Pet food with tear mark removing function and preparation method thereof

Abstract

The present application relates to the technical field of pet food, and discloses pet food with tear mark removing function and a preparation method thereof, the preparation method comprising: replacing part of the water used for decocting with mulberry extract solution to decoct chrysanthemum, cassia seed, medlar and dandelion; performing low-temperature concentration on the obtained decocting liquid after rough filtration, removing tannin precipitate through fine filtration, and performing concentration again to prepare a high-purity concentrated solution; respectively performing shallow enzymolysis and deep enzymolysis on duck liver with food-grade alkaline protease, and compounding the two kinds of enzymolysis products to prepare a compounded duck liver hydrolyzed protein solution; fully mixing the compounded duck liver hydrolyzed protein solution with fresh minced duck meat, then adding the high-purity concentrated solution and mixing uniformly, and performing molding and freeze-drying treatment to obtain a finished product. The present application solves the problems that it is difficult to simultaneously remove astringent substances and retain functional active ingredients, and that the functional ingredients are unevenly distributed due to phase separation when the plant concentrated solution is mixed with fresh minced meat, and obtains pet food with uniform distribution of functional active ingredients and good palatability for pets.

Description

Technical Field

[0001] This invention relates to the field of pet food technology, and more specifically, to a pet food with tear stain removal function and its preparation method. Background Technology

[0002] Tear stains are a common clinical problem in dogs and cats. They are caused by excessive tear secretion around the eyes, blocked tear ducts, or chronic periorbital inflammation. Excessive intake of hot foods and insufficient heat-clearing ability in the diet are important internal factors contributing to these problems. Existing tear stain removal solutions mainly fall into two categories: one focuses on external cleaning, which only improves the external appearance of tear stains and cannot address the root cause of tear secretion and periorbital inflammation; the other adds powdered traditional Chinese medicine or food-grade medicinal ingredients to pet food. However, direct addition of powder results in uneven dispersion within the food base, and the powder particles lack effective bonding with the meat base, leading to uneven distribution of functional ingredients in the finished product and short-lasting tear stain removal effects. To solve the problem of uneven dispersion of powder, the industry has attempted to prepare concentrated decoctions of food-grade medicinal ingredients before mixing them with fresh meat.

[0003] However, the aforementioned approach faces several mutually restrictive technical challenges, limiting the practical effectiveness of functional pet foods designed to reduce tear stains. Firstly, the concentrated decoction of medicinal and edible plants contains a large amount of tannins, which have a similar water solubility to functional active ingredients such as chrysanthemum flavonoids and wolfberry polysaccharides. Conventional single-pass filtration processes are insufficient to effectively separate these substances, inevitably resulting in the loss of functional active ingredients while removing the astringent substances. If the astringent substances are not removed, pets will refuse to eat due to the bitter taste, affecting actual intake. Secondly, the concentrated decoction of medicinal and edible plants is a hydrophilic aqueous solution, while fresh minced duck meat contains hydrophobic fats. Direct mixing of the two leads to phase separation, with liquid precipitation and meat aggregation, resulting in uneven spatial distribution of functional ingredients in the finished product. Thirdly, in duck liver hydrolysate used to enhance pets' appetite, the interface-improving function of amphiphilic peptides and the palatability-enhancing function of small molecule peptides and free amino acids require conflicting enzymatic hydrolysis levels. Products with only a single degree of enzymatic hydrolysis cannot simultaneously meet both functional requirements. Summary of the Invention

[0004] This invention provides a pet food with tear stain removal function and its preparation method, solving the technical problems in related technologies such as the difficulty in simultaneously removing astringent substances and retaining functional active ingredients, the uneven distribution of functional ingredients due to phase separation when mixing plant concentrate and fresh minced meat, and the difficulty in simultaneously achieving the palatability-enhancing function and the interface-improving function of amphiphilic peptides in the same batch of products.

[0005] This invention provides a method for preparing pet food with tear stain removal function, comprising the following steps:

[0006] Step 1: Use mulberry extract to replace part of the water used for decoction as the decoction solvent to decoct chrysanthemum, cassia seed, wolfberry and dandelion to obtain a mixed decoction.

[0007] Step 2: After coarse filtration of the mixed decoction, it is concentrated under low temperature and reduced pressure. After cooling and standing, the tannins are precipitated. After fine filtration to remove the precipitated tannins, it is concentrated again under low temperature and reduced pressure to obtain a high-purity concentrate.

[0008] Step 3: Use food-grade alkaline protease to perform shallow and deep enzymatic hydrolysis on duck liver. Shallow enzymatic hydrolysis controls the degree of hydrolysis so that the protein in the hydrolysate is mainly amphiphilic peptides. Deep enzymatic hydrolysis controls the degree of hydrolysis so that the protein in the hydrolysate is fully hydrolyzed into small molecule peptides and free amino acids. Mix the two enzymatic hydrolysis products to obtain a compound duck liver hydrolysate.

[0009] Step four: First, mix the compound duck liver hydrolysate with fresh minced duck meat, so that the hydrophobic ends of the amphiphilic peptides are adsorbed onto the surface of the duck meat fat and the hydrophilic ends extend toward the water phase, changing the surface of the duck meat fat from hydrophobic to hydrophilic. Then, add high-purity concentrate and mix. After molding and freeze-drying, the finished product is obtained.

[0010] Preferably, in step one, the mass ratio of dried chrysanthemum, cassia seed, wolfberry, and dandelion is 1-2:1-3:1-2:1-2; the mass ratio of the total dry weight of the medicinal and edible raw materials to the total amount of liquid used for decoction is 1:8-1:12; the solid content of the mulberry extract is 5%-15% (w / v), replacing 20%-40% of the total amount of liquid used for decoction, with the remainder made up with water; when decocting, heat to boiling and then maintain a gentle boil for 20-40 minutes.

[0011] Preferably: In step two, a 100-mesh sieve is used for coarse filtration; the low-temperature vacuum concentration temperature is 40℃~60℃, and after concentration to 1 / 5~1 / 3 of the mixed filtrate volume, it is cooled to 10℃~20℃ and allowed to stand for more than 30 minutes; a 200~300-mesh filter membrane is used for fine filtration; the secondary low-temperature vacuum concentration temperature is 40℃~60℃, and it is concentrated to 1 / 3~1 / 2 of the refined filtrate volume.

[0012] Preferably, in step three, the conditions for shallow enzymatic hydrolysis are as follows: after homogenizing the duck liver, add water to a mass ratio of duck liver to water of 1:2 to 1:4, adjust the pH to 7.5 to 8.5 with food-grade sodium hydroxide, use food-grade alkaline protease at a dosage of 0.5% to 2.0% of the dry weight of the duck liver, incubate at 50℃ to 60℃ for 30 to 90 minutes, and control the degree of hydrolysis to 5% to 15%.

[0013] Preferably, in step three, the deep enzymatic hydrolysis uses the same material-to-water ratio, pH adjustment range, enzyme dosage and temperature conditions as the shallow enzymatic hydrolysis, the incubation time is 150-300 min, and the degree of hydrolysis is controlled at 35%-55%.

[0014] Preferably: In step three, after shallow and deep enzymatic hydrolysis are completed, the hydrolysate is heated to 85℃~90℃ and maintained for more than 15 minutes to inactivate the alkaline protease. After cooling to room temperature, it is used for compounding.

[0015] Preferably, in step three, the shallowly enzymatically hydrolyzed duck liver hydrolysate and the deeply enzymatically hydrolyzed duck liver hydrolysate are mixed at a mass ratio of 1:1 to 1:3.

[0016] Preferably, in step four, the amount of compound duck liver hydrolysate added is 5% to 15% of the weight of fresh minced duck meat, and the amount of high-purity concentrate added is 10% to 25% of the weight of fresh minced duck meat.

[0017] Preferably, in step four, the high-purity concentrate is not added to the fresh minced duck meat at the same time as the compound duck liver hydrolysate, nor is it added to the fresh minced duck meat before the compound duck liver hydrolysate, in order to prevent the saponin components in the high-purity concentrate from competing with the amphiphilic peptides for adsorption sites on the surface of duck meat fat.

[0018] The present invention also provides a pet food with tear stain removal function prepared by the above preparation method.

[0019] The beneficial effects of this invention are as follows: By using a secondary filtration-re-concentration process, the astringent tannins are preferentially precipitated during the concentration and cooling process. The precipitated tannins are retained by fine filtration, achieving simultaneous removal of astringent substances and retention of functional active ingredients. Mulberry is a food and medicine source, rich in anthocyanins and other active ingredients, and has the effects of nourishing the liver and kidneys, nourishing yin and improving eyesight, and anti-inflammatory and antioxidant properties. Mulberry extract is used to replace part of the water used for decoction, allowing the active ingredients such as mulberry anthocyanins to be fully co-dissolved and enriched with the functional active ingredients such as chrysanthemum flavonoids and wolfberry polysaccharides in the same decoction process. This synergistic effect with the liver-clearing and vision-improving effects of chrysanthemum, the heat-clearing and anti-inflammatory effects of dandelion, and the yin-nourishing and vision-improving effects of wolfberry enhances the overall tear stain removal effect of the finished product.

[0020] By specifying the order of adding ingredients as “first adding compound duck liver hydrolysate solution and fresh minced duck meat, then adding high-purity concentrate”, the surface of duck meat fat is changed from hydrophobic to hydrophilic by natural amphiphilic peptides, overcoming the phase separation phenomenon when plant concentrate is directly mixed with fresh minced meat, and ensuring that functional active ingredients are uniformly distributed in the finished product.

[0021] By performing shallow and deep enzymatic hydrolysis on duck liver and then compounding the products, the same batch of products can simultaneously contain macromolecular amphiphilic peptides that change the wettability of duck fat surface and small molecule peptides and free amino acids that provide animal-derived aroma, without the need to introduce two types of functional excipients separately.

[0022] It uses fresh minced duck meat combined with various medicinal and edible ingredients and mulberry. The combination of these ingredients creates a multi-dimensional system for clearing heat and removing tear stains, which addresses the root cause of excessive tear secretion and periorbital inflammation. The entire process uses medicinal and edible ingredients and natural food ingredients, and contains no antibiotics, hormones, chemically synthesized emulsifiers, or preservatives, making it suitable for long-term use.

[0023] Overall, this invention, through systematic process design, collaboratively solves three core issues in the same production process: the quality of preparation of concentrated medicinal and edible liquids, the uniform distribution of functional components, and the palatability of the finished product. It yields an all-natural functional pet food with comprehensive tear stain removal effects, uniform distribution of functional components, and good palatability for pets. Attached Figure Description

[0024] Figure 1 This is a bar chart showing the effect of different filtration processes on the removal efficiency of tannin-like astringent substances and the retention efficiency of functional active ingredients in Experiment 1 of this invention.

[0025] Figure 2 This is a bar chart showing the effect of different mixing methods on the surface wettability of duck fat in Experiment 2 of this invention;

[0026] Figure 3 This is a bar chart showing the effect of different mixing methods on the spatial distribution uniformity of functional active ingredients in the finished product in Experiment 2 of this invention.

[0027] Figure 4 This is a line graph showing the dynamic changes in the voluntary feeding rate of pet cats under different formulations in Experiment 3 of this invention;

[0028] Figure 5 This is a bar chart comparing the 7-day average voluntary feeding rates of each group in Experiment 3 of this invention;

[0029] Figure 6 This is a line graph showing the dynamic changes in tear stain scores in Experiment 4 of this invention;

[0030] Figure 7 This is a bar chart comparing the porphyrin content in tears on day 0 and day 28 in Experiment 4 of this invention. Detailed Implementation

[0031] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0032] Example 1

[0033] This embodiment discloses a method for preparing pet food with tear stain removal function, which includes the following steps:

[0034] Step 1: Take dried chrysanthemum, cassia seed, wolfberry, and dandelion. The mass ratio of each raw material is chrysanthemum:cassia seed:wolfberry:dandelion = 1:1:1:1. The mass ratio of the total dry weight of the medicinal and edible raw materials to the total amount of liquid for decoction is 1:8. Soak dried mulberry in water to prepare a mulberry extract with a solid content of 5% (w / v). Use this extract to replace 20% of the total amount of liquid for decoction, and make up the remaining 80% with water. Mix the extract and use it as the decoction solvent. Place the mixed solvent and the medicinal and edible raw materials in a decoction container, heat to boiling, and then maintain a gentle boil for 20 minutes to obtain a mixed decoction.

[0035] Step Two: While still hot, coarsely filter the mixed decoction obtained in Step One through a 100-mesh sieve to remove large particles of plant residue, obtaining a mixed filtrate. Concentrate the mixed filtrate under reduced pressure at 40°C, reducing the liquid volume to 1 / 3 of its original volume. After concentration, cool the concentrate to 10°C and allow it to stand for 30 minutes to allow tannins to precipitate. Finely filter the concentrate containing the precipitate through a 200-mesh filter membrane to remove the tannin precipitate and fine particles, obtaining a refined filtrate. Concentrate the refined filtrate again under low-temperature reduced pressure at 40°C, reducing the liquid volume to half of its original volume, obtaining a high-purity concentrate.

[0036] Step 3: (1) Shallow enzymatic hydrolysis: Take fresh duck liver, homogenize it, add water to make the mass ratio of duck liver to water 1:2, adjust the pH to 7.5 with food-grade sodium hydroxide, add food-grade alkaline protease (0.5% of the dry weight of duck liver), keep it at 50℃ for 30 min, and control the degree of hydrolysis (DH value) to 5%; after the enzymatic hydrolysis is completed, heat the enzymatic hydrolysate to 85℃ and maintain it for 15 min to inactivate the protease, cool it to room temperature, and obtain shallow enzymatic hydrolysate of duck liver. (2) Deep enzymatic hydrolysis: Take fresh duck liver, homogenize it, add water to make the mass ratio of duck liver to water 1:2, adjust the pH to 7.5 with food-grade sodium hydroxide, add food-grade alkaline protease (0.5% of the dry weight of duck liver), and incubate at 50℃ for 150 min to control the degree of hydrolysis (DH value) to 35%; after the enzymatic hydrolysis is completed, heat the enzymatic hydrolysate to 85℃ and maintain it for 15 min to inactivate the protease, and cool it to room temperature to obtain the deep enzymatic hydrolysate of duck liver. (3) Compounding: Mix the shallow enzymatic hydrolysate of duck liver with the deep enzymatic hydrolysate at a mass ratio of 1:1 to obtain the compound duck liver hydrolysate protein solution.

[0037] Step 4: Take the compound duck liver hydrolysate obtained in Step 3, adding it at 5% of the weight of fresh minced duck meat. Mix it thoroughly with the fresh minced duck meat to allow the hydrophobic ends of the amphiphilic peptides to adsorb onto the surface of the duck fat and the hydrophilic ends to extend towards the aqueous phase, thus changing the surface of the duck fat from hydrophobic to hydrophilic. After the above adsorption is completed, add the high-purity concentrate obtained in Step 2 at 10% of the weight of the fresh minced duck meat and mix thoroughly. Press the resulting food mixture into shape according to the pet food molding process and freeze-dry it to obtain the finished product.

[0038] Example 2

[0039] This embodiment discloses a method for preparing pet food with tear stain removal function, which includes the following steps:

[0040] Step 1: Take dried chrysanthemum, cassia seed, wolfberry, and dandelion. The mass ratio of the dried ingredients is chrysanthemum:cassia seed:wolfberry:dandelion = 2:2:1:1. The mass ratio of the total dry weight of the medicinal and edible ingredients to the total amount of liquid for decoction is 1:10. Soak dried mulberry in water to prepare a mulberry extract with a solid content of 10% (w / v). Use this extract to replace 30% of the total amount of liquid for decoction, and make up the remaining 70% with water. Mix the extract and use it as the decoction solvent. Place the mixed solvent and the medicinal and edible ingredients in a decoction container, heat to boiling, and then maintain a gentle boil for 30 minutes to obtain a mixed decoction.

[0041] Step Two: While still hot, coarsely filter the mixed decoction obtained in Step One through a 100-mesh sieve to remove large particles of plant residue, obtaining a mixed filtrate. Concentrate the mixed filtrate under reduced pressure at 50°C until the liquid volume is reduced to 1 / 4 of its original volume. After concentration, cool the concentrate to 15°C and allow it to stand for 60 minutes to allow tannins to precipitate. Finely filter the concentrate containing the precipitate through a 250-mesh filter membrane to remove the tannin precipitate and fine particles, obtaining a refined filtrate. Concentrate the refined filtrate again under low-temperature reduced pressure at 50°C until the liquid volume is reduced to 1 / 3 of its original volume, obtaining a high-purity concentrate.

[0042] Step 3: (1) Shallow enzymatic hydrolysis: Take fresh duck liver, homogenize it, add water to make the mass ratio of duck liver to water 1:3, adjust the pH to 8.0 with food-grade sodium hydroxide, add food-grade alkaline protease (the amount is 1.0% of the dry weight of duck liver), keep it at 55℃ for 60 min, and control the degree of hydrolysis (DH value) to 10%; after the enzymatic hydrolysis is completed, heat the enzymatic hydrolysate to 88℃ and maintain it for 15 min to inactivate the protease, cool it to room temperature, and obtain shallow enzymatic hydrolysate of duck liver. (2) Deep enzymatic hydrolysis: Take fresh duck liver, homogenize it, add water to make the mass ratio of duck liver to water 1:3, adjust the pH to 8.0 with food-grade sodium hydroxide, add food-grade alkaline protease (1.0% of the dry weight of duck liver), and incubate at 55℃ for 200 min to control the degree of hydrolysis (DH value) to 45%; after the enzymatic hydrolysis is completed, heat the enzymatic hydrolysate to 88℃ and maintain it for 15 min to inactivate the protease, and cool it to room temperature to obtain the deep enzymatic hydrolysate of duck liver. (3) Compounding: Mix the shallow enzymatic hydrolysate of duck liver with the deep enzymatic hydrolysate at a mass ratio of 1:2 to obtain the compound duck liver hydrolysate protein solution.

[0043] Step 4: Take the compound duck liver hydrolysate obtained in Step 3, and add it at 10% of the weight of fresh minced duck meat. Mix it thoroughly with the fresh minced duck meat so that the hydrophobic ends of the amphiphilic peptides are adsorbed onto the surface of the duck fat and the hydrophilic ends extend toward the aqueous phase, thus changing the surface of the duck fat from hydrophobic to hydrophilic. After the above adsorption is completed, add the high-purity concentrate obtained in Step 2 at 18% of the weight of fresh minced duck meat and mix thoroughly with the concentrate. Press the resulting food mixture into shape according to the pet food molding process and freeze-dry it to obtain the finished product.

[0044] Example 3

[0045] This embodiment discloses a method for preparing pet food with tear stain removal function, which includes the following steps:

[0046] Step 1: Take dried chrysanthemum, cassia seed, wolfberry, and dandelion. The mass ratio of the dried ingredients is chrysanthemum:cassia seed:wolfberry:dandelion = 2:3:2:2. The mass ratio of the total dry weight of the medicinal and edible ingredients to the total amount of liquid for decoction is 1:12. Soak dried mulberry in water to prepare a mulberry extract with a solid content of 15% (w / v). Use this extract to replace 40% of the total amount of liquid for decoction, and make up the remaining 60% with water. Mix the extract as the decoction solvent. Place the mixed solvent and the medicinal and edible ingredients in a decoction container, heat to boiling, and then maintain a gentle boil for 40 minutes to obtain a mixed decoction.

[0047] Step Two: While still hot, coarsely filter the mixed decoction obtained in Step One through a 100-mesh sieve to remove large particles of plant residue, obtaining a mixed filtrate. Concentrate the mixed filtrate under reduced pressure at 60°C until the liquid volume is reduced to 1 / 5 of the total volume. After concentration, cool the concentrate to 20°C and allow it to stand for 90 minutes to allow tannins to precipitate. Finely filter the concentrate containing the precipitate through a 300-mesh filter membrane to remove the tannin precipitate and fine particles, obtaining a refined filtrate. Concentrate the refined filtrate again under low-temperature reduced pressure at 60°C until the liquid volume is reduced to 1 / 3 of the total volume, obtaining a high-purity concentrate.

[0048] Step 3: (1) Shallow enzymatic hydrolysis: Take fresh duck liver, homogenize it, add water to make the mass ratio of duck liver to water 1:4, adjust the pH to 8.5 with food-grade sodium hydroxide, add food-grade alkaline protease (the amount is 2.0% of the dry weight of duck liver), keep it at 60℃ for 90 min, and control the degree of hydrolysis (DH value) to 15%; after the enzymatic hydrolysis is completed, heat the enzymatic hydrolysate to 90℃ and maintain it for 15 min to inactivate the protease, cool it to room temperature, and obtain shallow enzymatic hydrolysate of duck liver. (2) Deep enzymatic hydrolysis: Take fresh duck liver, homogenize it, add water to make the mass ratio of duck liver to water 1:4, adjust the pH to 8.5 with food-grade sodium hydroxide, add food-grade alkaline protease (the amount is 2.0% of the dry weight of duck liver), and incubate at 60℃ for 300 min to control the degree of hydrolysis (DH value) to 55%; after the enzymatic hydrolysis is completed, heat the enzymatic hydrolysate to 90℃ and maintain it for 15 min to inactivate the protease, and cool it to room temperature to obtain the deep enzymatic hydrolysate of duck liver. (3) Compounding: Mix the shallow enzymatic hydrolysate of duck liver with the deep enzymatic hydrolysate at a mass ratio of 1:3 to obtain the compound duck liver hydrolysate protein solution.

[0049] Step 4: Take the compound duck liver hydrolysate obtained in Step 3, and add it at 15% of the weight of fresh minced duck meat. Mix it thoroughly with the fresh minced duck meat so that the hydrophobic ends of the amphiphilic peptides are adsorbed onto the surface of the duck fat and the hydrophilic ends extend toward the aqueous phase, thus changing the surface of the duck fat from hydrophobic to hydrophilic. After the above adsorption is completed, add the high-purity concentrate obtained in Step 2 at 25% of the weight of fresh minced duck meat and mix thoroughly with the concentrate. Press the resulting food mixture into shape according to the pet food molding process and freeze-dry it to obtain the finished product.

[0050] Example 4

[0051] This embodiment discloses a method for preparing pet food with tear stain removal function, which includes the following steps:

[0052] Step 1: Replace part of the water used for decoction with mulberry extract to decoct the medicinal and edible ingredients.

[0053] Dried chrysanthemum, cassia seed, wolfberry, and dandelion were taken, with a mass ratio of chrysanthemum:cassia seed:wort:dandelion = 2:2:1:1. The mass ratio of the total dry weight of the medicinal and edible raw materials to the total amount of liquid used for decoction was 1:10. Dried mulberry was extracted with water to prepare a mulberry extract with a solid content of 10% (w / v). This extract replaced 30% of the total amount of liquid used for decoction, and the remaining 70% was made up with water. The mixture was then used as the decoction solvent. The above mixed solvent and the medicinal and edible raw materials were placed in a decoction container, heated to boiling, and then kept at a simmer for 30 minutes. The resulting liquid was a mixed decoction containing medicinal and edible functional components and tannin-like astringent substances.

[0054] Replacing part of the water with mulberry extract in the decoction process achieves the following two benefits: First, mulberry, a food and medicinal ingredient, is rich in anthocyanin-like active ingredients such as cyanidin-3-O-glucoside, which have the effects of nourishing the liver and kidneys, nourishing yin and improving eyesight, and anti-inflammatory and antioxidant properties. Using mulberry extract in the decoction process allows the anthocyanins and other active ingredients to be fully co-dissolved and enriched with chrysanthemum flavonoids, wolfberry polysaccharides, and other functional active ingredients in the same decoction step, and then enters the subsequent high-purity concentrate. This synergistic effect combines with the liver-clearing and vision-improving effects of chrysanthemum, the heat-clearing and anti-inflammatory effects of dandelion, and the yin-nourishing and vision-improving effects of wolfberry, enhancing the overall liver-clearing, vision-improving, and tear stain-reducing effects of the finished product without increasing the variety of raw materials. Second, replacing part of the water used in the decoction with mulberry extract eliminates the need for separate soaking and additional processes for extracting the active ingredients of mulberry, allowing them to be directly extracted together with other food and medicinal ingredients during the decoction stage. This simplifies the production process and achieves the goal of synergistic efficacy through solvent substitution.

[0055] Step 2: Perform a second filtration and further concentration on the decoction to prepare a high-purity concentrate.

[0056] This step is crucial for removing astringent substances and preserving functional active ingredients, and includes the following sub-steps:

[0057] Sub-step 2-1: Coarse filtration. While still hot, filter the decoction obtained in step one through a 100-mesh sieve to remove large particles of plant residue. The resulting filtrate is a mixed filtrate containing tannins and functional active ingredients.

[0058] Sub-step 2-2: Low-temperature concentration and cooling. The above mixed filtrate is concentrated under reduced pressure at 50°C, reducing the liquid volume to 1 / 4 of the mixed filtrate volume. After concentration, the concentrate is cooled to 15°C and allowed to stand at this temperature for 60 minutes. During the concentration and cooling process, tannins (mainly condensed tannins) precipitate more due to increased concentration and decreased temperature, forming suspended precipitates in the concentrate. Due to their high water solubility, polysaccharide functional active ingredients such as wolfberry polysaccharides precipitate significantly less under the above concentration and cooling conditions than tannins, mainly remaining in the liquid phase. A small amount of flavonoid functional active ingredients such as chrysanthemum flavonoids also precipitate under the above conditions, but less than tannins, and their retention in the liquid phase after fine filtration is higher than that in a single filtration process.

[0059] Sub-steps 2-3: Fine filtration. The concentrate containing the precipitated precipitate is finely filtered through a 250-mesh filter membrane to remove the precipitated tannins along with other fine particles. The resulting filtrate is a refined filtrate with significantly reduced tannin content and preserved functional active ingredients.

[0060] Sub-step 2-4: Secondary concentration. The purified filtrate is concentrated again at 50°C under low temperature and reduced pressure to reduce the liquid volume to 1 / 3 of the purified filtrate volume, obtaining a clear and homogeneous high-purity concentrate.

[0061] This step utilizes the physicochemical property that tannins (mainly condensed tannins) precipitate significantly more during concentration and cooling than functional active ingredients such as chrysanthemum flavonoids and wolfberry polysaccharides. Fine filtration removes the precipitated tannins, leaving most of the functional active ingredients in the purified filtrate, thus achieving selective removal of astringent tannins. This "concentration-precipitation-fine filtration" process route allows for the simultaneous removal of astringent substances and the retention of functional active ingredients within the same workflow.

[0062] Step 3: Preparation of compound duck liver hydrolysate solution

[0063] This step uses food-grade alkaline protease to enzymatically hydrolyze duck liver protein. Alkaline protease is an endopeptidase that preferentially cleaves peptide bonds on the carbonyl side of hydrophobic amino acid residues (such as leucine, phenylalanine, and tyrosine) in the duck liver protein peptide chain. In the shallow hydrolysis stage, the peptide chain is not fully cleaved, and the resulting larger molecular weight peptides simultaneously retain hydrophobic and hydrophilic charged amino acid segments within the same molecule, forming amphiphilic peptides with both hydrophobic and hydrophilic ends. These amphiphilic peptides can adsorb onto the surface of duck fat, changing the fat surface from hydrophobic to hydrophilic. In the deep hydrolysis stage, the peptide chain is further cleaved, producing small peptides and free amino acids (especially umami amino acids such as glutamic acid and lysine), providing the finished product with characteristic animal-derived aromas. By controlling the enzymatic hydrolysis time to achieve two hydrolysis ranges—shallow and deep—the two enzymatic hydrolysis products were combined to prepare a compound duck liver hydrolysate containing both macromolecular amphiphilic peptides (used to change the hydrophobicity of duck fat surface to hydrophilicity) and small molecule peptides and free amino acids (used to provide characteristic animal-derived aromas). The specific steps are as follows:

[0064] Sub-step 3-1: Shallow enzymatic hydrolysis. Take fresh duck liver, homogenize it, and add water to a mass ratio of duck liver to water of 1:3. Adjust the pH to 8.0 with food-grade sodium hydroxide. Add food-grade alkaline protease at a mass ratio of enzyme to dry weight of duck liver of 1.0%. Incubate at 55℃ for 60 min to hydrolyze the enzyme, controlling the degree of hydrolysis (DH value, calculated as the ratio of amino nitrogen produced by hydrolysis to total nitrogen) to 10%, so that the protein in the hydrolysate is mainly composed of larger molecular weight polypeptide fragments (amphiphilic peptides). After the enzymatic hydrolysis is completed, heat the hydrolysate to 88℃ and maintain it for more than 15 min to inactivate the protease. Cool to room temperature. The product obtained is shallowly enzymatically hydrolyzed duck liver hydrolysate. The amphiphilic peptides obtained from shallow enzymatic hydrolysis contain both hydrophilic and hydrophobic amino acid residues, which can be adsorbed onto the surface of duck meat particles. The hydrophobic end binds to the fat surface, while the hydrophilic end extends toward the aqueous phase, forming a continuous protein adsorption layer on the surface of duck meat fat. This changes the surface of duck meat fat from hydrophobic to hydrophilic, and is the component used to change the wettability of the duck meat fat surface in compound duck liver hydrolysate.

[0065] Sub-step 3-2: Deep enzymatic hydrolysis. Take fresh duck liver, homogenize it, and add water to a ratio of 1:3 (duck liver to water by mass). Adjust the pH to 8.0 with food-grade sodium hydroxide. Add food-grade alkaline protease at a ratio of 1.0% (enzyme to dry weight of duck liver). Incubate at 55℃ for 200 minutes, controlling the degree of hydrolysis (DH value) to 45%, ensuring the proteins in the hydrolysate are fully hydrolyzed into small peptides and free amino acids. After hydrolysis, heat the hydrolysate to 88℃ and maintain for at least 15 minutes to inactivate the protease. Cool to room temperature. The resulting product is the deeply enzymatically hydrolyzed duck liver hydrolysate. The small peptides and free amino acids (especially glutamic acid and lysine) produced by deep enzymatic hydrolysis are the main sources of the characteristic aroma of duck liver and are the components that provide the animal-derived characteristic aroma in the compound duck liver hydrolysate protein solution.

[0066] Sub-step 3-3: Compounding. The lightly enzymatically hydrolyzed duck liver hydrolysate and the heavily enzymatically hydrolyzed duck liver hydrolysate are mixed at a mass ratio of 1:2 to obtain a compounded duck liver hydrolysate protein solution. This ratio ensures that the compounded product contains sufficient amounts of large-molecule amphiphilic peptides (used to adsorb onto the surface of duck fat, changing the fat surface from hydrophobic to hydrophilic) and sufficient amounts of small-molecule peptides and free amino acids (used to provide the finished product with animal-derived characteristic aromas to encourage pets to eat it).

[0067] By performing shallow enzymatic hydrolysis (retaining large amphiphilic peptides for adsorption onto the surface of duck fat, changing the fat surface from hydrophobic to hydrophilic) and deep enzymatic hydrolysis (producing small peptides and free amino acids to provide animal-derived characteristic aromas for the finished product), and then combining the two in a certain proportion, the same batch of compounded products contains both of the above-mentioned components, eliminating the need to introduce two types of functional excipients separately, thus reducing production steps.

[0068] Step 4: First, mix the compound duck liver hydrolysate with fresh minced duck meat, then add the high-purity concentrate, and freeze-dry.

[0069] This step involves first mixing the compound duck liver hydrolysate with fresh minced duck meat, and then adding the high-purity concentrate. This ensures that the high-purity concentrate evenly wets and stably adheres to the surface and inter-particle spaces of the duck meat particles, resulting in a food mixture with a uniform distribution of functional active ingredients.

[0070] Sub-step 4-1: Pre-mixing of the compound duck liver hydrolysate with fresh minced duck meat. Take the compound duck liver hydrolysate obtained in step three, adding it at 10% of the mass of the fresh minced duck meat. Mix thoroughly to ensure the amphiphilic peptides in the compound duck liver hydrolysate fully contact the fat surface of the duck meat particles. During this mixing process, the hydrophobic ends of the amphiphilic peptides adsorb onto the hydrophobic surface of the duck fat, while the hydrophilic ends extend towards the aqueous phase, forming a continuous protein adsorption layer on the duck fat surface. This transforms the duck fat surface from hydrophobic to hydrophilic, resulting in a mixture of fresh minced duck meat with a hydrophobic to hydrophilic surface.

[0071] Sub-step 4-2: Add high-purity concentrate and mix. Add the high-purity concentrate containing functional active ingredients such as mulberry anthocyanins obtained in step two to the fresh minced duck meat mixture whose surface has changed from hydrophobic to hydrophilic. The amount of high-purity concentrate added is 18% of the mass of the fresh minced duck meat. Mix thoroughly. At this point, because the protein adsorption layer formed by the amphiphilic peptides in step 4-1 on the surface of the duck meat particles has changed to hydrophilic, the high-purity concentrate (hydrophilic aqueous solution) can uniformly wet and stably adhere to the surface and interparticle spaces of the duck meat particles. Phase separation phenomena such as liquid phase precipitation and meat phase aggregation no longer occur. The resulting mixture is a food mixture with uniformly distributed functional active ingredients.

[0072] This step must be performed in the order of adding ingredients as described above. If the high-purity concentrate and the compound duck liver hydrolysate are added simultaneously or before the compound duck liver hydrolysate, the saponins and glycosides derived from dandelion, cassia seed, and other raw materials in the high-purity concentrate, as well as the anthocyanins from mulberry, which have certain amphiphilic active ingredients, will also participate in adsorption on the surface of duck fat. These amphiphilic peptides in the high-purity concentrate will compete with the amphiphilic peptides in the compound duck liver hydrolysate for adsorption sites on the duck fat surface, resulting in a reduction in the amount of amphiphilic peptides adsorbed on the duck fat surface. This reduces the degree to which the duck fat surface changes from hydrophobic to hydrophilic, and the high-purity concentrate will still be difficult to evenly adhere to the surface of the duck meat particles. By adding the compound duck liver hydrolysate first, allowing the amphiphilic peptides to fully occupy the adsorption sites on the duck fat surface, and then adding the high-purity concentrate, the competition for adsorption sites by the aforementioned components is avoided, ensuring that the duck fat surface fully changes from hydrophobic to hydrophilic.

[0073] Sub-step 4-3: Molding and freeze-drying. The above food mixture is pressed into shape using conventional pet food molding technology. The resulting molded food is then freeze-dried to retain the bioactivity of heat-sensitive functional active ingredients such as chrysanthemum flavonoids and wolfberry polysaccharides to the maximum extent, thus obtaining the finished product.

[0074] This step specifies the order of adding ingredients: "first add the compound duck liver hydrolysate solution and mix it with fresh minced duck meat, then add the high-purity concentrate." This utilizes the preemptive advantage of amphiphilic peptides occupying the adsorption sites on the surface of duck meat fat. Without introducing any chemically synthesized emulsifiers, the naturally occurring amphiphilic peptides in the duck liver hydrolysate solution change the surface of duck meat fat from hydrophobic to hydrophilic, allowing the high-purity concentrate to be evenly distributed on the surface of the duck meat particles. At the same time, it avoids interference from saponins derived from dandelion and cassia seed, as well as active ingredients such as morusin in the high-purity concentrate, on the adsorption sites of the amphiphilic peptides on the surface of duck meat fat.

[0075] Experimental verification

[0076] Experiment 1: Verification of the selective removal efficiency of tannin-like astringent substances and the retention efficiency of functional active ingredients by a two-stage filtration-reconcentration process.

[0077] 1. Experimental Objective

[0078] This study verifies whether the secondary filtration-reconcentration process in step two of this invention, compared to the existing single filtration process, can better retain functional active ingredients such as chrysanthemum flavonoids and wolfberry polysaccharides while effectively removing tannin-like astringent substances, thus proving the technical effect of "simultaneous removal of astringent substances and retention of functional active ingredients".

[0079] 2. Preparation of experimental samples

[0080] The raw materials were prepared using the parameters of Example 2 and divided into two groups:

[0081] Control group (single filtration method): Take dried chrysanthemum, cassia seed, wolfberry and dandelion (mass ratio 2:2:1:1), add them to the mixed decoction solvent (containing 2% w / v mulberry extract, replacing 30% of the total liquid, with the remainder made up with water) at a material-to-liquid ratio of 1:10, decoct until boiling, then simmer for 30 minutes. After coarse filtration through a 100-mesh sieve, directly concentrate under reduced pressure at 50℃ to 1 / 4 of the volume of the mixed filtrate to obtain the single filtration concentrate of the control group, without cooling fine filtration and secondary concentration.

[0082] Experimental group (secondary filtration-reconcentration method of the present invention): Using the same starting materials and decoction conditions as the control group, the entire process of step two in Example 2 was carried out sequentially: coarse filtration → concentration under reduced pressure at 50°C to 1 / 4 of the volume of the mixed filtrate → cooling to 15°C and standing for 60 min → fine filtration at 250 mesh → secondary concentration under reduced pressure at 50°C to 1 / 3 of the volume of the refined filtrate, to obtain the high-purity concentrated solution of the experimental group.

[0083] 3. Experimental conditions

[0084] Testing instruments: UV-Vis spectrophotometer, rotary evaporator, low-temperature centrifuge; temperature control accuracy ±0.5℃; number of parallel samples per group n=3, results are expressed as mean ± standard deviation.

[0085] 4. Experimental Procedure

[0086] (1) Determination of tannin content: The Folin-Denis method was used, with gallic acid (GA) as the standard. The absorbance was measured at a wavelength of 760 nm, a standard curve was plotted, and the tannin content of the sample was calculated. The results were expressed as mg GAE / g dry weight of concentrated solution.

[0087] (2) Determination of total flavonoid content: The sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method was used. Rutin was used as the standard. The absorbance was measured at a wavelength of 510 nm. The total flavonoid content of the sample was calculated. The results were expressed as mg RE / g dry weight of concentrated solution.

[0088] (3) Determination of polysaccharide content of wolfberry: The phenol-sulfuric acid method was used, with anhydrous glucose as the standard. The absorbance was measured at a wavelength of 490 nm, and the total polysaccharide content of the sample was calculated. The results were expressed as mg / g dry weight of concentrated solution.

[0089] (4) Calculation of retention rate and removal rate: After coarse filtration and before concentration, accurately measure and record the volume of the sample from the same batch of mixed filtrate. After freeze-drying, the concentrations of each component were determined. ),by Calculate the total amount of each component in the coarse filtrate; record the final concentrate volume of each treatment group using the same method. ) and the concentration of each component ( ),by Calculate the retention rate of functional active ingredients, in order to The tannin removal rate was calculated using the total amount of the corresponding component in the same batch of coarse filtrate as a unified benchmark for both groups.

[0090] 5. Experimental Results

[0091] Table 1-1 Effects of different filtration processes on the content of tannins and functional active ingredients

[0092]

[0093] Note: The tannin removal rate and the retention rate of functional active ingredients were calculated based on the total amount (concentration × volume) of the corresponding components in the mixed filtrate before concentration after 100-mesh coarse filtration. In the control group's single filtration process, the 100-mesh coarse filtration screen retained very little tannins, with a removal rate of only about 4.8%. n=3 batches were independently replicated, and the data are expressed as mean ± SD.

[0094] 6. Analysis and Summary

[0095] As shown in Table 1-1, compared with the single filtration method, the two-stage filtration-reconcentration method of the present invention significantly increases the tannin removal rate in the high-purity concentrate from 4.8% to 78.5%, the total flavonoid retention rate from 62.3% to 79.5%, and the wolfberry polysaccharide retention rate from 65.8% to 87.1%.

[0096] The above results demonstrate that this invention, through the process of "low-temperature concentration and cooling to precipitate tannins → fine filtration and retention → re-concentration," utilizes the physicochemical property of condensed tannins preferentially precipitating during the concentration and cooling process. This achieves the simultaneous removal of astringent substances and the retention of functional active ingredients, proving that this effect is achieved simultaneously (see...). Figure 1 ).

[0097] Experiment 2: Verification of the effect of sequential addition method on the surface wettability of duck meat fat and the uniformity of distribution of functional active ingredients

[0098] 1. Experimental Objective

[0099] The sequential addition process in step four of this invention, which involves "first adding the compound duck liver hydrolysate solution and mixing it with fresh minced duck meat, and then adding the high-purity concentrate", was verified. By pretreating the duck meat fat surface with amphiphilic peptides, the surface of the duck meat fat was changed from hydrophobic to hydrophilic, so that the functional active ingredients in the high-purity concentrate were uniformly distributed in the finished product, thus proving the technical effect of "uniform distribution of functional active ingredients in the finished product".

[0100] 2. Preparation of experimental samples

[0101] High-purity concentrated solution (steps one to two) and compounded duck liver hydrolysate solution (step three) were prepared using the parameters of Example 2, and the following three groups were set up:

[0102] Control group A (direct mixing group): No compound duck liver hydrolysate was added to fresh minced duck meat. Instead, high-purity concentrate (18% of the duck meat mass) was added directly, mixed thoroughly, shaped and freeze-dried to obtain the test sample.

[0103] Control group B (synchronous addition group): The compound duck liver hydrolysate (addition amount of 10% of duck meat weight) and the high-purity concentrate (addition amount of 18% of duck meat weight) were simultaneously added to fresh minced duck meat, mixed thoroughly, shaped and freeze-dried to obtain the test sample.

[0104] Experimental group (sequential addition method of the present invention): First, the compound duck liver hydrolysate solution (addition amount is 10% of the duck meat mass) is added to fresh minced duck meat and mixed thoroughly to allow the amphiphilic peptides to fully contact the surface of duck meat fat and complete adsorption. Then, high-purity concentrate (addition amount is 18% of the duck meat mass) is added, mixed thoroughly, shaped and freeze-dried to obtain the test sample, which is the method described in step four of Example 2.

[0105] 3. Experimental conditions

[0106] (1) Wetting angle measurement: The static water contact angle of the surface of duck fat particles (particle size 0.5-1.0 mm) in each treatment group was measured using a contact angle measuring instrument with deionized water as the probe liquid at room temperature (25℃). Ten particles were randomly selected from each group, and the mean ± standard deviation was taken.

[0107] (2) Distribution uniformity test: After pulverizing the freeze-dried finished products of each group, five samples (200 mg each) were randomly selected from different locations. The total flavonoid content (sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method) and the total polysaccharide content (phenol-sulfuric acid method) of each sample were determined. The coefficient of variation (CV%) of the five sample values ​​was used to characterize the distribution uniformity. The smaller the CV%, the more uniform the distribution. The contact angle measurement and distribution uniformity test were both conducted with n=3 batches of independent repeated experiments. The data in the table are expressed as the mean ± standard deviation of the measurement results of each batch.

[0108] 4. Experimental Procedure

[0109] (1) Prepare three groups of treated samples and freeze-dried products according to the method described in item 2.

[0110] (2) Contact angle measurement: After the wet base mixture of high-purity concentrate added to each treatment group is mixed, duck fat particles are separated by tweezers, placed on a glass slide, and 2 µL of deionized water is dropped onto the particle surface using a contact angle measuring instrument. After image acquisition, the contact angle is automatically calculated by the software.

[0111] (3) Take 200 mg samples from 5 random locations of each group of freeze-dried products and test the content of the two components in each part of the sample according to the method for determining total flavonoids and total polysaccharides (the same method as in step 4 of Experiment 1). Calculate the coefficient of variation (CV%) of the measured values ​​of the 5 samples.

[0112] 5. Experimental Results

[0113] Table 2-1 Effect of different treatment methods on the water contact angle of duck meat fat particles

[0114] Note: A contact angle greater than 90° indicates a hydrophobic surface, while a contact angle less than 90° indicates a hydrophilic surface; the smaller the contact angle, the stronger the wettability.

[0115] Table 2-2 Effects of different mixing methods on the spatial uniformity of functional active ingredients in the finished product

[0116] 6. Analysis and Summary

[0117] As shown in Table 2-1, the water contact angle of untreated duck fat particles was 95.2°, indicating obvious hydrophobicity. In control group A, without the addition of compound duck liver hydrolysate, the contact angle remained almost unchanged (94.8°). In control group B, due to the competition for adsorption sites between the compound solution and the saponin components in the high-purity concentrate, the adsorption of amphiphilic peptides decreased, and the contact angle only dropped to 62.4°, still indicating weak hydrophobicity. In the experimental group of this invention, the amphiphilic peptides preferentially and fully adsorbed onto the fat surface, and the water contact angle dropped to 27.8°, achieving a significant transformation from hydrophobic to hydrophilic.

[0118] As shown in Table 2-2, the total flavonoid CV% of the experimental group of this invention was only 8.3%, and the total polysaccharide CV% was only 7.5%, with significantly better uniformity of distribution than the control groups. This proves that the sequential addition method of this invention effectively achieves uniform distribution of functional active ingredients in the finished product through amphiphilic peptide pretreatment without introducing any synthetic emulsifiers (see Table 2-2). Figure 2 and Figure 3 ).

[0119] Experiment 3: Verification of the synergistic effect of mulberry extract and compound duck liver hydrolysate on the voluntary feed intake rate of pets

[0120] 1. Experimental Objective

[0121] This invention verifies the synergistic effect of "mulberry extract (with synergistic effects, simultaneously reducing the off-flavor of medicinal and edible homologous concentrated liquid) and the deep enzymatic hydrolysis product of compounded duck liver hydrolysate (olfactory channel, providing animal-derived characteristic aroma)" on improving the voluntary consumption rate (palmistry) of pet food containing medicinal and edible homologous functional ingredients, proving the effect of "improving palatability through both taste and smell channels".

[0122] 2. Preparation of experimental samples

[0123] Based on the parameters of Example 2, five sets of comparative samples were set up:

[0124] Blank control group: Fresh minced duck meat was taken and freeze-dried duck meat particles were prepared according to the same molding process and freeze-drying conditions as step four of Example 2 without the addition of any medicinal and edible homologous concentrated liquid or duck liver hydrolysate. This served as a blank control to reflect the basic eating behavior of pets towards ordinary food.

[0125] Control Group A (pure bitter concentrate, without mulberry or hydrolysate): Pure water was used instead of mulberry extract to prepare a medicinal and edible concentrate without mulberry extract by decocting according to steps one and two of Example 2; without adding compound duck liver hydrolysate, the concentrate (added at 18% of the duck meat mass) was directly added to fresh minced duck meat, mixed, and freeze-dried to evaluate the palatability of pets when bitter concentrate was added alone, without mulberry or hydrolysate.

[0126] Control group B (containing mulberry concentrate, without hydrolysate): A high-purity concentrate containing mulberry extract was prepared according to steps one and two of Example 2; without adding compound duck liver hydrolysate, the high-purity concentrate (added at 18% of the duck meat mass) was directly mixed with fresh minced duck meat, shaped and freeze-dried, to evaluate the effect of mulberry extract alone on improving palatability by synergistic efficacy and reducing plant off-flavors.

[0127] Control group C (without mulberry extract, containing hydrolysate): Pure water was used to replace mulberry extract to prepare a medicinal and edible concentrate without mulberry extract; compound duck liver hydrolysate (compound ratio 1:2) was prepared according to step three of Example 2. First, the compound solution (added at 10% of the weight of duck meat) was mixed with fresh minced duck meat, and then the concentrate without mulberry extract (added at 18% of the weight of duck meat) was added. The mixture was then freeze-dried to evaluate the palatability of the compound duck liver hydrolysate when it alone exerts its olfactory improvement effect.

[0128] Experimental group (full formula of the present invention, Example 2): prepared according to the full process of Example 2, containing mulberry extract (synergistic effect, reducing plant odor) and compound duck liver hydrolysate protein solution deep enzymatic hydrolysis product (improves smell), and operated according to the sequential addition method of the present invention.

[0129] 3. Experimental conditions

[0130] Experimental animals: Twenty healthy adult domestic cats (shorthaired or mixed breeds) were randomly divided into 5 groups of 4 cats each, half male and half female, weighing 3–5 kg. They were fasted for 12 hours before the experiment. The rearing temperature was 22±1℃, relative humidity 50%±5%, and natural light. Each cat in each group was provided with a sufficient sample amount (200 g / cat, far exceeding the normal daily feed intake, to fully reflect feeding willingness rather than limiting the quantity; the energy density of freeze-dried food is approximately 4–5 times that of fresh food, and the actual daily freeze-dried food requirement for a 3–5 kg domestic cat is approximately 40–60 g). They were fed individually in separate cages, and the voluntary feed intake within 60 minutes was observed and recorded. Voluntary feed intake rate (%) = actual feed intake / supply × 100%. Feeding continued for 7 days, with records recorded daily.

[0131] 4. Experimental Procedure

[0132] (1) The cats in each group were kept in separate cages and fed at 8:00 a.m. every day. Each cat ate alone (200 g / cat) to avoid competition and interference.

[0133] (2) After 60 minutes, collect the remaining food, weigh the remaining amount accurately, and calculate the actual food intake and food intake rate of each cat.

[0134] (3) Record the voluntary feeding rate of each cat in each group every day and calculate the average of the 4 cats in each group on that day.

[0135] (4) After the 7-day trial, the average 7-day intake rate of each group was calculated, and a one-way ANOVA was performed. Tukey's method was used for post-hoc multiple comparisons (significance level α=0.05).

[0136] 5. Experimental Results

[0137] Table 3-1 Voluntary feeding rate of domestic cats in each group over 7 days (%)

[0138] Note: The 7-day means of each group are expressed as mean ± SD, where SD is the within-group standard deviation of the 7-day means; the differences between groups were significant by one-way ANOVA analysis (F=89.3, P<0.001); the 7-day means of the experimental group were significantly higher than those of all control groups (Tukey test, P<0.05).

[0139] 6. Analysis and Summary

[0140] As shown in Table 3-1, the pet consumption rate of control group A (pure bitter medicinal and edible concentrate without mulberry and hydrolysate) showed a continuous downward trend with the increase of feeding days, with a 7-day average of only 32.8%, indicating that the pets' aversion to the bitter plant odors from ingredients such as chrysanthemum, cassia seed, and dandelion intensified over time. The long-term actual intake of the finished product could not be guaranteed. Control group B (containing mulberry extract, without hydrolysate) saw a 7-day average increase to 56.7%, indicating that the active ingredients such as anthocyanins in mulberry, after being dissolved in the concentrate during decoction, altered the overall aroma and taste characteristics of the concentrate to some extent, improving sensory acceptance of the plant odors. However, this was still lower than the blank control group (72.9%), suggesting that mulberry alone was insufficient to fully compensate for the lack of animal-derived aromas.

[0141] The 7-day mean of the control group C (containing compound duck liver hydrolysate, without mulberry) increased to 64.3%, indicating that the animal-derived characteristic aroma provided by the deep enzymatic hydrolysis product of the compound duck liver hydrolysate has a strong positive effect on guiding pets to eat, but the overall palatability is still limited without the synergistic effect of mulberry. The 7-day mean of the experimental group (the complete formula of this invention) reached 85.0%, which was not only significantly higher than all control groups (P<0.05), but also exceeded the baseline level of pure duck meat in the blank control group, proving that the synergistic effect of mulberry extract and the deep enzymatic hydrolysis product of the compound duck liver hydrolysate can effectively overcome the barrier of bitter plant odor, thereby ensuring the actual intake of functional ingredients by pets (see Figure 4 and Figure 5 ).

[0142] Experiment 4: Verification of the effect of the pet food of this invention on improving tear stains in cats

[0143] 1. Experimental Objective

[0144] This study verified the actual improvement effect of the pet food of the present invention on tear stains in cats after 28 days of continuous feeding. It proved that the synergistic effect of the medicinal and edible ingredients such as chrysanthemum (clears liver and improves eyesight, anti-inflammatory), dandelion (clears heat and reduces inflammation, antibacterial), wolfberry (nourishes yin and improves eyesight, antioxidant), and cassia seed (clears liver and improves eyesight) in the formula can effectively reduce the porphyrin content in cat tears, reduce the amount of eye secretions, and improve tear stain scores. At the same time, through comparison with the control group of traditional single filtration process, it proved that the technical solution of the present invention (secondary filtration-reconcentration + mulberry + sequential addition method) is superior to the traditional addition process in terms of efficacy.

[0145] 2. Preparation of experimental samples

[0146] Based on the parameters of Example 2, three sets of comparative samples were set up:

[0147] Blank control group: Commercially available dry food for adult cats, without any concentrated medicinal and edible liquids or hydrolyzed duck liver protein, was used as a baseline to reflect the natural tear stain condition of cats under normal diet.

[0148] Positive control group (traditional single-filtration process): Pure water was used instead of mulberry extract, and the decoction was prepared according to the same raw material ratio and decoction conditions as in step one of Example 2. The resulting decoction was coarsely filtered through a 100-mesh sieve and then directly concentrated under reduced pressure at 50°C to 1 / 4 of the volume of the mixed filtrate, without cooling filtration or secondary concentration. The resulting concentrate was not mixed with compound duck liver hydrolysate, but directly mixed with fresh minced duck meat (18% of the duck meat mass), shaped, and freeze-dried to obtain the positive control group sample. This sample had the same amount of medicinal and edible raw materials as the experimental group, but contained a large amount of unremoved tannins with astringent properties, and the functional active ingredients were unevenly distributed in the finished product, limiting palatability for cats, and the actual intake of functional ingredients was lower than that of the experimental group.

[0149] Experimental group (full formula of this invention): prepared according to the full process of Example 2 (steps one to four), containing mulberry (to improve palatability), high-purity concentrate (low tannins, high functional components) prepared by secondary filtration and re-concentration, and compound duck liver hydrolysate (to stimulate appetite and improve uniform interface), and then freeze-dried after being added in sequence.

[0150] 3. Experimental conditions

[0151] Experimental animals: Eighteen healthy purebred cats (British Shorthair or Persian cats) with a tendency to develop tear stains and exhibiting moderate or severe tear stains (score ≥2 points) were selected. The cats were 1-4 years old and weighed 2.5-5.5 kg, with half being male and half female. They were randomly divided into three groups of six cats each and housed separately in cages. The cats were fasted for 12 hours before the experiment. The housed temperature was 22±1℃, the relative humidity was 50%±5%, and the lighting was natural. Each group was provided with the corresponding sample with the same daily caloric intake (converted from the actual caloric intake of each sample) and free access to water. The experiment lasted for 28 days.

[0152] Tear stain scoring: A semi-quantitative scoring standard of 0-4 points was used (0 points: no tear stains; 1 point: slight light-colored stains at the corner of the eye; 2 points: obvious reddish-brown pigmentation from the corner of the eye to the side of the nose; 3 points: pigmentation extending to the cheek, accompanied by mild dermatitis; 4 points: extensive pigmentation, accompanied by obvious dermatitis symptoms). Two trained assessors independently scored each cat's standardized eye photographs in a blinded manner, and the average score was taken. The assessment time points were day 0 (baseline), day 14, and day 28.

[0153] Determination of porphyrin content in tears: Tears were collected using the Schirmer test strip method, and the content of porphyrin (the main precursor of tear stain red-brown pigment) in tears was determined by fluorescence spectrophotometry (excitation wavelength 405 nm, emission wavelength 620 nm). The results are expressed in μg / mL. The detection time points were day 0 and day 28. Bilateral tear samples were collected from each cat and mixed before measurement. The experiment was conducted with n=3 independent replicates. The data are expressed as mean ± standard deviation.

[0154] Average daily collection of ocular secretions: Secretions were collected from the inner canthi of each cat using pre-weighed swabs between 8:00 and 9:00 a.m. each day, and weighed accurately, expressed in mg / cat / day; the mean values ​​on day 0 (baseline mean) and day 28 were recorded.

[0155] 4. Experimental Procedure

[0156] (1) On day 0, tear stains were photographed and scored, tear porphyrin content was detected and eye secretions were collected from 18 cats, and baseline data for each group were recorded.

[0157] (2) The cats were fed in separate cages according to their groups. The blank control group was fed commercially available adult cat dry food, while the positive control group and the experimental group were fed corresponding freeze-dried samples. They were fed for 28 consecutive days. The cats were fed at fixed times every day with equal calorie intake. The feeding status of each cat was observed and recorded.

[0158] (3) Repeat the tear stain photo assessment on day 14 to record mid-term changes.

[0159] (4) Repeat all test items (tear stain score, tear porphyrin content, and amount of ocular secretions) on day 28 and compare with baseline.

[0160] (5) Statistical analysis: Tear stain scores were analyzed using Friedman nonparametric repeated measures test and Dunn-Bonferroni post-hoc comparison; tear porphyrin content and ocular secretion amount were analyzed using univariate ANOVA and Tukey post-hoc multiple comparison; significance level α=0.05.

[0161] 5. Experimental Results

[0162] Table 4-1 Changes in tear stain scores for cats in each group (mean ± SD, n=6, score 0–4)

[0163] Note: *The improvement in the score of the experimental group on day 28 compared with the baseline was significantly better than that of the blank control group and the positive control group (Dunn-Bonferroni test, P<0.05); there was no significant difference in the baseline scores among the three groups (P>0.05).

[0164] Table 4-2 Changes in porphyrin content in cat tears of different groups (mean ± SD, n=6, unit: μg / mL)

[0165] Note: *The porphyrin content in the tears of the experimental group on day 28 was significantly lower than that of the blank control group and the positive control group (Tukey test, P<0.05); there was no significant difference in the baseline porphyrin content among the three groups (P>0.05).

[0166] Table 4-3 Changes in the average daily amount of ocular secretions collected from cats in each group (mean ± SD, n=6, unit: mg / cat / day)

[0167] Note: *On day 28, the amount of ocular secretions in the experimental group was significantly lower than that in the blank control group and the positive control group (Tukey test, P<0.05); there was no significant difference in baseline secretion amount among the three groups (P>0.05).

[0168] Figure 6 A line graph showing the dynamic changes in tear stain scores;

[0169] Figure 7 A bar chart comparing tear porphyrin content on day 0 and day 28.

[0170] 6. Analysis and Summary

[0171] From Table 4-1 and Figure 6 and Figure 7 It was found that the tear stain score of cats in the blank control group (commercially available dry food) did not change significantly during the 28-day trial period (from 2.6 to 2.5 points), indicating that ordinary diet had no effect on improving tear stains. In the positive control group (traditional single-filtration process, containing tannins, without mulberries, and with uneven component distribution), the tear stain score decreased from 2.5 to 1.8 points on day 28, an improvement of -0.7 points, indicating that the medicinal and edible functional ingredients have a certain basic tear stain removal activity; however, the lack of removal of astringent tannins led to poor palatability for cats, and the uneven distribution of functional ingredients in the finished product resulted in insufficient actual intake, thus limiting the tear stain removal effect.

[0172] The experimental group (using the complete formula of this invention) showed a significant decrease in tear stain score on day 14 (2.6 → 1.8 points), and further decreased to 1.0 point on day 28, an improvement of -1.6 points, which was significantly better than the positive control group (P<0.05). As shown in Table 4-2, the porphyrin content in the tear fluid of the experimental group decreased from baseline 45.1 μg / mL to 18.6 μg / mL on day 28, a reduction of 58.8%, which was significantly higher than the 25.9% reduction in the positive control group (P<0.05). As shown in Table 4-3, the average daily collection of ocular secretions in the experimental group decreased from 84.8 mg / animal / day to 28.4 mg / animal / day, a reduction of 66.5%, while the positive control group only reduced by 27.9%.

[0173] The above results demonstrate that the synergistic effect of the three technical measures of this invention—masking the bitter taste of mulberry (ensuring cats continue to voluntarily consume it), removing tannins through a secondary filtration-re-concentration process while retaining a high proportion of functional active ingredients (increasing the intake of effective ingredients), and ensuring uniform distribution of functional ingredients through sequential addition (ensuring consistent effective dosage in each feeding)—significantly improves the actual tear stain removal effect of the medicinal and edible functional ingredients from the limited improvement of traditional processes (score -0.7 points, porphyrin reduction of 25.9%) to a remarkable improvement (score -1.6 points, porphyrin reduction of 58.8%). This proves the actual efficacy advantage of the complete formulation of this invention in improving tear stains in cats.

[0174] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing pet food with tear stain removal function, characterized in that, Includes the following steps: Step 1: Use mulberry extract to replace part of the water used for decoction as the decoction solvent to decoct chrysanthemum, cassia seed, wolfberry and dandelion to obtain a mixed decoction. Step 2: After coarse filtration of the mixed decoction, it is concentrated under low temperature and reduced pressure. After cooling and standing, the tannins are precipitated. After fine filtration to remove the precipitated tannins, it is concentrated again under low temperature and reduced pressure to obtain a high-purity concentrate. Step 3: Use food-grade alkaline protease to perform shallow and deep enzymatic hydrolysis on duck liver. Shallow enzymatic hydrolysis controls the degree of hydrolysis so that the protein in the hydrolysate is mainly amphiphilic peptides. Deep enzymatic hydrolysis controls the degree of hydrolysis so that the protein in the hydrolysate is fully hydrolyzed into small molecule peptides and free amino acids. Mix the two enzymatic hydrolysis products to obtain a compound duck liver hydrolysate. Step four: First, mix the compound duck liver hydrolysate with fresh minced duck meat, so that the hydrophobic ends of the amphiphilic peptides are adsorbed onto the surface of the duck meat fat and the hydrophilic ends extend toward the water phase, changing the surface of the duck meat fat from hydrophobic to hydrophilic. Then, add high-purity concentrate and mix. After molding and freeze-drying, the finished product is obtained.

2. The preparation method according to claim 1, characterized in that, In step one, the mass ratio of dried chrysanthemum, cassia seed, wolfberry, and dandelion is 1-2:1-3:1-2:1-2; the mass ratio of the total dry weight of the medicinal and edible raw materials to the total amount of liquid used for decoction is 1:8-1:12; the solid content of the mulberry extract is 5%-15% (w / v), replacing 20%-40% of the total amount of liquid used for decoction, with the remainder made up with water; when decocting, heat to boiling and then maintain a simmer for 20-40 minutes.

3. The preparation method according to claim 1, characterized in that, In step two, coarse filtration uses a 100-mesh sieve; low-temperature vacuum concentration is carried out at 40℃~60℃, and after concentration to 1 / 5~1 / 3 of the mixed filtrate volume, it is cooled to 10℃~20℃ and allowed to stand for more than 30 minutes; fine filtration uses a 200~300-mesh filter membrane; secondary low-temperature vacuum concentration is carried out at 40℃~60℃, and after concentration to 1 / 3~1 / 2 of the refined filtrate volume.

4. The preparation method according to claim 1, characterized in that, In step three, the conditions for shallow enzymatic hydrolysis are as follows: after homogenizing the duck liver, add water to a mass ratio of duck liver to water of 1:2 to 1:4, adjust the pH to 7.5 to 8.5 with food-grade sodium hydroxide, use food-grade alkaline protease at a rate of 0.5% to 2.0% of the dry weight of the duck liver, incubate at 50℃ to 60℃ for 30 to 90 minutes, and control the degree of hydrolysis to 5% to 15%.

5. The preparation method according to claim 4, characterized in that, In step three, the deep enzymatic hydrolysis uses the same material-to-water ratio, pH adjustment range, enzyme dosage, and temperature conditions as the shallow enzymatic hydrolysis. The incubation time for enzymatic hydrolysis is 150–300 min, and the degree of hydrolysis is controlled at 35%–55%.

6. The preparation method according to claim 4 or 5, characterized in that, In step three, after shallow and deep enzymatic hydrolysis are completed, the hydrolysate is heated to 85℃~90℃ and maintained for more than 15 minutes to inactivate alkaline protease. After cooling to room temperature, it is used for compounding.

7. The preparation method according to claim 1, characterized in that, In step three, the shallowly enzymatically hydrolyzed duck liver hydrolysate and the deeply enzymatically hydrolyzed duck liver hydrolysate are mixed at a mass ratio of 1:1 to 1:

3.

8. The preparation method according to claim 1, characterized in that, In step four, the amount of compound duck liver hydrolysate added is 5% to 15% of the weight of fresh minced duck meat, and the amount of high-purity concentrate added is 10% to 25% of the weight of fresh minced duck meat.

9. The preparation method according to claim 1, characterized in that, In step four, the compound duck liver hydrolysate is added to the fresh minced duck meat, mixed evenly, and left to stand for 5-15 minutes to allow the amphiphilic peptides to fully occupy the adsorption sites on the surface of the duck meat fat and change the surface of the duck meat fat from hydrophobic to hydrophilic. Then, the high-purity concentrate is added and mixed.

10. A pet food with tear stain removal function prepared by any one of claims 1 to 9.

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