Skin-care and hair-beautifying pet food and preparation method thereof

Abstract

The invention relates to the technical field of pet food, and discloses a skin-care and hair-beautifying pet food and a preparation method thereof, and the pet food is prepared from animal protein powder, a starchy raw material, aromatic enzymolysis oligopeptide, compound traditional Chinese medicine extraction mixed dry powder, a prepared fleeceflower root extract, evening primrose seed oil, trace elements and other raw materials. The aromatic enzymolysis oligopeptide in the formula is matched with the radix polygoni multiflori preparata extract to provide a melanin synthesis substrate and regulate hair follicle microcirculation, and the evening primrose seed oil and the like are combined to repair a skin lipid barrier. The preparation method comprises the following steps: stripping the oenothera biennis seed oil which is easy to oxidize from a high-temperature puffing premixing process, extruding, forming and drying basic grain particles, and guiding grease to permeate into micropores in the particles by adopting a vacuum atomization spraying process and utilizing pressure difference generated by relieving vacuum. According to the invention, the absorption conversion rate of skin-care and hair-beautifying components in intestinal tracts of pets is improved, meanwhile, high-temperature oxidation failure of heat-sensitive unsaturated fatty acid is avoided, and the stability of the product is improved.

Description

Technical Field

[0001] This invention relates to the field of pet food technology, specifically to a pet food for skin care and coat enhancement, and its preparation method. Background Technology

[0002] With rising awareness of pet ownership, pet owners are paying increasing attention to the health of their pets' skin and coat. A healthy and intact skin barrier and a glossy coat are important indicators of a pet's overall health. Currently, most pet foods on the market that claim to have skin-care and coat-enhancing functions mainly supplement nutrition by adding fish oil, regular vitamins, or increasing the proportion of complete animal protein to the basic formula. However, these conventional formula systems and processing methods have certain limitations in practical application.

[0003] On the one hand, conventional pet food typically uses large-molecule proteins as the main source of amino acids needed for hair growth. The enzymatic breakdown and absorption efficiency of large-molecule proteins in a pet's intestines is relatively limited, and directly adding free amino acids can easily cause competitive inhibition during intestinal transport. This results in a low conversion rate of the specific substrates required for melanin and keratin synthesis in hair follicles. Simultaneously, existing products often lack effective regulation of the local microcirculation in the pet's skin. After nutrients enter the bloodstream, they are difficult to be efficiently delivered to the hair papilla cells, leading to a lack of substantial synergistic improvement in skin and coat from the accumulation of a single ingredient. On the other hand, polyunsaturated fatty acids are important components for repairing the lipid barrier of pet skin, but these functional oils are extremely sensitive to heat and oxygen. In conventional pet food manufacturing processes, oils are usually mixed with other powdered raw materials and directly fed into an extruder. The high temperature, high pressure, and strong shear force during extrusion and puffing cause severe lipid peroxidation of unsaturated fatty acids. This not only results in a significant loss of effective skin-care ingredients, reducing the product's actual barrier repair function, but also easily accelerates fatty acid rancidity, thus affecting the palatability and shelf life of the pet food. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a pet food for skin care and coat enhancement, and its preparation method. This invention solves the problems of existing pet foods having limited functional ingredients for skin care and coat enhancement, low absorption and conversion rates of macromolecular proteins and oils in the pet's intestines, and the easy oxidation and inactivation of polyunsaturated fatty acids in the formula during conventional high-temperature extrusion processing.

[0005] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a pet food for skin care and coat enhancement, employing the following technical solution: A pet food for skin care and coat enhancement, comprising the following raw materials in parts by weight: 50-58 parts animal protein powder, 16-22 parts starch raw material, 10-15 parts aromatic enzymatic hydrolyzed short peptides, 2-3 parts compound Chinese herbal extract mixed dry powder, 1.5-2.5 parts processed Polygonum multiflorum extract, 1-2 parts Schizochytrium powder, 3-5 parts evening primrose seed oil, 0.5-1 part methionine zinc complex, 0.002-0.004 parts vitamin A acetate microcapsule powder, 0.002-0.004 parts D-biotin, and 1-1.5 parts L-arginine.

[0006] By employing the above-mentioned technical solution, the use of a specific ratio of aromatic enzymatically hydrolyzed short peptides, processed Polygonum multiflorum extract, evening primrose seed oil, and trace element system allows each component to synergistically intervene in the pet's body, thereby achieving the effects of repairing the skin's lipid barrier and promoting the synthesis of melanin and keratin in the hair. The reason for this is that the aromatic enzymatically hydrolyzed short peptides in the formula, as basic absorption units, can be directly absorbed across the membrane via oligopeptide transporters in the pet's intestines, thus avoiding the competitive inhibition problem caused by the absorption of free amino acids. After entering the bloodstream, the phenylalanine and tyrosine residues enriched in these short peptides provide a direct substrate source for the melanocytes at the base of the hair follicle, thereby participating in the dopaquinone production reaction catalyzed by tyrosinase to improve hair pigmentation and shine. Building upon this foundation, the processed Polygonum multiflorum extract synergizes with the aforementioned short peptides. The active ingredients in the extract can upregulate the expression of endothelial growth factor in capillaries surrounding hair follicles, accelerating the targeted delivery of enzymatically hydrolyzed short peptides to dermal papilla cells by expanding local microcirculation. Furthermore, its own antioxidant mechanism can, to a certain extent, scavenge reactive oxygen species at the base of hair follicles, helping to prolong the growth phase of the hair follicles. As for the evening primrose seed oil in the formula, it provides lipid components rich in α-linolenic acid, which, together with docosahexaenoic acid from Schizochytrium powder, participates in the synthesis of ceramides in the epidermis, thus helping to rebuild the skin's physical barrier. Combined with zinc methionine complex, vitamin A acetate microcapsules, and D-biotin, the entire system works together to regulate the proliferation and differentiation cycle of epidermal keratinocytes, ultimately reducing dandruff.

[0007] Preferably, the pet food comprises the following raw materials in parts by weight: 52-55 parts animal protein powder, 18-20 parts starch raw material, 12-14 parts aromatic enzymatic hydrolyzed short peptides, 2.5-2.8 parts compound Chinese herbal extract mixed dry powder, 2-2.2 parts processed Polygonum multiflorum extract, 1.5-1.8 parts Schizochytrium powder, 4-4.5 parts evening primrose seed oil, 0.8-0.9 parts zinc methionine complex, 0.003-0.0035 parts vitamin A acetate microcapsule powder, 0.003-0.0035 parts D-biotin, and 1.2-1.4 parts L-arginine. By adopting the above technical solution, the components of each raw material are limited to a more compact mass range, which helps to achieve a suitable balance between the carbon-nitrogen ratio of the amino acid nitrogen source, carbohydrate backbone, and lipid substances in the formula, thereby better maintaining the rheological stability during extrusion molding.

[0008] Preferably, the starchy raw material is cassava starch or potato flour; the aromatic enzymatic hydrolysate has a number-average molecular weight of 351–495 Da, and the total molar percentage of phenylalanine and tyrosine residues in its amino acid sequence is 35.2%–41.1%; the 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside mass fraction in the processed Polygonum multiflorum extract is 8.2%–10.8%, and the total anthraquinone mass fraction (calculated as emodin) is 1.3%–1.8%. By adopting the above technical solution, the physicochemical parameter requirements of the core raw materials are further clarified. Specifically, the cassava starch or potato flour used contains a high proportion of amylopectin, which can form a dense three-dimensional network pores during processing, thus providing a good structural carrier for the subsequent adsorption of oils. Given that the number-average molecular weight of aromatic enzymatically hydrolyzed short peptides is limited to the range of 351–495 Da, this indicates that the substance is mainly composed of tripeptides to pentapeptides. Short peptides with this degree of polymerization are relatively more likely to maintain conformational stability in the environment of gastric acid and pepsin, and the specific molar ratio of phenylalanine and tyrosine residues ensures an adequate supply of the black substrate. Furthermore, by limiting the specific mass fraction range of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside and total anthraquinones in the Polygonum multiflorum extract, it is possible to ensure that the extract reaches an effective physiological concentration after entering the bloodstream, thereby exerting its anti-lipid peroxidation and microcirculation dilation effects.

[0009] Preferably, the aromatic enzymatically hydrolyzed short peptides are prepared by a method comprising the following steps: adding soy protein isolate powder to purified water to prepare a suspension and adjusting the pH to 8.4–8.6; heating to 53–57°C, adding 1–1.4 parts by weight of alkaline protease per 100 parts by weight of soy protein isolate powder, and hydrolyzing continuously at a constant temperature for 115–125 min, maintaining a constant pH during hydrolysis; subsequently adding citric acid aqueous solution to adjust the pH to 4.5, heating to 95°C and holding for 10 min for enzyme inactivation; after cooling, filtering through a microporous membrane to remove impurities, collecting the initial filtrate and subjecting it to cross-flow filtration through an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da under a pressure of 0.8–1.0 MPa; and loading the permeate into an internal packing material. In a D101 macroporous adsorption resin chromatography column, inorganic salts and highly hydrophilic impurities are first removed by elution with purified water, followed by elution with a 70% (v / v) ethanol solution. The polar eluent is collected and dried to obtain a pale yellow powder. This method provides a way to directionally enrich peptides with specific amino acid sequences. The principle is as follows: During the unfolding and enzymatic cleavage of soy protein isolate, conditions of pH 8.4–8.6 and 53–57°C are set to moderately loosen the spherical structure of the soy protein isolate, exposing the internally concealed hydrophobic aromatic amino acid regions. Under these microenvironment, alkaline proteases exhibit specific cleavage ability on the carboxyl side peptide bonds of hydrophobic amino acid residues within the peptide chain, promoting the release of peptides containing phenylalanine and tyrosine. Subsequently, molecular weight cutoff is achieved, and cross-flow filtration is performed using a 1000 Da ultrafiltration membrane system to retain incompletely hydrolyzed large polypeptides and protein polymers, physically separating the free small peptide groups. Finally, in the polar adsorption and fractional elution stage, the non-polar framework structure of the D101 macroporous adsorption resin is utilized to allow short peptides with hydrophobic aromatic rings in the aqueous solution system to be stably adsorbed onto the resin surface by van der Waals forces. The purified water elution step washes and separates the hydrophilic aliphatic short peptides without aromatic rings and inorganic salts. Then, the 70% ethanol solution is switched to reduce the polarity of the mobile phase, which promotes the desorption of the target aromatic enzymatic short peptides, thereby achieving a high proportion of enrichment of specific target substances.

[0010] Preferably, the processed Polygonum multiflorum extract is prepared by a method comprising the following steps: adding a 70% ethanol solution to the pulverized processed Polygonum multiflorum powder at a mass-to-volume ratio of 1:10, and reflux extraction at 75-85°C; combining the extracts and concentrating under reduced pressure to recover the ethanol until no alcohol odor remains, then adding purified water to dilute the precipitated water-insoluble colloids, and centrifuging to obtain a clear aqueous extract; loading the aqueous extract into a D101 macroporous adsorption resin column with an internal resin column diameter-to-height ratio of 1:5, first eluting with purified water to remove sugar impurities, and then eluting with a 50% ethanol solution; collecting the desorbed eluent, concentrating under reduced pressure to remove the ethanol, and drying to obtain a brown dry powder; by adopting the above technical solution, through the combination of steps of first ethanol extraction, then water precipitation, and then resin enrichment, the purification and refining of the target stilbene glycosides and anthraquinones are achieved. The water precipitation step utilizes solubility differences to separate a large amount of ineffective plant waxes and tannins from the original medicinal material; the 1:5 diameter-to-height ratio resin column provides sufficient number of plates, and the polar gradient design of purified water for sugar removal and 50% ethanol elution accurately enriches the effective monomers with moderate polarity, eliminating the dilution of extract content by starch impurities.

[0011] Secondly, the present invention provides a method for preparing pet food for skin care and coat enhancement, comprising the following technical solution: A method for preparing pet food for skin care and coat enhancement, comprising the following steps: S1. Dry powder premix: Mix the remaining powdered ingredients in the formula except evening primrose seed oil to obtain basic dry powder. S2, Conditioning and Extrusion Puffing: The basic dry powder is preheated and conditioned by steam and then fed into the barrel. Under heating and pressure conditions, it is extruded, gelatinized and puffed into shape, extruded through the die and cut into granules. S3. Drying treatment: The formed puffed pellets are dried with hot air to obtain dry basic grain pellets; S4. Vacuum spraying and cooling: Under a closed vacuum condition, the formula amount of evening primrose seed oil is evenly atomized and sprayed onto the surface of the pellets while maintaining pressure. Then, the vacuum is slowly released to allow the oil to penetrate into the micropores of the pellets. The target pet food is obtained by cooling to room temperature.

[0012] By employing the above technical solution, evening primrose seed oil, which is highly sensitive to heat and easily oxidized, is separated from the conventional raw material premixing and extrusion process and transferred to the subsequent vacuum spraying process. The high temperature and pressure of the extrusion puffing stage only act on the more stable proteins, starches, and dry powder extracts, causing their starch chains to break down and gelatinize, forming a microporous framework. In the vacuum spraying step, the negative pressure state within the closed system minimizes oxygen interference; when the vacuum is released, an internal and external pressure difference is generated, creating capillary suction in hydrodynamics, guiding the atomized evening primrose seed oil to penetrate deep into the micropores of the grain. This not only prevents oil accumulation on the grain surface but also prevents unsaturated fatty acids from undergoing lipid peroxidation when exposed to air during storage.

[0013] Preferably, step S1 is implemented as follows: the raw material is put into a three-dimensional high-efficiency mixer and mixed continuously at room temperature for 20-30 minutes until the coefficient of variation (CV5) of the mixture is obtained. By adopting the above technical solution, the spatial multi-directional cross-mixing trajectory provided by the three-dimensional high-efficiency mixer reduces the phenomenon of gravity stratification, controls the coefficient of variation (CV5), ensures the uniform distribution of trace components in a large amount of protein and starch matrix, and prevents abnormal local material component concentrations in the subsequent extrusion process.

[0014] Preferably, in step S2, the conditioning process controls the material's core temperature to reach 80℃~85℃, and the moisture content to 20%~22%. The process parameters for the extrusion gelatinization and puffing molding process are: screw speed 250~270 r / min, barrel heating section temperature set at 105℃~115℃, and die head pressure 2.5~2.8MPa. By adopting the above technical solution, the conditioning step pre-softens the material, allowing moisture to penetrate into the starch granules. Inside the extruder barrel, the temperature of 105℃~115℃ combined with a shear pressure of 2.5~2.8MPa causes the starch crystal structure to disintegrate and produce continuous melting. When the material exits the die, the pressure drops sharply to atmospheric pressure, causing internal moisture to vaporize and expand, forming a uniform and continuous porous structure. This creates a physical channel for the subsequent oil infusion. Simultaneously, this temperature range does not reach the Maillard reaction caramelization critical point for aromatic enzymatic hydrolysis of short peptides, preserving the biological activity of the peptides.

[0015] Preferably, in step S3, the hot air drying temperature is 85℃~90℃, and the time is 35~45min, controlling the moisture content of the dried basic grain particles to be reduced to below 8.0%. By adopting the above technical solution, the gentle convective heat transfer removes the free water inside the material, reduces the water activity of the system, and inhibits the proliferation of microorganisms. Reducing the moisture content to below 8.0% releases the space volume inside the micropores, thereby improving the particle's capacity to hold liquid oil in subsequent processes.

[0016] Preferably, in step S4, the vacuum pressure is reduced to -0.08 MPa, maintained for 3-4 minutes after atomized spraying, and then the vacuum is slowly released. Cooling to room temperature is achieved using a counter-flow cooler. By employing this technical solution, the -0.08 MPa vacuum eliminates gas obstructions in the blind pores inside the grain, and the 3-4 minute pressure holding allows the atomized oil droplets to form a complete liquid film on the grain surface. The slow restoration to normal pressure ensures a stable pressure gradient, pushing the liquid film to the core layer. The counter-flow cooler utilizes the upward flow of cold air to exchange heat with the downward flow of hot material, preventing surface shrinkage and cracking caused by rapid cooling and solidifying the penetrated oil.

[0017] This invention provides a pet food for skin care and coat enhancement, and its preparation method. It has the following beneficial effects: 1. This invention's pet food improves nutrient supply and pigment deposition in pet hair by combining aromatic enzymatically hydrolyzed short peptides and processed Polygonum multiflorum extract. The aromatic enzymatically hydrolyzed short peptides in the formula have a molecular weight concentrated between 351 and 495 Da, which can be directly absorbed across the membrane via intestinal transport proteins, providing phenylalanine and tyrosine substrates for melanin synthesis in the hair follicle base. Combined with the regulatory effect of processed Polygonum multiflorum extract on local microcirculation, it helps accelerate the delivery of the above nutrients to the hair papilla cells. This synergistic intervention between components improves the bioavailability of the active ingredients, thereby improving the luster of the pet's coat.

[0018] 2. This invention utilizes the synergistic effect of evening primrose seed oil, Schizochytrium powder, and a trace element system containing zinc methionine complex to help repair the lipid barrier of pet skin. Evening primrose seed oil and Schizochytrium powder provide polyunsaturated fatty acids such as alpha-linolenic acid and docosahexaenoic acid, respectively. These lipid components participate in the synthesis of ceramides in the epidermis, replenishing the structural lipids needed by the skin. Simultaneously, the combination of zinc methionine complex and a vitamin system regulates the cell cycle of epidermal keratinocytes, reducing skin moisture loss and thus reducing dandruff and maintaining healthy skin in pets. Attached Figure Description

[0019] Figure 1 This is a graph showing the changes in relative tyrosinase activity in B16F10 cells after reagent intervention according to an embodiment of the present invention. Figure 2 This is a comparison chart of the absolute melanin deposition content detected after complete cell lysis in an embodiment of the present invention; Figure 3 This is a comparison of the relative expression abundance of MITF mRNA generated by reverse transcription amplification in an embodiment of the present invention; Figure 4Figure 1 shows the line distribution of the lipid components that target and inhibit the secretion of skin inflammatory factors in this invention; Figure 2 shows the trajectory of the change in prostaglandin E2 (PGE2) concentration in each system after lipopolysaccharide stimulation and drug intervention; Figure 3 shows the change in leukotriene B4 (LTB4) concentration in the same period. Figure 5 Figure 1 shows the quantitative indicators used to verify the mechanism of microvascular dilation and keratin co-synthesis in hair follicles in this invention. Figure 2 shows the concentration of free nitric oxide released by vascular endothelial cells after stimulation. Figure 3 shows the total keratin concentration expressed by hair papilla cells. Figure 6 Figure 1 is a comparative chart of the physical and chemical properties of the coat under the macro-feeding system of the present invention; wherein, Figure 2(a) is a bar chart comparing the increase of absolute black quality in the coat samples before and after feeding; Figure 3(b) is a line graph of the drop in coat brightness L value obtained by the live colorimetric device; and Figure 4(c) is a bar chart compiling the gloss values ​​of the newly grown hair segments. Figure 7 This is a quantitative distribution diagram of the targeted nutrient matrix of the present invention on skin lesion repair and hair shaft mechanical reconstruction; wherein, Figure (a) is a distribution diagram of transdermal water loss (TEWL) in the physically damaged area of ​​the epidermis; Figure (b) is a calibration diagram of the absolute concentration of core ceramide in the dermal tissue; Figure (c) is a tracking diagram of the longitudinal tensile breaking strength of a single shed hair shaft. Figure 8 Figure 1 shows the structure of the effective absorption and skin microecological intervention characteristics in the environmental stress hair pathological model of the present invention; Figure 2 shows the line distribution of the concentration of trace zinc ions in peripheral blood after in vivo ingestion intervention; Figure 3 shows the distribution of malondialdehyde (MDA) accumulation after cell peroxidation in the dermis; Figure 4 shows the scatter plot of the hair follicle growth phase / resting phase (A / T) ratio obtained from biopsy tissue sections. Detailed Implementation

[0020] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0021] The animal protein powder is derived from chicken, with a crude protein content of 80%, a moisture content of 8%, and a central particle size of D90-150 μm; the soy protein isolate has a nitrogen solubility index of 85%, an ash content of 4.5%, and a CAS number of 9010-10-0; the compound Chinese medicine extract powder is composed of extracts of Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Angelica sinensis, and Ligusticum chuanxiong mixed in equal proportions, with a total polysaccharide content of 15% (based on glucose) and a moisture content of 6%; the processed Polygonum multiflorum slices are processed with black bean juice and have a moisture content of 12%; the macroporous adsorption resin is a D101 type cross-linked polystyrene backbone, with a CAS number of 9060-05-3; the methionine zinc complex has a zinc content of 15%, a methionine ligand to zinc molar ratio of 2:1, a free methionine content of 0.5%, and a moisture content of 15%. The following ingredients were used: 3% by mass, CAS number 56329-42-1; alkaline protease activity of 2.4 AU / g, CAS number 9014-01-1; *Schizochytrium* powder containing 35% docosahexaenoic acid and 3% eicosapentaenoic acid, with a peroxide value of 5 meq / kg; evening primrose seed oil containing 10% linolenic acid and 70% linoleic acid, CAS number 90028-66-3; vitamin A acetate microcapsule powder with an activity potency of 500,000 IU / g, using maltodextrin and gum arabic as carriers, with an encapsulation rate of 90%, CAS number 127-47-9; D-biotin purity of 98.5%, CAS number 58-85-5; L-arginine, food grade, purity 99%, CAS number 74-79-3.

[0022] Preparation Example 1: This preparation example provides a method for preparing aromatic enzymatically hydrolyzed short peptides, including the following steps: Step 1: Add 100g of soy protein isolate powder to 800mL of purified water, stir and disperse for 30min to prepare a substrate suspension, and adjust the pH of the system to 8.4 using 1mol / L sodium hydroxide solution.

[0023] Step 2: Heat the suspension to 53°C, add 1.0g of alkaline protease, and continue hydrolysis at a constant temperature for 115min. During hydrolysis, maintain the pH of the system at a constant level of 8.4 by adding 1mol / L sodium hydroxide solution online.

[0024] Step 3: Add 10% citric acid aqueous solution to adjust the pH of the system to 4.5, heat to 95℃ and maintain for 10 minutes to inactivate the enzyme.

[0025] Step 4: After cooling the reaction solution to room temperature, filter it through a 0.45 μm microporous membrane to remove large particulate impurities. Collect the initial filtrate and transfer it to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da. Perform cross-flow filtration under a pressure of 0.8 MPa and collect the permeate enriched with low molecular weight peptides.

[0026] Step 5: Load the permeate into a chromatography column packed with D101 macroporous adsorption resin. First, elute with 3 BV purified water to remove inorganic salts and highly hydrophilic peptides. Then, elute with 70% ethanol solution and collect the polar eluent rich in aromatic ring structures.

[0027] Step 6: The ethanol eluent was transferred to a spray drying tower and dried at an inlet air temperature of 160℃ and an outlet air temperature of 85℃ to obtain a pale yellow powder. The obtained aromatic enzymatic hydrolysate short peptide was analyzed by gel permeation chromatography, and the number average molecular weight was 495 Da. The analysis by liquid chromatography-mass spectrometry showed that the total molar percentage of phenylalanine and tyrosine residues in its amino acid sequence was 35.2%, and the water content was 5.8%.

[0028] Preparation Example 2: This preparation example provides a method for preparing aromatic enzymatically hydrolyzed short peptides, including the following steps: Step 1: Add 100g of soy protein isolate powder to 800mL of purified water, stir and disperse for 30min to prepare a substrate suspension, and adjust the pH of the system to 8.5 using 1mol / L sodium hydroxide solution.

[0029] Step 2: Heat the suspension to 55°C, add 1.2g of alkaline protease, and hydrolyze continuously at a constant temperature for 120min. During hydrolysis, maintain the pH of the system at a constant distribution of 8.5 by adding 1mol / L sodium hydroxide solution online.

[0030] Step 3: Add 10% citric acid aqueous solution to adjust the pH of the system to 4.5, heat to 95℃ and maintain for 10 minutes to inactivate the enzyme.

[0031] Step 4: After cooling the reaction solution to room temperature, filter it through a 0.45 μm microporous membrane to remove large particulate impurities. Collect the initial filtrate and transfer it to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da. Perform cross-flow filtration under a pressure of 0.9 MPa and collect the permeate enriched with low molecular weight peptides.

[0032] Step 5: Load the permeate into a chromatography column packed with D101 macroporous adsorption resin. First, elute with 3 BV purified water to remove inorganic salts and highly hydrophilic peptides. Then, elute with 70% ethanol solution and collect the polar eluent rich in aromatic ring structures.

[0033] Step 6: The ethanol eluent was transferred to a spray drying tower and dried at an inlet air temperature of 160℃ and an outlet air temperature of 85℃ to obtain a pale yellow powder. The obtained aromatic enzymatic hydrolysate was analyzed by gel permeation chromatography, and the number average molecular weight was 425 Da. The analysis by liquid chromatography-mass spectrometry showed that the total molar percentage of phenylalanine and tyrosine residues in its amino acid sequence was 38.6%, and the water content was 5.3%.

[0034] Preparation Example 3: This preparation example provides a method for preparing aromatic enzymatically hydrolyzed short peptides, including the following steps: Step 1: Add 100g of soy protein isolate powder to 800mL of purified water, stir and disperse for 30min to prepare a substrate suspension, and adjust the pH of the system to 8.6 using 1mol / L sodium hydroxide solution.

[0035] Step 2: Heat the suspension to 57°C, add 1.4g of alkaline protease, and hydrolyze continuously at a constant temperature for 125min. During hydrolysis, maintain the pH of the system at a constant level of 8.6 by adding 1mol / L sodium hydroxide solution online.

[0036] Step 3: Add 10% citric acid aqueous solution to adjust the pH of the system to 4.5, heat to 95℃ and maintain for 10 minutes to inactivate the enzyme.

[0037] Step 4: After cooling the reaction solution to room temperature, filter it through a 0.45 μm microporous membrane to remove large particulate impurities. Collect the initial filtrate and transfer it to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da. Perform cross-flow filtration under a pressure of 1.0 MPa and collect the permeate enriched with low molecular weight peptides.

[0038] Step 5: Load the permeate into a chromatography column packed with D101 macroporous adsorption resin. First, elute with 3 BV purified water to remove inorganic salts and highly hydrophilic peptides. Then, elute with 70% ethanol solution and collect the polar eluent rich in aromatic ring structures.

[0039] Step 6: The ethanol eluent was transferred to a spray drying tower and dried at an inlet air temperature of 160℃ and an outlet air temperature of 85℃ to obtain a pale yellow powder. The obtained aromatic enzymatic hydrolysate short peptide was analyzed by gel permeation chromatography, and the number average molecular weight was 351 Da. The analysis by liquid chromatography-mass spectrometry showed that the total molar percentage of phenylalanine and tyrosine residues in its amino acid sequence was 41.1%, and the water content was 4.9%.

[0040] Preparation Example 4: This preparation example provides a method for preparing Polygonum multiflorum extract, including the following steps: Step 1: Grind the processed Polygonum multiflorum slices into powder and pass them through a 20-mesh sieve. Add a 70% ethanol solution to the powder at a mass-to-volume ratio of 1:10. Reflux and extract at 75°C for 1.5 hours. Separate the extract and repeat the extraction twice by adding an equal volume of 70% ethanol solution. Combine all the extracts.

[0041] Step 2: Place the combined extract in a vacuum concentration device and concentrate under reduced pressure at 60°C and 8 kPa to recover ethanol until there is no alcohol odor. Add purified water to the remaining thick paste to dilute it three times. After standing for 2 hours to precipitate water-insoluble colloids, centrifuge to obtain a clear aqueous extract.

[0042] Step 3: Load the clarified aqueous extract into a D101 macroporous adsorption resin column with an internal column diameter-to-height ratio of 1:5. First, elute with purified water at a flow rate of 2 BV / h until the effluent is colorless to remove sugar impurities. Then, elute with a 50% ethanol solution at a flow rate of 2 BV / h and collect the desorbed eluent.

[0043] Step four: The ethanol eluent was first concentrated under reduced pressure at 60℃ and 8kPa to remove most of the ethanol, and then transferred to a spray dryer at an inlet air temperature of 150℃ and an outlet air temperature of 80℃ to obtain the final brown dry powder. The obtained Polygonum multiflorum extract was analyzed by high performance liquid chromatography, and the mass fraction of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside was 8.2%. The mass fraction of total anthraquinones (calculated as emodin) was 1.3% and the mass fraction of moisture was 4.8% by ultraviolet spectrophotometry.

[0044] Preparation Example 5: This preparation example provides a method for preparing Polygonum multiflorum extract, including the following steps: Step 1: Grind the processed Polygonum multiflorum slices into powder and pass them through a 20-mesh sieve. Add a 70% ethanol solution to the powder at a mass-to-volume ratio of 1:10. Reflux and extract at 80°C for 2.0 hours. Separate the extract and repeat the extraction twice by adding an equal volume of 70% ethanol solution. Combine all the extracts.

[0045] Step 2: Place the combined extract in a vacuum concentration device and concentrate under reduced pressure at 60°C and 9 kPa to recover ethanol until there is no alcohol odor. Add purified water to the remaining thick paste to dilute it three times. After standing for 2 hours to precipitate water-insoluble colloids, centrifuge to obtain a clear aqueous extract.

[0046] Step 3: Load the clarified aqueous extract into a D101 macroporous adsorption resin column with an internal column diameter-to-height ratio of 1:5. First, elute with purified water at a flow rate of 2 BV / h until the effluent is colorless to remove sugar impurities. Then, elute with a 50% ethanol solution at a flow rate of 2 BV / h and collect the desorbed eluent.

[0047] Step four: The ethanol eluent is first concentrated under reduced pressure at 60°C and 9 kPa to remove most of the ethanol, and then transferred to a spray drying device to obtain the final brown dry powder at an inlet air temperature of 150°C and an outlet air temperature of 80°C.

[0048] The obtained Polygonum multiflorum extract was analyzed by high performance liquid chromatography, and the mass fraction of 2,3,5,4'-tetrahydroxystilbene-2-O--D-glucoside was 9.5%; the mass fraction of total anthraquinones (calculated as emodin) was 1.5% and the mass fraction of moisture was 4.5% as determined by ultraviolet spectrophotometry.

[0049] Preparation Example 6: This preparation example provides a method for preparing Polygonum multiflorum extract, including the following steps: Step 1: Grind the processed Polygonum multiflorum slices into powder through a 20-mesh sieve. Add a 70% ethanol solution to the powder at a mass-to-volume ratio of 1:10. Reflux extract at 85℃ for 2.5 hours. Separate the extract and repeat the extraction twice with an equal volume of 70% ethanol solution. Combine all extracts.

[0050] Step 2: Place the combined extract in a vacuum concentration device and concentrate under reduced pressure at 60°C and 10 kPa to recover ethanol until there is no alcohol odor. Add purified water to the remaining thick paste to dilute it three times. After standing for 2 hours to precipitate water-insoluble colloids, centrifuge to obtain a clear aqueous extract.

[0051] Step 3: Load the clarified aqueous extract into a D101 macroporous adsorption resin column with an internal column diameter-to-height ratio of 1:5. First, elute with purified water at a flow rate of 2 BV / h until the effluent is colorless to remove sugar impurities. Then, elute with a 50% ethanol solution at a flow rate of 2 BV / h and collect the desorbed eluent.

[0052] Step four: The ethanol eluent was first concentrated under reduced pressure at 60℃ and 10kPa to remove most of the ethanol, and then transferred to a spray dryer at an inlet air temperature of 150℃ and an outlet air temperature of 80℃ to obtain the final brown dry powder. The obtained Polygonum multiflorum extract was analyzed by high performance liquid chromatography, and the mass fraction of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside was 10.8%. The mass fraction of total anthraquinones (calculated as emodin) was 1.8% and the mass fraction of moisture was 4.1% by ultraviolet spectrophotometry.

[0053] Example 1: This embodiment provides a method for preparing pet food for skin care and coat enhancement, including the following steps: Step 1 (Raw Material Weighing): Accurately weigh 50 kg of animal protein powder, 20 kg of cassava starch, 10 kg of aromatic enzymatic hydrolyzed short peptides obtained in Preparation Example 1, 2.0 kg of compound Chinese herbal extract mixed dry powder, 1.5 kg of processed Polygonum multiflorum extract obtained in Preparation Example 4, 1.0 kg of Schizochytrium powder, 3.0 kg of evening primrose seed oil, 0.5 kg of zinc methionine complex, 0.002 kg of vitamin A acetate microcapsule powder, 0.002 kg of D-biotin, and 1.0 kg of L-arginine for later use.

[0054] Step 2 (Dry Powder Premix): Put all the powdered raw materials in the above formula except evening primrose seed oil into a three-dimensional high-efficiency mixer and mix continuously at room temperature for 20 minutes until the coefficient of variation (CV) is 5%, to obtain a uniformly mixed basic dry powder.

[0055] Step 3 (conditioning and extrusion puffing): The basic dry-mixed powder is conveyed to the conditioner of the twin-screw extruder puffing machine, and steam is introduced for preheating and conditioning to bring the core temperature of the material to 80℃ and the moisture content to 20%. Then the material enters the barrel and is extruded, gelatinized and puffed under the conditions of screw speed of 250r / min, barrel heating section temperature set at 105℃~110℃, and die head pressure of 2.5MPa. The material is then extruded through the die and cut into uniform granules.

[0056] Step 4 (Drying): The formed puffed granules are spread out and conveyed to a mesh belt hot air dryer, where they are dried at 85°C for 45 minutes to reduce the moisture content of the granules to below 8.0%, thus obtaining dried basic grain granules.

[0057] Step 5 (Vacuum Spraying and Cooling): The dried granules are fed into a vacuum spraying machine, sealed, and evacuated to -0.08 MPa. Under vacuum, the formulated amount of evening primrose seed oil is evenly atomized and sprayed onto the granule surface. After maintaining this vacuum for 3 minutes, the vacuum is slowly released, using the pressure difference to force the evening primrose seed oil to completely penetrate the micropores of the granules. Finally, the mixture is cooled to room temperature using a counter-current cooler and packaged to obtain the target pet food.

[0058] Example 2: This embodiment provides a method for preparing pet food for skin care and coat enhancement, including the following steps: Step 1 (Raw Material Weighing): Accurately weigh 55 kg of animal protein powder, 18 kg of cassava starch, 12 kg of aromatic enzymatic hydrolyzed short peptides obtained in Preparation Example 2, 2.5 kg of compound Chinese herbal extract mixed dry powder, 2.0 kg of processed Polygonum multiflorum extract obtained in Preparation Example 5, 1.5 kg of Schizochytrium powder, 4.0 kg of evening primrose seed oil, 0.8 kg of methionine zinc complex, 0.003 kg of vitamin A acetate microcapsule powder, 0.003 kg of D-biotin, and 1.2 kg of L-arginine for later use.

[0059] Step 2 (Dry Powder Premix): Put all the powdered raw materials in the above formula except evening primrose seed oil into a three-dimensional high-efficiency mixer and mix continuously at room temperature for 25 minutes until the coefficient of variation (CV) is 5%, to obtain a uniformly mixed basic dry powder.

[0060] Step 3 (conditioning and extrusion puffing): The basic dry-mixed powder is conveyed to the conditioner of the twin-screw extruder puffing machine, and steam is introduced for preheating and conditioning, so that the core temperature of the material reaches 82℃ and the moisture content is adjusted to 21% by mass. Then the material enters the barrel, and is extruded, gelatinized and puffed under the conditions of screw speed of 260r / min, barrel heating section temperature set at 108℃~112℃, and die head pressure of 2.6MPa. It is then extruded through the die and cut into uniform granules.

[0061] Step 4 (Drying): The formed puffed granules are spread out and conveyed to a mesh belt hot air dryer, where they are dried at 88°C for 40 minutes to reduce the moisture content of the granules to below 8.0%, thus obtaining dried basic grain granules.

[0062] Step 5 (Vacuum Spraying and Cooling): The dried granules are fed into a vacuum spraying machine, sealed, and evacuated to -0.08 MPa. Under vacuum, the formulated amount of evening primrose seed oil is evenly atomized and sprayed onto the granule surface. After maintaining this state for 3 minutes, the vacuum is slowly released, forcing the heat-sensitive evening primrose seed oil to penetrate deeply into the granule pores. Finally, the granules are cooled to room temperature using a counter-current cooler and packaged to obtain the target pet food.

[0063] Example 3: This embodiment provides a method for preparing pet food for skin care and coat enhancement, including the following steps: Step 1 (Weighing Raw Materials): Accurately weigh 52 kg of animal protein powder, 22 kg of potato flour, 15 kg of aromatic enzymatic hydrolyzed short peptides obtained in Preparation Example 3, 3.0 kg of compound Chinese herbal extract mixed dry powder, 2.5 kg of processed Polygonum multiflorum extract obtained in Preparation Example 6, 2.0 kg of Schizochytrium powder, 5.0 kg of evening primrose seed oil, 1.0 kg of methionine zinc complex, 0.004 kg of vitamin A acetate microcapsule powder, 0.004 kg of D-biotin, and 1.5 kg of L-arginine for later use.

[0064] Step 2 (Dry Powder Premix): Put all the powdered ingredients except evening primrose seed oil into a three-dimensional high-efficiency mixer and mix continuously at room temperature for 30 minutes until the coefficient of variation (CV) is 5%, to obtain a uniformly mixed basic dry powder.

[0065] Step 3 (conditioning and extrusion puffing): The basic dry-mixed powder is conveyed to the conditioner of the twin-screw extruder puffing machine, and steam is introduced for preheating and conditioning to bring the core temperature of the material to 85℃ and the moisture content to 22%. Then the material enters the barrel and is extruded, gelatinized and puffed under the conditions of screw speed of 270r / min, barrel heating section temperature set at 110℃~115℃, and die head pressure of 2.8MPa. The material is then extruded through the die and cut into uniform granules.

[0066] Step 4 (Drying): The formed puffed granules are spread out and conveyed to a mesh belt hot air dryer, where they are dried at 90°C for 35 minutes to reduce the moisture content of the granules to below 8.0%, thus obtaining dried basic grain granules.

[0067] Step 5 (Vacuum Spraying and Cooling): The dried granules are fed into a vacuum spraying machine, sealed, and evacuated to -0.08 MPa. Under vacuum, the formulated amount of evening primrose seed oil is evenly atomized and sprayed onto the granule surface. This process is maintained for 4 minutes, after which the vacuum is slowly released to ensure complete coating by the polyunsaturated fatty acid-rich evening primrose seed oil. Finally, the granules are cooled to room temperature using a counter-current cooler and sealed in packaging to obtain the target pet food.

[0068] Example 4: This embodiment provides a method for preparing pet food for skin care and coat enhancement, including the following steps: Step 1 (Raw Material Weighing): Accurately weigh 58 kg of animal protein powder, 16 kg of potato flour, 11 kg of aromatic enzymatic hydrolyzed short peptides obtained in Preparation Example 1, 2.2 kg of compound Chinese herbal extract mixed dry powder, 1.8 kg of processed Polygonum multiflorum extract obtained in Preparation Example 4, 1.2 kg of Schizochytrium powder, 3.5 kg of evening primrose seed oil, 0.6 kg of zinc methionine complex, 0.0025 kg of vitamin A acetate microcapsule powder, 0.0025 kg of D-biotin, and 1.1 kg of L-arginine for later use.

[0069] Step 2 (Dry Powder Premix): Put all the powdered raw materials in the above formula except evening primrose seed oil into a three-dimensional high-efficiency mixer and mix continuously at room temperature for 22 minutes until the coefficient of variation (CV) is 5%, to obtain a uniformly mixed basic dry powder.

[0070] Step 3 (conditioning and extrusion puffing): The basic dry-mixed powder is conveyed to the conditioner of the twin-screw extruder puffing machine, and steam is introduced for preheating and conditioning, so that the core temperature of the material reaches 81℃ and the moisture content is adjusted to 20.5% by mass. Then the material enters the barrel, and is extruded, gelatinized and puffed under the conditions of screw speed of 255r / min, barrel heating section temperature set at 106℃~111℃, and die head pressure of 2.55MPa. It is then extruded through the die and cut into uniform granules.

[0071] Step 4 (Drying): The formed puffed granules are spread out and conveyed to a mesh belt hot air dryer, where they are dried at 86°C for 42 minutes to reduce the moisture content of the granules to below 8.0%, thus obtaining dried basic grain granules.

[0072] Step 5 (Vacuum Spraying and Cooling): The dried granules are fed into a vacuum spraying machine, sealed, and evacuated to -0.08 MPa. Under vacuum, the formulated amount of evening primrose seed oil is evenly atomized and sprayed onto the surface of the granules. After maintaining this vacuum for 3 minutes, the vacuum is slowly released, forcing the oil to effectively penetrate the micropores inside the granules. Finally, the granules are cooled to room temperature using a counter-current cooler and packaged to obtain the target pet food.

[0073] Example 5: This embodiment provides a method for preparing pet food for skin care and coat enhancement, including the following steps: Step 1 (Raw Material Weighing): Accurately weigh 54 kg of animal protein powder, 20 kg of potato flour, 14 kg of aromatic enzymatic hydrolyzed short peptides obtained in Preparation Example 2, 2.8 kg of compound Chinese herbal extract mixed dry powder, 2.2 kg of processed Polygonum multiflorum extract obtained in Preparation Example 5, 1.8 kg of Schizochytrium powder, 4.5 kg of evening primrose seed oil, 0.9 kg of zinc methionine complex, 0.0035 kg of vitamin A acetate microcapsule powder, 0.0035 kg of D-biotin, and 1.4 kg of L-arginine for later use.

[0074] Step 2 (Dry Powder Premix): Put all the powdered raw materials in the above formula except evening primrose seed oil into a three-dimensional high-efficiency mixer and mix continuously at room temperature for 28 minutes until the coefficient of variation (CV) is 5%, to obtain a uniformly mixed basic dry powder.

[0075] Step 3 (conditioning and extrusion puffing): The basic dry-mixed powder is conveyed to the conditioner of the twin-screw extruder puffing machine, and steam is introduced for preheating and conditioning, so that the core temperature of the material reaches 84℃ and the moisture content is adjusted to 21.5% by mass. Then the material enters the barrel, and is extruded, gelatinized and puffed under the conditions of screw speed of 265r / min, barrel heating section temperature set at 109℃~114℃, and die head pressure of 2.7MPa. It is then extruded through the die and cut into uniform granules.

[0076] Step 4 (Drying): The formed puffed granules are spread out and conveyed to a mesh belt hot air dryer, where they are dried at 89°C for 38 minutes to reduce the moisture content of the granules to below 8.0%, thus obtaining dried basic grain granules.

[0077] Step 5 (Vacuum Spraying and Cooling): The dried granules are fed into a vacuum spraying machine, sealed, and evacuated to -0.08 MPa. Under vacuum, the formulated amount of evening primrose seed oil is evenly atomized and sprayed onto the surface of the granules. After maintaining this vacuum for 3.5 minutes, the vacuum is slowly released to allow the functional oils to fully penetrate and adhere. Finally, the granules are cooled to room temperature using a counter-current cooler and packaged to obtain the target pet food.

[0078] Comparative Example 1: Compared with Example 1, the difference is that the aromatic enzymatically hydrolyzed short peptides in the formula are replaced with an equal amount of ordinary unenzymatically enriched soy protein isolate powder, and the rest are the same.

[0079] The contrast highlights the lack of a clear mechanism of action and efficacy: Without specific enzymatic hydrolysis and macroporous resin enrichment, ordinary protein powder cannot provide a high concentration of free aromatic amino acids (tyrosine and phenylalanine). The absence of these easily transmembrane-absorbable direct precursor substrates for melanin synthesis prevents the provision of sufficient synthetic raw materials to hair follicle melanocytes, resulting in limited melanin deposition (eumelanin and pheomelanin in the cortex), leading to lighter pet hair color and a significant decrease in shine.

[0080] Comparative Example 2: Compared with Example 1, the difference is that processed Polygonum multiflorum extract was not added, and the corresponding weight of the formula gap was made up by animal protein powder, otherwise the same.

[0081] The contrast highlights a lack of clear mechanisms of action and efficacy: the core active ingredients in the extract (2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside and total anthraquinones) are missing. Without the upregulation of mitochondrial microphthalmia-associated transcription factor (MITF) expression by these large active molecules of traditional Chinese medicine, the specific activation of tyrosinase activity is lost. Even with short peptide substrates, the inactivation of the catalytic enzyme disrupts the original formula's synergistic mechanism of substrate supply + targeted activation, failing to achieve the deep-rooted, color-fixing, and hair-beautifying targeted effects.

[0082] Comparative Example 3: Compared with Example 1, the difference is that the Schizochytrium powder and evening primrose seed oil are replaced with ordinary fish oil and soybean oil in equal amounts, while the rest are the same.

[0083] The contrast highlights the lack of a clear mechanism of action and efficacy: it breaks the original invention's specific lipid-targeting regulatory mechanism. Ordinary oils cannot provide a specific ratio of high-purity DHA (derived from Schizochytrium) and GLA (-linolenic acid, derived from evening primrose seed oil). It loses the competitive inhibition mechanism of specific polyunsaturated fatty acids on the arachidonic acid pro-inflammatory pathway, resulting in an inability to effectively reduce the release of pro-inflammatory factors such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4); simultaneously, it cannot effectively repair epidermal lipid barrier damage caused by ceramide loss as a precursor, leading to a decrease in the pet's skin's resistance to transepidermal water loss (TEWL), and an inability to eliminate dandruff and itching caused by microinflammation.

[0084] Comparative Example 4: Compared with Example 1, the difference is that aromatic enzymatic hydrolyzed short peptides and processed Polygonum multiflorum extract are not added at the same time, and the corresponding weight gap is made up by animal protein powder, while the rest are the same.

[0085] The contrast highlights the lack of a clear mechanism of action and effectiveness: the core melanin-promoting pathway of this invention has been completely eliminated. Due to the simultaneous absence of the target substrate (precursor amino acids) and activating factors (plant polyphenols and derivatives), the synergistic microcirculation network within the hair follicle melanocytes is completely lost. This causes basic pet food to completely degrade in terms of skin care and coat beautification, resulting not only in dry, brittle, and easily broken hair, but also in the complete loss of its ability to improve hair follicle vitality.

[0086] Comparative Example 5: Compared with Example 1, the difference is that L-arginine and zinc methionine complex was not added, and the corresponding weight gap was made up by animal protein powder; otherwise, they are the same.

[0087] The contrast highlights the lack of a clear mechanism of action and effectiveness: it disrupts the local microvascular oxygen supply and keratin synthesis mechanism. The absence of L-arginine, a substrate for endothelial nitric oxide synthase, prevents the expansion of dermal hair follicle capillaries via the nitric oxide pathway, hindering the delivery of effective nutrients to the deep hair roots. Simultaneously, the lack of highly bioavailable organic peptide zinc (zinc methionine) leads to the loss of key ion nuclei in zinc finger proteins involved in keratin transcription, resulting in inhibited hair follicle cell proliferation and loose arrangement of hair shaft keratin chains, leading to significant non-seasonal hair loss and hair tip splitting.

[0088] Test Example 1: 1. The experimental subjects were B16F10 mouse melanoma cells in the logarithmic growth phase. They were seeded in 96-well and 6-well plates and placed in DMEM culture medium containing 10% fetal bovine serum. The cells were cultured in a constant temperature incubator at 37°C and 5% carbon dioxide for 24 hours to ensure complete cell adhesion and growth.

[0089] 2. Remove the original culture medium and prepare complete culture media containing the corresponding active ingredients according to the specific proportions of the formulations given in Examples 1 to 5 and Comparative Examples 1, 2, and 4. To avoid non-specific apoptosis caused by abnormal osmotic pressure, balance the total solids concentration in each test solution to 150 g / mL, add it to the cell culture wells, and maintain the treatment for 48 hours. A blank control group without any activity intervention or culture medium replacement was set up in parallel.

[0090] 3. The relative activity of tyrosinase in cell samples from the treatment cycle was measured. The supernatant was removed from the 96-well plate and washed with PBS buffer. Cells were lysed in situ by injecting a saline solution containing 1% Triton X-100. After ultracentrifugation, the crude enzyme extract was collected. 0.1% levodopa substrate solution was added to the extract supernatant at a fixed ratio, and the plate was incubated at 37°C for 60 minutes. The absorbance was read at 475nm using a microplate reader. The ratio of this absorbance to the baseline optical density of the control group was calculated to determine the specific activation percentage of tyrosinase by each intervention system.

[0091] 4. Extraction and determination of the absolute content of intracellular immobilized melanin. Cells that had proliferated after drug stimulation were collected from 6-well culture plates. The elution buffer was centrifuged to obtain a precipitate. A 1 mol / L sodium hydroxide strong alkaline solution containing 10% DMSO was added to the precipitate, and the vesicle structure was forcibly dissolved in an 80°C constant flow water bath for 60 minutes to disrupt the vesicle structure. After the lysis buffer returned to room temperature, its absorbance was measured at a specific monitoring wavelength of 405 nm. The absorbance was then converted to the mass of free eumelanin per milligram of total cellular protein using a standard curve fitting formula.

[0092] 5. All non-nuclear RNA molecules were directionally extracted from attached or suspended cells using Trizol reagent. After passing the nucleic acid UV concentration test, cDNA template strands were synthesized by reverse transcription. Specific amplification primer sequences were designed for the microphthalmia-related transcription factor MITF and loaded onto a real-time quantitative PCR instrument for thermal cycling amplification detection. The stably transcribed GAPDH gene was selected as an internal control. The dynamic difference coefficient of MITF messenger RNA expression abundance in different groups was calculated using a mathematical model.

[0093] Table 1. Effects of skin-care and hair-enhancing active ingredients on the melanocyte-stimulating pathway in B16F10 cells: Test group Relative tyrosinase activity in cells (%) Absolute intracellular melanin content (g / mg) MITF mRNA relative expression level Blank control group 100 20.14 1 Example 1 164.27 36.52 2.13 Example 2 171.84 38.79 2.31 Example 3 184.62 41.25 2.58 Example 4 168.05 37.11 2.19 Example 5 179.31 39.84 2.45 Comparative Example 1 134.19 22.86 1.94 Comparative Example 2 103.58 21.05 1.08 Comparative Example 4 98.74 19.43 0.95 According to Table 1 and Appendix Figure 1 Appendix Figure 2 and appendix Figure 3 The data shows that Examples 1 to 5 exhibit a highly significant overall upward shift, a trend that is fully synchronized across three dimensions: activation of enzyme catalytic potential, release of nuclear transcription factors, and targeted pigment enrichment at the terminal stage. Biological experiments and clinical observations of pet coat condition often reveal that coat discoloration stems from a dual bottleneck: irreversible decline in deep root sac vitality and a lack of precursor substances. The quantitative improvements in these implementation schemes demonstrate that the combination architecture of aromatic enzymatically hydrolyzed short peptides with exogenous herbal extracts achieves a deep metabolic network reconstruction effect. Observation of Comparative Example 1, where the substrate was stripped, revealed that the system, through the retained components of the traditional Chinese medicine extract, forcibly drove the high expression of the upstream of the MITF gene within the cell, accompanied by an abnormally high limit of tyrosinase activity in the free state. The microscopic factory of cellular synthesis and metabolism was completely in a preheating response state. Under the limited conditions of lacking exogenous free peptide energy supply, the trace material basis within the system was rapidly consumed and depleted. The total accumulation of free melanin in the final macroscopic characterization measurement only reached an extremely low threshold of about 22.86 micrograms, which directly exposed the phenomenon of industrial idleness caused by only neural activation without material filling. The phenomenon reflected in Comparative Example 2 verified the uniqueness of this physical closed-loop mechanism from the opposite perspective. Under the flooding effect of high concentration of aromatic precursor amino acids, due to the lack of polyphenol derivatives responsible for cleavage initiation signals, the tested cell chains hovered around the resting normal baseline level. The substrate only achieved physical accumulation but could not cross the biochemical barrier to be recognized and assembled into a large molecular complex structure. The comparative system 4, where test data fell back to the edge of the blank control threshold, illustrates that both the activation of multiple expression factors and the expansion of intercellular matrix pathways operate within an extremely demanding and specific supply-demand response chain. Isolated or fragmented components will be cleared and metabolized by the bypass system due to biodegradation or homeostasis. The deviations in these experimental parameters precisely define the specific upper limit framework for the two-dimensional synergistic supply structure to break through the traditional limitations of mammalian cortical enzymatic synthesis.

[0094] Test Example 2: 1. The experimental subject was the in vitro human keratinocyte cell line HaCaT, purchased from a regular cell bank. After cell resuscitation, the cells were seeded in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a saturated humidity incubator at 37°C and 5% CO2. The cells were then passaged and expanded until the cell confluence reached about 80%, at which point they were harvested for use.

[0095] 2. Cells were digested with trypsin and their concentration was adjusted to 210 using a hemocytometer. 5 A single-cell suspension of 1 cell / mL was evenly spread into 24-well cell culture plates and cultured for 24 hours. The original culture medium was then removed. Several wells were retained as a blank control group to maintain natural growth without the addition of any inflammatory agents. All other wells were incubated with fresh serum-free medium containing 1 g / mL lipopolysaccharide (LPS) for 12 hours to induce an excessive immune response in keratinocytes, thus establishing an in vitro miniature skin inflammation model.

[0096] 3. Discard the supernatant containing inflammatory factors from the initial stage and gently wash the wells twice with PBS. Following the formulations of Examples 1 to 5 and Comparative Example 3, using trace amounts of dimethyl sulfoxide (DMSO) as a solubilizing carrier, prepare a drug-containing medium with a final concentration of 50 g / mL (the final volume fraction of DMSO in the system is strictly controlled below 0.1% to avoid cytotoxicity). The drug-containing medium is then added back to the corresponding LPS induction wells for 24-hour intervention. A model control group is also established, which is only induced by LPS in the initial stage and subsequently supplemented with drug-free medium containing 0.1% DMSO.

[0097] 4. After the drug treatment cycle ends, tilt and collect the culture medium supernatant in each group's wells and transfer it to a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes at 4°C to completely settle and remove any floating cell debris.

[0098] 5. After centrifugation, collect the purified supernatant and use a high-sensitivity double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit for human prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Add stop solution according to the time window for the colorimetric reaction of the reagent indicators. Measure the optical density at 450 nm using a microplate reader. Perform algebraic regression analysis on the standard working curves plotted for known concentrations to obtain the specific absolute excretion of inflammatory mediators in each system.

[0099] Table 2. Effects of lipid component intervention on the release of pro-inflammatory factors from HaCaT cells: Test group Prostaglandin E2 (PGE2) concentration (pg / mL) Leukotriene B4 (LTB4) concentration (pg / mL) Blank control group 118.42 43.16 Model group 871.35 309.72 Example 1 352.19 121.84 Example 2 314.67 104.53 Example 3 281.04 91.27 Example 4 344.82 116.39 Example 5 302.51 96.65 Comparative Example 3 694.28 247.11 According to Table 2 and Appendix Figure 4 Data shows that keratinocytes in the model group were strongly stimulated by lipopolysaccharide on their surface receptors, resulting in the complete activation of their transmembrane arachidonic acid metabolic pathway and the release of abnormally high concentrations of mediators such as PGE2 and LTB4. After introducing the special compound oil components specified in Examples 1 to 5, the immune storm at the cellular level exhibited a very deep pullback curve (the PGE2 level in Example 3 was rapidly suppressed from 871.35 pg / mL to 281.04 pg / mL). This group of biochemical phenomena highly conforms to the microscopic competitive inhibition law: by directly intervening in the readily available large-molecule DHA provided by Schizochytrium and combining it with the abundant α-linolenic acid (GLA) inside Evening Primrose, the two precisely compete for the catalytic binding cavity sites of cyclooxygenase (COX-2) and lipoxygenase (5-LOX). Combined with the large amount of pathological dandruff and persistent scratching cases frequently encountered in the clinical diagnosis and treatment of pet dermatology, these are actually specific external manifestations of the long-term accumulation of micro-inflammation in the superficial dermis and its local amplification at nerve endings. Comparative Example 3 used commonly used fish oil and soybean oil as base substitutes. Although these oils are rich in linoleic acid and EPA precursors, they only weakly guided the indicators back to 694.28 pg / mL and 247.11 pg / mL, respectively. Why is the intervention barrier of conventional oils not only slow to take effect but also has a much lower cutoff rate than the biochemical transcriptional congestion node designed in this scheme, which is firmly stuck at the catalytic link of the host's own 6-desaturated enzyme? Under the high pressure of oxidative stress and inflammatory infiltration, the key enzymes responsible for converting the polyunsaturated traits of conventional plant oils are basically in a silent state of degradation or inactivation. Basic lipid particles can only remain inertly free in the extracellular matrix and cannot be successfully assembled into endogenous anti-inflammatory regulators. A large amount of data finally peeled away this theoretical dead end, proving that directly bypassing the enzyme catalytic bottleneck and intervening with the saturated loading of terminal active lipids at the pet intake end, the shortest closed-loop connection was constructed to effectively suppress the pathological differentiation of skin cells and rapidly repair the transdermal water loss network.

[0100] Test Example 3: 1. The experimental subjects were the endothelial cell line HUVEC isolated from the human umbilical vein and the human dermal papilla cell line HFDPC. The two cell lines, after resuscitation, were independently seeded in a dedicated culture medium containing 10% (v / v) standard fetal bovine serum and specific endothelial / mesenchymal growth factor. The cells were cultured at 37°C with 5% (v / v) carbon dioxide until the cell population reached 75% to 85% confluence, at which point enzymatic digestion and passage were performed.

[0101] 2. Collect suspension cells in the logarithmic growth phase, and mix HUVECs and HFDPCs at a ratio of 1.5 x 10⁻⁶ cells per well. 4Cells were individually spread into two 96-well cell culture plates at a density of 100 g / mL and placed for 24 hours to ensure stable cell phenotype and complete adhesion. The core components to be tested in Examples 1 to 5 and Comparative Example 5 were extracted according to the pre-defined proportions and uniformly prepared into a test solution matrix with a final mass concentration of 100 g / mL. This matrix replaced the original culture system, providing exogenous intervention to the cells for 24 hours. Simultaneously, normal culture medium was retained to maintain the well positions for replacement, forming a basic control system.

[0102] 3. Quantitative determination of microvascular dilation mediators in HUVEC cell populations. After the intervention cycle, the supernatant culture medium was extracted and placed in clean centrifuge tubes. Equal volumes of Griess reagent A and B were added for a colorimetric coupling reaction. The mixture was incubated at room temperature in the dark for 15 minutes to allow the azo derivatives to form. The optical absorbance of each sample at a specific wavelength of 540 nm was measured using a spectrophotometer. The accurate concentration equivalent of free nitric oxide (NO) metabolites in the system was calculated by referring to the pre-prepared endogenous sodium nitrite standard curve equation.

[0103] 4. Extraction and detection of HFDPC keratin synthesis levels, which control hair shaft physical toughness, were performed. After emptying and washing the culture plate, pre-cooled RIPA high-efficiency lysis buffer was injected, and the cell membrane bilayer skeleton was thoroughly broken down by sonication. Then, the plate was subjected to high-speed refrigerated centrifugation at 12000 rpm at 4°C. The clear supernatant enriched with total protein was transferred to the wells of an ELISA plate pre-coated with anti-human pankeratin-specific antibody. Horseradish peroxidase-labeled secondary antibody was added according to the double-antibody sandwich immunosorbent assay protocol. The reaction was terminated by TMB substrate conversion and perfusion with a small amount of sulfuric acid within a specified time. The optical density value of the ELISA plate at 450 nm was read to determine the actual compensatory expression quality of total keratin within the cells.

[0104] Table 3. Cellular physicochemical indicators under the intervention of the microvascular oxygen supply and keratin synergistic synthesis mechanism: Test group HUVEC nitric oxide release concentration (mol / L) HFDPC total keratin expression concentration (ng / mL) Blank control group 12.83 46.12 Example 1 47.91 134.58 Example 2 52.37 143.21 Example 3 55.84 161.09 Example 4 49.15 139.73 Example 5 53.08 148.66 Comparative Example 5 15.42 51.37 According to Table 3 and Appendix Figure 5 The data showed that the metabolic activity of the untreated control cells remained consistently low. In Examples 1-5, the addition of L-arginine and zinc methionine increased endothelial cell NO levels to approximately four times the baseline value, indicating enhanced eNOS substrate supply and improved microvascular dilation. Non-seasonal hair loss is common in clinical practice and is often associated with atrophy of the microcirculation at the base of the hair follicle, leading to impaired nutrient delivery.

[0105] Further data showed that the keratin content in the example group reached approximately 160 ng / mL, significantly higher than that in the control group. The hypothesized mechanism is that zinc methionine enters the cell nucleus in a highly bioavailable form, participates in the construction of zinc finger protein structures, and promotes keratin gene transcription and translation.

[0106] In Comparative Example 5, the absence of L-arginine and zinc methionine meant that even with sufficient amino acid precursors in the culture medium, cells were unable to effectively synthesize keratin. This indicates that simply supplementing protein raw materials cannot directly improve hair growth. Only by improving local microcirculation and activating underlying transcriptional mechanisms with zinc can hair follicles truly synthesize keratin. This result suggests that hair densification depends on unobstructed microcirculation and zinc finger-mediated transcriptional synergy, rather than simply increasing the dosage of exogenous proteins or trace elements.

[0107] Test Example 4: 1. A total of 90 healthy Poodles and Pomeranians in their physiologically active period (aged 2 to 5 years, excluding those in pregnancy and lactation) were selected from a designated pet breeding base. These dogs exhibited typical signs of degeneration visible to the naked eye in the initial screening cross-section: extreme fading of the coat, dry and split ends, and overall roughness and loss of luster. The dogs were then transferred to a standard, isolated cage environment and uniformly fed a basic extruded food without functional additives for 7 days to allow for a transition in gastrointestinal tolerance and stress response. Subsequently, they were randomly divided into 9 independent parallel groups of 10 dogs each, corresponding to Examples 1 to 5, Comparative Examples 1, 2, and 4, and a blank control group that continued to be fed the basic food.

[0108] 2. Thoroughly eliminate any additional sources of nutrition in the environment, and initiate a 60-day fully enclosed single-daily diet feeding cycle for each group. The temperature and humidity of the overall rearing environment are strictly controlled by a computer module (room temperature 22-26℃, relative humidity 50%-60%), and a cyclical day-night artificial lighting system is maintained. During the feeding period, a clean, constant water supply is ensured throughout the day. Researchers weigh and record the remaining feed in the troughs for each group daily to monitor average daily feed intake, thereby eliminating systematic errors caused by insufficient nutrient intake due to variations in the physical properties or palatability of the dry food.

[0109] 3. At the baseline of day 0 and the final acceptance point of day 60 of the dietary intervention, approximately 2 grams of newly grown hair samples were taken from the dog's back at fixed coordinates (precisely defined as a 5 cm x 5 cm square) using medical clippers. The hair surface was repeatedly washed with ether and double-distilled water using ultrasound to remove free sebum and environmental dust, then dried in a vacuum chamber until constant weight. Using a precision analytical balance, 50 mg of the treated hair fragment was added to a mixed strong alkaline solution containing 1 mol / L sodium hydroxide and 10% dimethyl sulfoxide, and forcibly digested in a constant-temperature boiling water bath at 100°C for 2 hours until the hair shaft keratin skeleton was completely disintegrated and lost. After centrifugation to remove the supernatant, the absorbance was measured at 405 nm using a spectrophotometer. The absolute melanin mass (mg / g) of solidified melanin entrained in each gram of original hair sample was then calculated, determining the absolute precipitation difference between the two time points.

[0110] 4. Conduct live colorimetric testing directly aligned with the physiological dimensions of pet fur. A portable Lab colorimeter, rigorously calibrated with a standard white board, was used. The fur around the dog's back was combed and spread out at the designated sampling area, ensuring the colorimeter's optical probe was firmly engaged with the fur and that no ambient light leakage occurred. Five measurements were taken at different angles on a single live dog under a calibrated D65 standard light source, and the average value was recorded as the luminance parameter L (this value reflects the reflected light brightness; a lower value indicates the fur color is closer to extreme black). The absolute decrease in L before and after feeding (= before feeding - after feeding) was statistically analyzed to calculate the actual depth of the fur's blackness transformation.

[0111] 5. A multi-angle gloss meter was set up to conduct physical optical reflectance tests at a standard geometric angle of incidence of 60°. The test aperture was strictly aligned with the growth texture of the hair shaft and cuticles of the sample, avoiding exposed skin areas and underlying vellus hair. The gloss unit (GU) was calculated from the specular reflection light flux captured by the probe, and the physical optical scattering convergence ability caused by the density of the hair shaft under different compatibility interventions was quantified and fixed.

[0112] Table 4. Macroscopic data on changes in the physical and chemical properties of pet coats during the clinical feeding cycle: Test group Absolute increase in pigment deposition (mg / g) The decrease in chromatographic brightness L ( ) Glossiness (GU) of the coat at the end of the feeding period Blank control group 0.84 0.63 33.15 Example 1 14.22 8.94 57.42 Example 2 16.59 9.81 61.27 Example 3 19.86 12.05 67.89 Example 4 15.71 9.42 59.03 Example 5 17.65 10.76 63.54 Comparative Example 1 3.58 2.15 38.61 Comparative Example 2 4.12 2.47 40.08 Comparative Example 4 1.15 0.94 34.22 According to Table 4 and Appendix Figure 6 The data shows that after 60 days of feeding with the original diet, the coat of the model group showed obvious degenerative changes: low melanin content, decreased brightness L value (as low as 12.05), and low surface gloss (GU), indicating insufficient pigment deposition and loose keratin structure.

[0113] In Examples 1-5, the test dogs showed a significant increase in melanin within the same period, with the L value remaining at a low level, while the gloss level increased to above 57 GU. This improvement was not due to the absence of high-fat components in the exogenous oil coating experiment, and although supplementing the control group with fish oil or lecithin may have a temporary effect on sebum secretion, it failed to improve problems such as hair breakage, pigment cavities at the hair tips, or loose stratum corneum structure.

[0114] The comparative groups performed poorly: Comparative Example 2 retained the plant extract but removed short peptides, resulting in melanin levels of only 4.12 mg / g; Comparative Example 3 and other schemes lacking key amino acids or plant ligand combinations showed overall performance close to the model group. This indicates that a single component is unlikely to penetrate the intestinal barrier effectively to reach the hair follicles.

[0115] Further analysis showed that the short peptides and Polygonum multiflorum extract in the examples could remain stable in the acidic environment of the stomach and were absorbed through a methionine-zinc synergistic mechanism. After entering the hair papilla, they participated in melanin synthesis and keratin repair, reducing the porosity of the hair shaft medulla and making the structure denser. This ultimately resulted in improved microstructure and enhanced macroscopic gloss (up to 67.89 GU), confirming the effective control of hair degeneration by multi-component synergistic intervention in long-term feeding.

[0116] Test Example 5: 1. Experimental subjects were selected from 90 adult Beagles from a designated breeding center who exhibited similar physical characteristics and non-specific seasonal coat shedding and mild dandruff. Before the full-process experiment, the subjects were fed a standardized, bland base diet for two weeks to eliminate metabolic interference from the previous diversified diet. Nine independent feeding matrices were randomly assigned to map the blank control group, the model group, the groups associated with Examples 1 to 5, and two comparative examples (Comparative Example 3 and Comparative Example 5) with specific pairings removed.

[0117] 2. Except for the blank control group which remained in its natural state, in all other groups, a rectangular area from the dorsal neck region to the midline of the scapula was selected from the subject's back before the formal introduction of the target diet. The surface hair was shaved off, and standardized D-Squame polyurethane keratinocyte peeling tape was used to repeatedly apply and peel the exposed skin with equal pressure 15 times. This physical removal of the top layer of the keratinocyte's protective layer established an acute percutaneous water loss pathological model, inducing compensatory exudation in the underlying microvessels. After confirming that the modeling indicators met the standards, a 45-day single-track feeding period with the corresponding formula functional diet was initiated.

[0118] 3. At the end of the observation period, the subjects were transferred to a constant temperature and humidity measurement room with no convective wind interference and the temperature and humidity locked. The subjects were left to stand for 30 minutes to allow the metabolic heat flow in their bodies to settle. The open-top transdermal water loss meter was then activated, and the probe was gently pressed vertically above the original damaged coordinate area of ​​the model to capture the water vapor density gradient change data in the microenvironment. The transdermal water loss (TEWL) at the corresponding point was then calculated and recorded.

[0119] 4. A minimally invasive biopsy was performed on the tissue surrounding the damaged dermis. 100 mg of skin tissue was weighed and immersed in a pre-cooled chloroform-methanol mixture. The intercellular lipid substrate was fully extracted using high-frequency ultrasonic water bath agitation. The organic phase sequence was combined by centrifugation, concentrated to a fixed volume using nitrogen blowing, and then injected into a high-performance liquid chromatograph for elution and separation of standard substances. The relative cumulative concentration of total ceramides per unit mass of tissue was determined by integrating the peak areas using the external standard method.

[0120] 5. Collect back hair shaft samples from each test unit that naturally shed or were gently combed during their hair growth cycle. Extract the mid-section fibers from unstructured hair follicles and place them in a standard mechanical laboratory environment with equilibrated humidity for 24 hours. Mount them onto a micro-force testing platform for monofilament tensile strength, applying longitudinal stress at a constant tensile displacement deformation rate of 5 mm / min until irreversible fracture occurs in the hair shaft. The equipment control terminal records the absolute breaking strength value at the moment of yielding.

[0121] Table 5. Biochemical and physical tracking indicators under the skin lesion repair and hair shaft biomechanical reconstruction system: Test group <![CDATA[Transdermal water loss (g / hm 2） > Epidermal tissue ceramide concentration (g / mg) Breaking strength of a single hair (cN) Blank control group 12.34 45.18 16.52 Model group 38.67 18.25 11.04 Example 1 16.42 39.71 23.18 Example 2 15.18 42.06 25.64 Example 3 13.05 47.33 28.91 Example 4 15.89 40.12 24.03 Example 5 14.26 44.95 26.85 Comparative Example 3 29.84 24.37 17.62 Comparative Example 5 22.15 33.68 13.25 According to Table 5 and Appendix Figure 7 Data shows that after the epidermal stratum corneum of the model group dogs was damaged, the transdermal water loss reached 38.67 g / hm. 2 This indicates that the skin's moisture barrier has been damaged, and such microscopic physical gaps are often the basis for infectious dermatitis.

[0122] The compound formulation in the example group effectively controlled water loss, restoring the water loss rate in the damaged area to normal levels. Data from the intercellular matrix indicated that this repair effect did not rely on physical coating of the body surface, but rather on the endogenous absorption of DHA and evening primrose extract, providing substrates for ceramide synthesis, thereby reconstructing the lipid structure within the tissue and sealing off water loss pathways. In contrast, Comparative Example 3, which only supplemented with trace elements, lacked specific fatty acids and could not effectively promote endogenous lipid synthesis, resulting in a water loss rate as high as nearly 30 g / hm². 2 .

[0123] Furthermore, the skin barrier's water retention capacity directly affects the physical toughness of the hair shaft. Mechanical testing showed that the tensile breaking strength of a single hair shaft in the example group reached 28.91 cN, improving hair brittleness; while the comparative example 5, lacking zinc ions and the synergistic effect of short peptide chains, had a breaking strength of only 13.25 cN. This is mainly because the high concentration of ceramides within the hair follicle helps keratin chains bind tightly, thereby improving hair dryness and hollowness caused by nutritional deficiencies or epidermal cracking. These data validate the practical effectiveness of multi-target intervention in repairing skin damage and strengthening hair shaft performance.

[0124] Test Example 6: 1. Experimental subjects: Ninety young, medium-short-haired domestic cats (weighing 3.5 to 4.5 kg, aged 1.5 to 3 years) purchased from an experimental animal breeding facility were selected. All cats underwent a 14-day clinical screening before enrollment to exclude individuals carrying latent skin pathogens or exhibiting metabolic defects in their digestive system. The selected cats were randomly assigned to nine independent groups, including a blank control group, a model group, test groups corresponding to Examples 1 to 5, and comparative examples 1 and 2. All cats were isolated individually in a barrier system-equipped enclosure with constant temperature and humidity control and artificial spectral intervention.

[0125] 2. Establishment of an environmental stress-induced hair degeneration pathological model. Except for the blank control group, which was fed a basal diet under normal natural light and standard humidity (60%), all other groups were exposed to continuous high-intensity artificial cold light (16 hours of light exposure per day) and forced dry airflow (relative humidity controlled below 25%). Feeding them a basal dry cat food lacking functional lipids induced oxidative stress in the systemic skin microecology and hair follicle closure. Twenty days after modeling, a moderate proportion of the corresponding intervention preparation was mixed into the diet of each test group, and a 30-day continuous targeted feeding experiment was conducted under the same stress environment. Daily changes in food intake and fecal characteristics were recorded to calculate the effective intake threshold.

[0126] 3. Near the end of the testing period, blood was collected from the cephalic vein of the forelimb of each subject by venipuncture. Approximately 3 mL of whole blood was collected and injected into a coagulation-promoting tube. The sample was then centrifuged at 3000 rpm at 4°C to obtain the clear supernatant serum. A quantitative amount of serum matrix solution was extracted and subjected to microwave digestion with a mixture of nitric acid and perchloric acid as a strong oxidant to break down protein inclusions. The solution was then introduced into an inductively coupled plasma mass spectrometer (ICP-MS). Using a specific mass-to-charge ratio separation technique and referring to the international standard zinc element working curve, the background enrichment concentration of total zinc in the peripheral blood circulation of each subject was absolutely quantified.

[0127] 4. Under anesthesia, a full-thickness skin biopsy block approximately 3 mm in diameter was taken from the dorsal side of the test cat. The tissue specimen was flash-frozen in liquid nitrogen and then transferred to a glass homogenizer. Low-temperature phosphate buffer containing protease inhibitors was added in the appropriate ratio for mechanical homogenization. The clarified tissue homogenate was extracted, and based on the principle of the thiobarbituric acid (TBA) colorimetric reaction, malondialdehyde (MDA), the final product of lipid peroxidation, was induced to form a red condensate with the reagent under 95°C water bath conditions. The reaction solution was placed in a cuvette, and the optical density at the characteristic wavelength of 532 nm was read. The absolute accumulation of MDA per unit milligram of dermal tissue protein, representing the degree of cellular oxidative damage, was calculated.

[0128] 5. Microscopic measurements of the histological morphology of the hair growth cycle were performed. After fixation in paraformaldehyde and paraffin embedding, continuous sagittal sections approximately 5 μm thick were cut from the edge tissue of the biopsy and stained with hematoxylin and eosin (H&E) as usual. Under high magnification of an optical microscope, based on the morphology of the dermal papilla tip, the integrity of the inner root sheath, and the morphological characteristics of dermal papilla cells, the absolute number of hair follicles in the anagen and telogen phases within the section area was accurately distinguished and counted. The A / T ratio was calculated by dividing the A / T ratio by the A / T ratio, which served as a direct morphological parameter for assessing the proliferative activity of the hair follicle basal layer.

[0129] Table 6. Serum trace elements and skin metabolic characteristics after targeted intervention for environmental stress: Test group Peak concentration of free zinc ions in serum (g / dL) Accumulation of malondialdehyde (MDA) in the dermis (nmol / mg) anagen / telogen ratio Blank control group 86.15 3.42 2.68 Model group 80.47 8.95 0.74 Example 1 148.26 4.67 3.51 Example 2 154.09 4.12 4.05 Example 3 167.83 2.81 4.88 Example 4 151.62 4.36 3.79 Example 5 160.14 3.75 4.26 Comparative Example 1 95.38 6.82 1.83 Comparative Example 2 158.71 7.96 2.45 According to Table 6 and Figure 8 Data showed that individuals in the model group continuously exposed to dry, cold light environments exhibited significant signs of regression. Their dermal MDA accumulation increased to 8.95 nmol / mg, while the A / T ratio of the hair follicle's anagen (growth) to telogen (resting) phase decreased to 0.74, indicating that oxidative stress caused active hair follicles to prematurely enter the telogen phase. This condition is more common in urban pets that are constantly exposed to constant indoor temperatures and artificial light, easily accumulating free radicals and triggering non-seasonal hair loss.

[0130] The example group improved this metabolic state through the compatibility of nutrients. Taking Example 3 as an example, this system utilized an organic complexation mechanism to reduce the interference of phytic acid in the diet on mineral absorption, increasing the peripheral serum free zinc concentration to 167.83 g / dL. After zinc entered the dermal tissue via transport peptides, it acted synergistically with the glycosides retained in processed Polygonum multiflorum as a core component of superoxide dismutase synthesis. This combination neutralized free hydrogen peroxide on the cell membrane surface, thereby blocking lipid peroxidation and reducing MDA accumulation to the normal level of 2.81 nmol / mg. Observation of the comparative example without plant component intervention revealed that although its formulation could ensure the effective enrichment of trace elements in the blood, with a serum zinc concentration of 158.71 g / dL, lipid peroxidation inside the tissue could not be controlled due to the lack of corresponding antioxidant mediators. The MDA accumulation in this group remained high at 7.96 nmol / mg, and the presence of local inflammation made it difficult for resting hair follicles to restart cell differentiation. From the final morphological indicators, the combined intervention significantly improved the metabolic cycle of hair follicles; the A / T ratio in the example was increased to 4.88, confirming that dermal papilla cells regained their proliferative activity. This indicates that in addressing the physiological decline of the skin's basal layer caused by environmental degradation, simply increasing the absorption rate of trace elements is insufficient. It is necessary to combine the promotion of element absorption with free radical scavenging mechanisms to effectively reverse the stagnant state of hair follicles and improve hair quality.

Claims

1. A pet food for skin care and coat enhancement, characterized in that, The pet food is made from the following ingredients in parts by weight: 50-58 parts animal protein powder, 16-22 parts starchy raw materials, 10-15 parts aromatic enzymatic hydrolyzed short peptides, 2-3 parts compound Chinese herbal extract mixed dry powder, 1.5-2.5 parts processed Polygonum multiflorum extract, 1-2 parts Schizochytrium powder, 3-5 parts evening primrose seed oil, 0.5-1 part methionine zinc complex, 0.002-0.004 parts vitamin A acetate microcapsule powder, 0.002-0.004 parts D-biotin, and 1-1.5 parts L-arginine.

2. The pet food for skin care and coat enhancement according to claim 1, characterized in that, The pet food is made from the following preferred raw materials in parts by weight: 52-55 parts animal protein powder, 18-20 parts starchy raw material, 12-14 parts aromatic enzymatic hydrolysed short peptides, 2.5-2.8 parts compound Chinese herbal extract mixed dry powder, 2-2.2 parts processed Polygonum multiflorum extract, 1.5-1.8 parts Schizochytrium powder, 4-4.5 parts evening primrose seed oil, 0.8-0.9 parts methionine zinc complex, 0.003-0.0035 parts vitamin A acetate microcapsule powder, 0.003-0.0035 parts D-biotin, and 1.2-1.4 parts L-arginine.

3. The pet food for skin care and coat enhancement according to claim 1, characterized in that, The starchy raw material is cassava starch or potato flour; The aromatic enzymatically hydrolyzed short peptides have a number-average molecular weight of 351–495 Da, and the total molar percentage of phenylalanine and tyrosine residues in their amino acid sequences is 35.2%–41.1%. The mass fraction of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside in the prepared Polygonum multiflorum extract is 8.2% to 10.8%, and the mass fraction of total anthraquinones, calculated as emodin, is 1.3% to 1.8%.

4. The pet food for skin care and coat enhancement according to claim 1, characterized in that, The aromatic enzymatically hydrolyzed short peptides are prepared by a method including the following steps: Soy protein isolate powder was added to purified water to prepare a suspension and the pH was adjusted to 8.4–8.6; the temperature was raised to 53–57°C, and 1.0–1.4 parts by weight of alkaline protease were added per 100 parts by weight of soy protein isolate powder. Hydrolysis was carried out continuously at a constant temperature for 115–125 minutes, and the pH of the system was kept constant during hydrolysis; then citric acid aqueous solution was added to adjust the pH to 4.5, and the temperature was raised to 95°C and held for 10 minutes to inactivate the enzyme. After cooling, the solution is filtered through a microporous membrane to remove impurities. The initial filtrate is collected and subjected to cross-flow filtration through an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da under a pressure of 0.8–1 MPa. The permeate is then loaded into a chromatography column packed with D101 macroporous adsorption resin. First, purified water is used to remove inorganic salts and highly hydrophilic peptides, followed by elution with a 70% (v / v) ethanol solution. The polar eluent is collected and dried to obtain a pale yellow powder.

5. The pet food for skin care and coat enhancement according to claim 1, characterized in that, The prepared Polygonum multiflorum extract is obtained by a method including the following steps: Add a 70% ethanol solution to the pulverized processed Polygonum multiflorum powder at a mass-to-volume ratio of 1:10, and perform reflux extraction at 75–85°C. Combine the extracts and concentrate under reduced pressure to recover the ethanol until no alcohol odor remains. Add purified water to dilute the precipitated water-insoluble colloids, and centrifuge to obtain a clear aqueous extract. Load the aqueous extract into a D101 macroporous adsorption resin column with an internal column diameter-to-height ratio of 1:

5. First, elute with purified water to remove sugar impurities, and then elute with a 50% ethanol solution. Collect the desorbed eluent, concentrate under reduced pressure to remove the ethanol, and then dry to obtain a brown dry powder.

6. A method for preparing pet food for skin care and coat enhancement, comprising the pet food for skin care and coat enhancement according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Dry powder premix: Mix the remaining powdered ingredients in the formula except evening primrose seed oil to obtain basic dry powder. S2, Conditioning and Extrusion Puffing: The basic dry powder is preheated and conditioned by steam and then fed into the barrel. Under heating and pressure conditions, it is extruded, gelatinized and puffed into shape, extruded through the die and cut into granules. S3. Drying treatment: The formed puffed pellets are dried with hot air to obtain dry basic grain pellets; S4. Vacuum spraying and cooling: Under a closed vacuum condition, the formula amount of evening primrose seed oil is evenly atomized and sprayed onto the surface of the pellets while maintaining pressure. Then, the vacuum is slowly released to allow the oil to penetrate into the micropores of the pellets. The target pet food is obtained by cooling to room temperature.

7. The method for preparing a pet food for skin care and coat enhancement according to claim 6, characterized in that, The specific implementation method of step S1 is as follows: put the raw materials into a three-dimensional high-efficiency mixer and mix them continuously at room temperature for 20 to 30 minutes until the coefficient of variation (CV5) of the mixture is obtained.

8. The method for preparing a pet food for skin care and coat enhancement according to claim 6, characterized in that, In step S2, the conditioning process controls the material center temperature to reach 80-85℃ and adjusts the moisture content to 20%-22%; the process parameters for the extrusion gelatinization and puffing molding process are: screw speed 250-270 r / min, barrel heating section temperature set to 105℃-115℃, and die head pressure 2.5-2.8 MPa.

9. A method for preparing pet food for skin care and coat enhancement according to claim 6, characterized in that, In step S3, the hot air drying temperature is 85-90℃ and the time is 35-45 minutes, controlling the moisture content of the dried basic grain particles to be reduced to below 8.0%.

10. A method for preparing pet food for skin care and coat enhancement according to claim 6, characterized in that, In step S4, the pressure of the vacuum state is reduced to -0.08MPa, and after atomized spraying, it is maintained for 3 to 4 minutes before the vacuum is slowly released; the cooling to room temperature is achieved by using a counter-flow cooler.

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