Preparation method of crocodile hydrolyzed protein zinc skin care product

Abstract

The invention discloses a preparation method of a crocodile hydrolyzed protein zinc skin care product, and relates to the technical field of skin care. The method comprises the following steps: step a, pretreating raw materials: taking fresh crocodile meat and bones, cleaning, mechanically crushing until the particle size is less than 5mm, and adding water according to the ratio of 1: (3-5) to prepare homogenate; step b, performing three-step enzymolysis, namely performing enzymolysis by adopting alkaline protease, flavourzyme and zinc chelating enzyme in sequence; according to the constructed technology of three-step enzymolysis and three-stage membrane separation, accurate preparation of crocodile protein zinc is achieved, and the zinc chelating rate is increased to 90% or above through the gradient effect of alkaline protease, flavourzyme and zinc chelating enzyme and directional release of zinc binding sites and is remarkably higher than that of a traditional single-enzyme hydrolysis technology; meanwhile, a combined purification technology of a ceramic membrane, an ultrafiltration membrane and a nanofiltration membrane is adopted to replace chemical precipitation and organic solvent extraction, so that the completeness of active ingredients is reserved, the risk of environmental pollution is reduced, and the method conforms to the green manufacturing trend of food industry.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care, and particularly to a preparation method of an alligator hydrolyzed protein zinc skin care product. Background Art

[0002] Zinc is an essential trace element for the human body, participating in the regulation of the activities of multiple enzymes, the maintenance of immune function and protein synthesis. It plays an important role especially in the growth and development of children, the nutritional supplement of pregnant women and the antioxidant needs of middle-aged and elderly people. Currently, commercially available zinc supplements are mainly divided into inorganic zinc (such as zinc sulfate, zinc oxide) and organic zinc (such as zinc gluconate, zinc glycinate). Although inorganic zinc has a low cost, it has defects such as strong gastrointestinal irritation, low bioavailability (absorption rate is about 15 - 20%), and easy formation of precipitates with food components (such as phytic acid); although organic zinc has improved solubility and safety, the stability of its chelated form is insufficient, and the traditional chemical chelation method is prone to introducing organic solvent residues, making it difficult to meet the requirements of "green processes" for high-end foods.

[0003] Animal-derived protein zinc has become a research hotspot in recent years due to its advantages such as good biocompatibility and natural matching of zinc ion binding sites. Alligator muscle and bone are rich in collagen, elastin and natural zinc elements. The histidine and cysteine residues in its protein structure can form stable coordination bonds with zinc ions. In theory, a protein zinc complex with high biological activity can be prepared. However, traditional enzymatic hydrolysis processes (such as single-enzyme hydrolysis) are difficult to specifically cleave the specific peptide segments in alligator protein that bind to zinc ions, resulting in a low zinc chelation rate (generally <70%). In addition, the subsequent purification process relies on acid-base precipitation or organic solvent extraction, which is easy to damage active ingredients and cause environmental pollution. Moreover, when making chewable tablets with alligator hydrolyzed protein zinc, there are problems such as low biological activity of animal protein zinc, insufficient zinc chelation efficiency, organic solvent residues in the traditional chemical purification process, and damage to active ingredients.

[0004] Therefore, we provide a preparation method of an alligator hydrolyzed protein zinc skin care product to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an alligator hydrolyzed protein zinc skin care product, which solves the problems of low biological activity of animal protein zinc, insufficient zinc chelation efficiency, organic solvent residues in the traditional chemical purification process, and damage to active ingredients in the prior art through the cooperation of a directional three-step enzymatic hydrolysis technology and a three-stage membrane separation and purification process.

[0006] To solve the above technical problems, the present invention is realized through the following technical solutions: The present invention relates to a preparation method of an alligator hydrolyzed protein zinc skin care product, which comprises the following steps: Step a: Raw material pretreatment: Take fresh alligator meat and bones, wash them, and mechanically crush them to a particle size < 5 mm, and prepare a homogenate by adding water in a ratio of 1:3 to 5; Step b: Three-step enzymatic hydrolysis: Sequentially use alkaline protease, flavor protease, and zinc chelating enzyme for enzymatic hydrolysis, with a single-stage enzymatic hydrolysis time of 2 - 4 h and a total hydrolysis degree ≥ 25%; Step c: Fine purification: Remove macromolecular impurities through a ceramic membrane → Ultrafiltration to retain active peptide segments → Nanofiltration to desalt and concentrate the peptide-zinc complex, and combine with D301 resin to remove heavy metals to obtain a hydrolyzate with a zinc chelation rate ≥ 90%; Step d: Preparation of the preparation: Concentrate the purified solution under reduced pressure to a solid content of 40 - 50%, mix it with auxiliary materials such as microcrystalline cellulose, lactose, fructooligosaccharide, and magnesium stearate, granulate, dry, and then press tablets. The tableting pressure is 8 - 15 kN to obtain a chewable tablet containing 10 - 30 mg of zinc per tablet.

[0007] The present invention is further configured such that the zinc chelating enzyme is a zinc-dependent metalloprotease, and the hydrolysis degree is monitored in real time during the enzymatic hydrolysis process. By adjusting the enzyme addition amount and temperature, the proportion of polypeptides with a molecular weight < 1 kDa in the product is ≥ 60%.

[0008] The present invention is further configured such that the auxiliary materials further include a disintegrant cross-linked carboxymethylcellulose sodium (1 - 3%) and a flavoring agent natural vanilla essence (0.1 - 0.5%), and the total mass of the auxiliary materials accounts for 30 - 50% of the chewable tablet.

[0009] The present invention is further configured such that the chewable tablet comprises the following components: Alligator hydrolyzed protein zinc active ingredient: 30 - 50% (zinc content 5 - 15%, zinc chelation rate ≥ 90%); Filler: Microcrystalline cellulose 20 - 30%, lactose 15 - 25%; Functional auxiliary material: Fructooligosaccharide 5 - 10%, cross-linked carboxymethylcellulose sodium 1 - 3%; Lubricant: Magnesium stearate 0.5 - 1%; Flavoring agent: Natural vanilla essence 0.1 - 0.5%; The tablet hardness is 40 - 60 N, the disintegration time ≤ 15 min, and the dissolution degree (in a pH 6.8 buffer solution at 37 °C) ≥ 85% (30 min).

[0010] The present invention is further configured such that the alligator hydrolyzed protein zinc contains hydrophobic amino acid residues of Pro and Leu, accounting for 30 - 40% of the total amino acids, and has antioxidant activity (ORAC value ≥ 800 μmol TE / g).

[0011] The present invention is further configured such that the zinc exists in the form of polypeptide-chelated zinc. Verified by the Caco-2 cell model, compared with zinc sulfate, its small intestine absorption rate is increased by 40 - 60%.

[0012] The present invention is further configured such that in the prepared chewable tablets containing alligator hydrolyzed protein zinc, the alligator hydrolyzed protein zinc has zinc chelation properties, enhances the bioavailability of zinc through a polypeptide carrier, reduces irritation to the gastrointestinal tract, and is suitable for people in need of efficient zinc supplementation.

[0013] The present invention is further configured such that the chewable tablets release zinc-binding sites directionally through a three-step enzymatic hydrolysis process, making the concentration of free zinc ions in the product ≤ 5%, and avoiding the oxidative side effects of inorganic zinc.

[0014] A composite functional chewable tablet composition includes prepared alligator hydrolyzed protein zinc as the active ingredient, adding vitamin C (5 - 10%) and yeast β-glucan (2 - 5%). Through the synergistic effect of zinc and polypeptides, it promotes the proliferation of T lymphocytes.

[0015] A chewable tablet with intestinal protection function includes prepared alligator hydrolyzed protein zinc. The small peptides (<1 kDa) in the alligator hydrolyzed protein zinc account for ≥ 60% and can be directly absorbed through the intestinal oligopeptide transporter PEPT1. Compared with macromolecular proteins, the absorption rate is increased by more than 2 times.

[0016] The present invention has the following beneficial effects: 1. The "three-step enzymatic hydrolysis + three-stage membrane separation" process constructed by the present invention realizes the precise preparation of alligator protein zinc. Through the gradient action of alkaline protease, flavor protease, and zinc chelating enzyme, zinc-binding sites are released directionally, and the zinc chelation rate is increased to more than 90%, significantly higher than the traditional single-enzyme hydrolysis process. At the same time, the combined purification technology of ceramic membrane, ultrafiltration membrane, and nanofiltration membrane is used to replace chemical precipitation and organic solvent extraction, which not only retains the integrity of active ingredients but also reduces the risk of environmental pollution, meeting the trend of green manufacturing in the food industry.

[0017] 2. The present invention clearly defines key indicators, such as the proportion of polypeptides with a molecular weight <1 kDa ≥ 60%, the proportion of zinc chelation form ≥ 90%, combined with the proportion of hydrophobic amino acids (≥ 35%) and antioxidant activity (ORAC value ≥ 800 μmol TE / g), ensuring the efficient absorption and biological functions of protein zinc. At the formulation level, by optimizing the excipient formula (microcrystalline cellulose, fructooligosaccharide, etc.) and process parameters (granulation particle size, tableting pressure), indicators such as tablet hardness, disintegration time, and dissolution degree are superior to industry standards, ensuring the quality stability of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments will be briefly introduced below.

[0019] Figure 1 It is the overall flowchart of a preparation method for an alligator hydrolyzed protein zinc skin care product; Figure 2 It is a flowchart of raw material pretreatment in the preparation method of a crocodile hydrolyzed protein zinc skin care product; Figure 3 It is a flowchart of three-step enzymatic hydrolysis in the preparation method of a crocodile hydrolyzed protein zinc skin care product.

[0020] Figure 4 It is a flowchart of fine purification in the preparation method of a crocodile hydrolyzed protein zinc skin care product.

[0021] Figure 5 It is a flowchart of preparation of preparations in the preparation method of a crocodile hydrolyzed protein zinc skin care product. Specific embodiments

[0022] Next, the technical solutions in the embodiments of the present invention will be described in conjunction with the accompanying drawings in the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0023] Example 1 Please refer to Figure 1 , a preparation method of a crocodile hydrolyzed protein zinc skin care product, comprising the following steps: Step a: Raw material pretreatment: Take fresh crocodile meat and bones, wash and mechanically crush them to a particle size <5 mm, and prepare a homogenate by adding water at a ratio of 1:3 - 5 (w / v); Step b: Three-step enzymatic hydrolysis: Sequentially use alkaline protease (pH 8 - 9, 50 °C, enzyme addition amount 1 - 3%), flavor protease (pH 6 - 7, 45 °C, enzyme addition amount 1 - 2%), and zinc chelating enzyme (pH 7 - 8, 55 °C, enzyme addition amount 0.5 - 1.5%) for enzymatic hydrolysis. The single-stage enzymatic hydrolysis time is 2 - 4 h, and the total hydrolysis degree ≥ 25%; Step c: Fine purification: Remove macromolecular impurities through a ceramic membrane (50 nm) → Ultrafiltration (10 kDa) to retain active peptide segments → Nanofiltration (200 Da) for desalination and concentration of the peptide-zinc complex, and combine with D301 resin to remove heavy metals to obtain a hydrolysis solution with a zinc chelation rate ≥ 90%; Step d: Preparation of preparations: Concentrate the purified solution under reduced pressure to a solid content of 40 - 50%, mix it with excipients [microcrystalline cellulose (20 - 30%), lactose (15 - 25%), fructooligosaccharide (5 - 10%), magnesium stearate (0.5 - 1%)], granulate (particle size 0.5 - 1 mm), dry (40 - 50 °C, water content ≤ 5%), and then press tablets. The tableting pressure is 8 - 15 kN to obtain chewable tablets containing 10 - 30 mg of zinc per tablet.

[0024] Zinc chelatase is a zinc-dependent metalloprotease. During the enzymatic hydrolysis process, the degree of hydrolysis is monitored in real time. By adjusting the enzyme addition amount and temperature, the proportion of polypeptides with a molecular weight < 1 kDa in the product is ≥ 60%. The excipients also include croscarmellose sodium (1 - 3%) as a disintegrant and natural vanilla essence (0.1 - 0.5%) as a flavoring agent. The total mass of the excipients accounts for 30 - 50% of the chewable tablets. The chewable tablets contain the following components: active ingredient of crocodile hydrolyzed protein zinc: 30 - 50% (zinc content 5 - 15%, zinc chelation rate ≥ 90%); filler: microcrystalline cellulose 20 - 30%, lactose 15 - 25%; functional excipient: fructooligosaccharide 5 - 10%, croscarmellose sodium 1 - 3%; lubricant: magnesium stearate 0.5 - 1%; flavoring agent: natural vanilla essence 0.1 - 0.5%; the hardness of the tablets is 40 - 60 N, the disintegration time ≤ 15 min, and the dissolution rate (in 37°C, pH 6.8 buffer solution) ≥ 85% (30 min). Crocodile hydrolyzed protein zinc contains hydrophobic amino acid residues such as Pro and Leu, accounting for 30 - 40% of the total amino acids, and has antioxidant activity (ORAC value ≥ 800 μmol TE / g). Zinc exists in the form of polypeptide-chelated zinc. Verified by the Caco-2 cell model, compared with zinc sulfate, its small intestine absorption rate is increased by 40 - 60%.

[0025] In the preparation of chewable tablets with crocodile hydrolyzed protein zinc, crocodile hydrolyzed protein zinc has zinc chelation characteristics, improves the bioavailability of zinc through polypeptide carriers, reduces irritation to the gastrointestinal tract, and is suitable for people who need to supplement zinc efficiently. The chewable tablets release zinc binding sites directionally through a three-step enzymatic hydrolysis process, making the concentration of free zinc ions in the product ≤ 5% and avoiding the oxidation side effects of inorganic zinc.

[0026] A composite functional chewable tablet composition includes the prepared crocodile hydrolyzed protein zinc as the active ingredient, adding vitamin C (5 - 10%) and yeast β-glucan (2 - 5%). Through the synergistic effect of zinc and polypeptides, it promotes the proliferation of T lymphocytes.

[0027] A chewable tablet with intestinal protection function includes the prepared crocodile hydrolyzed protein zinc. The proportion of small peptides (< 1 kDa) in crocodile hydrolyzed protein zinc is ≥ 60%, which can be directly absorbed through the intestinal oligopeptide transporter PEPT1. Compared with macromolecular proteins, the absorption rate is increased by more than 2 times Example 2 Please refer to Figures 2 - 4 , a preparation method of a crocodile hydrolyzed protein zinc skin care product, including the following steps: Raw material pretreatment: fresh Nile crocodile (Crocodylus niloticus) muscle and bones were selected, and after blood and fat treatment, they were crushed to a particle size of ≤3 mm using a double-blade crusher, deionized water was added at a solid-liquid ratio of 1:4 (w / v), and homogenized using a high-speed homogenizer (10000 rpm, 5 min); Three-step enzymatic hydrolysis: ① The first stage: adjust the pH of the homogenate to 8.5, add 2% of the substrate mass of alkaline protease (Novozymes Alcalase 2.4L), and enzymolyze in a 50℃ water bath for 3 hours until the degree of hydrolysis (DH) reaches 15-20%; ② The second stage: cool down to 45℃, adjust the pH to 6.5, add 1.5% flavor protease (Flavourzyme500MG), and perform enzymolysis for 2h to remove bitter peptides; ③The third stage: raise the temperature to 55°C, adjust the pH to 7.5, add 1% zinc chelatase (derived from Bacillus subtilis, EC3.4.24.23), direct enzymatic hydrolysis for 2 hours, monitor DH ≥ 25% in real time, and release zinc ion binding sites; Fine purification: ① Inactivate the enzyme: incubate in 85℃ water bath for 10min to terminate the enzyme activity, and centrifuge (4000rpm, 20min) to remove the residue; ②Membrane separation: 50nm ceramic membrane (cross-flow velocity 3m / s, operating pressure 0.2MPa) to remove macromolecular impurities → 10kDa ultrafiltration membrane (molecular weight cutoff 10000Da) to enrich active peptides → 200Da nanofiltration membrane (operating pressure 1.5MPa) to desalt and concentrate, so that the zinc chelation rate is ≥92%; ③ Ion exchange: D301 macroporous weakly alkaline anion resin (column diameter-to-height ratio 1:5, flow rate 2BV / h) is used to adsorb heavy metal ions to make the lead and arsenic content ≤0.1mg / kg; Preparation of formulations: ① Concentration and drying: The purified solution was concentrated under reduced pressure (60°C, vacuum degree -0.08MPa) to a solid content of 45%, and spray-dried (inlet temperature 180°C, outlet temperature 85°C) to obtain protein zinc powder; ② Mixed tableting: Mix protein zinc powder and excipients [microcrystalline cellulose 25%, lactose 20%, oligofructose 8%, cross-linked carboxymethyl cellulose sodium 2%, magnesium stearate 0.8%, natural vanilla flavor 0.3%] through an 80-mesh sieve, granulate using a swing granulator (sieve mesh size 1 mm), air-dried at 50°C to a moisture content of ≤4%, and tableted using a rotary tablet press (tabletting pressure 12 kN, die diameter 8 mm) to obtain round convex tablets with a single tablet weight of 0.5 g and a zinc content of 20 mg.

[0028] The enzymatic specificity of zinc chelatase is to preferentially cleave peptide bonds containing histidine and cysteine residues, resulting in a molecular weight distribution of zinc-containing polypeptides in the product as follows: <1 kDa accounts for 65 - 75%, 1 - 3 kDa accounts for 20 - 25%, and ≥3 kDa < 5%. During the membrane separation process, a spiral wound composite membrane (rejection rate ≥95% for NaCl) is used in the nanofiltration stage, such that the sodium chloride content in the final product is ≤0.5% (w / w) and the conductivity is ≤100 μS / cm.

[0029] The chewable tablets contain the following components in mass percentages: Active ingredient: Hydrolyzed alligator protein zinc 35 - 45% (zinc content 8 - 12%, proportion of zinc chelated form ≥90%, among which the proportion of polypeptides <1 kDa ≥65%); Filler: Microcrystalline cellulose 22 - 28%, lactose 18 - 22%; Functional excipient: Fructooligosaccharide 6 - 10% (regulating intestinal flora), cross-linked carboxymethylcellulose sodium 1.5 - 2.5% (disintegrant); Additive: Magnesium stearate 0.5 - 1.0% (lubricant), natural vanilla essence 0.2 - 0.4% (flavoring agent); The hardness of the tablets is 50 - 60 N (Monsanto hardness tester), the disintegration time is ≤12 min (disintegration tester method in Chinese Pharmacopoeia 2025 edition), the dissolution rate at 30 min (pH 6.8 phosphate buffer solution, 37 °C) is ≥90%, among which the dissolution rate of zinc ions is ≥85%.

[0030] In the amino acid composition of hydrolyzed alligator protein zinc, the total proportion of Pro (proline), Leu (leucine), and Ala (alanine) is ≥40%, among which the proportion of hydrophobic amino acids (Pro + Leu + Ile + Val) is ≥35%. The measured ORAC value is ≥900 μmol TE / g (oxygen radical absorbance capacity assay). The zinc chelated form is a polypeptide-zinc coordination complex, which is detected by Fourier transform infrared spectroscopy (FT-IR). Zinc ions form coordination bonds with the carbonyl group (C=O) and amino group (-NH2) in the polypeptide. Compared with zinc sulfate, its transmembrane transport rate in the Caco-2 cell model is increased by 55% (P < 0.05).

[0031] In the preparation of crocodile hydrolyzed protein zinc chewable candies, crocodile hydrolyzed protein zinc has zinc chelation properties, with a zinc chelation rate ≥ 90% and a free zinc ion concentration ≤ 3%, reducing the irritation to the gastric mucosa (animal experiments show that the incidence of gastric ulcers is reduced by 70% compared to the zinc sulfate group). The small molecule peptide carrier promotes the intestinal absorption of zinc. Mediated by the PEPT1 oligopeptide transporter, the peak time of blood zinc concentration is shortened by 1.5 h compared to inorganic zinc, and the bioavailability is increased by 60% (human trial, n = 50). The chewable candies are suitable for the following groups of people: (1) Children (4 - 12 years old) in need of zinc supplementation: Take 1 tablet daily for 8 consecutive weeks, and the serum zinc concentration increases by 25 - 35%; (2) Pregnant women: Through the mild absorption characteristics of polypeptide zinc, nausea and vomiting caused by traditional inorganic zinc are avoided, and the zinc supplementation compliance is increased by 40%; (3) Middle-aged and elderly people: Combining the antioxidant function of zinc, the serum MDA (malondialdehyde) level is reduced by 15 - 20%, and the activity of immune cells is enhanced (the CD4+ cell count increases by 10%).

[0032] A composite functional chewable candy composition includes the prepared crocodile hydrolyzed protein zinc. With crocodile hydrolyzed protein zinc (30 - 40%) as the core ingredient, the following functional ingredients are compounded: Vitamin C (10 - 15%): Synergistically promotes collagen synthesis with zinc and enhances the antioxidant capacity (the ORAC value shows a 15% synergistic effect); Yeast β-glucan (3 - 5%): Activates intestinal immune cells, jointly regulates the Th1 / Th2 immune balance with zinc, and increases the serum IgG concentration by 20%; Taurine (2 - 4%): Promotes the distribution of zinc in the retina and central nervous system, and is suitable for people for vision care.

[0033] A chewable candy with intestinal protection function includes the prepared crocodile hydrolyzed protein zinc. The oligopeptides (< 1 kDa) in crocodile hydrolyzed protein zinc have a mucosal repair effect and improve intestinal health through the following mechanisms: (1) Promotes the expression of tight junction proteins (ZO-1, Occludin) in intestinal epithelial cells and repairs intestinal mucosal damage caused by alcohol / antibiotics (cell experiments show a repair rate ≥ 40%); (2) Oligofructose and hydrolyzed peptides synergistically regulate the intestinal flora, increasing the number of Bifidobacterium and Lactobacillus by 3 times and reducing the number of Enterobacteriaceae by 50% (verified by fecal flora 16S rRNA sequencing).

[0034] Example 3 Preparation of crocodile hydrolyzed protein zinc essence skin care products I. Raw material preparation Crocodile raw materials Select 500g of fresh crocodile meat and bones from legal sources and qualified quarantine. When rinsing, place the raw materials in a stainless steel mesh basket, and gently scrub the surface with a soft brush under running drinking water to ensure that blood, residual scales and other impurities are completely removed. Mechanical crushing uses a laboratory-grade high-speed tissue crusher (model: JJ-2, power: 300W, speed range: 8000-24000r / min), and the raw materials are placed in the crusher in batches. The crushing time is controlled at 30-40 seconds each time. After crushing, pass through a standard inspection sieve with a pore size of 5mm to ensure that all particles are less than 5mm in size. The crushed raw materials are temporarily stored in a sterilized sealed container and placed in a 4℃ refrigerator for standby use.

[0035] Enzyme preparations Alkaline protease: Alcalase 2.4L alkaline protease produced by Novozymes was used, with enzyme activity ≥2.4AU / g. Before use, a 10% (w / v) enzyme solution was prepared with deionized water according to the product instructions.

[0036] Flavor protease: Flavourzyme 500MG flavor protease produced by Bioengineering Co., Ltd. was used, with an enzyme activity of ≥500U / g, and was also prepared into a 10% (w / v) enzyme solution.

[0037] Zinc chelatase: a zinc-dependent metalloproteinase purchased from Sigma-Aldrich, product number: Z1234. The amount added is accurately weighed according to 2% of the mass of the substrate (crocodile meat and bones), dissolved in deionized water and used immediately.

[0038] Other accessories Hyaluronic acid: Use food-grade sodium hyaluronate produced by Bloomage Biotechnology, with a molecular weight of 1-1.5 million Da and a specification of 25g / bag. Take 20g for later use.

[0039] Niacinamide: Purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., purity ≥ 99%, specification 100g / bottle, weigh 15g. At the same time, prepare appropriate amount of preservatives that meet the requirements of cosmetic production (such as phenoxyethanol, added at 0.5-1%) and food-grade flavors (select according to preference, added at 0.1-0.3%). 2. Preparation process Three-step enzymatic hydrolysis Transfer the crushed alligator raw materials into a stainless-steel reactor (volume: 2 L) equipped with a stirring device and a pH and temperature control system. Add deionized water at twice the mass of the raw materials, start stirring (rotation speed: 100 r / min), adjust the pH to 8.5 using 1 mol / L NaOH solution, and control the temperature of the reaction system at 50°C ± 1°C through a constant-temperature water bath. Add the prepared alkaline protease solution. During the enzymatic hydrolysis process, measure the pH every 30 minutes and promptly adjust the pH with 1 mol / L NaOH solution to keep it within the range of 8 - 9. After 2 hours of enzymatic hydrolysis, quickly raise the temperature to 90°C and keep it warm for 10 minutes for enzyme inactivation treatment.

[0040] Cool the solution after enzyme inactivation to 45°C, adjust the pH to 6.5 using 1 mol / L HCl solution, add the flavor protease solution, and also keep stirring (rotation speed: 80 r / min). During the enzymatic hydrolysis process, monitor the pH every 30 minutes and fine-tune it with 1 mol / L HCl or NaOH solution to maintain the pH between 6 - 7. After 2 hours of enzymatic hydrolysis, raise the temperature to 90°C again and keep it warm for 10 minutes for enzyme inactivation.

[0041] Cool the solution after the second enzyme inactivation to 55°C, adjust the pH to 7.5 using 1 mol / L NaOH solution, add the zinc chelating enzyme solution, adjust the stirring speed to 60 r / min, measure and adjust the pH every 20 minutes to keep the pH stable between 7 - 8. After 2 hours of enzymatic hydrolysis, raise the temperature to 95°C and keep it warm for 15 minutes for thorough enzyme inactivation, and then cool the enzymatic hydrolysate to room temperature. During the enzymatic hydrolysis process, take samples every 1 hour and determine the degree of hydrolysis by the Kjeldahl method. Ensure that after a total enzymatic hydrolysis time of 6 hours, the degree of hydrolysis reaches more than 25%.

[0042] Fine purification Ceramic membrane filtration: Pass the cooled enzymatic hydrolysate through a ceramic membrane filtration system (membrane material: alumina, pore size: 50 nm, model: CMF - 50). Control the operating pressure at 0.1 - 0.2 MPa and keep the temperature at 25 - 30°C. During the filtration process, periodically backwash the membrane with deionized water to prevent membrane fouling, and collect the filtered clear liquid.

[0043] Ultrafiltration: Use an ultrafiltration device (membrane material: polysulfone, molecular weight cut-off: 10 kDa, model: UF-10K) to perform ultrafiltration on the clear liquid after ceramic membrane filtration. The operating pressure is 0.2 - 0.3 MPa, the temperature is controlled at 20 - 25 °C, collect the permeate, and the retentate can be subjected to secondary ultrafiltration or otherwise treated. Nanofiltration: Introduce the ultrafiltration permeate into a nanofiltration device (membrane material: aromatic polyamide, molecular weight cut-off: 200 Da, model: NF-200), with an operating pressure of 0.5 - 0.8 MPa and the temperature maintained at 20 - 25 °C for desalination and concentration. At the same time, load D301 ion exchange resin (pretreatment method: first soak in 2 - 4 mol / L HCl solution for 2 - 4 hours, wash with water until neutral, then soak in 2 - 4 mol / L NaOH solution for 2 - 4 hours, wash with water until neutral) into the ion exchange column. The nanofiltration concentrate passes through the ion exchange column to further remove impurities, collect the effluent, and detect the zinc chelation rate to ensure it reaches over 90%.

[0044] Post-treatment Vacuum evaporation and concentration: Transfer the solution after nanofiltration and ion exchange to a rotary evaporator (model: RE-52AA, water bath temperature: 60 - 70 °C, vacuum degree: -0.08 - -0.09 MPa) for vacuum evaporation and concentration until the solid content of the solution reaches over 30%. Stir continuously during the concentration process to prevent local overheating.

[0045] Spray drying: Pump the concentrated solution through a peristaltic pump into a spray dryer (model: LPG-5, inlet temperature: 180 °C ± 5 °C, outlet temperature: 80 °C ± 3 °C, feeding rate: 20 - 30 mL / min) for spray drying, collect the dried crocodile hydrolyzed protein zinc powder, and store it sealed in a desiccator.

[0046] III. Preparation of Skin Care Essence In a clean stainless steel container, add 200 mL of deionized water, start stirring (rotation speed: 150 r / min), slowly add 25 g of crocodile hydrolyzed protein zinc powder, and continue stirring for 30 minutes to ensure complete dissolution of the powder.

[0047] Add 20 g of hyaluronic acid and 15 g of niacinamide in sequence, and continue stirring for 1 hour to fully dissolve and mix the excipients evenly.

[0048] According to needs, add an appropriate amount of preservative (phenoxyethanol) and fragrance, adjust the pH to between 5.5 - 6.5 with 1 mol / L HCl or NaOH solution, finally make up the volume to 500 mL with deionized water. After stirring evenly, transfer the essence to a sterilized glass bottle and store it sealed to obtain the essence containing crocodile hydrolyzed protein zinc that can be directly used on the skin.

[0049] Example 4 Preparation and Performance Detection of Crocodile Hydrolyzed Protein Zinc Chewable Candies I. Raw Material Pretreatment Raw material selection: Take 50 kg of deboned muscle and 20 kg of bones from fresh Nile crocodiles (Crocodylus niloticus), remove visible fat and connective tissue, rinse 3 times with deionized water until no blood seeps out, and drain and weigh.

[0050] Crushing and homogenization: Put the muscle and bones into a double-knife crusher (rotating speed 2000 rpm) respectively, crush until the particle size is ≤ 3 mm, combine them, add 280 L of deionized water according to the solid-liquid ratio of 1:4 (w / v), transfer to a high-speed homogenizer (10000 rpm, 5 min) for treatment to obtain a uniform milky homogenate.

[0051] II. Three-step Enzymatic Hydrolysis Process The first stage: Hydrolysis by alkaline protease Adjust the pH of the homogenate to 8.5 (1M NaOH), add 1.4 kg of alkaline protease (Novozymes Alcalase 2.4L, enzyme activity 2.4 AU / g), hydrolyze with stirring in a water bath at 50°C (150 rpm) for 3 h, monitor the degree of hydrolysis (DH) by formaldehyde titration every 30 min during this period, and terminate the first stage when DH reaches 18%.

[0052] The second stage: Debittering by flavor protease Cool down to 45°C, adjust the pH to 6.5 with 1M HCl, add 1.05 kg of flavor protease (Flavourzyme 500MG, enzyme activity 500 U / g), continue enzymatic hydrolysis for 2 h, and confirm that the bitter peptide segments are completely removed through the bitter threshold test (sensory evaluation).

[0053] The third stage: Directed cleavage by zinc chelating enzyme Raise the temperature to 55°C, adjust the pH to 7.5 (Tris-HCl buffer), add 0.7 kg of zinc chelating enzyme (derived from Bacillus subtilis, EC3.4.24.23, enzyme activity 1000 U / mg), hydrolyze for 2 h, and monitor in real time until DH reaches 28%, effectively releasing zinc ion binding sites (exposure of histidine and cysteine residues).

[0054] III. Fine Purification Process Inactivation of enzymes and solid-liquid separation The enzyme hydrolysate is heated in a water bath at 85°C for 10 min to inactivate the enzymes, transferred to a centrifuge (4000 rpm, 20 min) to remove bone residues, and about 250 L of supernatant is collected.

[0055] Three-stage membrane separation Ceramic membrane filtration: The supernatant was passed through a 50 nm alumina ceramic membrane (cross-flow velocity 3 m / s, operating pressure 0.2 MPa) to retain and remove macromolecular impurities larger than 50 nm (such as nucleic acids and cell debris), obtaining 230 L of filtrate; Ultrafiltration enrichment: The filtrate was treated with a 10 kDa polysulfone ultrafiltration membrane (operating pressure 0.3 MPa) to retain peptide segments with a molecular weight above 10 kDa, and 200 L of active components with a molecular weight ≤ 10 kDa were collected; Nanofiltration concentration: Further passed through a 200 Da aromatic polyamide nanofiltration membrane (operating pressure 1.5 MPa) to remove inorganic salts and small molecule impurities, and concentrated to a volume of 50 L. The zinc chelation rate was detected by atomic absorption spectrometry (AAS) to reach 92.5%.

[0056] Ion exchange for impurity removal The concentrated solution was passed through a D301 macroporous weakly basic anion resin column (column diameter to height ratio 1:5, packing volume 10 L) to remove heavy metal ions at a flow rate of 2 BV / h. The lead and arsenic contents were detected to be < 0.05 mg / kg, meeting the "National Food Safety Standard" (GB2762).

[0057] IV. Preparation of the preparation Concentration and drying The purified liquid was transferred to a vacuum concentration kettle (60 °C, vacuum degree -0.08 MPa) and concentrated to a solid content of 45% (about 25 kg). After passing through a spray drying tower (inlet temperature 180 °C, outlet temperature 85 °C, feeding rate 5 L / h), 20 kg of milky white powder was obtained, with a moisture content of 3.2% (GB5009.3).

[0058] Mixing, granulating and tableting Auxiliary material formula (based on a total input of 100 kg): Hydrolyzed protein zinc powder from alligator: 40 kg Filler: Microcrystalline cellulose 25 kg, lactose 20 kg Functional auxiliary material: Fructooligosaccharide 8 kg, cross-linked carboxymethyl cellulose sodium 2 kg Additive: Magnesium stearate 0.8 kg, natural vanilla essence 0.2 kg The above materials were passed through an 80-mesh sieve and mixed evenly, and 5% ethanol aqueous solution was added to make soft materials. Granules were made by a rocking granulator (1 mm sieve mesh), dried at 50 °C under forced air for 4 h until the moisture content ≤ 4%, and after sizing, circular convex tablets of 0.5 g / tablet were pressed by a rotary tablet press (punch die diameter 8 mm, tableting pressure 12 kN), and a total of 200,000 tablets were made.

[0059] V. Product performance testing Analysis of active ingredients Zinc content: Detected by AAS method, each tablet contains 19.8 mg of zinc, and the zinc chelation rate is 92.3% (verified by ultrafiltration centrifugation method, free zinc ions < 3%); Molecular weight distribution: Shown by SDS-PAGE electrophoresis, polypeptides with a molecular weight < 1 kDa account for 72%, 1 - 3 kDa account for 23%, and ≥ 3 kDa account for 5%; Amino acid composition: Shown by HPLC detection, the total proportion of Pro (18%), Leu (12%), and Ala (10%) is 40%, the proportion of hydrophobic amino acids is 38%, and the ORAC value is 920 μmol TE / g.

[0060] Quality indicators of the preparation Physical properties: Tablet hardness is 55 N (Monsanto hardness tester), disintegration time is 10 min (disintegration tester method in the 2025 edition of the Chinese Pharmacopoeia), dissolution rate at 30 min (pH 6.8 buffer solution) is 91.2%, and the dissolution rate of zinc ions is 87.5%; Safety: Total number of colonies < 100 CFU / g, coliform group < 10 MPN / 100 g (GB4789.2 / 3), heavy metal residues comply with the GB17399 standard.

[0061] Bioavailability verification Caco-2 cell model: Compared with the zinc sulfate group, the transmembrane transport rate of zinc ions increased by 58% (P < 0.01), confirming the efficient absorption mediated by the PEPT1 oligopeptide transporter; Human trial (n = 50, 2 tablets per day for 4 consecutive weeks): Serum zinc concentration increased from (7.6 ± 1.2) μmol / L to (9.8 ± 1.5) μmol / L, with a promotion rate of 28.9%, and there were no reports of gastrointestinal discomfort (such as the incidence of nausea and vomiting was 0% vs 22% in the zinc sulfate group).

[0062] VI. Conclusion In this example, through the synergistic effect of directional three-step enzymatic hydrolysis and three-stage membrane separation technology, a high-chelation-rate (92.3%) and high-bioavailability crocodile hydrolyzed protein zinc chewable candy was successfully prepared. Its preparation parameters (hardness, disintegration time, dissolution rate) are all superior to industry standards, and it is significantly superior to traditional inorganic zinc preparations in terms of zinc supplementation efficiency and safety.

[0063] Example 5 Preparation and effect verification of a composite functional crocodile hydrolyzed protein zinc chewable candy I. Raw material and formula adjustment Core raw material: Use the crocodile hydrolyzed protein zinc powder prepared in Example 1 (zinc chelation rate 92%, small molecule peptides accounting for 70%), with a dosage of 35 kg.

[0064] Composite functional components: Vitamin C (food grade, ascorbic acid): 12 kg Yeast β-glucan (purity ≥ 90%): 4 kg Taurine: 3 kg Excipient system: Filler: Microcrystalline cellulose 28 kg, lactose 18 kg Disintegrant: Croscarmellose sodium 2.5 kg Lubricant: Magnesium stearate 0.8 kg Flavoring agent: Natural vanilla essence 0.2 kg Sweetener: Fructooligosaccharide 10 kg II. Preparation process optimization Mixing pretreatment: Pass the alligator hydrolyzed protein zinc, vitamin C, β-glucan, and taurine through an 80-mesh sieve respectively, and mix them evenly in proportion to form an active ingredient premix.

[0065] Granulation and tabletting: Mix the premix with excipients (such as microcrystalline cellulose and lactose) for the second time, add 5% ethanol aqueous solution to make soft materials, and granulate through a rocking granulator (sieve mesh aperture 1 mm).

[0066] Dry the granules at 45 °C under forced air for 6 h until the moisture content drops to ≤ 3.5%. After sizing, press them into oval tablets of 0.6 g / tablet through a rotary tablet press (punch die diameter 8 mm, pressure 13 kN), and the total output is about 167,000 tablets.

[0067] III. Product performance testing Component analysis: Zinc content: Each tablet contains 18 mg of zinc, and the zinc chelation rate is 91.8%; Functional components: Vitamin C content is 72 mg / tablet, β-glucan is 24 mg / tablet, and taurine is 18 mg / tablet; Synergistic activity: The ORAC value of the compound formula is increased to 1050 μmol TE / g, which is 14% higher than that of single protein zinc.

[0068] Quality indicators: Hardness: 58 N, disintegration time 11 min, dissolution rate (pH 6.8 buffer solution) at 30 min ≥ 90%; Microbiological detection: Total number of colonies < 50 CFU / g, meeting the GB17399 standard.

[0069] IV. Functional verification experiment Intestinal protection effect: In vitro experiment: Using the Caco-2 cell model, simulating alcohol damage (treated with 100 mM ethanol), after adding the hydrolyzate of this product (final concentration 0.5 mg / mL), the expression level of tight junction protein ZO-1 increased by 42% compared with the control group (P < 0.05), and the secretion of inflammatory factor IL-6 decreased by 35%.

[0070] Animal experiment: Establishing an intestinal flora disorder model by intragastric administration of antibiotics (amoxicillin, 100 mg / kg) to SD rats. The experimental group was fed with the feed containing this product (zinc content 20 mg / kg). After 2 weeks, fecal flora sequencing showed that the abundance of Bifidobacterium increased by 2.8 times and Enterobacter decreased by 60%.

[0071] Immunomodulatory effect: Human trial: Recruiting 60 healthy volunteers aged 45 - 60 years, randomly divided into the experimental group (2 tablets per day) and the placebo group. After continuous administration for 8 weeks, the following were detected: The serum IgG concentration in the experimental group increased from (12.5 ± 1.2) g / L to (14.8 ± 1.5) g / L, an increase of 18.4%; The peripheral blood CD4+ T cell count increased by 12%, significantly higher than that of the placebo group (P < 0.01).

[0072] V. Conclusion In this example, by adding vitamin C, β-glucan and taurine, a crocodile hydrolyzed protein zinc chewable candy with dual functions of intestinal repair and immunomodulation was prepared. The experimental results show that the composite formula not only retains the zinc supplementation effect with high bioavailability, but also enhances the antioxidant and immunomodulatory activities through the synergistic effect among the components.

[0073] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not elaborate all the details, nor limit the invention to the specific embodiments described. The present specification selects and specifically describes these embodiments to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention.

Claims

1. A preparation method of a crocodile hydrolyzed protein zinc skin care product, characterized in that: It includes the following steps: Step a: Raw material pretreatment: Take fresh alligator meat and bones, wash them, mechanically crush them to a particle size < 5 mm, and prepare a homogenate by adding water at a ratio of 1:3 - 5. Step b: Three-step enzymatic hydrolysis: Sequentially use alkaline protease, flavor protease, and zinc chelating enzyme for enzymatic hydrolysis. The single-stage enzymatic hydrolysis time is 2 - 4 h, and the total degree of hydrolysis is ≥ 25%. Step c: Fine purification: Remove macromolecular impurities through a ceramic membrane → Ultrafiltration to retain active peptide segments → Nanofiltration for desalination and concentration of the peptide-zinc complex, and combine with D301 resin to remove heavy metals to obtain a hydrolyzate with a zinc chelation rate ≥ 90%. Step d: Preparation of the preparation: Concentrate the purified liquid under reduced pressure to a solid content of 40 - 50%, mix it with excipients such as microcrystalline cellulose, lactose, fructooligosaccharide, and magnesium stearate, granulate, dry, and then press tablets. The tableting pressure is 8 - 15 kN to obtain a chewable tablet containing 10 - 30 mg of zinc per tablet.

2. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 1, characterized in that: The zinc chelating enzyme is a zinc-dependent metalloprotease. During the enzymatic hydrolysis process, the degree of hydrolysis is monitored in real time, and by adjusting the enzyme addition amount and temperature, the proportion of polypeptides with a molecular weight < 1 kDa in the product is ≥ 60%.

3. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 1, characterized in that: The excipients also include a disintegrant cross-linked carboxymethylcellulose sodium (1 - 3%) and a flavoring agent natural vanilla essence (0.1 - 0.5%). The total mass of the excipients accounts for 30 - 50% of the chewable tablet.

4. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 1, characterized in that: The chewable tablet is prepared by any of the methods of claims 1 - 3 and contains the following components: Active ingredient of alligator hydrolyzed protein zinc: 30 - 50% (zinc content 5 - 15%, zinc chelation rate ≥ 90%); Filler: Microcrystalline cellulose 20 - 30%, lactose 15 - 25%; Functional excipient: Fructooligosaccharide 5 - 10%, cross-linked carboxymethylcellulose sodium 1 - 3%; Lubricant: Magnesium stearate 0.5 - 1%; Flavoring agent: Natural vanilla essence 0.1 - 0.5%; The hardness of the tablet is 40 - 60 N, the disintegration time ≤ 15 min, and the dissolution rate (in 37°C, pH 6.8 buffer solution) ≥ 85% (30 min).

5. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 4, characterized in that: The alligator hydrolyzed protein zinc contains hydrophobic amino acid residues of Pro and Leu, accounting for 30 - 40% of the total amino acids, and has antioxidant activity (ORAC value ≥ 800 μmol TE / g).

6. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 4, characterized in that: The zinc exists in the form of polypeptide chelated zinc. Verified by the Caco-2 cell model, compared with zinc sulfate, its small intestine absorption rate is increased by 40 - 60%.

7. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 4, characterized in that: In the preparation of the chewable tablet with alligator hydrolyzed protein zinc, the alligator hydrolyzed protein zinc has zinc chelation characteristics, improves the bioavailability of zinc through a polypeptide carrier, reduces irritation to the gastrointestinal tract, and is suitable for people in need of efficient zinc supplementation.

8. The preparation method of a crocodile hydrolyzed protein zinc skin care product according to claim 7, characterized in that: The chewable tablet releases zinc binding sites directionally through a three-step enzymatic hydrolysis process, making the free zinc ion concentration in the product ≤ 5%, avoiding the oxidation side effects of inorganic zinc.

9. A composite functional compressed candy composition according to any one of claims 1-8, comprising crocodile hydrolyzed protein zinc prepared by any one of the methods of claims 1-2, characterized in that: The alligator hydrolyzed protein zinc is used as an active ingredient, adding vitamin C (5 - 10%) and yeast β-glucan (2 - 5%). Through the synergistic effect of zinc and polypeptides, it promotes the proliferation of T lymphocytes.

10. A chewable candy with intestinal protection function according to any one of claims 1-8, including the crocodile hydrolyzed protein zinc prepared by any one of the methods of claims 1-2, characterized in that: The proportion of small peptides (< 1 kDa) in the alligator hydrolyzed protein zinc is ≥ 60%, which can be directly absorbed through the intestinal oligopeptide transporter PEPT1. Compared with macromolecular proteins, the absorption rate is increased by more than 2 times.

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