Peptide composition for improving tear stains of pets and preparation method thereof

Abstract

A peptide composition for improving tear stains of pets belongs to the field of pet health, is used for improving tear stains of pets, and is characterized by comprising 20-30 parts of sea cucumber peptide, 5-10 parts of casein phosphopeptides, 10-20 parts of soybean peptide, 20-30 parts of corn oligopeptide, 10-20 parts of wheat oligopeptide and 10-20 parts of mussel peptide, the amino acid sequence of common mussel peptide is His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe, and the amino acid sequence of the common mussel peptide is His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe. The amino acid sequence of the perna viridis peptide is Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr, and the perna viridis peptide has the effects of effectively improving the metabolism of ferriporphyrin, inhibiting the activity of saccharomycetes and reducing the formation of tear stains of pets.

Description

Technical Field

[0001] The present invention relates to the field of pet nutrition and health, and particularly to a peptide composition for improving pet tear stains and a preparation method thereof. Background Art

[0002] With the rise of the pet economy and the improvement of the pet's family status, pet owners hope that their pets are not only healthy but also beautiful. However, many pets have the problem of tear stains, such as Teddy, Bichon Frise, Pomeranian, Golden Retriever among dogs, and Persian cat, Garfield, Maine Coon among cats, which seriously affect the appearance and health of pets and reduce the quality of pet life.

[0003] The overflow of pet tears onto the fur causes the fur to be dyed and form tear stains. Tear stains are one of the common and difficult problems in the breeding process of dogs and cats. It is believed that there are various reasons for the appearance of tear stains, including congenital reasons (light-colored fur, short nose, large curvature of the nasolacrimal duct, etc.), as well as factors such as eyelashes, eye or ear diseases, nasolacrimal duct obstruction, environmental or drug stimulation, diet, etc. In response to these reasons, people have tried various means, including surgery, drugs, wiping, improving the environment, changing food, etc. to treat tear stains, with a relatively high investment cost, but the actual effect is still not good.

[0004] The main reason for the fur to be dyed by tear stains is the iron porphyrin in the tears. After the tears stay on the fur, the iron in the porphyrin is oxidized by the air and dyes the fur rust-colored, forming rust-colored tear stains. In addition, at the place where the fur is wetted by the tears, it is very easy to be infected with yeast, and the growth and reproduction of yeast further deepen the fur dyeing, forming brown or dark brown tear stains. Therefore, developing a product with universality and effective in improving pet tear stains is very meaningful for solving the pain points of pet owners and protecting the health of pets, and has broad market prospects. Summary of the Invention

[0005] In view of the above pet tear stain problem, the present invention provides a peptide composition for improving pet tear stains and a preparation method thereof.

[0006] The technical solutions adopted by the present invention to achieve the above object are as follows:

[0007] A mytilus edulis peptide, characterized in that its amino acid sequence is His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe.

[0008] A preparation method of a mytilus edulis peptide, characterized by including

[0009] S11. Crushing and homogenizing mytilus edulis, adding pure water and mixing evenly, with a material-to-water ratio of 1:(5-20), a temperature of 45-60 °C, and a pH of 6.0-8.0 to obtain a material liquid;

[0010] S12. Add compound protease accounting for 0.2 - 1% of the volume of the feed liquid, and then stir and enzymatically hydrolyze for 3 - 5 h. The ratio of compound protease is trypsin:papain:neutral protease = (2 - 4):(1 - 2):(1 - 2);

[0011] S13. After the enzymatic hydrolysis is completed, inactivate the enzyme in the feed liquid, and then centrifuge to obtain the supernatant;

[0012] S14. Separate the supernatant through a membrane device with a molecular weight cut-off of 1000 Da, take the permeate, concentrate it, and then spray-dry to obtain mussel peptides.

[0013] Furthermore, the molecular weight of the mussel peptides is 896 Da, and the content is ≥ 1.5%;

[0014] Furthermore, in step S13, the feed liquid is heated to 85°C and maintained for 15 min.

[0015] A kind of green-lipped mussel peptide, characterized in that its amino acid sequence is Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr.

[0016] A preparation method of a kind of green-lipped mussel peptide, characterized by including

[0017] S21. Break and homogenize green-lipped mussels, add pure water and mix evenly. The ratio of feed to water is 1:(5 - 20), the temperature is 45 - 60°C, and the pH is 6.0 - 8.0 to obtain the feed liquid;

[0018] S22. Add compound protease accounting for 0.2 - 1% of the volume of the feed liquid, and then stir and enzymatically hydrolyze for 3 - 5 h. The ratio of compound protease is trypsin:papain:neutral protease = (2 - 4):(1 - 2):(1 - 2);

[0019] S23. After the enzymatic hydrolysis is completed, inactivate the enzyme in the feed liquid, and then centrifuge to obtain the supernatant;

[0020] S24. Separate the supernatant through a membrane device with a molecular weight cut-off of 1000 Da, take the permeate, concentrate it, and then spray-dry to obtain green-lipped mussel peptides.

[0021] Furthermore, the molecular weight of the green-lipped mussel peptides is 1082 Da, and the content is ≥ 1.5%;

[0022] Furthermore, in step S23, the feed liquid is heated to 85°C and maintained for 15 min.

[0023] A peptide composition for improving pet tear stains, characterized by including 20 - 30 parts of sea cucumber peptides, 5 - 10 parts of casein phosphopeptides, 10 - 20 parts of soy peptides, 20 - 30 parts of corn oligopeptides, 10 - 20 parts of wheat oligopeptides, and 10 - 20 parts of mussel peptides.

[0024] Further, the sea cucumber peptide is prepared in the following manner:

[0025] S31. The sea cucumber is broken and homogenized, pure water is added and mixed evenly, the ratio of material to water is 1:(5-20), the temperature is 45-60°C, and the pH is 7.0-9.0 to obtain a material liquid;

[0026] S32. To the material liquid, 0.2-1% of compound protease based on the volume of the material liquid is added, and then stirred and enzymatically hydrolyzed for 3-5 h. The ratio of the compound protease is trypsin: alkaline protease: neutral protease = (3-5):(1-3):(1-3);

[0027] S33. After the enzymatic hydrolysis is completed, the temperature of the material liquid is raised to 85°C, and it is kept for 15 min to inactivate the enzyme, and then centrifuged to obtain a supernatant;

[0028] S34. The supernatant is separated by a membrane device with a molecular weight cut-off of 3000 Da, and the permeate is taken, concentrated and spray-dried to obtain sea cucumber peptide.

[0029] Further, the sea cucumber in step S31 is preferably selected from Apostichopus japonicus and Thelenota ananas;

[0030] Further, the proportion of peptide segments with a molecular weight less than 1000 Da in the sea cucumber peptide is ≥90%;

[0031] Further, the dosage form of the composition can be powder, granule, tablet, capsule, pill, oral liquid.

[0032] Further, the peptide composition for improving pet tear stains includes any one of mussel peptides such as green-lipped mussel peptide and blue mussel peptide.

[0033] Tear stains are caused by the overflow of a pet's tears onto the fur, which in turn causes fur discoloration. The culprit for fur discoloration is iron porphyrin in the tears. After the tears stay on the hair, the iron in the porphyrin is oxidized by the air, dyeing the hair rust color, forming rust-colored tear stains. In addition, at the place where the hair is wetted by the tears, it is very easy to be infected with yeast, and the growth and reproduction of yeast further deepen the hair discoloration, forming brown and dark brown tear stains. Therefore, starting from iron porphyrin is the fundamental way to solve the problem of tear stains and is more universal.

[0034] In the body, red blood cells are gradually damaged and aged by various stimuli. Usually, the life cycle of canine red blood cells is about 100 days, and that of cats is only about 70 days. The aged red blood cells are recognized and phagocytosed by macrophages, and then degraded and release iron porphyrin, and the iron porphyrin is further decomposed into Fe 2+, CO, and biliverdin. The free Fe ions are bound and transported to the liver for storage. The liver is one of the main storage organs of iron. After a series of physiological reactions and metabolism, the iron porphyrin produced by the decomposition of senescent red blood cells is usually excreted from the body through feces and urine, but pets such as cats and dogs can also excrete it through tears and saliva. When there are problems in the iron porphyrin metabolism process, pets are prone to develop tear stains; conversely, if the iron porphyrin metabolism is restored to normal, the tear stain problem of pets will be improved and solved.

[0035] Sea cucumber peptides have an activating effect on macrophages, can increase the phagocytosis of macrophages on senescent red blood cells, accelerate the normal release and metabolism of iron porphyrin, and at the same time can enhance the immunity of pets and help pets resist diseases and infections.

[0036] Soybean peptides can form chelates with iron ions, which is beneficial to the absorption and utilization of iron by the body. Casein phosphopeptides have a strong activity of promoting the absorption and utilization of Fe 2+ and play a role in the process of iron porphyrin decomposition and the binding of free Fe ions.

[0037] Corn oligopeptides have the function of protecting the liver. It can promote the detoxification function of the liver, reduce the damage of toxic and harmful chemicals to the liver, and at the same time can reduce the fatty degeneration of liver cells and improve the degree of liver tissue damage.

[0038] Wheat oligopeptides contain oligomethionine, which helps to improve the ability of the liver to store iron.

[0039] Mussel peptides have anti-inflammatory activity and contain antibacterial active peptide segments, and have a very good effect on tear stains caused by yeast infections.

[0040] Advantages of the present invention:

[0041] 1. The present invention provides a product for solving pet tear stains with universality, low cost, and high efficiency. The raw materials of the composition of the present invention are easily available, easy to industrialize, safe for consumption, with clear mechanisms and remarkable effects, and have broad market development prospects.

[0042] 2. The present invention starts from the metabolism of releasing iron porphyrin from senescent red blood cells, and through activating macrophages, promoting the absorption and utilization of iron by the body, and enhancing the iron storage ability of the liver, multiple aspects work together synergistically to fundamentally solve the tear stain problem caused by the oxidation of iron porphyrin due to the overflow of tears. At the same time, it can also have antibacterial and anti-inflammatory effects, enhance the immunity of pets, and prevent further infection by yeast and aggravation of tear stains.

[0043] The invention provides an antibacterial active peptide segment from mussel peptide, wherein the amino acid sequence of the antibacterial active peptide segment in the purple mussel peptide is His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe, and the amino acid sequence of the antibacterial active peptide segment in the green mussel peptide is Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr. After retrieval of online databases BIOPEP and EROP-Moscow, the above sequences are all new active peptides. DETAILED DESCRIPTION

[0044] Example 1: A peptide composition for improving pet tear marks, comprising: 20 parts of sea cucumber peptide, 7 parts of casein phosphopeptide, 20 parts of soybean peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of purple mussel peptide.

[0045] The preparation method is:

[0046] S1. Preparation of sea cucumber peptide: The sea cucumber was crushed and homogenized, and pure water was added to mix, the material-water ratio was 1:10, the material temperature was 55°C, the pH was 8.0, and 0.5% of the composite protease volume of the material was added and stirred for enzymolysis for 4 hours, and the ratio of the composite protease was trypsin: alkaline protease: neutral protease = 4:2:1. After the enzymolysis was completed, the material was heated to 85°C, kept for 15 minutes to kill the enzyme, and then centrifuged, and the supernatant was separated by a membrane device with a molecular weight cutoff of 3000Da, and the permeate was concentrated and spray-dried to obtain the sea cucumber peptide.

[0047] S2. Preparation of purple mussel peptide: crush the purple mussel and homogenize it, add pure water to mix, the material-water ratio is 1:10, the material temperature is 55°C, the pH is 8.0, add 0.5% of the material volume of the composite protease and stir for enzymolysis for 4 hours, the ratio of the composite protease is trypsin: papain: neutral protease = 3:2:1. After the enzymolysis is completed, the material is heated to 85°C, kept for 15 minutes to kill the enzyme, and then centrifuged, the supernatant is separated by a membrane device with a molecular weight cutoff of 5000Da, and the permeate is concentrated and spray-dried to obtain the purple mussel peptide.

[0048] S3. Ingredient packaging: accurately weigh 20 parts of sea cucumber peptide, 7 parts of casein phosphopeptide, 20 parts of soybean peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of purple mussel peptide, mix them in a mixer for 15 minutes, and then use an automatic packaging machine to package to obtain a powder product.

[0049] After testing, it was found that the peptide segments less than 1000Da accounted for 95.27% of the sea cucumber peptides.

[0050] Purple mussel peptide contains His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe active peptide segment with a content of 1.63%.

[0051] Example 2: A peptide composition for improving pet tear stains, the components of which include: 20 parts of sea cucumber peptide from Apostichopus japonicus, 7 parts of casein phosphopeptide, 20 parts of soybean peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of mussel peptide from Perna viridis.

[0052] Except for the following differences, the rest are the same as in Example 1.

[0053] The differences are as follows: 1. The sea cucumber peptide raw material is changed from Apostichopus japonicus to Apostichopus japonicus var. nanhaiensis.

[0054] 2. The mussel peptide raw material is changed from Mytilus edulis to Perna viridis.

[0055] After detection, the proportion of peptide segments less than 1000 Da in the sea cucumber peptide from Apostichopus japonicus var. nanhaiensis is 91.49%.

[0056] The mussel peptide from Perna viridis contains the active peptide segment Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr, and the content is 1.82%.

[0057] Comparative Example 1: A peptide composition for improving pet tear stains, the components of which include: 20 parts of sea cucumber powder, 7 parts of casein phosphopeptide, 20 parts of soybean peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of Mytilus edulis powder.

[0058] The preparation method is as follows:

[0059] S1. Preparation of sea cucumber powder: The sea cucumber is directly crushed to obtain sea cucumber powder.

[0060] S2. Preparation of Mytilus edulis powder: The Mytilus edulis is directly crushed to obtain Mytilus edulis powder.

[0061] S3. Weighing and packaging: Accurately weigh 20 parts of sea cucumber powder, 7 parts of casein phosphopeptide, 20 parts of soybean peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of Mytilus edulis powder, place them in a mixer and mix for 15 minutes, and then package them with an automatic packaging machine to obtain a powder product.

[0062] Comparative Example 2: A peptide composition for improving pet tear stains, the components of which include: 20 parts of sea cucumber peptide from Apostichopus japonicus var. nanhaiensis powder, 7 parts of casein phosphopeptide, 20 parts of soybean peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of mussel peptide from Perna viridis powder.

[0063] The preparation method is as follows:

[0064] S1. Preparation of sea cucumber peptide from Apostichopus japonicus var. nanhaiensis powder: The sea cucumber peptide from Apostichopus japonicus var. nanhaiensis is directly crushed to obtain sea cucumber peptide from Apostichopus japonicus var. nanhaiensis powder.

[0065] S2. Preparation of mussel peptide from Perna viridis powder: The mussel peptide from Perna viridis is directly crushed to obtain mussel peptide from Perna viridis powder.

[0066] S3. Ingredient Packaging: Accurately weigh 20 parts of sea cucumber powder, 7 parts of casein phosphopeptide, 20 parts of soy peptide, 20 parts of corn oligopeptide, 15 parts of wheat oligopeptide, and 20 parts of green mussel powder. Place them in a mixer and mix for 15 minutes, then use an automatic packaging machine to package and obtain a powder product.

[0067] Experimental Verification 1: Verify the anti-yeast activity of the antibacterial peptide segments in purple mussel peptide and green mussel peptide through the antibacterial circle experiment.

[0068] Dissolve the purple mussel peptide in Example 1 in water, separate and purify to obtain the His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe active peptide segment, and freeze-dry to obtain the purple mussel antibacterial peptide sample.

[0069] Dissolve the green mussel peptide in Example 2 in water, separate and purify to obtain the Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr active peptide segment, and freeze-dry to obtain the green mussel antibacterial peptide sample.

[0070] Weigh the purple mussel antibacterial peptide and green mussel antibacterial peptide samples respectively, add sterile pure water to dissolve and mix them evenly, and prepare antibacterial peptide solutions with concentrations of 5mg / mL and 10mg / mL.

[0071] Heat and dissolve the prepared medium, cool it to 45 - 50°C, then add the Saccharomyces cerevisiae suspension (1×105 - 106 CFU / mL) to the medium at an addition amount of 1%, mix evenly, measure 20 mL and pour it into a sterilized petri dish, gently shake it to make it evenly spread, and set it aside after solidification.

[0072] Place 3 sterile Oxford cups equidistantly on the petri dish containing yeast, gently press them, and slowly add 200 μL of the antibacterial peptide solution to each Oxford cup respectively. Transfer the petri dish to a 4°C refrigerator for pre-diffusion for 4 hours, then take it out and culture it in a constant temperature incubator at 28 ± 1°C for 18 - 24 hours until the antibacterial circle is clear. Measure the diameter of the antibacterial circle, take the average value, and record the data.

[0073] The results are shown in Table 1. The experiment shows that both the His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe active peptide segment of purple mussel peptide and the Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr active peptide segment of green mussel peptide have good inhibitory activity against yeast, and the antibacterial activity increases with the increase of concentration.

[0074] Table 1. Anti-yeast activity results of purple mussel peptide and green mussel peptide

[0075]

[0076]

[0077] Experimental verification 2: The products obtained in Examples 1 and 2 and Comparative Examples 1 and 2 of the present invention were used as samples, and their effects on improving tear stains were verified through pet tests.

[0078] Eight healthy pet cats with relatively serious tear stain problems were selected and randomly divided into 4 groups (Example 1 and 2 groups and Comparative Examples 1 and 2 groups), with 2 cats in each group. After numbering, the tests were carried out. Before the start, an initial score was given to each pet cat, and then they were fed according to the plan in Table 3 for 3 weeks. Subsequently, the changes in tear stains were observed and scored and recorded, and the scoring criteria were all carried out according to Table 2.

[0079] Table 2. Scoring table for the tear stain situation of pet cats

[0080]

[0081] Table 3. Pet feeding plans for each group

[0082]

[0083] The test results are shown in Table 4. The results show that during the feeding process of the pet cats in Example 1 group and Example 2 group, the frequency, color, smell, inflammation around the eye sockets, and hair condition of the tear stains have been significantly improved; among the 4 pet cats in Comparative Example 1 group and Comparative Example 2 group, 3 had improved tear stain results and 1 had no improvement, showing an obvious gap compared with Example 1 group and Example 2 group. In summary, the present invention performs excellently in improving the tear stains of pet cats, and there is a synergistic effect among the original components of the formulation of the present invention.

[0084] Table 4. Tear stain scoring situations of each group of pets before and after feeding

[0085]

[0086] Although the description of the present invention is through specific embodiments, the protection scope of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. A purple mussel peptide, characterized in that: Its amino acid sequence is His-Gly-Ala-Asp-Gly-Val-Pro-Pro-Phe.

2. A method for preparing the blue mussel peptide according to claim 1, characterized in that: include S11. Crush the purple mussels and add pure water to mix, the material-water ratio is 1: (5-20), the temperature is 45-60°C, the pH is 6.0-8.0, and a feed solution is obtained; S12. After adding 0.2-1% of the volume of the feed solution of a composite protease, stirring and enzymolysis for 3-5 hours, the ratio of the composite protease is trypsin: papain: neutral protease = (2-4): (1-2): (1-2); S13. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and then centrifuged to obtain a supernatant; S14. The supernatant is separated by a membrane device with a molecular weight cutoff of 1000Da, and the permeate is concentrated and spray-dried to obtain the blue mussel peptide.

3. The purple mussel peptide according to claim 1, characterized in that Preferably, the purple mussel peptide has a molecular weight of 896Da and a content of ≥1.5%; Preferably, in step S13, the temperature of the feed solution is raised to 85° C. and maintained for 15 minutes.

4. A perilla leaf peptide, characterized in that: Its amino acid sequence is Asp-Ser-Ser-Leu-Pro-Pro-Ser-Arg-Pro-Thr.

5. A method for preparing the green mussel peptide according to claim 4, characterized in that: include S21. Crushing the green mussel into a homogenate, adding pure water and mixing, the material-water ratio is 1: (5-20), the temperature is 45-60°C, the pH is 6.0-8.0, and a feed solution is obtained; S22. After adding 0.2 to 1% of the volume of the feed solution of the composite protease, stirring and enzymolysis for 3 to 5 hours, the ratio of the composite protease is trypsin: papain: neutral protease = (2 to 4): (1 to 2): (1 to 2); S23. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and then centrifuged to obtain a supernatant; S24. The supernatant is separated by a membrane device with a molecular weight cutoff of 1000Da, and the permeate is concentrated and spray-dried to obtain the green mussel peptide.

6. The green mussel peptide according to claim 4, characterized in that Preferably, the green mussel peptide has a molecular weight of 1082Da and a content of ≥1.5%; Preferably, in step S23, the temperature of the feed solution is raised to 85° C. and maintained for 15 minutes.

7. A peptide composition for improving pet tear stains, characterized in that: The invention comprises 20-30 parts of sea cucumber peptide, 5-10 parts of casein phosphopeptide, 10-20 parts of soybean peptide, 20-30 parts of corn oligopeptide, 10-20 parts of wheat oligopeptide and 10-20 parts of mussel peptide.

8. The peptide composition for improving pet tear stains according to claim 7, characterized in that: The sea cucumber peptide is prepared based on the following method: S31. The sea cucumber was crushed and homogenized, and pure water was added and mixed, the material-water ratio was 1: (5-20), the temperature was 45-60 ° C, the pH was 7.0-9.0, and the liquid was obtained; S32. Add 0.2-1% of the volume of the composite protease to the feed solution and stir for enzymatic hydrolysis for 3-5 hours. The ratio of the composite protease is trypsin: alkaline protease: neutral protease = (3-5): (1-3): (1-3); S33. After the enzymatic hydrolysis is completed, the temperature of the feed solution is raised to 85°C, and the enzyme is inactivated for 15 min, followed by centrifugation to obtain a supernatant; S34. The supernatant is separated by a membrane device with a molecular weight cutoff of 3000Da, and the permeate is concentrated and spray-dried to obtain sea cucumber peptide.

9. The peptide composition for improving pet tear stains according to claim 8, characterized in that: Preferably, the sea cucumber in step S31 is preferably selected from sea cucumbers and sea cucumbers; Preferably, the sea cucumber peptides with a peptide segment smaller than 1000Da account for ≥90%; Preferably, the composition can be in the form of powder, granules, tablets, capsules, pills, or oral liquid.

10. The peptide composition for improving pet tear stains according to claim 7, characterized in that: The invention comprises any one of the mussel peptides in claims 1-2 and claims 4-5.

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