active ingredients such as small molecule collagen peptides, retains the traditional efficacy of donkey-hide gelatin, provides high-quality raw materials for the development of targeted products, and clarifies its application value in the fields of nourishing yin and blood, and promoting lactation.

[0031] The present invention also provides an application of the above-mentioned donkey-hide gelatin extract in the preparation of products that nourish yin and blood and promote lactation.

[0032] The product of the present invention preferably includes donkey-hide gelatin extract and prepared rehmannia root extract, wherein the weight ratio of donkey-hide gelatin extract and prepared rehmannia root extract is preferably (2-5):1, more preferably 3:1; and also includes pharmaceutically or health-product-acceptable excipients.

[0033] The preferred method for preparing the Rehmannia glutinosa extract of the present invention includes: pulverizing Rehmannia glutinosa and passing it through a 100-200 mesh sieve to obtain Rehmannia glutinosa powder; adding water at a material-to-liquid ratio of 1:(10-15) g / mL; sterilizing; inoculating 1%-3% of the weight of Rehmannia glutinosa powder with Kluyveromyces yeast; aerobic fermentation at 25-30℃, pH 5.5-6, and oxygen flow rate of 1-2 vvm (using a ring aerator for uniform aeration) for 36-42 h (sterilization not required); turning off the aeration device; introducing high-purity nitrogen (99.99%) into the fermentation system to replace oxygen for 30 min; inoculating 3%-5% of the weight of Rehmannia glutinosa powder with Lactobacillus rhamnosus; anaerobic fermentation at 28-32℃ and pH 6-6.5 for 48-72 h; filtering; sterilizing the filtrate by filtering it through a 0.22 μm filter membrane; and freeze-drying to obtain the Rehmannia glutinosa extract. Among them, Pichia kluyveri CICC is the Kluyveri yeast. ® 32947, Lactobacillus rhamnosus CICC ® 6241, all purchased from the China Industrial Microbial Culture Collection Center; the optimal viability of *Kluyveromyces kluyveromyces* was (4-7) × 10⁻⁶. 8 5 × 10⁻⁶ cells / g, more preferably 5 × 10⁻⁶ cells / g. 8 The optimal viability of *Lactobacillus rhamnosus* is (1-3) × 10⁻⁶ cells / g. 9 pcs / g, more preferably 2×10 9 per g.

[0034] This invention utilizes microbial transformation to convert the effective components of Rehmannia glutinosa from macromolecules to small molecules, thereby improving bioavailability and making its yin-nourishing and lactation-promoting effects more direct. The metabolites (organic acids, polysaccharides, vitamins, etc.) of yeast and lactic acid bacteria synergistically work with the components of Rehmannia glutinosa itself, providing multiple functions including nourishing yin, replenishing blood, clearing the meridians, and regulating the body. It reduces the cloying nature of the formula, lessening the burden on the spleen and stomach, making it suitable for postpartum women who are weak, have yin deficiency, and experience poor spleen and stomach function during lactation. The Rehmannia glutinosa extract prepared through a two-step fermentation process using Kluyveromyces kluyveromyces and Lactobacillus rhamnosus can significantly enhance its comprehensive effects of nourishing yin and moisturizing dryness, replenishing blood and promoting lactation, and clearing the meridians and promoting lactation through component optimization, activity enhancement, and multi-target regulation.

[0035] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] Unless otherwise specified, the following embodiments are all conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] The donkey-hide gelatin tablets used in the examples and comparative examples were purchased from Shandong Dong'e Bainiantang Donkey-hide Gelatin Biological Products Co., Ltd.; the collagen hydrolase activity was 300,000 U / g, purchased from Guangzhou Chenyang Biotechnology Co., Ltd.; the fig protease activity was 100,000 U / g, purchased from Guangzhou Longde Biotechnology Co., Ltd.; the bromelain activity was 100,000 U / g, purchased from Xi'an Yatu Biotechnology Co., Ltd.; and the Kluyveri yeast was Pichia kluyveri CICC. ® 32947, Lactobacillus rhamnosus CICC ® 6241, the brewing yeast is Saccharomyces cerevisiae CICC ® 30245, Bifidobacterium adolescentis (Bifidobacterium adolescentis CICC) ® All 6178 samples were purchased from the China Industrial Microbial Culture Collection Center.

[0039] Example 1 Purification methods of donkey-hide gelatin S1. Preprocessing The donkey-hide gelatin slices were crushed and passed through a 700-mesh sieve to obtain donkey-hide gelatin powder. The powder was mixed with the extract at a material-to-liquid ratio of 1:35 g / mL. The mixture was then ultrasonically extracted for 25 min at a temperature of 28℃, a power of 350W, and a frequency of 28kHz. The mixture was then filtered to obtain the filtrate. The method for preparing the extract is as follows: glacial acetic acid is added to water and stirred at 350 rpm for 4 min; L-cysteine ​​is added and stirred at 350 rpm for 8 min; sodium chloride is added and stirred at 350 rpm for 4 min to obtain the extract. The extract contained 0.15 mol / L of L-cysteine, 1.5% acetic acid (volume fraction), and 0.15 mol / L of sodium chloride.

[0040] S2. Complex enzyme hydrolysis Add 1% of the weight of the compound enzyme of donkey-hide gelatin powder to the filtrate, and enzymatically hydrolyze it at 37℃ and pH 6.5 for 4 hours to inactivate the enzyme. Centrifuge at 4℃ and 8000 rpm for 20 minutes to obtain the supernatant. The complex enzyme consists of collagen hydrolase and fig protease in a weight ratio of 5:1.

[0041] S3. Ultrafiltration membrane filtration The supernatant was filtered using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 4 kDa at a temperature of 30°C and a pressure of 0.2 MPa. The permeate was collected and freeze-dried under vacuum until the water content was 3 wt% to obtain the donkey-hide gelatin extract.

[0042] Example 2 Purification methods of donkey-hide gelatin S1. Preprocessing The donkey-hide gelatin slices were crushed and passed through a 600-mesh sieve to obtain donkey-hide gelatin powder. The powder was mixed with the extract at a material-to-liquid ratio of 1:30 g / mL. The mixture was then ultrasonically extracted for 30 min at a temperature of 25℃, a power of 300W, and a frequency of 25kHz. After filtration, the filtrate was obtained. The preparation method of the extract is as follows: glacial acetic acid is added to water and stirred at 300 rpm for 5 min, L-cysteine ​​is added and stirred at 300 rpm for 10 min, sodium chloride is added and stirred at 300 rpm for 5 min. The extract contained 0.1 mol / L of L-cysteine, 1% acetic acid (volume fraction), and 0.1 mol / L of sodium chloride.

[0043] S2. Complex enzyme hydrolysis Add 0.5% of the weight of the compound enzyme of donkey-hide gelatin powder to the filtrate, and enzymatically hydrolyze it at 35℃ and pH 6 for 5 hours to inactivate the enzyme. Centrifuge at 4℃ and 8000 rpm for 20 minutes to obtain the supernatant. The complex enzyme consists of collagen hydrolase and fig protease in a weight ratio of 3:1.

[0044] S3. Ultrafiltration membrane filtration The supernatant was filtered using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 4 kDa at a temperature of 28°C and a pressure of 0.1 MPa. The permeate was collected and freeze-dried under vacuum to a water content of 4 wt% to obtain donkey-hide gelatin extract.

[0045] Example 3 Purification methods of donkey-hide gelatin S1. Preprocessing The donkey-hide gelatin slices were crushed and passed through an 800-mesh sieve to obtain donkey-hide gelatin powder. The powder was mixed with the extract at a material-to-liquid ratio of 1:40 g / mL. The mixture was then ultrasonically extracted for 20 minutes at a temperature of 30℃, a power of 400W, and a frequency of 30kHz. The mixture was then filtered to obtain the filtrate. The preparation method of the extract is as follows: glacial acetic acid is added to water and stirred at 400 rpm for 3 min, L-cysteine ​​is added and stirred at 400 rpm for 5 min, sodium chloride is added and stirred at 400 rpm for 3 min. The extract contained 0.2 mol / L L-cysteine, 2% acetic acid (volume fraction), and 0.2 mol / L sodium chloride.

[0046] S2. Complex enzyme hydrolysis Add 1.5% of the weight of the compound enzyme of donkey-hide gelatin powder to the filtrate, and enzymatically hydrolyze it at 40℃ and pH 7 for 3 hours to inactivate the enzyme. Centrifuge at 4℃ and 8000 rpm for 20 minutes to obtain the supernatant. The complex enzyme consists of collagen hydrolase and fig protease in a weight ratio of 7:1.

[0047] S3. Ultrafiltration membrane filtration The supernatant was filtered using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 4 kDa at a temperature of 32°C and a pressure of 0.3 MPa. The permeate was collected and freeze-dried under vacuum until the water content was 2 wt% to obtain donkey-hide gelatin extract.

[0048] Example 4 Preparation of Yin-nourishing and lactation-promoting composition (1) Preparation of Rehmannia glutinosa extract Rehmannia glutinosa powder was pulverized and passed through a 150-mesh sieve. Water was added at a material-to-liquid ratio of 1:12 g / mL. After sterilization, 2% (by weight) of Kluyveromyces yeast was inoculated into the powder. Aerobic fermentation was carried out at 28℃, pH 5.8, and an oxygen flow rate of 1.5 vvm (using a ring aerator for uniform aeration) for 40 hours. The aeration device was then turned off, and high-purity nitrogen (99.99%) was introduced into the fermentation system to replace the oxygen for 30 minutes. Then, 4% (by weight) of Lactobacillus rhamnosus was inoculated into the powder. Anaerobic fermentation was carried out at 30℃ and pH 6.2 for 60 hours. The mixture was filtered, and the filtrate was sterilized by filtration through a 0.22 μm filter membrane. The filtrate was then freeze-dried under vacuum until the water content reached 3 wt%, yielding the Rehmannia glutinosa extract. The activity of *Pichia pastoris* was 5 × 10⁻⁶. 8 The bacterial activity of *Lactobacillus rhamnosus* was 2 × 10⁶ cells / g. 9 per g.

[0049] (2) Preparation of Yin-nourishing and lactation-promoting composition The extract of donkey-hide gelatin from Example 1 and the extract of Rehmannia glutinosa were mixed at a weight ratio of 3:1 to obtain a yin-nourishing and lactation-promoting composition.

[0050] Example 5 Preparation of Yin-nourishing and lactation-promoting composition (1) Preparation of Rehmannia glutinosa extract The prepared Rehmannia root was pulverized and passed through a 100-mesh sieve to obtain prepared Rehmannia powder. Water was added at a material-to-liquid ratio of 1:10 g / mL. After sterilization, 1% of the weight of the prepared Rehmannia powder was inoculated with Kluyveromyces yeast. Aerobic fermentation was carried out at 25℃, pH 5.5, and oxygen flow rate of 1 vvm (uniform aeration using a ring aerator) for 42 hours. After the aeration device was turned off, high-purity nitrogen (99.99%) was introduced into the fermentation system to replace the oxygen for 30 minutes. Then, 3%-5% of the weight of prepared Rehmannia powder was inoculated with Lactobacillus rhamnosus. Anaerobic fermentation was carried out at 28℃ and pH 6 for 72 hours. After filtration, the filtrate was filtered through a 0.22μm filter membrane for sterilization and then freeze-dried under vacuum to a water content of 3wt% to obtain the prepared Rehmannia extract. The activity of *Pichia pastoris* was 7 × 10⁻⁶. 8 The bacterial activity of *Lactobacillus rhamnosus* was 3 × 10⁶ cells / g. 9 per g.

[0051] (2) Preparation of Yin-nourishing and lactation-promoting composition The extracts of donkey-hide gelatin and Rehmannia glutinosa from Example 2 were mixed at a weight ratio of 2:1 to obtain a yin-nourishing and lactation-promoting composition.

[0052] Example 6 Preparation of Yin-nourishing and lactation-promoting composition (1) Preparation of Rehmannia glutinosa extract Rehmannia glutinosa powder was pulverized and passed through a 200-mesh sieve. Water was added at a material-to-liquid ratio of 1:15 g / mL. After sterilization, 3% (by weight) of Kluyveromyces oryzae (by weight of the Rehmannia glutinosa powder) was inoculated into the system. Aerobic fermentation was carried out at 30℃, pH 6, and an oxygen flow rate of 2 vvm (using a ring aerator for uniform aeration) for 36 hours. The aeration device was then turned off, and high-purity nitrogen (99.99%) was introduced into the fermentation system to replace the oxygen for 30 minutes. Then, 5% (by weight of the Rehmannia glutinosa powder) of Lactobacillus rhamnosus was inoculated into the system. Anaerobic fermentation was carried out at 32℃ and pH 6.5 for 48 hours. The mixture was filtered, and the filtrate was sterilized by filtration through a 0.22 μm filter membrane. The filtrate was then freeze-dried under vacuum until the water content reached 4 wt%, yielding the Rehmannia glutinosa extract. Among them, the activity of Pichia kluivekia was 4×10⁻⁶. 8 CFU / g, Lactobacillus rhamnosus activity 1×10⁻⁶ 9 per g.

[0053] (2) Preparation of Yin-nourishing and lactation-promoting composition The extracts of donkey-hide gelatin and Rehmannia glutinosa from Example 3 were mixed at a weight ratio of 5:1 to obtain a yin-nourishing and lactation-promoting composition.

[0054] Comparative Example 1 Unlike Example 1, the preparation method of the extract in step S1 is as follows: anhydrous citric acid is added to water and stirred at 350 rpm for 8 min, and sodium citrate is added and stirred at 350 rpm for 12 min; the concentration of citric acid in the extract is 0.1 mol / L and the concentration of sodium citrate is 0.1 mol / L.

[0055] Comparative Example 2 Unlike Example 1, L-cysteine ​​in the extract of step S1 was discarded. The extract was prepared by adding glacial acetic acid to water and stirring at 350 rpm for 4 min, then adding sodium chloride and stirring at 350 rpm for 4 min to obtain the extract. The volume fraction of acetic acid in the extract was 1.5%, and the concentration of sodium chloride was 0.15 mol / L.

[0056] Comparative Example 3 Unlike Example 1, S2. The enzymatic hydrolysis of the compound enzyme is as follows: 1% of the weight of the compound enzyme of donkey-hide gelatin powder is added to the filtrate, and the mixture is enzymatically hydrolyzed at 37°C and pH 6.5 for 4 hours to inactivate the enzyme. The mixture is then centrifuged at 4°C and 8000 rpm for 20 minutes to obtain the supernatant. The complex enzyme is composed of collagen hydrolase and bromelain in a weight ratio of 5:1.

[0057] Comparative Example 4 The difference from Example 4 is that the donkey-hide gelatin extract of Example 1 was replaced with the donkey-hide gelatin extract of Comparative Example 1.

[0058] Comparative Example 5 The difference from Example 4 is that the donkey-hide gelatin extract of Example 1 was replaced with the donkey-hide gelatin extract of Comparative Example 2.

[0059] Comparative Example 6 The difference from Example 4 is that the donkey-hide gelatin extract of Example 1 was replaced with the donkey-hide gelatin extract of Comparative Example 3.

[0060] Comparative Example 7 The difference from Example 4 is that step (1) is as follows: the prepared Rehmannia powder is pulverized and passed through a 150-mesh sieve to obtain prepared Rehmannia powder. Water is added at a material-to-liquid ratio of 1:12 g / mL. After sterilization, 2% of the weight of prepared Rehmannia powder is inoculated with brewer's yeast. After aerobic fermentation for 40 hours at 28°C, pH 5.8, and oxygen flow rate of 1.5 vvm (uniform aeration using a ring aerator), the aeration device is turned off, and high-purity nitrogen (99.99%) is introduced into the fermentation system to replace oxygen for 30 minutes. Then, 4% of the weight of prepared Rehmannia powder is inoculated with Bifidobacterium adolescentis. After anaerobic fermentation for 60 hours at 30°C and pH 6.2, the mixture is filtered. The filtrate is filtered and sterilized using a 0.22 μm filter membrane and then freeze-dried under vacuum until the water content is 3 wt% to obtain prepared Rehmannia extract. The activity of the brewer's yeast was 5×10⁻⁶. 8 The bacterial activity of Bifidobacterium adolescentis was 2 × 10⁶ cells / g.9 per g.

[0061] Experimental Example 1 Animal experiments on nourishing yin and replenishing blood SPF-grade female SD rats, weighing 200±20g (8 weeks old), were selected and acclimatized for one week in an environment with a temperature of 22±2℃, humidity of 50±5%, 12h light-dark cycle, and free access to food and water (standard feed + purified water).

[0062] The animals were divided into 13 groups: blank control group, model group, positive control group and experimental group (Example 1 group, Rehmannia glutinosa extract group (Rehmannia glutinosa extract from step (1) of Example 4), Example 4 group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, comparative example 5 group, comparative example 6 group and comparative example 7 group), with 10 animals in each group.

[0063] Except for the blank control group, rats in other groups were subcutaneously injected with acetylphenylhydrazine on days 1, 3, and 5. The injection dose on day 1 was 40 mg / kg, and the injection dose on days 3 and 5 was 20 mg / kg. On days 2, 4, 6, and 7, rats were intraperitoneally injected with cyclophosphamide at a dose of 25 mg / kg. The blank control group was injected with an equal volume of physiological saline.

[0064] On day 8, at 9 a.m. each day, the experimental group was administered the corresponding donkey-hide gelatin extract / rehmannia extract / combination by gavage at a dose of 500 mg / kg. The blank control group and the model group were administered an equal volume of physiological saline by gavage. The positive control group was administered Jitai'an Siwu Granules by gavage at a dose of 150 mg / kg. The gavage was continued for 7 days.

[0065] Sixty minutes after administration on day 14, blood was collected from the orbital cavity of rats in each group to obtain peripheral blood. The percentages of red blood cells (RBC), hemoglobin (HGB), and reticulocytes (RET) in the peripheral blood of rats in each group were measured using a fully automated blood cell analyzer. The specific results are shown in Table 1.

[0066] Table 1. Peripheral blood RBC, HGB, and RET levels in rats of each group

[0067] Table 1 shows that the RBC, HGB, and RET levels in the model group were significantly lower than those in the blank group, consistent with the characteristics of the blood deficiency model, indicating successful model establishment. The RBC, HGB, and RET levels in the Example 4 group were close to those in the positive control group and superior to those in the Example 1 group (donkey-hide gelatin extract alone) and the Rehmannia glutinosa extract group (Rehmannia glutinosa extract alone), demonstrating the synergistic effect of donkey-hide gelatin extract and Rehmannia glutinosa extract in nourishing Yin and replenishing blood. The RBC, HGB, and RET levels in Comparative Examples 1, 2, and 3 were all lower than those in Example 1, and the RBC, HGB, and RET levels in Comparative Examples 4-6 were also lower than those in Example 4, indicating that selecting appropriate extracts and compound enzymes has a positive effect on ensuring the Yin-nourishing and blood-replenishing effects of donkey-hide gelatin extract. The RBC, HGB, and RET levels in Comparative Example 7 were lower than those in Example 4, indicating that the fermentation combination of *Pichia pastoris* and *Lactobacillus rhamnosus* is more conducive to the generation of active ingredients in the Rehmannia glutinosa extract.

[0068] Experimental Example 2 Lactating animal experiments SPF-grade healthy pregnant SD rats, weighing 250±30g, were selected at 16-18 days of gestation. They were kept in an environment with a temperature of 22±2℃, humidity of 50±5%, 12h light-dark cycle, and free access to food and water (standard feed + purified water). They were fed and watered separately during pregnancy. After parturition, the number of pups in each litter was randomly adjusted to 6.

[0069] The rats were divided into 13 groups: blank control group, model group, positive control group and experimental group (Example 1 group, Rehmannia glutinosa extract group (Rehmannia glutinosa extract from step (1) of Example 4), Example 4 group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, comparative example 5 group, comparative example 6 group and comparative example 7 group). Each group contained 10 lactating rats. There were no significant differences in health status and weight among the rats in each group.

[0070] Except for the blank control group, rats in other groups were injected subcutaneously with bromocriptine at a dose of 1 mg / kg every day for 3 consecutive days starting from the 3rd day after delivery, while the blank control group was injected with an equal volume of physiological saline.

[0071] Meanwhile, starting from the 3rd day after delivery, at 9:00 AM every day, the experimental group was administered the corresponding donkey-hide gelatin extract / rehmannia extract / combination at a dose of 500 mg / kg by gavage, the blank control group and the model group were administered the same volume of physiological saline by gavage, and the positive control group was administered Baiyunfeng Compound Wangbuliuxing Tablets by gavage at a dose of half a tablet per animal, for 7 consecutive days.

[0072] The weight of each litter of pups was measured on day 3 postpartum (M0) and day 7 postpartum (M1) after gavage, and the weight gain rate of each litter of pups in each group was calculated.

[0073] Weight gain rate (%) = (M1-M0) / M0×100%.

[0074] The amount of milk secreted by each group of lactating rats was recorded on days 3 and 7 after gavage.

[0075] The method for detecting single-time milk yield was as follows: pups were separated from their mothers at 10:00 AM, 2:00 PM, and 6:00 PM, and placed in clean cages at 32°C. After fasting for 2 hours (emptying stomach contents), the weight of each pup was recorded (M2). The pups were then returned to their mothers and allowed to suckle for 30 minutes in a quiet environment at 25°C. Immediately after nursing, the pups were removed, and the weight of each pup was re-weighed (M3). The single-time milk yield (g) of each pup was calculated as M3 - M2. The single-time milk yields of 8 pups in each group were then added together to obtain the single-time milk yield of each lactating rat. Measurements were taken once a day at 10:00 AM, 2:00 PM, and 6:00 PM, and the average value was taken.

[0076] The weight gain rate of each group of young rats and the amount of milk secreted per lactating rat in a single feeding are shown in Table 2.

[0077] Table 2. Weight gain rate and single milk yield of each group of juvenile mice

[0078] Table 2 shows that the weight gain rate and milk production on day 7 of the model group were significantly lower than those of the control group, indicating that bromocriptine effectively inhibited lactation and the hypogalactia model was successfully established. The weight gain rate and single-time milk production of the mice in Example 4 were close to those of the positive control group and superior to those of Example 1 (extracted donkey-hide gelatin alone) and the Rehmannia glutinosa extract group (extracted Rehmannia glutinosa alone), demonstrating the synergistic effect of the extracted donkey-hide gelatin and Rehmannia glutinosa extract in improving lactation. The weight gain rate and single-time milk production of mice in Comparative Examples 1, 2, and 3 were all lower than those in Example 1, and the data for Comparative Examples 4-6 were also lower than those in Example 4, indicating that selecting appropriate extracts and compound enzymes has a positive effect on ensuring lactation by the extracted donkey-hide gelatin. The weight gain rate and single-time milk production of mice in Comparative Example 7 were lower than those in Example 4, indicating that the fermentation combination of Kluyveromyces oryzae and Lactobacillus rhamnosus is more conducive to the generation of lactation-promoting active components by the Rehmannia glutinosa extract.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for purifying donkey-hide gelatin, characterized in that, The process includes the following steps: crushing the donkey-hide gelatin slices, mixing them with the extract, extracting them by ultrasound, filtering them, adding a compound enzyme to the filtrate for enzymatic hydrolysis, centrifuging them, filtering the supernatant through an ultrafiltration membrane, collecting the permeate and drying it to obtain the donkey-hide gelatin extract. The extract contains 0.1-0.2 mol / L of L-cysteine, 1%-2% of acetic acid, and 0.1-0.2 mol / L of sodium chloride.

2. The method for purifying donkey-hide gelatin according to claim 1, characterized in that, The material is pulverized to a fineness below 600 mesh.

3. The method for purifying donkey-hide gelatin according to claim 1, characterized in that, The mass-to-volume ratio of the donkey-hide gelatin to the extract is 1:(30-40)g / mL.

4. The method for purifying donkey-hide gelatin according to claim 1, characterized in that, The ultrasonic extraction temperature is 25-30℃, the power is 300-400W, the frequency is 25-30kHz, and the time is 20-30min.

5. The method for purifying donkey-hide gelatin according to claim 1, characterized in that, The compound enzyme is composed of collagen hydrolase and fig protease in a weight ratio of (3-7):1, and the amount of compound enzyme added is 0.5%-1.5% of the weight of the donkey-hide gelatin.

6. The method for purifying donkey-hide gelatin according to claim 1 or 5, characterized in that, The enzymatic hydrolysis of the compound enzyme is carried out at a temperature of 35-40℃, a pH value of 6-7, and a time of 3-5 hours.

7. A donkey-hide gelatin extract obtained by the donkey-hide gelatin purification method according to any one of claims 1-6.

8. The application of the donkey-hide gelatin extract according to claim 7 in the preparation of products that nourish yin and blood and promote lactation.

9. The application according to claim 8, characterized in that, The products include donkey-hide gelatin extract and Rehmannia glutinosa extract.

10. The application according to claim 9, characterized in that, The Rehmannia glutinosa extract is obtained by microbial fermentation of Rehmannia glutinosa.

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