An enzymatic hydrolyzed sheep liver powder and its preparation method and its application in dog and cat food.

Abstract

This invention discloses an enzymatically hydrolyzed sheep liver powder and its preparation method, as well as its application in dog and cat food, belonging to the field of enzymatic hydrolysate preparation technology. Enzymatically hydrolyzed sheep liver powder is obtained by sequentially hydrolyzing sheep liver with trypsin, papain, and endogenous lysosomal proteases. This enzymatically hydrolyzed sheep liver powder possesses functional activities such as enhancing immunity, improving anemia, resisting stress, protecting the kidneys, improving athletic performance, enhancing digestion, alleviating joint damage, improving coat quality, reducing lipids and aiding weight loss, and delaying aging. Compared to traditional sheep hydrolysates prepared solely with exogenous proteases, this enzymatically hydrolyzed sheep liver powder has a higher content of nine active peptides, specifically: immune peptide TEAPLNPK, blood-building peptide VDGVLLPK, anti-stress and kidney-protecting peptide MYPGIADR, athletic peptide GPMGPR, digestive peptide KEEIFGPVQQ, joint-protecting peptide GPPGPV, coat-enhancing peptide GPMGPS, lipid-lowering peptide KFPLDQL, and anti-aging peptide AGPHFNPL.

Description

Technical Field

[0001] This invention relates to the field of enzymatic hydrolysate preparation technology, specifically to a method for preparing enzymatically hydrolyzed sheep liver powder and its application in dog and cat food. Background Technology

[0002] With the continuous growth of the number of pet dogs and cats in modern society, pet owners are paying increasing attention to their dogs' and cats' health, mainly focusing on the following aspects: immunity, anemia, stress, exercise capacity, digestion, joint health, coat health, weight loss, and aging. Peptides are small-molecule proteins composed of multiple amino acids linked by peptide bonds. Compared to large-molecule proteins, they exhibit better functional activity and have been widely used in dog and cat food. Lamb liver contains 20%–25% protein and is rich in lysine, arginine, and valine, making it a good protein source for preparing bioactive peptides. Currently, the preparation of bioactive peptides from lamb liver mainly relies on enzymatic hydrolysis by exogenous proteases, such as neutral proteases and alkaline proteases (e.g., optimization of the preparation process of lamb liver antioxidant peptides based on Plackett-Burman design and response surface methodology, 2017; lamb liver peptide nutritional formulation and efficient extraction process based on low-temperature enzymatic hydrolysis technology, 2025). However, these exogenous proteases have strong non-specific enzymatic hydrolysis capabilities, and relying entirely on exogenous protease hydrolysis can lead to excessive degradation of active oligopeptides, resulting in a limited variety and low activity of the product's functional activities.

[0003] Sheep liver is rich in endogenous proteases, such as lysosomal proteases. Currently, there are no reported studies on using endogenous proteases in sheep liver to perform mild enzymatic hydrolysis to prepare multifunctional enzymatically hydrolyzed sheep liver powder for dogs and cats. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing enzymatically hydrolyzed sheep liver powder and its application in dog and cat food.

[0005] In a first aspect, the present invention provides an enzymatically hydrolyzed sheep liver powder comprising bioactive peptides, the bioactive peptides including polypeptides with amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.9.

[0006] The amino acid sequences of the polypeptides shown in SEQ ID NO.1 to SEQ ID NO.9 are as follows: SEQ ID NO.1: TEAPLNPK; SEQ ID NO.2: VDGVLLPK; SEQ ID NO.3: MYPGIADR; SEQ ID NO.4: GPMGPR; SEQ ID NO.5: KEEIFGPVQQ; SEQ ID NO.6: GPPGPV; SEQ ID NO.7: GPMGPS; SEQ ID NO.8: KFPLDQL; SEQ ID NO.9: AGPHFNPL.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned enzymatically hydrolyzed sheep liver powder, comprising the following steps:

[0008] (1) Isolation of endogenous lysosomes: After long-term warming and rapid thawing of frozen sheep liver, water is added, the mixture is crushed into a paste, trypsin and lipase are added for enzymatic hydrolysis, and the supernatant liquid after centrifugation is the endogenous lysosome fluid. The precipitate and liquid fractions are stored separately for later use.

[0009] (2) Papain tissue dissociation: Take the precipitate after centrifugation in step (1) and mix it with minced mutton in a certain proportion. Add water to mix and crush into a paste. Then add papain to treat and dissociate the meat tissue. After the process is completed, centrifuge and keep the liquid part.

[0010] (3) Endogenous lysosomal protease hydrolysis: Take the liquid portions after centrifugation in steps (1) and (2) and mix them in proportion for endogenous lysosomal protease hydrolysis. Phospholipase is added at the same time as hydrolysis. After hydrolysis is completed, boil to stop the hydrolysis. After concentration and drying, enzymatically hydrolyzed sheep liver powder is obtained.

[0011] In step (1) above, the temperature for long-term warming is -5~-1℃ and the time is 24~48 h; the temperature for rapid melting is 30~40℃ and the time is 20~30 min; and the amount of water added is 1~2 mL / g of frozen lamb liver.

[0012] In step (1) above, trypsin and lipase are used for enzymatic hydrolysis. The amount of trypsin added is 5~10 g / kg of frozen sheep liver, and the enzyme activity of trypsin is 200,000~300,000 U / g. The amount of lipase added is 1~5 g / kg of frozen sheep liver, and the enzyme activity of lipase is 100,000~200,000 U / g. The enzymatic hydrolysis pH is 7.5~8.5, the enzymatic hydrolysis temperature is 35~45℃, and the enzymatic hydrolysis time is 0.5~1 h.

[0013] In step (2) above, the precipitate after centrifugation in step (1) is mixed with minced mutton in a certain proportion, specifically in a mass ratio of 8:2 to 10:0, and the amount of water added is 1 to 2 mL / g of the mixture.

[0014] The minced mutton used in step (2) above can be replaced by other mutton products such as mutton intestines and mutton heart.

[0015] In step (2) above, the papain treatment is carried out at a specific amount of 5~10 g / kg of tissue homogenate, with a papain activity of 100,000~200,000 U / g. The pH during treatment is 6.0~7.0 and the temperature is 50~60℃. The treatment ends when the pH drops to 4.0~4.5.

[0016] In step (3) above, the liquid portions after centrifugation in steps (1) and (2) are mixed in proportion, specifically in a volume ratio of 1:2 to 2:1.

[0017] In step (3) above, endogenous lysosomal protease is used for enzymatic hydrolysis at a pH of 4.0-4.5, a temperature of 35-45℃, and a hydrolysis time of 1-2 h; the amount of phospholipase added is 0.1-0.5 g / L, and the phospholipase activity is 2000-5000 U / g.

[0018] A third aspect of the present invention provides the application of the enzymatically hydrolyzed sheep liver powder prepared by the above method in dog and cat food.

[0019] Furthermore, the application method is to feed the enzymatically hydrolyzed sheep liver powder prepared by the above method to dogs and cats directly or mix it into dog or cat food (or snacks), or use it as a raw material component in the production and processing of dog or cat food (or snacks).

[0020] Furthermore, the above-mentioned enzymatically hydrolyzed sheep liver powder can be used for other pets such as foxes, hamsters, and sugar gliders.

[0021] The present invention has the following beneficial effects:

[0022] (1) The lysosomes in sheep liver are rapidly released by long-term warming, rapid melting and enzymatic hydrolysis (trypsin, lipase). Long-term warming of frozen sheep liver at -5~-1℃ (the maximum ice crystal formation zone) for 24~48 h can promote the gradual migration and fusion of the original small ice crystals in sheep liver to form large ice crystals, effectively destroying the structure of sheep liver cells and promoting the release of lysosomes in the cells; when rapidly thawing at 30~40℃, a large "osmotic pressure difference" can be formed inside and outside sheep liver cells, which also promotes the release of lysosomes in the cells; the combined use of trypsin and lipase can effectively promote the release of substances in the cell membrane. Trypsin can enzymatically destroy the intercellular connection proteins (such as occlusive proteins, closure proteins, cadherins and chain proteins) to disperse cells, and lipase can hydrolyze the phospholipid structure of the cell membrane, thereby destroying the stability of the membrane structure and promoting the release of lysosomes.

[0023] (2) Papain is used to process meat tissue in meat paste to achieve rapid dissociation of tissue and release of soluble proteins. Papain has a high efficiency in dissociating common proteins such as collagen and actomyosin in meat tissue, which rapidly decomposes connective tissue, collagen fibers and muscle fibers in meat, exposing the internal structure of the tissue and making the texture loose. A large amount of soluble proteins are dissolved, providing more substrates for subsequent enzymatic hydrolysis of sheep liver lysosomal proteases.

[0024] (3) Soluble proteins and peptides are further hydrolyzed using endogenous lysosomal proteases from sheep liver to prepare enzymatically hydrolyzed sheep liver powder with multiple functional activities. The supernatant obtained by centrifugation during sheep liver pretreatment is rich in lysosomal proteases (including cathepsins, cysteine ​​proteases, aspartic proteases, and matrix metalloproteinases, etc.), which can specifically recognize amino acid sequences and perform enzymatic digestion. Compared with the complete use of exogenous proteases (such as neutral proteases and alkaline proteases) for enzymatic digestion, the segmented mild enzymatic digestion method (papain and endogenous lysosomal proteases from sheep liver digestion in sequence) will not cause excessive degradation of active peptides, thus resulting in a high content and strong activity of active peptides in the final enzymatically hydrolyzed sheep liver powder. Phospholipase is also added during the enzymatic digestion process, which can effectively decompose phospholipids on the lysosomal membrane, thereby disrupting the structural stability of the lysosomal membrane and promoting the full release of proteases within the lysosome, thus improving the efficiency of lysosomal protease hydrolysis.

[0025] The enzymatically hydrolyzed sheep liver powder prepared according to the method of this invention not only contains active peptides with amino acid sequences SEQ ID NO.1~SEQ ID NO.9, but also the total mass content of these nine active peptides in the enzymatically hydrolyzed sheep liver powder is 5.22~5.78 times higher than that of amniotic hydrolysate prepared by traditional exogenous protease hydrolysis. The enzymatically hydrolyzed sheep liver powder exhibits different functional activities in dogs and cats, and the activity effect is better than that of amniotic hydrolysate. Specific activity test results are as follows:

[0026] (1) Both the immunopeptide TEAPLNPK (SEQ ID NO.1) and enzymatically hydrolyzed sheep liver powder can enhance the immunity of dogs and cats. The immunopeptide TEAPLNPK (SEQ ID NO.1) can effectively increase the total number of serum lymphocytes in dogs and cats with low immunity, reaching 3.45 and 4.19×10^9 / L, respectively, which are 1.91 and 1.72 times that of the amniotic fluid hydrolysate group. Enzymatically hydrolyzed sheep liver powder can effectively increase the total number of serum lymphocytes in dogs and cats with low immunity, reaching 2.67~2.85 and 3.38~3.71×10^9 / L, respectively, which are 1.48~1.57 and 1.39~1.53 times that of the amniotic fluid hydrolysate group.

[0027] (2) Both the blood-enriching peptide VDGVLLPK (SEQ ID NO.2) and enzymatically hydrolyzed sheep liver powder have the effect of improving anemia in dogs and cats. Blood-enriching peptide VDGVLLPK (SEQ ID NO.2) can effectively increase the hemoglobin content in the whole blood of anemic dogs and cats to 139 and 122 g / L, respectively, which are 1.53 and 1.44 times that of the amniotic fluid hydrolysate group. Enzymatically hydrolyzed sheep liver powder can effectively increase the hemoglobin content in the whole blood of anemic dogs and cats to 114~123 and 102~109 g / L, respectively, which are 1.25~1.35 and 1.20~1.28 times that of the amniotic fluid hydrolysate group.

[0028] (3) Both the anti-stress renal protective peptide MYPGIADR (SEQ ID NO.3) and enzymatically hydrolyzed sheep liver powder have the effect of relieving stress response and protecting the kidneys in dogs and cats. The anti-stress renal protective peptide MYPGIADR (SEQ ID NO.3) can effectively reduce the cortisol content in the serum of dogs and cats to 322 and 441 g / L, respectively, which are 20.30% and 13.19% lower than the amniotic hydrolysate group, respectively; at the same time, the blood-enriching peptide VDGVLLPK (SEQ ID NO.2) can increase the proliferation rate of renal epithelial cells in dogs and cats by 23.9% and 21.8%, respectively, which are 2.69 and 2.83 times that of the amniotic hydrolysate group, respectively. Enzymatic hydrolysis of sheep liver powder effectively reduced the cortisol levels in the serum of dogs and cats to 351-369 and 469-481 g / L, respectively, which were 8.66%-13.12% and 5.31%-7.68% lower than those in the amniotic hydrolysate group, respectively. At the same time, enzymatic hydrolysis of sheep liver powder increased the proliferation rate of renal epithelial cells in dogs and cats by 11.1%-15.6% and 15.2%-18.3%, respectively, which were 1.25-1.75 and 1.97-2.38 times higher than those in the amniotic hydrolysate group, respectively.

[0029] (4) Both the exercise peptide GPMGPR (SEQ ID NO.4) and enzymatically hydrolyzed sheep liver powder improved the athletic performance of dogs and cats. The exercise peptide GPMGPR (SEQ ID NO.4) effectively increased the running endurance of dogs and the jumping height of cats by 24.6 min and 0.94 m, respectively, which were 1.47 and 1.31 times that of the amniotic fluid hydrolysate group. Enzymatically hydrolyzed sheep liver powder effectively increased the running endurance of dogs and the jumping height of cats by 19.7~22.4 min and 0.86~0.94 m, respectively, which were 1.18~1.34 and 1.19~1.31 times that of the amniotic fluid hydrolysate group.

[0030] (5) Both the digestive peptide KEEIFGPVQQ (SEQ ID NO.5) and enzymatically hydrolyzed sheep liver powder improved the intestinal digestive capacity of dogs and cats. The digestive peptide KEEIFGPVQQ effectively increased the intestinal peristalsis frequency of dogs and cats to 7.7 and 6.8 times / min, respectively, which were 1.38 and 1.58 times that of the amniotic fluid hydrolysate group. Enzymatically hydrolyzed sheep liver powder effectively increased the intestinal peristalsis frequency of dogs and cats to 6.8~7.2 and 5.8~6.1 times / min, respectively, which were 1.21~1.29 and 1.35~1.42 times that of the amniotic fluid hydrolysate group.

[0031] (6) Both the joint-protective peptide GPPGPV (SEQ ID NO. 6) and enzymatically hydrolyzed sheep liver powder have the effect of relieving the symptoms of arthritis in dogs and cats. The joint-protective peptide GPPGPV (SEQ ID NO. 6) can effectively reduce the joint swelling of dogs and cats with arthritis, reducing it to 1.12 and 1.11, respectively, which are 15.15% and 18.38% lower than the amniotic fluid hydrolysate group, respectively. Enzymatically hydrolyzed sheep liver powder can effectively reduce the joint swelling of dogs and cats with arthritis, reducing it to 1.19~1.24 and 1.18~1.25, respectively, which are 6.06%~9.85% and 8.09%~13.24% lower than the amniotic fluid hydrolysate group, respectively.

[0032] (7) SEQ ID NO.7: GPMGPS (hair-enhancing peptide) and enzymatically hydrolyzed sheep liver powder both improve the quality of canine and cat hair. SEQ ID NO.7: GPMGPS (hair-enhancing peptide) can effectively increase the peak breaking force of canine and cat hair to 0.31 and 0.28 N, respectively, which are 1.72 and 1.87 times that of the amniotic fluid hydrolysate group. Enzymatically hydrolyzed sheep liver powder can effectively increase the peak breaking force of canine and cat hair to 0.23~0.26 and 0.19~0.23 N, respectively, which are 1.28~1.44 times and 1.27~1.53 times that of the amniotic fluid hydrolysate group.

[0033] (8) Both the lipid-lowering peptide KFPLDQL (SEQ ID NO.8) and enzymatically hydrolyzed sheep liver powder have lipid-lowering and weight-loss effects on dogs and cats. The lipid-lowering peptide KFPLDQL (SEQ ID NO.8) can effectively reduce the serum cholesterol levels in dogs and cats to 6.2 and 4.1 mmol / L, respectively, which are 23.46% and 38.81% lower than those in the amniotic fluid hydrolysate group, respectively. Enzymatically hydrolyzed sheep liver powder can effectively reduce the serum cholesterol levels in dogs and cats to 6.8~7.1 and 4.9~5.3 mmol / L, respectively, which are 11.11%~16.05% and 20.90%~26.87% lower than those in the amniotic fluid hydrolysate group, respectively.

[0034] (9) Both the anti-aging peptide AGPHFNPL (SEQ ID NO.9) and enzymatically hydrolyzed sheep liver powder have the effect of delaying aging in dogs and cats. The anti-aging peptide AGPHFNPL (SEQ ID NO.9) can effectively reduce the serum β-galactosidase content in dogs and cats to 24.8 and 28.5 ng / mL, respectively, which are 32.61% and 32.46% lower than those in the amniotic fluid hydrolysate group, respectively. Enzymatically hydrolyzed sheep liver powder can effectively reduce the serum β-galactosidase content in dogs and cats to 28.2~31.7 and 32.8~35.3 ng / mL, respectively, which are 13.86%~23.37% and 16.35%~22.27% lower than those in the amniotic fluid hydrolysate group, respectively. Attached Figure Description

[0035] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0036] Figure 1 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide TEAPLNPK (SEQ ID NO. 1) mixture (C).

[0037] Figure 2 The mass spectrometry analysis results are for the synthesized polypeptide TEAPLNPK (SEQ ID NO.1).

[0038] Figure 3 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide VDGVLLPK (SEQ ID NO. 2) mixture (C).

[0039] Figure 4 The mass spectrometry analysis results are for the synthesized polypeptide VDGVLLPK (SEQ ID NO.2).

[0040] Figure 5 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide MYPGIADR (SEQ ID NO. 3) mixture (C).

[0041] Figure 6 The mass spectrometry analysis results are for the synthesized polypeptide MYPGIADR (SEQ ID NO.3).

[0042] Figure 7Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide GPMGPR (SEQ ID NO. 4) mixture (C).

[0043] Figure 8 The mass spectrometry analysis results are for the synthetic polypeptide GPMGPR (SEQ ID NO.4).

[0044] Figure 9 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide KEEIFGPVQQ (SEQ ID NO. 5) mixture (C).

[0045] Figure 10 The mass spectrometry analysis results are for the synthesized polypeptide KEEIFGPVQQ (SEQ ID NO.5).

[0046] Figure 11 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide GPPGPV (SEQ ID NO. 6) mixture (C).

[0047] Figure 12 The mass spectrometry analysis results are for the synthesized polypeptide GPPGPV (SEQ ID NO.6).

[0048] Figure 13 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide GPMGPS (SEQ ID NO. 7) mixture (C).

[0049] Figure 14 The results are from the mass spectrometry analysis of the synthetic polypeptide GPMGPS (SEQ ID NO.7).

[0050] Figure 15 Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate of sheep liver powder No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate of sheep liver powder No. 2-synthetic polypeptide KFPLDQL (SEQ ID NO. 8) mixture (C).

[0051] Figure 16 The mass spectrometry analysis results are for the synthesized polypeptide KFPLDQL (SEQ ID NO.8).

[0052] Figure 17Liquid chromatography chromatograms of traditional amniotic hydrolysate sample solution (A), enzymatic hydrolysate sample solution No. 2 prepared by the method of the present invention (B), and enzymatic hydrolysate No. 2-synthetic polypeptide AGPHFNPL (SEQ ID NO. 9) mixture (C).

[0053] Figure 18 The mass spectrometry analysis results are for the synthesized polypeptide AGPHFNPL (SEQ ID NO.9). Detailed Implementation

[0054] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention. Those skilled in the art can make non-essential improvements and adjustments to the invention based on the above description.

[0055] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The source, trade name, and components of the reagents used, if necessary, are indicated upon their first appearance, and subsequent use of the same reagents, unless otherwise specified, are identical to the initial indication.

[0056] In the examples, the lamb liver, minced lamb, trypsin, and papain were commercially available.

[0057] Preparation method of amniotic hydrolysate in the examples:

[0058] 100 g of frozen sheep liver was thawed at room temperature, cut into small pieces, and mixed with 200 mL of water. The mixture was then homogenized using a high-speed tissue homogenizer. The pH was adjusted to 7.0 and the temperature to 50℃. 1 g of neutral protease (100,000 U / g) was added to the homogenized tissue solution for enzymatic hydrolysis. After 4 hours of hydrolysis, the hydrolysis was terminated by boiling. The hydrolysate was centrifuged, and the liquid fraction was collected. This liquid fraction was then concentrated under reduced pressure and freeze-dried to obtain the amniotic hydrolysate.

[0059] Liquid chromatography detection method for peptides in the examples:

[0060] (1) Sample preparation

[0061] 1) Amniotic fluid sample solution: Take amniotic fluid and add it to water at a ratio of 1:10 (g / mL). Shake and mix at 40℃ for 30 min. After centrifugation, keep the supernatant, dilute it with an equal volume of water, and filter it through a 0.22 μm filter membrane for testing.

[0062] 2) Enzymatic hydrolysis of sheep liver powder: Add enzymatic hydrolyzed sheep liver powder to water at a ratio of 1:10 (g / mL), shake and mix at 40℃ for 30 min, centrifuge and keep the supernatant, dilute with an equal volume of water, and filter through a 0.22 μm filter membrane for testing.

[0063] 3) Enzymatic hydrolysis of sheep liver powder-synthetic polypeptide mixture: Take an equal volume of enzymatic hydrolysis of sheep liver powder sample and synthetic polypeptide solution (1 mg / mL), mix them, and filter through a 0.22 μm filter membrane for testing.

[0064] (2) Detection conditions for liquid chromatography

[0065] Column: Shim-pack GIST C 18 -AQ column, 250 mm×4.6 mm, 5 μm;

[0066] Mobile phase: Solution A: trifluoroacetic acid + acetonitrile = 0.05 + 99.95 (volume ratio); Solution B: trifluoroacetic acid + water = 0.10 + 99.90 (volume ratio);

[0067] Flow rate: 1 mL / min;

[0068] Column temperature: 20℃;

[0069] Injection volume: 8 μL;

[0070] Detection wavelength: 220 nm;

[0071] Gradient elution conditions: see Table 1. When the proportion of the mobile phase changes, the gradient changes.

[0072] Table 1 Gradient elution conditions

[0073]

[0074] Example 1:

[0075] The enzymatically hydrolyzed sheep liver powder disclosed in this invention contains active peptides with the following amino acid sequences:

[0076] SEQ ID NO.1: TEAPLNPK (immunopeptide);

[0077] SEQ ID NO.2: VDGVLLPK (blood-replenishing peptide);

[0078] SEQ ID NO.3: MYPGIADR (Anti-stress kidney-protecting peptide);

[0079] SEQ ID NO.4: GPMGPR (kinetic peptide);

[0080] SEQ ID NO.5: KEEIFGPVQQ (digestive peptide);

[0081] SEQ ID NO.6: GPPGPV (joint-protective peptide);

[0082] SEQ ID NO.7: GPMGPS (Hair Enhancement Peptide);

[0083] SEQ ID NO.8: KFPLDQL (lipid-lowering peptide);

[0084] SEQ ID NO.9: AGPHFNPL (anti-aging peptide).

[0085] Example 2:

[0086] The present invention discloses a method for preparing enzymatically hydrolyzed sheep liver powder, comprising the following steps:

[0087] (1) Take 100 g of frozen lamb liver, thaw it (-1℃, 48 h) and (40℃, 20 min), cut it into small pieces and mix it with 200 mL of water. Then, use a high-speed tissue homogenizer to homogenize it into a paste. Adjust the pH to 8.5 and the temperature to 35℃. Then, add 0.5 g of trypsin (300,000 U / g) and 0.1 g of lipase (200,000 U / g) for enzymatic hydrolysis for 1 h. After the enzymatic hydrolysis is completed, centrifuge the hydrolysate. Store the liquid portion after centrifugation in a refrigerator for later use. Mix the precipitate with minced lamb meat at a mass ratio of 8:2. Take 100 g of the mixture and add it to 200 mL of distilled water. After homogenizing it with a high-speed tissue homogenizer, add 1 g of papain (100,000 U / g) to the homogenized liquid. Adjust the pH to 6.0 and the temperature to 60℃. Continue the treatment until the pH drops to 3.5. After centrifugation, keep the liquid portion. The liquid fraction obtained from the first centrifugation and refrigeration was mixed with the liquid fraction obtained from the second centrifugation at a volume ratio of 2:1. The pH of the mixture was adjusted to 4.0 and the temperature to 45°C. Phospholipase (phospholipase activity 2000 U / g) was then added at 0.5 g / L, and enzymatic hydrolysis was carried out for 2 hours while maintaining the pH and temperature. The enzymatic hydrolysis was then terminated by boiling. The mixture was concentrated under reduced pressure and freeze-dried to obtain enzymatically hydrolyzed sheep liver powder No. 1.

[0088] (2) Take 100 g of frozen sheep liver, thaw it (-5℃, 24 h) and (30℃, 30 min), cut it into small pieces and mix it with 100 mL of water. Then, homogenize it into a paste using a high-speed tissue homogenizer. Adjust the pH to 7.5 and the temperature to 45℃. Add 1 g of trypsin (200,000 U / g) and 0.5 g of lipase (100,000 U / g) for enzymatic hydrolysis for 0.5 h. After hydrolysis, centrifuge the hydrolysate. Store the liquid portion after centrifugation in a refrigerator for later use. After centrifugation, take 100 g of the precipitate and mix it with 100 mL of distilled water. Homogenize the mixture using a high-speed tissue homogenizer. Add 0.5 g of papain (200,000 U / g) to the homogenized tissue solution, adjust the pH to 7.0 and the temperature to 50℃, and continue treatment until the pH drops to 4.5. After centrifugation, retain the liquid portion. The liquid fraction obtained from the first centrifugation and refrigeration was mixed with the liquid fraction obtained from the second centrifugation at a volume ratio of 1:2. The pH of the mixture was adjusted to 4.5 and the temperature to 35°C. Phospholipase (phospholipase activity 5000 U / g) was then added at 0.1 g / L, and enzymatic hydrolysis was carried out for 1 h while maintaining the pH and temperature. The enzymatic hydrolysis was then terminated by boiling. The mixture was concentrated under reduced pressure and freeze-dried to obtain enzymatically hydrolyzed sheep liver powder No. 2.

[0089] (3) Take 100 g of frozen lamb liver, thaw it (-3℃, 36 h) and (35℃, 25 min), cut it into small pieces and mix it with 150 mL of water. Then, use a high-speed tissue homogenizer to homogenize it into a paste. Adjust the pH to 8.0 and the temperature to 40℃. Add 0.75 g of trypsin (250,000 U / g) and 0.3 g of lipase (150,000 U / g) for enzymatic hydrolysis for 0.75 h. After hydrolysis, centrifuge the hydrolysate. Store the liquid portion after centrifugation in a refrigerator for later use. Mix the precipitate with minced lamb meat at a mass ratio of 9:1. Take 100 g of the mixture and add it to 150 mL of distilled water. Homogenize the mixture using a high-speed tissue homogenizer. Add 0.75 g of papain (150,000 U / g) to the homogenized liquid. Adjust the pH to 6.5 and the temperature to 55℃. Continue processing until the pH drops to 4.0. After centrifugation, retain the liquid portion. The liquid fraction obtained from the first centrifugation and refrigeration was mixed with the liquid fraction obtained from the second centrifugation at a volume ratio of 1:1. The pH of the mixture was adjusted to 4.2 and the temperature to 40°C. Phospholipase (phospholipase activity 3500 U / g) was then added at 0.3 g / L, and enzymatic hydrolysis was carried out for 1.5 h while maintaining the pH and temperature. The enzymatic hydrolysis was then terminated by boiling. The mixture was concentrated under reduced pressure and freeze-dried to obtain enzymatically hydrolyzed sheep liver powder No. 3.

[0090] Example 3:

[0091] Molecular weight distribution detection

[0092] The molecular weight distribution of enzymatically hydrolyzed sheep liver powder was determined according to the GPC / UV detection method in the appendix of the national standard GB 31645-2018. The mass contents of peptides with a molecular weight less than 3000 Da in enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 were 85%, 87%, and 82%, respectively, while the mass contents of small molecule peptides and amino acids with a molecular weight less than 1000 Da were 39%, 45%, and 42%, respectively.

[0093] Example 4:

[0094] Identification and screening of active peptides

[0095] (1) Identification by LC-MS / MS mass spectrometry

[0096] The enzymatically hydrolyzed sheep liver powder was dissolved in double-distilled water and then transferred to a 5 kD ultrafiltration tube. It was centrifuged at 12000 r / min for 10 min at 4°C to collect peptide samples smaller than 5 kD. Dithiothreitol solution was added to the peptide samples to bring the final concentration to 10 mmol / L, and the samples were reduced in a 56°C water bath for 1 h. Iodoacetic acid solution was then added to bring the final concentration to 50 mmol / L. After reacting in the dark for 40 min, the samples were desalted using a desalting column and the solvent was evaporated in a vacuum centrifuge at 45°C to obtain the sample to be tested.

[0097] The samples to be tested were analyzed by LC-MS / MS. The capillary liquid chromatography conditions were as follows: analytical column: 150 μm id x 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100 Å; mobile phase A: pure water (containing 0.1% (v / v) formic acid), mobile phase B: 80% acetonitrile (containing 0.1% (v / v) formic acid), flow rate: 600 nL / min, analysis time for each component: 66 min. The mass spectrometry conditions were as follows: primary mass spectrometry parameters: Resolution: 70000, AGCtarget: 3e6, Maximum IT: 100 ms, Scanrange: 300 to 1800 m / z; secondary mass spectrometry parameters: Resolution: 75000, AGCtarget: 1e5, Maximum IT: 50 ms, TopN: 20, NCE / stepped NCE: 28.

[0098] The raw mass spectrometry files obtained after LC-MS / MS detection were searched using Maxquant (1.6.2.10) in the Uniprot protein database (sheep-derived protein database, Capra hircus library) to obtain candidate active peptides and their amino acid sequences.

[0099] (2) Screening of bioactive peptides

[0100] The online prediction tool Peptide Ranker was used to predict the potential bioactivity of candidate bioactive peptides; a higher score indicates a higher probability of bioactivity. Bioactive peptides with scores >0.8 were screened from enzymatically hydrolyzed sheep liver powder samples 1-3. Among these, bioactive peptides with scores >0.8 shared by samples 1-3 included peptides with amino acid sequences SEQ ID No. 1-SEQ ID No. 9 (mass spectrometry analysis results of these peptides are shown below). Figure 2 , 4 (As shown in numbers 6, 8, 10, 12, 14, 16, and 18).

[0101] Example 5:

[0102] Peptide synthesis

[0103] The candidate peptides screened in Example 1 were synthesized by Genscript Biotech Inc. The specific method is as follows: the carboxyl group of the first amino acid was covalently linked to the carrier resin, and then, using the amino group of this amino acid as the reaction starting point, an acylation reaction was carried out with the carboxyl group of the adjacent amino acid to form a peptide bond; this process was repeated until the target peptide was synthesized; the Fmoc protecting group was removed and the resin was dried; then, the peptide was cleaved with 6 times the resin volume of cleavage buffer (97.50% TFA + 2.50% H2O, volume percentage), and the precipitate was washed 3 times with anhydrous diethyl ether to obtain crude peptide; the peptide with a purity of more than 95% was obtained by high performance liquid chromatography.

[0104] Example 6:

[0105] Liquid chromatography detection

[0106] The total mass content of active peptides with amino acid sequences SEQ ID NO.1~SEQ ID NO.9 in amniotic hydrolysate and enzymatically hydrolyzed sheep liver powder was determined by liquid chromatography. The results are shown in Table 2. Compared with amniotic hydrolysate prepared by traditional methods (using exogenous proteases completely), the total mass content of active peptides with amino acid sequences SEQ ID NO.1~SEQ ID NO.9 in enzymatically hydrolyzed sheep liver powder prepared by the method of the present invention (segmented mild enzymatic hydrolysis method) is higher, ranging from 1.93% to 2.14%, which is 5.22~5.78 times that of amniotic hydrolysate (0.37%). The nine bioactive peptides with amino acid sequences SEQ ID NO.1 to SEQ ID NO.9 were not only present simultaneously in the enzymatically hydrolyzed sheep liver powder, but their contents were also higher than those in the amniotic fluid hydrolysate. The contents of these nine bioactive peptides in the enzymatically hydrolyzed sheep liver powder reached 0.11%, 0.13%, 0.27%, 0.16%, 0.10%, 0.12%, 0.19%, 0.17%, and 0.21%, respectively, which were more than twice the contents in the amniotic fluid hydrolysate.

[0107] Table 2. Mass content of nine bioactive peptides in amniotic hydrolysate and enzymatically hydrolyzed sheep liver powder

[0108]

[0109] Example 7:

[0110] like Figure 1 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains an immunopeptide (SEQ ID NO.1: TEAPLNPK), and according to... Figure 1 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0111] The effects of the samples on the total lymphocyte count in dogs and cats were evaluated through feeding tests in dogs and cats. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease hydrolysis; (2) enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence TEAPLNPK (SEQ ID No. 1).

[0112] Sixty dogs with low immunity (total lymphocyte count in whole blood below 1.5×10^9 / L) were recruited to participate in the test. The breeds included Corgi, Teddy, Schnauzer, Bichon Frise, Pomeranian, Chihuahua, Beagle, French Bulldog, Miniature Pinscher, and Chinese Rural Dog. They were evenly divided into 6 groups according to breed: control group, amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and synthetic peptide group. In the control group, each dog was fed dog food at a dose of 30 g / kg Body Weight (BW) per day. In the amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3, each dog was fed dog food at a dose of 30 g / kg BW per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per day, respectively. In the synthetic peptide group, each dog was fed dog food at a dose of 30 g / kg BW per day, and simultaneously supplemented with the TEAPLNPK (SEQ ID No. 1) synthetic peptide at a dose of 10 mg / kg Body Weight (kg BW) per day, mixed with their food. The experiment lasted 21 days, and whole blood samples were collected from the dogs before the experiment and on day 21 to determine the total lymphocyte count.

[0113] Sixty cats with low immunity (total lymphocyte count in whole blood below 1.5×10^9 / L) were recruited to participate in the test. The breeds included American Shorthair, Tabby, British Shorthair, Tortoiseshell, Silver Shaded, Blue Cat, Garfield, Chinese Rural Cat, Golden Shaded, and Maine Coon. They were evenly divided into 6 groups according to breed: control group, amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and synthetic peptide group. In the control group, each cat was fed cat food at a dose of 15 g / kg BW per day. In the amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3, each cat was fed cat food at a dose of 15 g / kg BW per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per day, respectively. In the synthetic peptide group, each cat was fed cat food at a dose of 15 g / kg BW per day, and simultaneously supplemented with the TEAPLNPK (SEQ ID No. 1) synthetic peptide at a dose of 10 mg / kg BW per day, respectively. The experiment lasted 21 days, and whole blood samples were collected from the cats before and on day 21 of the experiment to determine the total lymphocyte count.

[0114] The results of the total lymphocyte count in canine and feline serum are shown in Table 3. In the canine feeding experiment, the total lymphocyte count in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group significantly increased compared to before the experiment, reaching 1.81, 2.74, 2.85, 2.67, and 3.45 × 10^9 / L, respectively, which were 2.70, 3.81, 4.13, 4.31, and 5.85 times higher than before the experiment. In the cat feeding experiment, the total lymphocyte count in the cat serum hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group significantly increased compared to before the experiment, reaching 2.43, 3.53, 3.71, 3.38, and 4.19 times higher than before the experiment, respectively, which were 2.13, 3.27, 3.64, 3.05, and 3.64 times higher than before the experiment.

[0115] The results show that the synthetic peptide with sequence TEAPLNPK (SEQ ID No. 1) and the enzymatically hydrolyzed sheep liver powder containing this peptide can effectively increase the total number of serum lymphocytes in dogs and cats with low immunity. Their effects are better than those of amniotic fluid hydrolysate. The synthetic peptide with sequence TEAPLNPK (SEQ ID No. 1) and the enzymatically hydrolyzed sheep liver powder containing this peptide lay a good foundation for improving the immune performance of dogs and cats.

[0116] Table 3 Results of total lymphocyte count in canine and feline serum

[0117]

[0118] Example 8:

[0119] like Figure 3 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains a blood-building peptide (SEQ ID NO.2: VDGVLLPK), and according to... Figure 3As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0120] The effects of the samples on the hemoglobin content of dogs and cats were evaluated by feeding tests in dogs and cats. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease hydrolysis; (2) enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence VDGVLLPK (SEQ ID No. 2).

[0121] Sixty anemic dogs (whole blood hemoglobin levels below 80 g / L) were recruited for the test. Breeds included Schnauzers, Corgis, Poodles, Yorkshire Terriers, Bichon Frises, Maltese, Pomeranians, Shiba Inus, Miniature Pinschers, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with a synthetic peptide containing sequence VDGVLLPK (SEQ ID No. 2) at a dose of 10 mg / kg BW per dog per day, also administered in their food. The experiment lasted 21 days, and whole blood samples were collected from dogs before and on day 21 of the experiment to test hemoglobin levels.

[0122] Sixty anemic cats (whole blood hemoglobin levels below 80 g / L) were recruited for the test. Breeds included Golden Shaded, Tabby, British Shorthair, Devon, Silver Shaded, Chinese Domestic Cat, Siamese, Russian Blue, Ragdoll, and Maine Coon. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence VDGVLLPK (SEQ ID No. 2) at a dose of 10 mg / kg BW per cat per day, respectively. The experiment lasted 21 days, and whole blood samples were collected from dogs before and on day 21 of the experiment to test hemoglobin levels.

[0123] The results of the whole blood hemoglobin content tests in dogs and cats are shown in Table 4. In the dog feeding experiment, the whole blood hemoglobin content in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group increased significantly compared with the pre-experiment levels, reaching 91, 116, 114, 123, and 139 g / L, respectively, which were 1.36, 1.84, 1.87, 2.24, and 2.73 times higher than the pre-experiment levels. In the cat feeding experiment, the whole blood hemoglobin content in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group increased significantly compared with the pre-experiment levels, reaching 85, 105, 102, 109, and 122 g / L, respectively, which were 2.07, 2.44, 2.00, 2.32, and 2.60 times higher than the pre-experiment levels.

[0124] The results show that the synthetic peptide with sequence VDGVLLPK (SEQ ID No. 2) and the enzymatically hydrolyzed sheep liver powder containing this peptide can effectively increase the hemoglobin content in the whole blood of dogs and cats. Their effects are better than those of amniotic fluid hydrolysate. The synthetic peptide with sequence VDGVLLPK (SEQ ID No. 2) and the enzymatically hydrolyzed sheep liver powder containing this peptide can help improve the anemia status of anemic dogs and cats.

[0125] Table 4. Results of hemoglobin content test in whole blood of dogs and cats

[0126]

[0127] Example 9:

[0128] like Figure 5 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains an anti-stress and kidney-protective peptide (SEQ ID NO. 3: MYPGIADR), and according to... Figure 5 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0129] The effects of the samples on the anti-stress effects of dogs and cats were evaluated through dog and cat feeding tests. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease enzymatic hydrolysis; (2) enzymatic hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence MYPGIADR (SEQ ID No. 3).

[0130] Test 1: Stress Resistance Test

[0131] Sixty healthy dogs were recruited for the test. Breeds included Pomeranians, Corgis, Miniature Pinschers, French Bulldogs, Yorkshire Terriers, Poodles, Bichon Frises, Schnauzers, Shiba Inus, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with the synthetic peptide sequence MYPGIADR (SEQ ID No. 3) at a dose of 10 mg / kg BW per dog per day, respectively. Before the trial and on day 21 of the trial, serum samples were collected from dogs after they were introduced to the new environment to measure cortisol levels.

[0132] Sixty healthy cats were recruited for the test. Breeds included Tabby, British Shorthair, Devon, Chinese Domestic Cat, Ragdoll, Silver Shaded, Russian Blue, Garfield, Golden Shaded, and Maine Coon. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence MYPGIADR (SEQ ID No. 3) at a dose of 10 mg / kg BW per cat per day, respectively. Before the trial and on day 21 of the trial, serum samples were collected from the cats after they were introduced to the new environment to measure cortisol levels.

[0133] Test 2: Kidney Cell Proliferation Test

[0134] The feline kidney epithelial cells (CRFK) and canine kidney epithelial cells (MDCK[NBL-2]) used in the test were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0135] Cat or canine kidney epithelial cells were used at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 1 / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h until cell adhesion was achieved. The culture medium was discarded, and medium containing the test sample (100 μg / mL) was added. After incubation for 24 h, 100 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance (OD) was measured at 450 nm. 450The control group received a culture medium without the test sample, and the blank group received no CCK-8 solution. Cell proliferation rate was calculated using the formula: Cell proliferation rate (%) = {[(Experimental group OD)} 450 Value - Blank group OD 450 (value) / (control group OD) 450 Value - Blank group OD 450 [Value) - 1} × 100%.

[0136] The results of serum cortisol levels in dogs and cats after environmental changes are shown in Table 5. In the dog feeding experiment, the serum cortisol levels in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic peptide group were significantly lower after environmental changes compared to before the experiment, reaching 404, 365, 351, 369, and 322 ng / mL, respectively, representing decreases of 6.70%, 21.84%, 21.83%, 18.18%, and 28.60% compared to before the experiment. In the cat feeding experiment, the serum cortisol levels in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic peptide group were significantly lower after environmental changes compared to before the experiment, reaching 508, 475, 481, 469, and 451 ng / mL, respectively, representing decreases of 8.96%, 17.10%, 15.47%, 16.55%, and 20.40% compared to before the experiment.

[0137] The results of the proliferation tests on canine and feline renal epithelial cells are shown in Table 6. Amniotic hydrolysate, enzymatically hydrolyzed sheep liver powder (No. 1-3), and synthetic peptides promoted the proliferation of canine renal epithelial cells, with proliferation rates reaching 8.9%, 13.3%, 11.1%, 15.6%, and 23.9%, respectively. Amniotic hydrolysate, enzymatically hydrolyzed sheep liver powder (No. 1-3), and synthetic peptides promoted the proliferation of feline renal epithelial cells, with proliferation rates reaching 7.7%, 18.3%, 16.9%, 15.2%, and 21.8%, respectively.

[0138] The results showed that the synthetic peptide with sequence MYPGIADR (SEQ ID No. 3) and the enzymatically hydrolyzed sheep liver powder containing this peptide could effectively reduce serum cortisol levels in dogs and cats after environmental changes and promote the proliferation of renal epithelial cells in dogs and cats. Moreover, the effects of reducing serum cortisol levels and promoting the proliferation of renal epithelial cells in dogs and cats were better than those of amniotic fluid hydrolysate. The synthetic peptide with sequence MYPGIADR (SEQ ID No. 3) and the enzymatically hydrolyzed sheep liver powder containing this peptide can help alleviate the stress response of dogs and cats during environmental changes such as moving, transportation, and boarding, and can also protect the kidneys.

[0139] Table 5. Results of serum cortisol levels in dogs and cats after environmental changes.

[0140]

[0141] Table 6 Results of renal epithelial cell proliferation assay in dogs and cats

[0142]

[0143] Example 10:

[0144] like Figure 7 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains a kinetic peptide (SEQ ID NO.4: GPMGPR), and according to... Figure 7 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains only trace amounts of this active peptide.

[0145] The effects of the samples on running endurance in dogs and jumping ability in cats were evaluated through feeding tests in dogs and cats. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease hydrolysis; (2) enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence GPMGPR (SEQ ID No. 4).

[0146] Sixty healthy dogs were recruited for the test. Breeds included Chinese Rural Dogs, Poodles, French Bulldogs, Maltese, Schnauzers, Corgis, Bichon Frises, Pomeranians, Shiba Inus, and Miniature Pinschers. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with the synthetic peptide sequence GPMGPR (SEQ ID No. 4) at a dose of 10 mg / kg BW per dog per day, respectively. Exercise capacity tests were conducted before and 21 days after the experiment. Dogs were placed on the front half of a treadmill conveyor belt at a speed of 15 km / h. A line was marked 15 cm from the rear of the treadmill. The test was stopped when the dog's hind paw touched the line for the third time, indicating insufficient stamina. The time from start to stop was recorded as the running time for the dog's fitness test, used to assess the dog's running endurance.

[0147] Sixty healthy cats were recruited for the test. Breeds included Ragdoll, Tabby, Chinese Domestic Cat, Garfield, British Shorthair, Devon, Golden Shaded, Silver Shaded, Russian Blue, Ragdoll, and Maine Coon. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence GPMGPR (SEQ ID No. 4) at a dose of 10 mg / kg BW per cat per day, respectively. Before and 21 days after the experiment, a stable, non-slip, height-adjustable obstacle was selected for the jump test. The vertical height from the moment the cat's hind legs pushed off the ground to the landing point was used as the standard. After successfully completing 3 jumps at each height, the cat was considered to have the jumping ability for that height. The height was then appropriately increased until the cat could not complete 6 consecutive jumps. The highest height the cat jumped was recorded to evaluate its jumping ability.

[0148] The results of the canine running endurance and cat jumping height tests are shown in Table 7. In the canine feeding experiment, the running endurance of dogs in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic peptide group increased significantly compared with the pre-experiment levels, reaching 16.7, 20.3, 19.7, 22.4, and 24.6 min, respectively, which were 1.27, 1.48, 1.53, 1.66, and 1.74 times higher than the pre-experiment levels. In the cat feeding experiment, the jumping height of cats in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic peptide group increased significantly compared with the pre-experiment levels, reaching 0.72, 0.86, 0.88, 0.86, and 0.94 m, respectively, which were 1.00, 1.19, 1.24, 1.30, and 1.42 times higher than the pre-experiment levels.

[0149] The results show that the synthetic peptide with sequence GPMGPR (SEQ ID No. 4) and the enzymatically hydrolyzed sheep liver powder containing this peptide can effectively improve the running endurance of dogs and the jumping ability of cats, and the improvement effect is better than that of amniotic fluid. The synthetic peptide with sequence GPMGPR (SEQ ID No. 4) and the enzymatically hydrolyzed sheep liver powder containing this peptide play a promoting role in improving the athletic ability of dogs and cats.

[0150] Table 7 Results of dog running endurance and cat jumping height tests

[0151]

[0152] Example 11:

[0153] like Figure 9As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains a digestive peptide (SEQ ID NO.5: KEEIFGPVQQ), and according to... Figure 9 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0154] The effects of the samples on intestinal peristalsis in dogs and cats were evaluated through feeding tests in dogs and cats. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease hydrolysis; (2) enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence KEEIFGPVQQ (SEQ ID No. 5).

[0155] Sixty healthy dogs were recruited for the test. Breeds included Schnauzers, Corgis, Yorkshire Terriers, French Bulldogs, Poodles, Bichon Frises, Pomeranians, Shiba Inus, Miniature Pinschers, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with the synthetic peptide sequence KEEIFGPVQQ (SEQ ID No. 5) at a dose of 10 mg / kg BW per dog per day, respectively. Before and 21 days after the experiment, canine intestinal peristalsis was detected using a portable veterinary color Doppler ultrasound system.

[0156] Sixty healthy cats were recruited for the test. Breeds included Golden Shaded, Tabby, Siamese, British Shorthair, Devon, Silver Shaded, Russian Blue, Chinese Domestic Cat, Ragdoll, and Maine Coon. They were evenly divided into six groups based on breed, sex, and age: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence KEEIFGPVQQ (SEQ ID No. 5) at a dose of 10 mg / kg BW per cat per day, respectively. Before and 21 days after the experiment, the intestinal peristalsis of cats was detected using a portable veterinary color Doppler ultrasound system.

[0157] The results of the intestinal peristalsis frequency tests in dogs and cats are shown in Table 8. In the dog feeding experiment, the intestinal peristalsis frequency of dogs in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group increased significantly compared with the pre-experimental values, reaching 5.6, 7.2, 7.2, 6.8, and 7.7 times / min, respectively, which were 1.08, 1.26, 1.31, 1.33, and 1.45 times higher than the pre-experimental values. In the cat feeding experiment, the intestinal peristalsis frequency of cats in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group increased significantly compared with the pre-experimental values, reaching 4.3, 6.1, 5.6, 5.8, and 6.8 times / min, respectively, which were 1.05, 1.42, 1.31, 1.38, and 1.48 times higher than the pre-experimental values.

[0158] The results show that the synthetic peptide with sequence KEEIFGPVQQ (SEQ ID No. 5) and the enzymatically hydrolyzed sheep liver powder containing this peptide can effectively increase the frequency of intestinal peristalsis in dogs and cats, and the effect is better than that of amniotic fluid hydrolysate. This helps to improve the intestinal digestive capacity of dogs and cats.

[0159] Table 8 Results of intestinal motility frequency test in dogs and cats

[0160]

[0161] Example 12:

[0162] like Figure 11 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains a joint-protective peptide (SEQ ID NO. 6: GPPGPV), and according to Figure 11 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0163] The effects of the samples on joint swelling in dogs and cats with arthritis were evaluated through dog and cat feeding tests. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease enzymatic hydrolysis; (2) enzymatic hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence GPPGPV (SEQ ID No. 6).

[0164] Sixty dogs with arthritis (joint swelling exceeding 1.3) were recruited for the test. Breeds included Schnauzers, Maltese, Corgis, Poodles, Bichon Frises, Chihuahuas, Pomeranians, Shiba Inus, Miniature Pinschers, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with the synthetic peptide sequence GPPGPV (SEQ ID No. 6) at a dose of 10 mg / kg BW per dog per day, respectively. Swelling of the knee joint in dogs was measured before the experiment and 21 days after the experiment. A suture was wrapped around the swollen knee joint, and its length was measured with a ruler. The average knee joint circumference of the normal group dogs was measured before the experiment and recorded as C0. The joint circumference measured during the experiment was recorded as C1. Swelling degree = (C1 / C0).

[0165] Sixty cats with arthritis (joint swelling exceeding 1.3) were recruited for the test. Breeds included Golden Shaded, Tabby, British Shorthair, Devon, Siamese, Eastern Shorthair, Silver Shaded, Russian Blue, Ragdoll, and Maine Coon. They were evenly divided into six groups based on breed, sex, and age: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence GPPGPV (SEQ ID No. 6) at a dose of 10 mg / kg BW per cat per day, respectively. Swelling of the cat's knee joint was measured before the experiment and 21 days after the experiment. A suture was wrapped around the swollen knee joint, and its length was measured with a ruler. The average knee joint circumference of the normal group cats was measured before the experiment and recorded as C0. The joint circumference measured during the experiment was recorded as C1. Swelling degree = (C1 / C0).

[0166] The results of joint swelling tests in dogs and cats with arthritis are shown in Table 9. In the canine feeding experiment, the joint swelling in the amniotic fluid group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic peptide group was significantly reduced compared to before the experiment, decreasing to 1.32, 1.21, 1.24, 1.19, and 1.12, respectively; representing reductions of 4.35%, 12.32%, 12.68%, 12.50%, and 17.04% compared to before the experiment. In the cat feeding experiment, the joint swelling in the amniotic fluid group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic peptide group was significantly reduced compared to before the experiment, decreasing to 1.36, 1.18, 1.21, 1.25, and 1.11, respectively; representing reductions of 5.56%, 17.48%, 14.18%, 13.19%, and 20.14% compared to before the experiment.

[0167] The results indicate that the synthetic polypeptide of sequence GPPGPV (SEQ ID No. 6) and the enzymatically hydrolyzed sheep liver powder containing this peptide can effectively alleviate joint swelling in dogs and cats with arthritis, and the allergic effect is better than that of amniotic fluid. The synthetic polypeptide of sequence GPPGPV (SEQ ID No. 6) and the enzymatically hydrolyzed sheep liver powder containing this peptide play an important functional role in alleviating the symptoms of arthritis in dogs and cats.

[0168] Table 9. Results of Joint Swelling Test in Dogs and Cats with Arthritis

[0169]

[0170] Example 13:

[0171] like Figure 13 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains hair-enhancing peptide (SEQ ID NO.7: GPMGPS), and according to... Figure 13 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0172] The effect of the samples on the peak breaking force of dog and cat hair was evaluated by feeding tests in dogs and cats. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease hydrolysis; (2) enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence GPMGPS (SEQ ID No. 7).

[0173] Sixty healthy dogs were recruited for the test. Breeds included Schnauzers, Corgis, Poodles, Bichon Frises, Pomeranians, Chihuahuas, French Bulldogs, Shiba Inus, Miniature Pinschers, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with the synthetic peptide sequence GPMGPS (SEQ ID No. 7) at a dose of 10 mg / kg BW per dog per day, also supplemented with the synthetic peptide sequence GPMGPS (SEQ ID No. 7). Before and 21 days after the test, dog hair was clipped from the back of the dog, and the hairs were picked out and fixed vertically to the clamp of the tensile testing instrument to measure the peak breaking force of the hair.

[0174] Sixty healthy cats were recruited for the test. Breeds included Golden Shaded, Tabby, British Shorthair, Devon, Silver Shaded, Eastern Shorthair, Russian Blue, Garfield, Ragdoll, and Maine Coon. They were evenly divided into six groups based on breed, sex, and age: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence GPMGPS (SEQ ID No. 7) at a dose of 10 mg / kg BW per cat per day, respectively. Before and 21 days after the test, cat hair was cut from the back of the cat, and the hairs were picked out and fixed vertically to the clamp of the tensile testing instrument to measure the peak breaking force of the hair.

[0175] The peak breaking force of canine and cat hair is shown in Table 10. In the canine feeding experiment, the peak breaking force of canine hair in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group increased significantly compared with the pre-experiment values, reaching 0.18, 0.24, 0.26, 0.23, and 0.31 N, respectively, which are 1.2, 1.71, 2.17, 1.64, and 2.38 times higher than the pre-experiment values. In the cat feeding experiment, the peak breaking force of cat hair in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group increased significantly compared with the pre-experiment values, reaching 0.15, 0.22, 0.23, 0.19, and 0.28 N, respectively, which are 1.36, 1.83, 1.77, 1.73, and 2.55 times higher than the pre-experiment values.

[0176] The results indicate that the synthetic peptide with sequence GPMGPS (SEQ ID No. 7) and the enzymatically hydrolyzed sheep liver powder containing this peptide both have a good effect on improving the peak tensile strength of dog and cat hair, and the improvement effect is better than that of amniotic fluid hydrolysate. The synthetic peptide with sequence GPMGPS (SEQ ID No. 7) and the enzymatically hydrolyzed sheep liver powder containing this peptide play an important role in improving the quality of dog and cat hair.

[0177] Table 10 Results of Peak Force Test on Dog and Cat Hair Breaking

[0178]

[0179] Example 14:

[0180] like Figure 15 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains a lipid-lowering peptide (SEQ ID NO.8: KFPLDQL), and according to... Figure 15 As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0181] The effects of the samples on cholesterol levels in dogs and cats were evaluated through feeding tests. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease hydrolysis; (2) enzymatically hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide with the sequence KFPLDQL (SEQ ID No. 8).

[0182] Sixty obese dogs (with a body condition score of 7 or higher) were recruited for the test. Breeds included Schnauzers, Corgis, Poodles, Yorkshire Terriers, Bichon Frises, Pomeranians, French Bulldogs, Shiba Inus, Miniature Pinschers, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with a synthetic peptide containing sequence KFPLDQL (SEQ ID No. 8) at a dose of 10 mg / kg BW per dog per day, also supplemented with this peptide. Before and 21 days after the experiment, whole blood was collected from dogs and serum was prepared. The cholesterol content in the serum was measured according to the instructions of the cholesterol content kit.

[0183] Sixty obese cats (with a body condition score of 7 or higher) were recruited for the test. Breeds included Golden Shaded, Tabby, British Shorthair, Devon, Garfield, Eastern Shorthair, Silver Shaded, Russian Blue, Ragdoll, and Maine Coon. They were evenly divided into six groups based on breed, sex, and age: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence KFPLDQL (SEQ ID No. 8) at a dose of 10 mg / kg BW per cat per day, respectively. Whole blood was collected from cats before the experiment and 21 days after the experiment to prepare serum. The cholesterol content in the serum was measured according to the instructions of the cholesterol content kit.

[0184] The results of serum cholesterol level tests in dogs and cats are shown in Table 11. In the dog feeding experiment, the serum cholesterol levels in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group were significantly lower than before the experiment, decreasing to 8.1, 7.1, 7.2, 6.8, and 6.2 mmol / L, respectively, representing decreases of 4.71%, 21.98%, 18.18%, 20.93%, and 29.55% compared to before the experiment. In the cat feeding experiment, the serum cholesterol levels in the amniotic fluid hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group were significantly lower than before the experiment, decreasing to 6.7, 5.3, 5.1, 4.9, and 4.1 mmol / L, respectively, representing decreases of 6.94%, 26.39%, 28.17%, 33.78%, and 43.06% compared to before the experiment.

[0185] The results indicate that the synthetic peptide with sequence KFPLDQL (SEQ ID No. 8) and the enzymatically hydrolyzed sheep liver powder containing this peptide can effectively help reduce serum cholesterol levels in dogs and cats, and the effect of assisting in reducing cholesterol levels is better than that of amniotic fluid. The synthetic peptide with sequence KFPLDQL (SEQ ID No. 8) and the enzymatically hydrolyzed sheep liver powder containing this peptide help dogs and cats reduce lipids and lose weight.

[0186] Table 11 Results of serum cholesterol levels in dogs and cats

[0187]

[0188] Example 15:

[0189] like Figure 17 As shown in -B, the enzymatically hydrolyzed sheep liver powder prepared according to the method of the present invention contains an anti-aging peptide (SEQ ID NO.9: AGPHFNPL), and according to... Figure 17As shown in -A, the amniotic hydrolysate prepared by the traditional exogenous protease hydrolysis method contains almost no active peptide.

[0190] The effects of the samples on the β-galactosidase content of aging dogs and cats were evaluated through dog and cat feeding tests. The specific samples were: (1) amniotic hydrolysate prepared by traditional exogenous protease enzymatic hydrolysis; (2) enzymatic hydrolyzed sheep liver powder No. 1 to No. 3 prepared in Example 2; and (3) a synthetic polypeptide (anti-aging peptide) with the sequence AGPHFNPL (SEQ ID No. 9).

[0191] Sixty senior dogs (over 7 years old) were recruited for the test. Breeds included Schnauzers, Corgis, Poodles, Maltese, Bichon Frises, Pomeranians, Shiba Inus, Yorkshire Terriers, Miniature Pinschers, and Chinese Rural Dogs. They were evenly divided into six groups according to breed: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed dog food at a dose of 30 g / kg BW per dog per day. The amniotic fluid hydrolysate group and the enzymatically hydrolyzed sheep liver powder groups 1-3 were fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per dog per day, respectively. The synthetic peptide group was fed dog food at a dose of 30 g / kg BW per dog per day, and simultaneously supplemented with the synthetic peptide sequence AGPHFNPL (SEQ ID No. 9) at a dose of 10 mg / kg BW per dog per day, respectively. Before and 21 days after the experiment, whole blood was collected from dogs and serum was prepared. The β-galactosidase content in the serum was detected according to the instructions of the β-galactosidase content kit.

[0192] Sixty senior cats (over 7 years old) were recruited for the test. Breeds included Golden Shaded, Tabby, British Shorthair, Devon, Silver Shaded, Eastern Shorthair, Chinese Domestic Cat, Russian Blue, Ragdoll, and Maine Coon. They were evenly divided into six groups based on breed, sex, and age: a control group, an amniotic fluid hydrolysate group, enzymatically hydrolyzed sheep liver powder groups 1-3, and a synthetic peptide group. The control group was fed cat food at a dose of 15 g / kg BW per cat per day. The amniotic fluid hydrolysate group and enzymatically hydrolyzed sheep liver powder groups 1-3 were fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with amniotic fluid hydrolysate and enzymatically hydrolyzed sheep liver powder groups 1-3 at a dose of 1 g / kg BW per cat per day, respectively. The synthetic peptide group was fed cat food at a dose of 15 g / kg BW per cat per day, and simultaneously supplemented with the synthetic peptide sequence AGPHFNPL (SEQ ID No. 9) at a dose of 10 mg / kg BW per cat per day, respectively. Before and 21 days after the experiment, whole blood was collected from cats and serum was prepared. The β-galactosidase content in the serum was detected according to the instructions of the β-galactosidase content kit.

[0193] The results of serum β-galactosidase levels in dogs and cats are shown in Table 12. In the dog feeding experiment, the serum β-galactosidase levels in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group were significantly lower than before the experiment, decreasing to 36.8, 29.2, 28.2, 31.7, and 24.8 ng / mL, respectively, representing decreases of 5.88%, 23.76%, 26.94%, 18.51%, and 33.87% compared to before the experiment. In the cat feeding experiment, the serum β-galactosidase levels in the amniotic hydrolysate group, the enzymatically hydrolyzed sheep liver powder groups 1-3, and the synthetic polypeptide group were significantly lower than before the experiment, decreasing to 45.2, 34.3, 35.3, 32.8, and 28.5 ng / mL, respectively, representing decreases of 7.25%, 21.28%, 23.43%, 26.29%, and 36.81% compared to before the experiment.

[0194] The results showed that the synthetic peptide with sequence AGPHFNPL (SEQ ID No. 9) and the enzymatically hydrolyzed sheep liver powder containing this peptide could effectively reduce the serum β-galactosidase content in dogs and cats, and the reduction effect was better than that of amniotic fluid hydrolysate. The synthetic peptide with sequence AGPHFNPL (SEQ ID No. 9) and the enzymatically hydrolyzed sheep liver powder containing this peptide showed good anti-aging effects in dogs and cats.

[0195] Table 12 Results of serum β-galactosidase content test in dogs and cats

[0196]

[0197] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An enzymatically hydrolyzed sheep liver powder, characterized in that, The enzymatically hydrolyzed sheep liver powder includes active peptides with amino acid sequences of SEQ ID NO.1 to SEQ ID NO.9, specifically the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.9 as follows: SEQ ID NO.1: TEAPLNPK; SEQ ID NO.2: VDGVLLPK; SEQ ID NO.3: MYPGIADR; SEQ ID NO.4: GPMGPR; SEQ ID NO.5: KEEIFGPVQQ; SEQ ID NO.6: GPPGPV; SEQ ID NO.7: GPMGPS; SEQ ID NO.8: KFPLDQL; SEQ ID NO.9: AGPHFNPL.

2. The method for preparing enzymatically hydrolyzed sheep liver powder according to claim 1, characterized in that, Follow these steps: (1) Isolation of endogenous lysosomes: After long-term warming and rapid thawing of frozen sheep liver, water was added, the mixture was crushed into a paste, and trypsin and lipase were added for enzymatic hydrolysis. After centrifugation of the hydrolysis product, the supernatant was the endogenous lysosome fluid. The precipitate and liquid fractions were stored separately for later use. (2) Papain tissue dissociation: Take the precipitate after centrifugation in step (1) and mix it with minced mutton in a certain proportion. Add water to mix and crush into a paste. Then add papain to treat and dissociate the meat tissue. After the process is completed, centrifuge and keep the liquid part. (3) Endogenous lysosomal protease hydrolysis: Take the liquid portions after centrifugation in steps (1) and (2) and mix them in proportion for endogenous lysosomal protease hydrolysis. Phospholipase is added at the same time as hydrolysis. After hydrolysis is completed, boil to stop the hydrolysis. After concentration and drying, hydrolyzed sheep liver powder is obtained.

3. The method for preparing enzymatically hydrolyzed sheep liver powder according to claim 2, characterized in that, In step (1) endogenous lysosome separation, the temperature for long-term warming is -5~-1℃ and the time is 24~48 h; the temperature for rapid melting is 30~40℃ and the time is 20~30 min; the amount of water added is 1~2 mL / g of frozen sheep liver; trypsin and lipase are used for enzymatic hydrolysis, the amount of trypsin added is 5~10 g / kg of frozen sheep liver, the activity of trypsin is 200,000~300,000 U / g, the amount of lipase added is 1~5 g / kg of frozen sheep liver, the activity of lipase is 100,000~200,000 U / g, the pH of enzymatic hydrolysis is 7.5~8.5, the temperature of enzymatic hydrolysis is 35~45℃, and the time of enzymatic hydrolysis is 0.5~1 h.

4. The method for preparing enzymatically hydrolyzed sheep liver powder according to claim 2, characterized in that, In step (2) of papain tissue dissociation, the precipitate after centrifugation in step (1) is mixed with minced mutton in a ratio of 8:2 to 10:0 by mass, and water is added at a rate of 1 to 2 mL / g of the mixture. Papain is then added at a rate of 5 to 10 g / kg of the tissue mixture, with an enzyme activity of 100,000 to 200,000 U / g. The pH during treatment is 6.0 to 7.0 and the temperature is 50 to 60°C. Treatment ends when the pH drops to 4.0 to 4.

5.

5. The method for preparing enzymatically hydrolyzed sheep liver powder according to claim 2, characterized in that, In step (3) endogenous lysosomal protease hydrolysis, the liquid portions after centrifugation in steps (1) and (2) are mixed in a specific ratio of 1:2 to 2:1 by volume. The endogenous lysosomal protease hydrolysis is carried out at a pH of 4.0 to 4.5, a temperature of 35 to 45°C, and a hydrolysis time of 1 to 2 hours. The amount of phospholipase added is 0.1 to 0.5 g / L, and the phospholipase activity is 2000 to 5000 U / g.

6. The enzymatically hydrolyzed sheep liver powder according to claim 1, characterized in that, The application of the enzymatically hydrolyzed sheep liver powder in dog and cat food.

7. The enzymatically hydrolyzed sheep liver powder according to claim 1, characterized in that, It can be fed directly to dogs or cats, or mixed into dog food, cat food, dog treats, or cat treats, or used as a raw material component in the production and processing of dog food, cat food, dog treats, or cat treats.

8. The enzymatically hydrolyzed sheep liver powder according to claim 1, characterized in that, The enzymatically hydrolyzed sheep liver powder is used for other pets such as foxes, hamsters, and sugar gliders.

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