A method for preparing lipid-lowering oyster peptides by low-frequency ultrasound field-assisted enzymatic hydrolysis

Abstract

The present invention relates to the field of polypeptide technology. To develop oyster protein resources, a method for preparing lipid-lowering oyster peptides by low-frequency ultrasound-assisted enzymatic hydrolysis is disclosed. The method comprises the following steps: high-temperature denaturation of shelled and visceral-removed oyster tissue, protein extraction, low-frequency ultrasound-assisted enzymatic hydrolysis with pepsin, secondary enzymatic hydrolysis with trypsin and flavor protease, inactivation, collection of molecules of 500 to 1000 Da, and subsequent separation and purification. The method utilizes pepsin enzymatic hydrolysis, followed by secondary enzymatic hydrolysis with trypsin and flavor protease, and employs intermittent low-frequency ultrasound-assisted enzymatic hydrolysis throughout the entire process. The resulting oyster peptides have a high protein content, a low molecular weight, are easily absorbed and utilized by the human body, are stable in the gastrointestinal environment, and have a high yield.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a method for preparing lipid-lowering oyster peptides by low-frequency ultrasonic field-assisted enzymatic hydrolysis. Background Art

[0002] In recent years, with the continuous improvement of living standards, Chinese residents have shifted from a traditional plant-based diet to a high-fat, low-carbohydrate diet, resulting in a year-on-year increase in the incidence of obesity. Obesity has become a global public health issue garnering widespread attention. Numerous studies have demonstrated that obesity is the most common risk factor for metabolic syndrome and a significant contributor to diabetes, cardiovascular and cerebrovascular disease, and various cancers. Currently, medications for the treatment of obesity are primarily categorized as non-centrally acting weight-loss drugs, centrally acting weight-loss drugs, and hypoglycemic drugs. Long-term use can cause adverse reactions such as liver damage, oily stools, palpitations, and hand tremors. However, functional food substances have not been shown to exhibit adverse reactions when used to alleviate or prevent obesity. Therefore, discovering bioactive peptides with lipid-lowering properties from food proteins is an alternative approach to weight loss and lipid-lowering treatments for obese individuals.

[0003] Oyster protein and its hydrolysate have been reported to have a variety of biological activities, such as lowering blood pressure and anti-thrombotic effects, but its lipid-lowering activity has not been reported or published. In order to improve the utilization of oyster protein, an alkali dissolution and acid precipitation method is usually used to extract and prepare a higher content of oyster protein. However, the oyster protein prepared by this method has a large water-soluble particle size. Based on conventional enzymatic hydrolysis methods, its hydrolysis degree and enzymatic hydrolysis efficiency are relatively low, which ultimately leads to a low production of the target functional polypeptide. The polypeptide preparation technology of this patent uses high-temperature denatured oyster tissue protein, intermittent low-frequency ultrasonic field to assist pepsin enzymatic hydrolysis, and trypsin and flavor protease dual enzyme secondary enzymatic hydrolysis, which improves the protein hydrolysis rate and the productivity of the target polypeptide, while also improving the utilization of the enzyme. This patent uses a low-frequency ultrasonic field to assist enzymatic hydrolysis. After searching, there are no reports of this method being used without enzymatic hydrolysis. It has the advantages of innovation and high efficiency. Summary of the Invention

[0004] To develop oyster resources, the present invention aims to provide a method for preparing lipid-lowering oyster peptides. The oyster peptides are obtained by subjecting the oyster raw materials to high-temperature denaturation and enzymatic hydrolysis. The oyster peptides prepared by the present invention have a small molecular weight, high yield, and high activity, and can significantly reduce the body weight of mice fed a high-fat diet.

[0005] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0006] A method for preparing lipid-lowering oyster peptides by low-frequency ultrasound field-assisted enzymatic hydrolysis comprises the following steps:

[0007] (1) After shelling and removing the viscera of the oysters, the remaining tissue was heated at 85-95°C for 10-15 min and homogenized with distilled water at a solid-liquid ratio of 1:5 g / mL to 1:8 g / mL;

[0008] (2) Oyster protein was extracted by alkali dissolution and acid precipitation. The alkali dissolution conditions were as follows: adding NaOH to the oyster homogenate to adjust the pH to 10.0-11.0, magnetic stirring for 1-3 h, and centrifugation to obtain the supernatant. The acid precipitation conditions were as follows: adjusting the pH to 5.0-5.4 with HCl, and centrifuging to obtain the protein precipitate.

[0009] (3) adding distilled water to the protein precipitate obtained in step (2) at a solid-liquid ratio of 1:2 g / mL to 1:4 g / mL to obtain a homogenate, adding pepsin to the homogenate for enzymatic hydrolysis, and then performing a secondary enzymatic hydrolysis with trypsin and flavor protease, with intermittent low-frequency ultrasound field assisting the enzymatic hydrolysis throughout the process;

[0010] (4) The final enzymatic hydrolyzate of step (3) was heated at 100°C for 10-15 minutes to inactivate the enzyme, and then centrifuged at 4500-6000 rpm for 30-50 minutes to obtain the supernatant;

[0011] (5) The supernatant obtained in the filtration step (4) is filtered using a nanofiltration membrane to retain molecules with a molecular weight of 500 to 1000 Da, and the filtrate is collected and freeze-dried to obtain polypeptide powder.

[0012] As a preferred embodiment of the present invention, step (2) is specifically as follows: adjusting the pH to 10.0-11.0 with NaOH, intermittently homogenizing for 5-8 minutes, and magnetically stirring for 2-3 hours at 4-8°C; then centrifuging at 8000-10000 r / min at 4-8°C for 15-20 minutes to obtain the supernatant; adjusting the pH to 5.0-5.4 with HCl, and centrifuging at 8000-10000 r / min at 4-8°C for 15-20 minutes to obtain the precipitate;

[0013] As a preferred embodiment of the present invention, in step (3), pepsin is added for enzymolysis, the pH value of the protein solution is maintained at 1.5-2.0, the enzymolysis time is 2-4 hours, the enzymolysis temperature is 36-38° C., and the amount of pepsin added is 2.5-3.5wt% of the protein content in the protein solution; trypsin and flavor protease are then added for secondary enzymolysis, the pH value of the homogenate is maintained at 8.0-9.0, the enzymolysis time is 4-8 hours, the enzymolysis temperature is 36-38° C., the amount of trypsin added is 2.5-3.5wt% of the protein content in the protein solution, and the amount of flavor protease added is 0.3-0.8wt% of the protein content in the protein solution; the activity of pepsin is 1500-2500 U / g, the activity of trypsin is 50,000-80,000 U / g, and the activity of flavor protease is 30,000-50,000 U / g.

[0014] As a preferred embodiment of the present invention, the low-frequency ultrasonic method in step (3) is: intermittent low-frequency ultrasonic field ultrasound is used throughout the enzymatic hydrolysis process, the ultrasonic frequency is 40, 60, and 80 kHz synchronous ultrasound, the ultrasonic power is 10 to 15 W, and the ultrasonic mode is: within one ultrasonic cycle, then ultrasound for 30 to 50 seconds, and intermittent for 60 to 80 seconds, and the above ultrasonic cycle is repeated until the enzymatic hydrolysis is completed.

[0015] The present invention also provides an oyster peptide prepared by the above method.

[0016] The present invention also provides the use of the oyster peptide in preparing a drug or preparation with lipid-lowering function.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention uses low-power ultrasound to assist enzymatic hydrolysis, which significantly improves the utilization rate of the enzyme, increases the polypeptide yield by 11.78%, and increases the hydrolysis degree by 15.60%, thereby greatly reducing the production cost.

[0019] (2) The oyster polypeptide prepared by the present invention has a significantly improved fat-reducing function. The prepared polypeptide has the advantages of high protein content, small molecular weight, easy absorption, stability in the gastrointestinal environment, and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a production process flow chart of the present invention.

[0021] Figure 2 The effect of oyster peptide on body weight, liver and epididymal fat weight of mice.

[0022] Figure 3 The effect of oyster peptide on the liver tissue structure of mice.

[0023] Figure 4 The effect of oyster peptide on serum biochemical indicators of mice.

[0024] Figure 5 The effect of oyster peptide on the expression of SREBP-1c and ACC1 genes related to fat synthesis in the liver of mice. DETAILED DESCRIPTION

[0025] The present invention will be further described and illustrated below in conjunction with specific embodiments. The embodiments are merely illustrative of the present disclosure and do not limit its scope. The technical features of the various embodiments of the present invention may be combined accordingly, provided that there is no conflict between them.

[0026] Unless otherwise specified, the raw materials used in the present invention can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are all conventional methods in the art.

[0027] Calculation of peptide yield

[0028]

[0029] Protein hydrolysis rate determination

[0030] Dissolve 0.1% (w / w) 2,4,6-trinitrobenzenesulfonic acid (TNBS) reagent in water. All undigested samples, digested samples and standard solutions are dissolved in 0.1% (w / w) SDS. Add 2.0 mL of 0.2125 M sodium phosphate buffer, pH 8.2, to a test tube, and then add 0.25 mL of the sample or standard solution of the same concentration. Add 2.0 mL of TNBS reagent to each centrifuge tube, then mix on a shaker and incubate in a dark water bath at 50°C for 60 minutes. After covering and incubating, add 4.0 mL of 0.1 M HCl to each tube to terminate the reaction. Then cool at room temperature for 30 minutes, and finally measure the absorbance at 340 nm using a spectrophotometer. Calculate the protein hydrolysis rate as follows:

[0031]

[0032] AN1: nitrogen content of undigested substrate;

[0033] AN2: nitrogen content of the matrix in the sample after digestion;

[0034] Npb: The nitrogen content of peptide bonds in the matrix protein. The unit is mg / g protein.

[0035] Example 1

[0036] After shelling and removing the oysters, the remaining tissue was heated at 90°C for 10 minutes. NaOH was added to the oyster homogenate at a material-liquid ratio of 1:5 g / mL to adjust the pH to 12.0. The mixture was intermittently homogenized for 5 minutes, magnetically stirred at 4°C for 3 hours, and centrifuged at 10,000 rpm for 15 minutes at 4°C. The supernatant was collected, the pH adjusted to 5.4 with HCl, and the pellet was collected after an additional centrifugation at 10,000 rpm for 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3 g / mL to homogenize, and pepsin with an enzyme activity of 1500 U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50,000 U / g and flavor protease with an enzyme activity of 30,000 U / g were added. Intermittent low-frequency ultrasonic field was used to assist enzymatic hydrolysis throughout the process, with ultrasonic frequencies of 40, 60, and 80 kHz, ultrasonic power of 10 W, ultrasonic mode of 30 s and intermittent 60 s, the pH value of the homogenate was maintained at 8.2 during ultrasonic treatment, the enzymatic hydrolysis time was 3 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0037] Comparative Example 1

[0038] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3 g / mL to homogenize, and pepsin with an enzyme activity of 1500 U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50,000 U / g and flavor protease with an enzyme activity of 30,000 U / g were added. Intermittent low-frequency ultrasonic field was used to assist enzymatic hydrolysis throughout the process, with ultrasonic frequencies of 40 and 80 kHz, ultrasonic power of 10 W, ultrasonic mode of 30 s and intermittent 60 s. The pH value of the homogenate was maintained at 8.2 during ultrasonic treatment, the enzymatic hydrolysis time was 3 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0039] Comparative Example 2

[0040] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3 g / mL to homogenize, and pepsin with an enzyme activity of 1500 U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50,000 U / g and flavor protease with an enzyme activity of 30,000 U / g were added. Intermittent low-frequency ultrasonic field was used to assist enzymatic hydrolysis throughout the process, with ultrasonic frequencies of 40 and 60 kHz, ultrasonic power of 10 W, ultrasonic mode of 30 s and 60 s intervals, the pH value of the homogenate was maintained at 8.2 during ultrasonic treatment, the enzymatic hydrolysis time was 3 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0041] Comparative Example 3

[0042] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3 g / mL to homogenize, and pepsin with an enzyme activity of 1500 U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50,000 U / g and flavor protease with an enzyme activity of 30,000 U / g were added. Intermittent low-frequency ultrasonic field was used to assist enzymatic hydrolysis throughout the process, with ultrasonic frequencies of 60 and 80 kHz, ultrasonic power of 10 W, ultrasonic mode of 30 s and intermittent 60 s, the pH value of the homogenate was maintained at 8.2 during ultrasonic treatment, the enzymatic hydrolysis time was 3 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0043] Comparative Example 4

[0044] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3g / mL to homogenize, and pepsin with an enzyme activity of 1500U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50000U / g and flavor protease with an enzyme activity of 30000U / g were added. Intermittent low-frequency ultrasonic field was used to assist enzymatic hydrolysis throughout the process, with an ultrasonic frequency of 80kHz, an ultrasonic power of 10W, an ultrasonic mode of 30s for 30s and an interval of 60s. During the ultrasonic treatment, the pH value of the homogenate was maintained at 8.2, the enzymatic hydrolysis time was 3h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0045] Comparative Example 5

[0046] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3 g / mL to homogenize, and pepsin with an enzyme activity of 1500 U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50,000 U / g and flavor protease with an enzyme activity of 30,000 U / g were added. Intermittent low-frequency ultrasonic field-assisted enzymatic hydrolysis was used throughout the process, with an ultrasonic frequency of 60 kHz, an ultrasonic power of 10 W, an ultrasonic mode of 30 s and an interval of 60 s. During the ultrasonic treatment, the pH value of the homogenate was maintained at 8.2, the enzymatic hydrolysis time was 3 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0047] Comparative Example 6

[0048] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The obtained protein precipitate was added to distilled water at a solid-liquid ratio of 1:3 g / mL to homogenize, and pepsin with an enzyme activity of 1500 U / g was added to the homogenate for enzymatic hydrolysis, the pH value of the homogenate was maintained at 1.5, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of pepsin added was 1.5wt% of the protein content in the homogenate, and then trypsin with an enzyme activity of 50,000 U / g and flavor protease with an enzyme activity of 30,000 U / g were added. Intermittent low-frequency ultrasonic field-assisted enzymatic hydrolysis was used throughout the process, with an ultrasonic frequency of 40 kHz, an ultrasonic power of 10 W, an ultrasonic mode of 30 s and an interval of 60 s. During the ultrasonic treatment, the pH value of the homogenate was maintained at 8.2, the enzymatic hydrolysis time was 3 h, the enzymatic hydrolysis temperature was 36-38°C, the amount of trypsin added was 1.5wt% of the protein content in the homogenate, and the amount of flavor protease added was 0.3wt% of the protein content in the homogenate. The resulting enzymatic hydrolysate was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered using a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and lyophilized to obtain polypeptide powder.

[0049] Comparative Example 7

[0050] After shelling and removing the viscera, the remaining tissue was heated at 90°C for 10 minutes and homogenized with distilled water at a 1:5 g / mL material-to-liquid ratio. The oyster homogenate was adjusted to pH 12.0 by adding NaOH, then intermittently homogenized for 5 minutes. The mixture was magnetically stirred at 4°C for 3 hours, centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The pH was then adjusted to 5.4 with HCl, and the pellet was collected by centrifugation at 10,000 rpm for another 15 minutes at 4°C. The resulting protein precipitate was homogenized with distilled water at a solid-liquid ratio of 1:3 g / mL. Pepsin (1500 U / g) was then added to the homogenate for enzymatic hydrolysis. The pH of the homogenate was maintained at 1.5. The hydrolysis time was 4 hours at a temperature of 36-38°C. The amount of pepsin added was 1.5% by weight of the protein content in the homogenate. Trypsin (50,000 U / g) and flavor protease (30,000 U / g) were then added. The pH of the homogenate was maintained at 8.2. The hydrolysis time was 3 hours at a temperature of 36-38°C. The amount of trypsin added was 1.5% by weight of the protein content in the homogenate, and the amount of flavor protease added was 0.3% by weight of the protein content in the homogenate. The resulting hydrolyzate was heated at 100°C for 10 minutes to inactivate the enzymes. The supernatant was then collected by centrifugation at 4500 rpm for 30 minutes. The supernatant obtained by filtration is filtered using a nanofiltration membrane to retain molecules with a molecular weight of 500 to 1000 Da, and is freeze-dried to obtain polypeptide powder.

[0051] The results are shown in Table 1

[0052] Table 1

[0053]

[0054] According to Table 1, compared with Comparative Example 7, Example 1 has a degree of hydrolysis increased by 15.60% and a polypeptide yield increased by 11.78%, indicating that the low-frequency ultrasonic field can improve the hydrolysis rate of protein and the productivity of the target polypeptide, thereby greatly improving the utilization efficiency of the enzyme. Compared with the other comparative examples, the protein hydrolysis rate and the yield of the target polypeptide using a combination of multiple low-frequency ultrasonic frequencies are higher than those using a single low-frequency ultrasonic frequency. The combined low-frequency ultrasound produces more cavitation effects than a single low-frequency ultrasound. The cavitation effect can instantly produce high temperature, high pressure, high energy density shock waves, microjets, etc., which can affect the conformation of the enzyme molecules, thereby improving enzyme activity and ultimately improving the hydrolysis rate of protein and the productivity of the target polypeptide.

[0055] Pharmacodynamic study of oyster peptides of the present invention on obesity model of mice induced by high-fat diet

[0056] Twenty-four healthy male SPF C57BL / 6J mice, aged 4-6 weeks and weighing 15-18g, were housed in separate cages with free access to water. The mice were maintained at a temperature of 25±1°C, a humidity of 50±3%, and a 12-h light-dark cycle. After one week of normal diet, all mice except the normal group were fed a high-fat diet (containing 49.5g breeding mouse feed, 20.4g lard, 15g sucrose, 12.3g casein, 2g premix, and 0.8g maltodextrin per 100g) and randomly divided into three groups of 8 mice each. The experimental groups were divided into the following groups: ① Normal group: normal diet + sterile water; ② High-fat model group: high-fat diet + sterile water; ③ Example 1 group: high-fat diet + 500mg / kg BW / day of the oyster peptide obtained in Example 1. In addition to the diet, the oyster peptide group was gavage-administered every morning, while the normal and model groups received equal amounts of sterile water. The dosage was adjusted according to body weight every day for 16 weeks.

[0057] During the feeding process, pay attention to the observation of the animal's mental state, diet, activity, feces color and characteristics and other general conditions, weigh the animals weekly, and check the effects of feeding high-fat feed and gavage of drugs on the animal's physical condition.

[0058] HE staining of pathological sections:

[0059] Mice were sacrificed, and the livers were removed, all miscellaneous tissues removed, and the blood was removed by rinsing with pre-chilled PBS. The sections were then blotted dry with filter paper and fixed in 4% paraformaldehyde. After dehydration, wax embedding, sectioning, spreading, attaching, and baking, the sections were stained. Through the adsorption and absorption of hematoxylin (HE), the acidic nuclei of cells are stained blue by alkaline hematoxylin, and the alkaline cytoplasm is stained red by acidic eosin, resulting in a blue nucleus and a red cytoplasm.

[0060] Determination of biochemical indicators in serum:

[0061] After the final dose, mice were weighed and sacrificed. Blood was collected from the heart and allowed to rest at 4°C for 2 hours. The blood was then centrifuged at 4000 rpm for 15 minutes. The supernatant was stored at -20°C. TC, TG, LDL-C, and HDL-C levels were measured according to the instructions in the kit. The liver was removed, the blood was rinsed with PBS, and then blotted dry with filter paper. The liver and epididymal fat were weighed.

[0062] Real-time fluorescence quantitative PCR was used to determine the expression levels of SPEBP-1c and ACC1 genes related to fat synthesis in mouse liver:

[0063] Take 80-100 mg of mouse liver tissue stored in a freezer, freeze it in liquid nitrogen, and grind it. Collect it and place it in a sterile, enzyme-free centrifuge tube. Add 1 mL of pre-chilled Trizol and gently pipette until the liquid is clear. Let it stand at room temperature for 5 minutes. Add 200 μL of chloroform and shake vigorously for 15 seconds. Let it stand at room temperature for 5 minutes. Centrifuge it at 12,000 rpm at 4°C for 15 minutes. Aspirate the upper aqueous phase and transfer it to a new sterile centrifuge tube. Add an equal amount of isopropanol. Vortex thoroughly to mix, then continue centrifugation at 12,000 rpm at 4°C for 15 minutes. Discard the supernatant and thoroughly wash the RNA pellet with 1 mL of pre-chilled 75% ethanol to remove impurities. Then centrifuge it at 12,000 rpm at 4°C for 5 minutes, discard the excess supernatant, and place the RNA pellet on a clean bench to air dry. Once the RNA pellet is dry, dissolve it in 60-100 μL of DEPC water. The ODA260 / A280 and ODA260 / A230 values ​​and the concentration of the extracted RNA samples were determined using a nucleic acid protein quantifier. Finally, the purity of the RNA samples was tested by agarose gel electrophoresis.

[0064] The RNA with tested purity was used for reverse transcription, and cDNA was obtained by performing specific operations according to the instructions of the TIANGEN reverse transcription kit.

[0065] Dilute the cDNA of different samples to the same concentration and configure the fluorescence quantitative reaction system according to the kit instructions. Mix the reaction solution evenly and place it in the fluorescence quantitative instrument amplification reaction system. After the reaction is completed, perform quantitative analysis. Use the internal reference gene β-actin as a blank control and press 2 -△△Ct The target gene mRNA expression level was calculated by the method.

[0066] Experimental results:

[0067] 1. Effects of oyster peptide on body weight, liver weight, and epididymal fat weight in mice

[0068] The experimental results are as follows Figure 2 As shown, the body weight, liver weight, and epididymal fat weight of the high-fat model group were significantly different from those of the normal group. The body weight, liver weight, and epididymal fat weight of the mice in Example 1 group were reduced to some extent. These experimental results suggest that oyster peptides can reduce the body weight, liver weight, and epididymal fat weight of mice to a certain extent.

[0069] 2. Effects of oyster peptides on mouse liver tissue structure

[0070] The experimental results are as follows Figure 3 As shown in the figure, compared with the normal group, the high-fat model group showed disordered hepatic cord arrangement, obvious vacuoles in the hepatocytes, and localized inflammatory cell infiltration. Oyster peptide can significantly improve these pathological conditions.

[0071] 3. Effects of oyster peptides on serum biochemical indicators in mice

[0072] The experimental results are as follows Figure 4 As shown, the total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels in the high-fat model group were significantly different from those in the normal group. Oyster peptides significantly reduced the levels of TC, TG, and LDL-C in the mouse serum, and increased the level of HDL-C, with significant differences compared to the model group. These results suggest that oyster peptides can significantly improve obesity and dyslipidemia caused by high-fat diets.

[0073] 4. Effects of oyster peptide on the expression of SREBP-1c and ACC1 genes related to liver fat synthesis in mice

[0074] The experimental results are as follows Figure 5 As shown, compared with the normal group, the high-fat model group showed upregulated expression of SREPF-1c and ACC1, indicating that high-fat diet consumption disrupts triglyceride metabolism in the liver and leads to lipid accumulation. In contrast, the expression of SREPF-1c and ACC1 was significantly downregulated in the Example 1 group. These results suggest that oyster peptides exert their triglyceride-lowering effects by acting on SREPF-1c and ACC1.

[0075] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. Persons skilled in the art will readily appreciate that variations and modifications may be made without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A method for preparing lipid-lowering oyster peptides by low-frequency ultrasound field-assisted enzymatic hydrolysis, characterized in that: The following steps are involved: (1) After shelling and removing the viscera of the oysters, the remaining tissue was heated at 85-95°C for 10-15 min and homogenized with distilled water at a solid-liquid ratio of 1:5 g / mL to 1:8 g / mL; (2) Oyster protein was extracted by alkali dissolution and acid precipitation. The alkali dissolution conditions were as follows: adding NaOH to the oyster homogenate to adjust the pH to 10.0-11.0, magnetic stirring for 1-3 h, and centrifugation to obtain the supernatant. The acid precipitation conditions were as follows: adjusting the pH to 5.0-5.4 with HCl, and centrifuging to obtain the protein precipitate. (3) adding the protein precipitate obtained in step (2) to distilled water at a solid-liquid ratio of 1:2 g / mL to 1:4 g / mL to obtain a protein solution, adding pepsin to the protein solution for enzymatic hydrolysis, and then performing a secondary enzymatic hydrolysis with trypsin and flavor protease, with intermittent low-frequency ultrasound field assisting the enzymatic hydrolysis throughout the process; (4) The final enzymatic hydrolyzate of step (3) was heated at 100°C for 10-15 minutes to inactivate the enzyme, and then centrifuged at 4500-6000 rpm for 30-50 minutes to obtain the supernatant; (5) filtering the supernatant obtained in step (4) using a nanofiltration membrane to retain molecules with a molecular weight cutoff of 500 to 1000 Da, collecting the filtrate, and lyophilizing to obtain polypeptide powder; Step (3) specifically comprises: adding pepsin for enzymolysis, maintaining the pH value of the protein solution at 1.5-2.0, the enzymolysis time at 2-4 hours, the enzymolysis temperature at 36-38° C., the amount of pepsin added being 2.5-3.5wt% of the protein content in the protein solution, and then adding trypsin and flavor protein for secondary enzymolysis, maintaining the pH value of the homogenate at 8.0-9.0, the enzymolysis time at 4-8 hours, the enzymolysis temperature at 36-38° C., the amount of trypsin added being 2.5-3.5wt% of the protein content in the protein solution, the amount of flavor protein added being 0.3-0.8wt% of the protein content in the protein solution, the activity of pepsin being 1500-2500 U / g, the activity of trypsin being 50000-80000 U / g, and the activity of flavor protein being 30000-50000 U / g.

2. The method according to claim 1, characterized in that Step (2) is specifically as follows: adjusting the pH to 10.0-11.0 with NaOH, intermittently homogenizing for 5-8 minutes, and magnetically stirring for 2-3 hours at 4-8°C; then centrifuging at 8000-10000 r / min at 4-8°C for 15-20 minutes to obtain the supernatant; adjusting the pH to 5.0-5.4 with HCl, and centrifuging at 8000-10000 r / min at 4-8°C for 15-20 minutes to obtain the precipitate.

3. The method according to claim 1, characterized in that In step (3), the low-frequency ultrasound method is as follows: the enzymatic hydrolysis is carried out using intermittent low-frequency ultrasound field, the ultrasound frequency is 40, 60, and 80 kHz synchronous ultrasound, the ultrasound power is 10 to 15 W, and the ultrasound mode is: within one ultrasound cycle, then ultrasound for 30 to 50 seconds, and intermittent for 60 to 80 seconds, and the above ultrasound cycle is repeated until the enzymatic hydrolysis is completed.

4. Use of the oyster peptide prepared by the method according to any one of claims 1 to 3 in the preparation of a drug with lipid-lowering function.

5. Use of the oyster peptide prepared by the method according to any one of claims 1 to 3 in the preparation of a lipid-lowering preparation.

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