Application of sea cucumber intestine flower polypeptide in preparation of pet blood pressure nursing product

Abstract

The invention relates to application of sea cucumber intestine flower polypeptide in preparation of pet blood pressure nursing products, and belongs to the technical field of marine biological product processing and bioactive peptide. The invention provides application of sea cucumber intestine and flower polypeptides in preparation of pet blood pressure nursing products. The sea cucumber intestine and flower polypeptides are one or more of sea cucumber intestine and flower polypeptides with amino acid sequences as shown in SEQ ID NO.1-4. The sea cucumber intestine flower polypeptide provided by the invention is a food-borne antihypertensive peptide, has the characteristics of small molecular weight, good water solubility, easy absorption, no toxicity and strong activity, and has important significance for realizing high-valued utilization of a sea cucumber processing byproduct intestine flower and enriching the variety of pet blood pressure nursing products.

Description

TECHNICAL FIELD

[0001] The application relates to an application of a sea cucumber intestinal flower polypeptide in the preparation of a pet blood pressure care product and belongs to the technical field of marine biological product processing and bioactive peptides. BACKGROUND

[0002] It should first be noted that the background section disclosed herein merely aims to increase the understanding of the overall background of the application and should not necessarily be considered as recognizing or admitting, in any form, that this information forms the prior art that is already known to those of ordinary skill in the art.

[0003] With the continuous growth of the number of pet breeding and the extension of the life span of dogs and cats, hypertension has become one of the common chronic circulatory system diseases of dogs and cats. Studies have shown that hypertension in dogs and cats is often related to factors such as chronic kidney disease, endocrine disease, cardiovascular disease and obesity. Long-term high blood pressure can cause damage to target organs, including retinal hemorrhage or detachment, myocardial hypertrophy, further deterioration of kidney function and nervous system abnormalities, which seriously affect the quality of life of pets and even endanger life.

[0004] At present, the clinical treatment of hypertension in dogs and cats mainly relies on drug intervention, such as calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors and vasodilators. Although the above drugs can control blood pressure to a certain extent, long-term use may cause adverse reactions such as hypotension, electrolyte imbalance, gastrointestinal reactions and increased renal function burden, and some pets have poor compliance, difficulty in administration and other problems. Therefore, the development of new antihypertensive care functional ingredients with higher safety, better palatability and long-term application is an important research direction in the field of pet health management.

[0005] Angiotensin converting enzyme is a key regulatory enzyme in the renin-angiotensin system, which can catalyze the conversion of angiotensin I to angiotensin II with strong vasoconstrictor effect, and degrade bradykinin with vasodilator effect, thereby playing a core role in blood pressure regulation.

[0006] In recent years, studies have found that ACE inhibitory peptides derived from natural protein hydrolysates have the advantages of small molecular weight, good biocompatibility, high safety, long-term use as functional foods or nutritional intervention means, and have shown good application prospects in the field of cardiovascular health. Therefore, at present, the development of ACE inhibitory peptides with ACE inhibitory activity to block the renin-angiotensin system is a safe and effective way for long-term care of pet blood pressure, which can make up for the shortcomings of existing drug treatment in terms of long-term use safety and palatability.

[0007] Sea cucumbers, a precious marine tonic, are widely utilized due to the rich collagen, polysaccharides, and other nutrients in their body walls. However, their viscera (including intestines) are often discarded as processing byproducts, causing serious resource waste and environmental pressure. Sea cucumber intestines, as a core component of the viscera, are rich in protein, polypeptides, minerals, and bioactive substances, possessing extremely high nutritional value and development potential. Traditional methods of processing sea cucumber intestines often involve simple drying and pulverization or direct discarding, which not only fails to release their internal active ingredients but also results in products that are difficult for the human body to absorb, leading to extremely low bioavailability.

[0008] Existing enzymatic hydrolysis technologies for processing marine biological raw materials often suffer from problems such as incomplete hydrolysis due to the use of a single enzyme, significant loss of active ingredients, and unstable product functionality. For example, hydrolyzing sea cucumber intestines with a single protease easily produces large protein fragments that are difficult to digest and absorb, and fails to fully activate the ACE-inhibiting peptides. Furthermore, some processes suffer from drawbacks such as demanding hydrolysis conditions, complex subsequent purification steps, and high production costs, limiting the industrial development and application of sea cucumber intestines.

[0009] Therefore, developing an enzymatic hydrolysis process based on sea cucumber intestine flowers that has high enzymatic hydrolysis efficiency and retains the ACE inhibitory active ingredients is of great significance for realizing the high-value utilization of sea cucumber processing by-products and enriching the variety of pet blood pressure care products. Summary of the Invention

[0010] To address the aforementioned issues, this paper provides an application of sea cucumber intestinal flower polypeptide in the preparation of pet blood pressure care products. The sea cucumber intestinal flower polypeptide is one or more of the sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO. 1~4. The provided sea cucumber intestinal flower polypeptide is a food-derived hypotensive peptide with characteristics such as small molecular weight, good water solubility, easy absorption, non-toxicity, and strong activity. It is of great significance for realizing the high-value utilization of sea cucumber processing by-products and enriching the variety of pet blood pressure care products, and has broad application prospects and research value.

[0011] This application provides the application of sea cucumber intestinal flower polypeptide in the preparation of pet blood pressure care products, wherein the sea cucumber intestinal flower polypeptide is one or more of the sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO.1~4.

[0012] Optionally, the sea cucumber intestinal flower polypeptide is one or two of the sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.4.

[0013] Optionally, the pet blood pressure care product is an oral product.

[0014] This application provides a method for preparing an enzymatic hydrolysate containing sea cucumber intestinal peptides with amino acid sequences as shown in SEQ ID NO. 1~4, the preparation method comprising the following steps: 1) Select fresh sea cucumber intestines, rinse them, crush and grind them to obtain sea cucumber intestine paste; 2) After adding water to the sea cucumber intestine slurry, adjust the pH to 7.2~7.5, and add serine endopeptidase and trypsin at a temperature of 46±5℃ for the first stage of enzymatic hydrolysis for 2~5 h. The mass ratio of serine endopeptidase to trypsin is (1.0~2.0):1. 3) Adjust the pH of the system to 6.3~6.5, add cysteine ​​protease and fig protease at 53±5℃ for the second stage of enzymatic hydrolysis for 1~4h, the mass ratio of cysteine ​​protease to fig protease is 1:(1.5~3). 4) After the enzymatic hydrolysis is completed, the complex enzymatic hydrolysate system is heated to above 100℃ to inactivate the enzyme activity; 5) After cooling to room temperature, filter through an 80-120 mesh sieve to remove residue and obtain crude peptide solution; 6) Filter the crude peptide solution through an ultrafiltration membrane and collect the permeate with a molecular weight less than 1 kDa to obtain the final product.

[0015] This application provides an oral product for pet blood pressure care, comprising the following components by weight: The ingredients include 28-35 parts chicken meal, 8-12 parts fish meal, 15-20 parts brown rice, 8-12 parts corn, 6-10 parts pea protein, 6-10 parts of the above-mentioned sea cucumber intestine peptides, 4-7 parts animal fat, 1-3 parts cellulose powder, 0.5-1.5 parts inulin, and 0.02-0.05 parts antioxidant.

[0016] Optional steps include the following: S1. Grind the chicken powder, fish powder, brown rice, corn and pea protein into particles with a diameter not exceeding 0.8 mm, and control the moisture content of each raw material to not exceed 10 wt%. Weigh each raw material according to weight and set aside. S2. The raw materials from step S1, along with sea cucumber intestine peptides, cellulose powder, inulin, and antioxidants, are added to a mixer and mixed. Then, steam and water are injected for conditioning, controlling the moisture content of the materials to be between 22% and 26% wt%. S3. The material obtained in step S2 is extruded and shaped using an extruder. The shaped granules are then dried, and the moisture content of the granules is controlled to be no higher than 10 wt%. S4. Spray animal fat onto the surface of the granules and let them cool to obtain the final product.

[0017] Optionally, the extrusion molding conditions in step S3 are: barrel temperature of 90~125℃, die temperature of 125~145℃, screw speed of 300~380rpm, and cavity pressure of 6~9MPa.

[0018] Optionally, in step S2, the mixing speed is 40~60 rpm, the mixing time is 4~6 min, the conditioning temperature is 80~95℃, and the conditioning time is 60~120 s.

[0019] Optionally, in step S3, the drying temperature after extrusion molding is 65~90℃, and the drying time is 25~35min.

[0020] Optionally, the spraying temperature in step S4 is 35~45℃.

[0021] Optionally, the sea cucumber intestinal flower polypeptide is one or two of the sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.4.

[0022] The beneficial effects of this application include, but are not limited to: 1. The application of the sea cucumber intestinal flower polypeptide in the preparation of pet blood pressure care products according to this application. The sea cucumber intestinal flower polypeptide provided is a food-derived antihypertensive peptide with the characteristics of small molecular weight, good water solubility, easy absorption, non-toxicity and strong activity. It is of great significance for realizing the high-value utilization of sea cucumber processing by-products and enriching the variety of pet blood pressure care products, and has broad application prospects and research value.

[0023] 2. The application of the sea cucumber intestinal flower polypeptide of this application in the preparation of pet blood pressure care products, wherein the sea cucumber intestinal flower polypeptide is the sea cucumber intestinal flower polypeptide with the amino acid sequence shown in SEQ ID NO.1~4, wherein the sea cucumber intestinal flower polypeptide shown in SEQ ID NO.1 and SEQ ID NO.4 exhibits good in vitro ACE inhibitory activity and has good biocompatibility, can improve vascular endothelial function, and plays a role in blood pressure care. Furthermore, through search, it was found that the sea cucumber intestinal flower polypeptide provided in this application has not been reported in the existing ACE inhibitory peptide database.

[0024] 3. The application of the sea cucumber intestinal flower polypeptide in the preparation of pet blood pressure care products according to this application also provides a method for extracting the above-mentioned sea cucumber intestinal flower polypeptide from sea cucumber intestinal flower, which enriches the application of sea cucumber intestinal flower in pet functional food and realizes the high-value utilization of sea cucumber processing by-products. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 The results show the in vitro ACE activity inhibition rate of the peptide SEQ ID NO.1 involved in Example 3 of this application; Figure 2 The results show the in vitro ACE activity inhibition rate of the peptide SEQ ID NO.2 involved in Example 3 of this application; Figure 3 The results show the in vitro ACE activity inhibition rate of the peptide SEQ ID NO.3 involved in Example 3 of this application; Figure 4 The results show the in vitro ACE activity inhibition rate of the peptide SEQ ID NO.4 involved in Example 3 of this application; Figure 5 The results of the toxicity test of the sea cucumber intestinal flower ACE inhibitory peptides of SEQ ID NO.1 and SEQ ID NO.4 involved in Example 5 of this application on rat umbilical vein endothelial cells are as follows. Detailed Implementation

[0026] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0027] It should be noted that the sea cucumber intestine peptides with amino acid sequences as shown in SEQ ID NO.1~4 provided in this application can be obtained not only by enzymatic hydrolysis of sea cucumber intestines, but also by artificial synthesis. For example, solid-phase synthesis of the short peptides in this application is a relatively mature technology and is a routine operation for those skilled in the art.

[0028] The present application solution will be described below through specific embodiments.

[0029] Example 1 Fresh sea cucumber intestines were selected, and the fascia and impurities were removed. After rinsing with water, the intestines were crushed and ground to obtain sea cucumber intestine paste. Water was added to the sea cucumber intestine paste to make the material-to-liquid ratio 1:5, and the pH was adjusted to 7.2-7.5. The first stage of enzymatic hydrolysis was carried out at 46℃ with the addition of serine endopeptidase from Bacillus subtilis and trypsin from porcine pancreas. The mass ratio of serine endopeptidase to trypsin was 1.5:1, and the total addition amount was 2.5 wt% of the substrate dry weight. The reaction was carried out for 3 h, with shaking every 20 min. Subsequently, the pH of the system was adjusted to 6.3-6.5, and the second stage of enzymatic hydrolysis was carried out at 53℃ with the addition of cysteine ​​protease from papaya and fig protease. The mass ratio of cysteine ​​protease to fig protease was 1:2, and the addition amount was 3.0 wt% of the substrate dry weight. The reaction was continued for 2 h, with shaking every 20 min. After the hydrolysis, a compound enzymatic hydrolysate was obtained. After the hydrolysis, the compound enzymatic hydrolysate system was heated to 110℃ and kept at that temperature for 30 minutes. The enzyme activity was inactivated by filtration, cooled to room temperature, and then filtered through an 80-120 mesh sieve to remove incompletely hydrolyzed tissue residues, yielding a crude peptide solution. The crude peptide solution was then sequentially filtered through 10kDa and 1kDa ultrafiltration membranes, and the permeate with a molecular weight less than 1kDa was collected to obtain a concentrated solution of sea cucumber intestinal flower ACE inhibitory peptides. The concentrated solution of sea cucumber intestinal flower ACE inhibitory peptides was enriched by column chromatography on a strong cation exchange resin SP Sepharose Fast Flow, followed by gradient elution with 1.0mol / L NaCl. Finally, the eluent was concentrated and freeze-dried to obtain a lyophilized powder containing sea cucumber intestinal flower ACE inhibitory peptides.

[0030] Example 2 The lyophilized powder containing ACE-inhibiting peptides from sea cucumber intestines, obtained through freeze-drying, was analyzed by LC-MS / MS. After separation by high-performance liquid chromatography, the samples were analyzed by Q Exactive mass spectrometry. The mass spectrometry data were retrieved from relevant databases using MaxQuant software to obtain peptide identification results and quantitative analysis results of the corresponding peptides in the lyophilized powder. PeptideRanker was used to analyze the obtained peptide sequences to predict the bioactivity potential of each peptide. Finally, peptides with potentially high bioactivity, good water solubility, and no toxicity were screened. Several peptides with ACE-inhibiting activity were finally selected from numerous peptides, as shown in Table 1 below.

[0031] Table 1. Peptides with ACE inhibitory activity

[0032] Example 3 To accurately verify the efficacy of the screened ACE-inhibiting peptides, SEQ ID NO. 1-4 peptides derived from sea cucumber intestine were artificially synthesized using the Fmoc solid-phase synthesis method. The in vitro ACE-inhibiting activity of each peptide was then verified. Before activity verification, the SEQ ID NO. 1-4 peptides were subjected to two stages of simulated gastric and intestinal digestion, and their ACE-inhibiting activity after digestion was measured.

[0033] In the gastric digestion stage: a fixed amount of the peptide to be tested was weighed into a centrifuge tube, and ultrapure water was added to prepare a peptide solution with a concentration of 2% (w / v). Then, the peptide solution was mixed with simulated gastric juice (3000 U / ml pepsin, 41.5 mM NaCl, 7.6 mM KCl, 0.75 mM KH2PO4, 28 mM NaHCO3, 0.18 mM MgCl2, 0.3 mM (NH4)2CO3, 0.18 mM CaCl2, pH adjusted to 2.8). The mixing volume was 15:1 by weight of the peptide to be tested and pepsin. The pH of the reaction system was adjusted to 2 with 1.2 M HCl solution, and then digested in a water bath at 37°C for 1 h. After the reaction was completed, 2 M NaOH solution was added to adjust the pH of the system to 8, and the gastric digestion stage was ended. Intestinal digestion stage: The solution after the gastric digestion stage was completed was mixed with simulated intestinal fluid (3 mg / mL trypsin, 34.2 mM NaCl, 6.3 mM KCl, 1.0 mM KH2PO4, 80 mM NaHCO3, 0.41 mM MgCl2, 0.8 mM CaCl2, pH adjusted to 7.5) at a ratio of 1:3 (v / v). The pH of the system was quickly adjusted to 6.9~7.1 with 1 M NaOH solution, and then digested in a water bath at 37℃ for 5 h. The intestinal digestion stage was ended by inactivating the enzymes in a boiling water bath.

[0034] The peptides to be tested, after undergoing two stages of simulated gastric and intestinal digestion, were then subjected to in vitro ACE inhibitory activity testing. In the in vitro ACE inhibitory activity experiment, the control group was pre-incubated at 37°C for 15 min after mixing 100 µL of BBS buffer and 30 µL of angiotensin-converting enzyme (ACE) solution, followed by addition of 60 µL of HHL (Hippuryl-L-histidyl-L-leucine) solution and incubation at 37°C for 15 min. After the reaction, 150 µL of 1M HCl solution was added to terminate the reaction. The content of hippuric acid (HA) in the mixture was then determined by high-performance liquid chromatography (HPLC). HA standard curve construction: A 0.1 mg / mL HA solution was prepared and successively diluted to 0.01, 0.02, 0.03, 0.05, 0.07, and 0.09 mg / mL. A standard curve was plotted with HA concentration on the x-axis and HA peak area on the y-axis. In the experimental group testing the ACE inhibitory activity of peptides, the BBS buffer solution needs to be replaced with the ACE inhibitory peptide solution to be tested, and then the in vitro ACE inhibitory activity of the peptide to be tested is measured. The ACE inhibitory peptide solution to be tested is prepared by dissolving the peptide to be tested in 0.15 mol / L BBS buffer solution and adjusting the pH to 8.3.

[0035] The formula for calculating the percentage inhibition of ACE enzyme activity is as follows: ACE inhibition rate (%) = (A1-A2) / A1*100%, where A1 is the peak area of ​​product HA in the control group and A2 is the peak area of ​​product HA after the addition of the peptide to be detected.

[0036] The results of the in vitro ACE activity inhibition rates of the tested peptides SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 are as follows: Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown.

[0037] according to Figures 1-4 The results showed that the sea cucumber intestinal flower peptides of SEQ ID NO.1~4 retained their in vitro ACE inhibitory activity after digestion, and the sea cucumber intestinal flower ACE inhibitory peptides of SEQ ID NO.1 and SEQ ID NO.4 had good in vitro ACE inhibitory activity, with an IC50 value of 1.5%. 50 The values ​​were 0.032 mM and 0.028 mM, respectively.

[0038] Example 4 The researchers further used rat vascular endothelial cells (RAOECs) to test the effect of the sea cucumber intestinal ACE inhibitory peptides (SEQ ID NO.1 and SEQ ID NO.4) on nitric oxide release from endothelial cells. RAOECs in good growth condition were used at a concentration of 5 x 10⁻⁶ cells / year. 3 After seeding 100 μL of cells per well into a 96-well plate and growing for 24 h, 20 μL of HEPES buffer was added to the wells of the control group and 20 μL of HEPES buffer containing the peptide to be detected was added to the wells of the experimental group. The cells were then cultured in an incubator for another 24 h.

[0039] Tests revealed that, compared to the blank control group without the addition of sea cucumber intestinal flower ACE inhibitory peptide, the group with the addition of sea cucumber intestinal flower ACE inhibitory peptide showed increased nitric oxide production in vascular endothelial cells. This indicates that the sea cucumber intestinal flower ACE inhibitory peptides of SEQ ID NO.1 and SEQ ID NO.4 have the physiological effect of increasing nitric oxide production in vascular endothelial cells, which can improve vascular endothelial function and play a role in blood pressure care.

[0040] Example 5 The cytotoxicity of the sea cucumber intestinal endothelial cells ACE inhibitory peptides of SEQ ID NO.1 and SEQ ID NO.4 was further determined using the CCK-8 assay. RUVEC cells from rat umbilical veins in good growth condition were used at a concentration of 5 x 10⁻⁶ cells / year. 5 After seeding 80 μL of cells per well into a 96-well plate and growing for 24 h, 20 μL of different concentrations of the peptide to be tested were added to the wells and cultured for another 24 h in an incubator. Then, 20 μL of CCK-8 (1 mg / mL) was added to each well for 2 h. The absorbance at 450 nm was measured using a microplate reader.

[0041] The cytotoxicity test results of the ACE inhibitory peptides from sea cucumber intestines (SEQ ID NO.1 and SEQ ID NO.4) are as follows: Figure 5 As shown, the results indicate that the sea cucumber intestinal flower ACE inhibitory peptides of SEQ ID NO.1 and SEQ ID NO.4 have no toxicity to rat umbilical vein endothelial cells RUVEC cells.

[0042] Example 6 The experimental animals were 12 male spontaneously hypertensive dogs, aged 8-9 months and weighing 9-10 kg. Before the experiment, the dogs were randomly divided into three groups: a control group, experimental group 1 (SEQ ID NO.1), and experimental group 2 (SEQ ID NO.4), with 4 dogs in each group. All dogs were housed in the same environment, with controlled light and darkness for 12 hours each day at a temperature of 25°C. They were acclimatized to a standard diet for two weeks prior to the experiment, during which blood pressure was measured to minimize stress during the experiment. The dogs were administered the drug orally for four consecutive weeks. The control group received sterile saline added to their standard diet, experimental group 1 received 10g of the SEQ ID NO.1 peptide added to their standard diet, and experimental group 2 received 10g of the SEQ ID NO.4 peptide added to their standard diet. All other feeding conditions were kept consistent. At the end of the four-week feeding period, systolic blood pressure was measured 8 hours after the last feeding and compared with the systolic blood pressure measured at the same time point before the four-week drug administration period. The rate of decrease in systolic blood pressure was calculated.

[0043] The results of the systolic blood pressure reduction rate in the control group, experimental group 1, and experimental group 2 are shown in Table 2 below.

[0044] Table 2 Results of the decrease in systolic blood pressure in experimental dogs

[0045] The experimental results showed that the systolic blood pressure of the experimental dogs in the control group did not show a significant downward trend after 4 weeks of feeding. However, after 4 weeks of feeding supplemented with SEQ ID NO.1 or SEQ ID NO.4 peptides, the systolic blood pressure of experimental group 1 dogs decreased from 172.12 mmHg to 154.29 mmHg, and the systolic blood pressure of experimental group 2 dogs decreased from 169.42 mmHg to 149.25 mmHg.

[0046] Example 7 This embodiment provides an oral product for pet blood pressure care, comprising the following components by weight: The mixture contains 28-35 parts chicken meal, 8-12 parts fish meal, 15-20 parts brown rice, 8-12 parts corn, 6-10 parts pea protein, 6-10 parts sea cucumber intestine peptides, 4-7 parts animal fat, 1-3 parts cellulose powder, 0.5-1.5 parts inulin, and 0.02-0.05 parts antioxidants. The sea cucumber intestine peptides are one or more of the sea cucumber intestine peptides with amino acid sequences as shown in SEQ ID NO.1-4. They can be obtained by enzymatic hydrolysis and purification of sea cucumber processing byproduct intestines, or they can be artificially synthesized by Fmoc solid-phase synthesis.

[0047] Furthermore, the preparation method of the oral product for pet blood pressure care is provided as follows: 1) Grind the chicken powder, fish powder, brown rice, corn, and pea protein into particles with a diameter of ≤0.8 mm. Set aside the cellulose powder and inulin directly. Control the moisture content of each raw material to ≤10 wt%. Weigh and measure the ingredients for later use. 2) Add the above raw materials, sea cucumber intestine peptides, cellulose powder, inulin and rosemary antioxidant to a twin-shaft paddle mixer. Mixing speed: 40~60 rpm, mixing time: 4~6 min. Then continue to spray steam and water into the mixture to make the material moisture content reach 22~26 wt%. Conditioning temperature: 80~95℃, conditioning time: 60~120s. 3) Extrusion molding is performed using a twin-screw extruder. The operating parameters are as follows: barrel zone 1 temperature: 90~105℃, barrel zone 2 temperature: 110~125℃, die head temperature: 125~145℃, screw speed: 300~380 rpm, cavity pressure: 6~9 MPa, and the die diameter can be set as needed. For example, the common die diameter for dog food is 6~10mm, and the common die diameter for cat food is 3~5mm. 4) The shaped granules are fed into a multi-layer belt hot air dryer. The drying temperature adopts a segmented cooling drying program: 90℃ for 10 min, 75℃ for 15 min, and 65℃ for 20 min. Finally, the moisture content of the finished product is controlled to be ≤10wt%. 5) Spray animal fat onto the dried granules in a vacuum roller sprayer. Spraying temperature: 35~45℃, spraying speed: 8~12rpm, spraying time: 2~4min, so that the fat is evenly adhered to the surface of the granules. 6) After the product is cooled, it is tested and screened, and finally sealed in an aluminum-plastic composite bag filled with nitrogen. The water activity of the finished product is controlled to be ≤0.6 to extend the shelf life of the product.

[0048] In summary, this application provides sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO.1~4, which have good in vitro ACE inhibitory activity and good biocompatibility. They can improve vascular endothelial function and play a role in blood pressure care. This is of great significance for realizing the high-value utilization of sea cucumber processing by-products and enriching the variety of pet blood pressure care products, and has broad application prospects and research value.

[0049] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. Use of a sea cucumber intestinal flower polypeptide in the preparation of a pet blood pressure care product, characterized in that, The sea cucumber intestinal flower polypeptide is one or more of sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO. 1~4.

2. Use according to claim 1, characterized in that, The sea cucumber intestinal flower polypeptide is one or two of sea cucumber intestinal flower polypeptides with amino acid sequences as shown in SEQ ID NO. 1 and SEQ ID NO.

4.

3. Use according to claim 1, characterized in that, The pet blood pressure care product is an oral product.

4. A method for preparing an enzymatic hydrolysate of a sea cucumber intestinal flower polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1-4, characterized in that, The preparation method comprises the following steps: 1) selecting fresh sea cucumber intestinal flowers, washing and crushing them to obtain sea cucumber intestinal flower slurry; 2) adding water to the sea cucumber intestinal flower slurry, adjusting the pH to 7.2~7.5, and adding endo-serine protease and trypsin for a first-stage enzymolysis reaction at a temperature of 46±5℃ for 2~5 h, the mass ratio of endo-serine protease to trypsin being (1.0~2.0):1; 3) adjusting the pH of the system to 6.3~6.5, adding cysteine protease and ficin for a second-stage enzymolysis reaction at a temperature of 53±5℃ for 1~4 h, the mass ratio of cysteine protease to ficin being 1:(1.5~3); 4) after the enzymolysis, the complex enzymolysis liquid system is heated to above 100℃ to inactivate the enzyme activity; 5) after cooling to room temperature, the residue is removed by filtering through an 80~120 mesh screen to obtain a crude peptide liquid; 6) the crude peptide liquid is filtered through an ultrafiltration membrane, and the permeate with a molecular weight of less than 1 kDa is collected.

5. A pet blood pressure care oral product, characterized by, The pet blood pressure care product is an oral product. The preparation method comprises the following steps:

6. The method for preparing the oral pet blood pressure care product as described in claim 5, characterized in that, 1) selecting fresh sea cucumber intestinal flowers, washing and crushing them to obtain sea cucumber intestinal flower slurry; 2) adding water to the sea cucumber intestinal flower slurry, adjusting the pH to 7.2~7.5, and adding endo-serine protease and trypsin for a first-stage enzymolysis reaction at a temperature of 46±5℃ for 2~5 h, the mass ratio of endo-serine protease to trypsin being (1.0~2.0):1; 3) adjusting the pH of the system to 6.3~6.5, adding cysteine protease and ficin for a second-stage enzymolysis reaction at a temperature of 53±5℃ for 1~4 h, the mass ratio of cysteine protease to ficin being 1:(1.5~3); 4) after the enzymolysis, the complex enzymolysis liquid system is heated to above 100℃ to inactivate the enzyme activity; 5) after cooling to room temperature, the residue is removed by filtering through an 80~120 mesh screen to obtain a crude peptide liquid; 7. The method for preparing the oral pet blood pressure care product according to claim 6, characterized in that, 6) the crude peptide liquid is filtered through an ultrafiltration membrane, and the permeate with a molecular weight of less than 1 kDa is collected.

8. The method of claim 6, wherein the pet blood pressure care oral preparation is prepared by mixing the following ingredients: The pet blood pressure care product is an oral product. ​ 9. The method for preparing the oral pet blood pressure care product according to claim 6, characterized in that, The preparation method comprises the following steps:

10. The method of claim 6, wherein the pet blood pressure care oral preparation is prepared by mixing the ingredients in the following order: 1) selecting fresh sea cucumber intestinal flowers, washing and crushing them to obtain sea cucumber intestinal flower slurry; 2) adding water to the sea cucumber intestinal flower slurry, adjusting the pH to 7.2~7.5, and adding endo-serine protease and trypsin for a first-stage enzymolysis reaction at a temperature of 46±5℃ for 2~5 h, the mass ratio of endo-serine protease to trypsin being (1.0~2.0):1; 3) adjusting the pH of the system to 6.3~6.5, adding cysteine protease and ficin for a second-stage enzymolysis reaction at a temperature of 53±5℃ for 1~4 h, the mass ratio of cysteine protease to ficin being 1:(1.5~3); 4) after the enzymolysis, the complex enzymolysis liquid system is heated to above 100℃ to inactivate the enzyme activity; 5) after cooling to room temperature, the residue is removed by filtering through an 80~120 mesh screen to obtain a crude peptide liquid; 6) the crude peptide liquid is filtered through an ultrafiltration membrane, and the permeate with a molecular weight of less than 1 kDa is collected. The pet blood pressure care product is an oral product. The preparation method comprises the following steps: 1) selecting fresh sea cucumber intestinal flowers, washing and crushing them to obtain sea cucumber intestinal flower slurry; 2) adding water to the sea cucumber intestinal flower slurry, adjusting the pH to 7.2~7.5, and adding endo-serine protease and trypsin for a first-stage enzymolysis reaction at a temperature of 46±5℃ for 2~5 h, the mass ratio of endo-serine protease to trypsin being (1.0~2.0):1; 3) adjusting the pH of the system to 6.3~6.5, adding cysteine protease and ficin for a second-stage enzymolysis reaction at a temperature of 53±5℃ for 1~4 h, the mass ratio of cysteine protease to ficin being 1:(1.5~3); 4) after the enzymolysis, the complex enzymolysis liquid system is heated to above 100℃ to inactivate the enzyme activity; 5) after cooling to room temperature, the residue is removed by filtering through an 80~120 mesh screen to obtain a crude peptide liquid; 6) the crude peptide liquid is filtered through an ultrafiltration membrane, and the permeate with a molecular weight of less than 1 kDa is collected. The pet blood pressure care product is an oral product. The preparation method comprises the following steps: 1) selecting fresh sea cucumber intestinal flowers, washing and crushing them to obtain sea cucumber intestinal flower slurry; 2) adding water to the sea cucumber intestinal flower slurry, adjusting the pH to 7.2~7.5, and adding endo-serine protease and trypsin for a first-stage enzymolysis reaction at a temperature of 46±5℃ for 2~5 h, the mass ratio of endo-serine protease to trypsin being (1.0~2.0):1; 3) adjusting the pH of the system to 6.3~6.5, adding cysteine protease and ficin for a second-stage enzymolysis reaction at a temperature of 53±5℃ for 1~4 h, the mass ratio of cysteine protease to ficin being 1:(1.5~3); 4) after the enzymolysis, the complex enzymolysis liquid system is heated to above 100℃ to inactivate the enzyme activity; 5) after cooling to room temperature, the residue is removed by filtering through an 80~120 mesh screen to obtain a crude peptide liquid; 6) the crude peptide liquid is filtered through an ultrafiltration membrane, and the permeate with a molecular weight of less than 1 kDa is collected. The pet blood pressure care product is an oral product. ​

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