Preparation and anti-intestinal inflammation application of abalone viscera-derived protein peptide
Through the bioactive peptide prediction system screening and enzymatic decomposition technology, high-purity abalone visceral protein peptide was prepared, which solved the problem of underutilization of abalone visceral resources, achieved the effect of anti-intestinal inflammation, and was verified through model animal experiments. Its efficacy shows the characteristics of green manufacturing and efficient process.
Patent Information
- Application Number
- CN202411381226.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-17
AI Technical Summary
The existing technology has not fully utilized the viscera resources of abalone, especially during the processing process, which has failed to develop its deep and high value, such as anti-cancer, antioxidant, anti-inflammatory, anti-coagulant and other health care effects, and the extraction process design and application Not fully studied.
AQS is used to screen a series of peptide sequences in abalone organs that have the activity of inhibiting intestinal inflammation by using the self-developed bioactive peptide prediction system. High-purity abalone visceral protein peptides are prepared through biological enzymatic decomposition, centrifugation, ultrafiltration and nanofiltration technologies. For the development of functional health care products for the anti-intestinal inflammation.
A high-purity abalone visceral protein peptide with specific effects against intestinal inflammation was obtained, which avoided the environmental and product damage of high-temperature cooking and chemical hydrolysis processes, achieved green manufacturing, excellent product quality, and verified its efficacy through model animal experiments. .
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Figure CN119144684A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the research field of utilization of seafood processing waste, and in particular to the preparation of abalone viscera-derived protein peptide and its application in resisting intestinal inflammation. Background Art
[0002] As one of the "eight treasures of seafood", abalone has extremely high nutritional value and is rich in trace elements, such as protein, amino acids, vitamins, etc. In addition to being edible, Chinese medicine theory believes that it also has the effects of enhancing immunity, regulating menstruation and moistening the intestines, and nourishing yin and tonifying yang. In addition, the viscera, which account for 20% to 30% of the weight of abalone, have high medical and biological value. The peptides they contain have antihypertensive, anti-inflammatory, and anti-cancer effects. However, abalone viscera are often not fully utilized during processing and are often discarded as waste, which also pollutes the environment. Therefore, the efficient development and utilization of abalone viscera resources is of great significance.
[0003] At present, many researchers have conducted research on abalone protein peptides, such as abalone protein peptides prepared from abalone organs (China Patent Publication No. CN 117426454 A), which uses bio-enzymatic hydrolysis and bio-fermentation technology to prepare abalone protein peptides that can fully preserve effective nutrients without being destroyed, and on this basis, abalone protein peptide functional beverages have been developed; another patent (China Patent Publication No. CN 107556364 B) uses high-temperature and high-pressure subcritical water (temperature between 100~374℃) to assist in processing and extract protein by enzymatic hydrolysis to prepare abalone protein peptides with biological activity. Based on the above patents, it can be seen that researchers mainly focus on the extraction rate, retention of nutrients, and easy operation of abalone visceral protein peptides. Its scope of application is mainly to provide raw materials for the preparation of marine aquatic condiments and functional health products. However, the deep-level high-value development of abalone visceral protein peptides, such as anti-cancer, anti-oxidation, anti-inflammatory, anti-coagulation, regulation of glucose and lipid metabolism and other health effects, as well as the development and application in the field of medicine, involves very little. As for the research and development of abalone visceral protein peptides with specific biological activity, no comprehensive research has been conducted from the aspects of extraction process design, optimization and application.
[0004] The invention adopts a self-developed bioactive peptide prediction system AQS to screen a series of peptide sequences with intestinal inflammation inhibiting activity in abalone organs; adopts a biological enzymatic hydrolysis method to enzymatically hydrolyze abalone viscera, controls enzymatic hydrolysis reaction conditions, and obtains a series of peptide mixtures; uses centrifugation, ultrafiltration and nanofiltration technology to obtain a high-purity functional peptide concentrated solution through single-molecule peptide targeted positioning separation; collects the active peptide solution concentrate, further freeze-dries it into a powder or directly uses the concentrate to dilute it into a liquid for subsequent preparation and development of functional health products against intestinal inflammation. Summary of the invention
[0005] The present invention aims to provide a preparation of abalone visceral protein peptide and its application in anti-intestinal inflammation. The abalone visceral protein peptide provided by the present invention has the specific effect of anti-intestinal inflammation in addition to the effects of ordinary abalone protein peptides, and also has the advantages of high purity and low heavy metal content.
[0006] To achieve the above object, the present invention adopts the following technical solution: A method for preparing abalone viscera-derived protein peptides is carried out according to the following steps: S1: Wash fresh abalone viscera, add deionized water, mince and homogenize to obtain abalone viscera homogenate; S2: filtering the abalone viscera homogenate obtained in step S1 through a 200-mesh sieve to obtain a filtrate; S3: hydrolyzing the filtrate obtained in step S2 with trypsin, and then inactivating the enzyme to obtain an enzymatic solution; S4: centrifuging the enzymatic hydrolyzate obtained in step S3 to obtain a supernatant; S5: ultrafiltration the supernatant obtained in step S4 using an ultrafiltration membrane to obtain an ultrafiltration membrane permeate; S6: performing nanofiltration treatment on the ultrafiltration membrane permeate obtained in step S5 using a nanofiltration membrane to obtain a nanofiltration membrane retained component; S7: pre-freeze the nanofiltration membrane retained fraction obtained in step S6, and freeze-dry it to obtain abalone viscera-derived protein peptides.
[0007] In the above step S1, the weight ratio of abalone viscera to deionized water is 1:2 to 1:4.
[0008] In the above step S3, the conditions for enzymolysis are: temperature of 37° C., pH of 7.0, enzyme substrate ratio of trypsin to abalone viscera of 4000-5000 U / g, concentration of abalone viscera in the enzymolysis system of 10 wt.%, and enzymolysis time of 3.5-5.5 h.
[0009] In the above step S5, the ultrafiltration membrane used in the ultrafiltration treatment has a molecular weight cutoff of 2000 Da.
[0010] In the above step S6, the molecular weight cut-off of the nanofiltration membrane used in the nanofiltration treatment is 200 Da.
[0011] Abalone viscera-derived protein peptide prepared by the above preparation method.
[0012] The use of the abalone viscera-derived protein peptide in the preparation of anti-intestinal inflammation drugs.
[0013] The above-mentioned anti-intestinal inflammatory drugs include drugs for treating colitis.
[0014] The significant advantages of the present invention are: The present invention provides a method for preparing abalone viscera-derived protein peptides, and their application in anti-intestinal inflammation. The preparation method can effectively avoid the damage and pressure disadvantages of high-temperature cooking and chemical hydrolysis processes to the environment and products, and the enzymatic hydrolysis process adopted meets the process characteristics of green manufacturing and environmental friendliness. The preparation of abalone viscera-derived protein peptides has the advantages of smooth process, simple operation, and high product quality. In addition, unlike other similar patents of similar abalone protein peptides, the present invention uses ultrafiltration membranes and nanofiltration membranes to intercept target molecular peptides with a molecular weight range close to the target molecular peptide during the preparation process based on the target molecular peptide pre-screened by AQS in the early stage, thereby obtaining high-purity functional peptides, and its efficacy has been verified by model animal intestinal inflammation experiments. It is expected to have broad prospects in improving the economic benefits of deep processing of abalone viscera and increasing the extension and development of gastrointestinal health functional products. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a curve chart of mouse body weight changes.
[0016] Figure 2 This is a graph of the DAI scoring results for mice. DETAILED DESCRIPTION
[0017] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0018] Embodiment 1: A method for preparing abalone viscera-derived protein peptides is carried out according to the following steps: S1: at 20° C., fresh abalone viscera were cleaned with deionized water and then drained, deionized water was added according to a weight ratio of abalone viscera to deionized water of 1:2, and the mixture was minced and homogenized to obtain abalone viscera pretreatment solution; S2: filtering the abalone viscera pretreatment liquid obtained in step S1 through a 200-mesh sieve to obtain a filtrate; S3: taking the filtrate obtained in step S2, adding purified water to make the substrate concentration 10wt.%, adjusting the pH to 7.0 with sodium hydroxide, adding trypsin at an enzyme dosage of 4500U / g substrate, performing enzymatic hydrolysis at 37°C for 4h, and then inactivating the enzyme at 100°C for 10min to obtain an enzymatic solution; S4: centrifuging the enzymatic hydrolysate obtained in step S3 at 8000 r / min for 10 min to obtain a supernatant; S5: The supernatant obtained in step S4 is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 2000Da, a temperature of room temperature, a pressure of 0.5MPa, and a flow rate of 1.2L / h to obtain an ultrafiltration membrane permeate; S6: The ultrafiltration membrane permeate obtained in step S5 is subjected to nanofiltration treatment using a nanofiltration membrane, wherein the molecular weight cutoff of the nanofiltration membrane is 200 Da, the temperature is room temperature, the pressure is 0.5 MPa, and the flow rate is 1.2 L / h, to obtain a nanofiltration membrane retained component; S7: pre-freeze the nanofiltration membrane retention component obtained in step S6 at -80°C for 2 hours, and then freeze-dry for 72 hours to obtain abalone visceral protein peptides.
[0019] Embodiment 2: A method for preparing abalone viscera-derived protein peptides is carried out according to the following steps: S1: at 25° C., fresh abalone viscera were cleaned with deionized water and then drained, deionized water was added in a weight ratio of 1:3 between the abalone viscera and deionized water, and the mixture was minced and homogenized to obtain abalone viscera pretreatment solution; S2: filtering the abalone viscera pretreatment liquid obtained in step S1 through a 200-mesh sieve to obtain a filtrate; S3: taking the filtrate obtained in step S2, adding purified water to make the substrate concentration 10wt.%, adjusting the pH to 7.0 with sodium hydroxide, adding trypsin at an enzyme dosage of 4000U / g substrate, performing enzymatic hydrolysis at 37°C for 3.5h, and then inactivating the enzyme at 100°C for 10min to obtain an enzymatic solution; S4: centrifuging the enzymatic hydrolyzate obtained in step S3 at 10000 r / min for 10 min to obtain a supernatant; S5: The supernatant obtained in step S4 is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 2000Da, a temperature of room temperature, a pressure of 0.3MPa, and a flow rate of 1.2L / h to obtain an ultrafiltration membrane permeate; S6: The ultrafiltration membrane permeate obtained in step S5 is subjected to nanofiltration treatment using a nanofiltration membrane, wherein the molecular weight cutoff of the nanofiltration membrane is 200 Da, the temperature is room temperature, the pressure is 0.3 MPa, and the flow rate is 1.2 L / h, to obtain a nanofiltration membrane retained component; S7: pre-freeze the nanofiltration membrane retention component obtained in step S6 at -80°C for 2 hours, and then freeze-dry for 72 hours to obtain abalone visceral protein peptides.
[0020] Embodiment 3: A method for preparing abalone viscera-derived protein peptides is carried out according to the following steps: S1: at 10° C., fresh abalone viscera were cleaned with deionized water and then drained, deionized water was added according to a weight ratio of abalone viscera to deionized water of 1:4, and the mixture was minced and homogenized to obtain abalone viscera pretreatment solution; S2: filtering the abalone viscera pretreatment liquid obtained in step S1 through a 200-mesh sieve to obtain a filtrate; S3: taking the filtrate obtained in step S2, adding purified water to make the substrate concentration 10wt.%, adjusting the pH to 7.0 with sodium hydroxide, adding trypsin at an enzyme dosage of 5000U / g substrate, performing enzymatic hydrolysis at 37°C for 5.5h, and then inactivating the enzyme at 100°C for 10min to obtain an enzymatic solution; S4: centrifuging the enzymatic hydrolysate obtained in step S3 at 12000 r / min for 10 min to obtain a supernatant; S5: The supernatant obtained in step S4 is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 2000Da, a temperature of room temperature, a pressure of 0.8MPa, and a flow rate of 1.2L / h to obtain an ultrafiltration membrane permeate; S6: The ultrafiltration membrane permeate obtained in step S5 is subjected to nanofiltration treatment using a nanofiltration membrane, wherein the molecular weight cutoff of the nanofiltration membrane is 200 Da, the temperature is room temperature, the pressure is 0.8 MPa, and the flow rate is 1.2 L / h, to obtain a nanofiltration membrane retained component; S7: pre-freeze the nanofiltration membrane retention component obtained in step S6 at -80°C for 2 hours, and then freeze-dry for 72 hours to obtain abalone visceral protein peptides.
[0021] Application examples: C57BL / 6 wild-type (WT) mice aged 5-7 weeks were purchased, regardless of gender, weighing 18-22g, and used for the experiment after pre-feeding for 4 days. During the pre-feeding period, they were allowed to drink purified water freely. The mice were randomly divided into 3 groups, namely, the drug administration group, the model control group and the blank control group, with 6 mice in each group. In the drug administration group and the model control group, purified water was replaced with 2.5wt.% polysaccharide sodium sulfate (DSS) solution on the first day of the experiment. After drinking for 5 days, the DSS solution was removed and replaced with purified water to establish a DSS-induced mouse colitis model; the blank control group did not receive any treatment. The drug administration group began to gavage the abalone visceral protein peptide prepared in Example 1 on the first day of drinking the DSS solution, with a dose of 250 mg / kg, and continued to be administered for 17 days; the model control group and the blank control group were given the same volume of PBS (pH=7.4) and purified water in the same way.
[0022] During the entire experimental period, each group of animals was weighed at the same time every day, and the percentage of weight loss (%) was calculated according to the following formula: Percentage of weight loss (%) = (1-W / W0) * 100%, where W is the weight of the animal measured every day, and W0 is the weight of the animal at the beginning of the experiment. The stool characteristics, fecal occult blood and other indicators of the animals were observed, and the disease activity index (DAI) score of each animal was calculated based on the average value of the three indicators of animal weight loss percentage, stool characteristics (diarrhea), and fecal occult blood; among them, the percentage of weight loss (weight unchanged is 0, 1-5 is 1 point, 5-10 is 2 points, 10-15 is 3 points, and greater than 15 is 4 points); stool characteristics (diarrhea) (normal is 0, loose stools are 2 points, and diarrhea is 4 points); fecal occult blood (normal is 0, positive occult blood is 2 points, and overt bleeding is 4 points).
[0023] Figure 1 This is a curve chart of the weight change of mice. As can be seen from the figure, the weight of mice in each group drinking DSS showed an overall downward trend throughout the experimental period. On the 5th day of the experiment, the weight of the model control group and the drug-treated group showed an obvious downward trend. The weight of the model control group decreased faster, reaching the lowest point on the 11th day (76.98% of the initial body weight), and the weight recovered slightly from the 12th day, while the weight loss of mice in the drug-treated group was significantly reduced, and the decline rate was slower, reaching the lowest point on the 12th day (90.80% of the initial body weight), and the weight began to recover on the 13th day. The results show that abalone visceral protein peptides can reduce the weight loss rate of DSS-induced colitis model mice to a certain extent.
[0024] Figure 2 This is the result of the DAI score of mice. As can be seen from the figure, the overall DAI score showed a trend of first rising and then falling. In the first 3 days after drinking DSS, the DAI scores of the model control group and the drug-treated group were low, and the index changes were not obvious; the DAI scores increased significantly from the 3rd day, indicating that the DSS-induced colitis model in mice was successfully established. The highest DAI value of the model control group reached (3.33). Although it showed a downward trend from the 9th day, the overall score was still high. Although the mice in the drug-treated group showed clinical symptoms, their DAI scores were low, with the highest value (2.22 points) on the 7th day, and the scores began to decline on the 8th day, indicating that abalone visceral protein peptides have good preventive and anti-inflammatory effects on the DSS-induced colitis model in mice.
[0025] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A method for preparing abalone visceral protein peptide, characterized in that: Follow these steps: S1: Wash fresh abalone viscera, add deionized water, mince and homogenize to obtain abalone viscera homogenate; S2: filtering the abalone viscera homogenate obtained in step S1 through a 200-mesh sieve to obtain a filtrate; S3: hydrolyzing the filtrate obtained in step S2 with trypsin, and then inactivating the enzyme to obtain an enzymatic solution; S4: centrifuging the enzymatic hydrolyzate obtained in step S3 to obtain a supernatant; S5: ultrafiltration the supernatant obtained in step S4 using an ultrafiltration membrane to obtain an ultrafiltration membrane permeate; S6: performing nanofiltration treatment on the ultrafiltration membrane permeate obtained in step S5 using a nanofiltration membrane to obtain a nanofiltration membrane retained component; S7: pre-freeze the nanofiltration membrane retained fraction obtained in step S6, and freeze-dry it to obtain abalone viscera-derived protein peptides.
2. The preparation method according to claim 1, characterized in that: In step S1, the weight ratio of abalone viscera to deionized water is 1:2 to 1:
4.
3. The preparation method according to claim 1, characterized in that: In step S3, the conditions for enzymolysis are: temperature of 37° C., pH of 7.0, an enzyme substrate ratio of trypsin to abalone viscera of 4000-5000 U / g, a concentration of abalone viscera in the enzymolysis system of 10 wt.%, and an enzymolysis time of 3.5-5.5 h.
4. The preparation method according to claim 1, characterized in that: In step S5, the ultrafiltration membrane used in the ultrafiltration treatment has a molecular weight cutoff of 2000 Da.
5. The preparation method according to claim 1, characterized in that: In step S6, the molecular weight cut-off of the nanofiltration membrane used in the nanofiltration treatment is 200 Da.
6. An abalone viscera-derived protein peptide prepared by the preparation method according to any one of claims 1 to 5.
7. Use of the abalone visceral protein peptide as claimed in claim 6 in the preparation of anti-intestinal inflammation drugs.
8. The use according to claim 7, characterized in that: The anti-intestinal inflammation drugs include drugs for treating colitis.