Earthworm yolk phospholipid peptide with blood lipid-lowering and memory improving functions, and preparation method and application thereof

Abstract

The application discloses a leydig's egg yolk phospholipid peptide with blood lipid-lowering and memory improving functions, and a preparation method and application thereof, and belongs to the technical field of bioactive peptide preparation. The preparation of the leydig's egg yolk phospholipid peptide with blood lipid-lowering and memory improving functions specifically comprises the following steps: mixing defatted leydig's powder and egg yolk phospholipid protein powder, adding the mixture into an enzymolysis tank, stirring uniformly, then adding bacillus subtilis protease and transglutaminase for enzymolysis, inactivating the enzyme, concentrating, and performing vacuum low-temperature spray drying to obtain the leydig's egg yolk phospholipid peptide product; the product has good dispersion stability, the raw materials are safe and non-toxic, a 'peptide-phospholipid' composite structure is constructed, activity reservation and synergistic effect are realized, and the problems of poor stability and single efficacy of leydig's peptide are solved; the product can be used for preparing functional food with blood lipid-lowering and / or memory improving functions, and is suitable for people with hyperlipidemia accompanied by cognitive function decline.

Description

Technical Field

[0001] This invention relates to an earthworm egg yolk phospholipid peptide with lipid-lowering and memory-improving functions, its preparation method and application, belonging to the field of bioactive peptide preparation technology. Background Technology

[0002] Hyperlipidemia and cognitive decline have become two major linked diseases threatening the health of middle-aged and elderly people. They influence each other, creating a vicious cycle: dyslipidemia leads to changes in cerebral hemodynamics and vascular endothelial damage, accelerating neuronal degeneration; while cognitive decline affects patient adherence to lipid management. Therefore, developing natural active substances that combine the dual effects of lowering blood lipids and improving memory has significant clinical value and market potential.

[0003] Earthworm, a traditional Chinese medicine, has been shown to regulate lipid metabolism through its extracts containing lumbrokinase and active peptides. These components can improve blood lipid levels by inhibiting cholesterol synthesis and promoting the clearance of low-density lipoprotein. Egg yolk phospholipids are recognized as brain-boosting components; their phosphatidylcholine can be converted into the neurotransmitter acetylcholine, directly participating in the learning and memory process. However, current technologies often use a simple mixture of earthworm active peptides and egg yolk phospholipid peptides, which has the following drawbacks: firstly, the two proteins have inconsistent degrees of hydrolysis, and simple physical mixing leads to poor dispersion stability; secondly, they lack specific interactions and cannot form a functional synergy, thus failing to fully realize their lipid-lowering and brain-boosting effects.

[0004] Therefore, how to achieve functional synergy between earthworm peptides and egg yolk phospholipids through structural modification and process innovation, while improving product stability and bioactivity, has become a pressing technical challenge. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an earthworm egg yolk phospholipid peptide with dual functions of lowering blood lipids and improving memory. By specifically modifying earthworm active peptides with egg yolk phospholipids, a "peptide-phospholipid" composite structure is constructed. At the same time, the preparation process is optimized to achieve activity retention and synergistic effect, thus solving the problems of poor stability and single efficacy of earthworm peptides.

[0006] To achieve the above objectives, the following technical solution is provided: The first objective of this invention is to provide an earthworm egg yolk phospholipid peptide that has the effects of lowering blood lipids and improving memory. The earthworm egg yolk phospholipid peptide is obtained by enzymatic hydrolysis of defatted earthworm powder and egg yolk phospholipid protein powder by Bacillus subtilis protease and transglutaminase.

[0007] In one embodiment, the earthworm egg yolk phospholipid peptide product has a protein content of >70% and a peptide content of >50%.

[0008] The second objective of this invention is to provide a method for preparing earthworm egg yolk phospholipid peptides that lower blood lipids and improve memory, comprising the following: Defatted earthworm powder and egg yolk phospholipid protein powder are mixed and added to an enzymatic hydrolysis tank. Water is added and stirred evenly. Then, Bacillus subtilis protease and transglutaminase are added for enzymatic hydrolysis, enzyme inactivation, concentration, and vacuum low-temperature spray drying to obtain the earthworm egg yolk phospholipid peptide product.

[0009] In one embodiment, the defatted earthworm powder is a commercially available product or is obtained by defatting fresh earthworms.

[0010] In one embodiment, the defatted earthworm powder is specifically prepared by: washing fresh earthworms, freezing, crushing, sieving, and drying them to obtain dry earthworm powder; and then defatting the dry earthworm powder using subcritical methods to obtain defatted earthworm powder.

[0011] In one embodiment, the freezing conditions are: -20℃ to -40℃, and the freezing time is 4 to 8 hours.

[0012] In one embodiment, the sieve mesh size is 40 to 80 mesh.

[0013] In one embodiment, the drying is spray drying, with an inlet temperature of 180~200℃ and an outlet temperature of 80~95℃.

[0014] In one embodiment, the moisture content of the earthworm powder is ≤8%.

[0015] In one embodiment, the supercritical carbon dioxide degreasing temperature is 40~50℃ and the time is 120~180min.

[0016] In one embodiment, the egg yolk phospholipid protein powder is a commercially available product or a homemade product.

[0017] In one embodiment, the mass ratio of defatted earthworm powder to egg yolk phospholipid protein powder is 1~3:1; preferably 2:1.

[0018] In one embodiment, the mass ratio of defatted earthworm powder and egg yolk phospholipid protein powder to water is 1:10~15; preferably 1:10.

[0019] In one embodiment, the amount of Bacillus subtilis protease added is 2-3% of the total mass of the substrate (defatted earthworm powder + egg yolk phospholipid protein powder), and the enzyme activity is 300,000 U / g.

[0020] In one embodiment, the amount of transglutaminase added is 0.2-0.3% of the total mass of the substrate (defatted earthworm powder + egg yolk phospholipid protein powder), and the enzyme activity is 120 U / g.

[0021] In one embodiment, the enzymatic hydrolysis conditions are: 40~70℃, time 4~8h, and pH 7~9.

[0022] In one embodiment, the enzyme inactivation is performed by flash evaporation at 90-100°C for 10-20 seconds.

[0023] In one embodiment, the concentration is carried out in a double-effect evaporator to a concentration of 30-40 Brix.

[0024] In one embodiment, the first effect temperature of the double-effect concentrator is 70~75℃ and the vacuum degree is 0.07~0.08 MPa, and the second effect temperature is 60~65℃ and the vacuum degree is 0.06~0.07 MPa.

[0025] In one embodiment, the inlet temperature of the vacuum low-temperature spray dryer is 110~120℃, the outlet temperature is 50~65℃, and the vacuum degree is 0.05~0.06 MPa.

[0026] The third objective of this invention is to provide an application of the earthworm egg yolk phospholipid peptide described above in the preparation of functional foods.

[0027] In one embodiment, the functional food can be formulated into dosage forms such as powder, tablets, capsules, and oral liquids as needed.

[0028] In one embodiment, the powder is packaged directly into individual small bags, each containing 1-2 g of earthworm egg yolk phospholipid peptide, which can be directly mixed with water or added to foods such as milk and porridge.

[0029] In one embodiment, the tablets / capsules are prepared by mixing the finished product with excipients such as microcrystalline cellulose and lactose, compressing it into tablets or filling it into capsules, with each tablet / capsule containing 0.5 g of active ingredient, and taken 2-4 times daily.

[0030] In one embodiment, the oral liquid is prepared by dissolving the finished product in deionized water, adding an appropriate amount of sweetener (such as steviol glycosides), etc., and each 10 mL vial contains 1 g of active ingredient.

[0031] Beneficial effects: The earthworm egg yolk phospholipid peptide prepared by this invention, which has the functions of lowering blood lipids and improving memory, has the following advantages compared with the prior art: (1) This invention achieves the structural fusion of earthworm active peptide and egg yolk phospholipid peptide for the first time through transglutamine covalent linkage. Earthworm peptide can specifically reduce blood lipid indicators such as low-density lipoprotein cholesterol, while phosphatidylcholine in egg yolk phospholipid peptide can enhance neurotransmitter synthesis. The two form a "lipid-lowering-brain-boosting" synergistic mechanism, which solves the problem of limited efficacy of single components. Experimental verification shows that the dual efficacy is better than using alone or physical mixing. (2) Significantly improved stability: By enzymatically hydrolyzing earthworm and egg yolk phospholipids, the problem of poor uniformity and insufficient dispersion caused by simple physical mixing of the two peptides was solved; (3) Safety and wide applicability: The raw materials are earthworm, which is both food and medicine, and egg yolk phospholipid protein, which is safe and reliable; the preparation process does not use harmful chemical reagents, and the product can be used in health food and medicine. It is suitable for long-term use by people with hyperlipidemia accompanied by cognitive decline. Attached Figure Description

[0032] Figure 1 The images show the dispersion of the earthworm egg yolk phospholipid peptide prepared in Example 1 and the product prepared in Comparative Example 1 in water. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0034] The testing method involved in this invention: 1. Polypeptide content detection: Refer to the method in Appendix B of GB / T 22492-2008 Soybean Peptide Powder; 2. Protein content detection: Refer to GB 5009.5-2016 Determination of protein in food; The source of raw materials involved in this invention: The egg yolk phospholipid protein powder was purchased from Guangzhou Baiyunshan Hanfang Modern Pharmaceutical Co., Ltd., and the product complies with the Q / GZHF0067S-2026 standard for "Egg Yolk Phospholipid Protein".

[0035] Bacillus subtilis protease was purchased from Wuhan Xinhua Yang Biotechnology Co., Ltd., with an enzyme activity of 300,000 U / g.

[0036] The transglutaminase was purchased from Jiangsu Yiming Biotechnology Co., Ltd., with an enzyme activity of 120 U / g.

[0037] Example 1 A method for preparing earthworm egg yolk phospholipid peptides with lipid-lowering and memory-improving effects includes the following steps: (1) Pretreatment of earthworms: Fresh Eisenia fetidae was taken, washed and frozen at -20℃ for 5 h, crushed and passed through a 40-mesh sieve, and then spray-dried at an inlet temperature of 185℃ and an outlet temperature of 85℃ to obtain earthworm powder; the earthworm powder was sent into a supercritical carbon dioxide extraction tank, and the extraction temperature was controlled at 50℃ and the extraction time was 120 min, with a total solvent to raw material mass ratio of 5:1; after extraction, the residual solvent was removed by vacuum evaporation to obtain defatted earthworm powder; (2) Targeted compound enzymatic hydrolysis: Weigh the defatted earthworm powder and egg yolk phospholipid protein powder obtained in step (1) (defatted earthworm powder: egg yolk phospholipid protein powder mass ratio 2:1), put them into the enzymatic hydrolysis tank, add deionized water at a material-to-liquid mass ratio of 1:10, and stir evenly; add 2% Bacillus subtilis protease (enzyme activity 300000U / g) and 0.2% transglutaminase (enzyme activity 120 U / g) of the total mass of substrate (defatted earthworm powder + egg yolk phospholipid protein powder), and enzymatically hydrolyze for 5 h at 50℃ and pH 7.2; (3) Enzyme inactivation and concentration: The enzyme hydrolysate obtained in step (2) is flash-evaporized at 95°C for 18 s, and then sent to a double-effect evaporator. The first effect is 72°C and the vacuum degree is 0.075 MPa, and the second effect is 63°C and the vacuum degree is 0.065 MPa. The concentration is carried out to 40 Brix to obtain the concentrated solution. (4) Drying and finished product: The concentrate is subjected to vacuum low-temperature spray drying with an inlet temperature of 115℃, an outlet temperature of 58℃ and a vacuum degree of 0.055 MPa. After drying, it is passed through an 80-mesh sieve to obtain the earthworm egg yolk phospholipid peptide product.

[0038] Example 2 The difference from Example 1 is that the mass ratio of defatted earthworm powder to egg yolk phospholipid protein powder in the directional compound enzymatic hydrolysis step is 1:1, while other parameters and conditions are the same as in Example 1.

[0039] Example 3 The difference from Example 1 is that the mass ratio of defatted earthworm powder to egg yolk phospholipid protein powder in the directional compound enzymatic hydrolysis step is 3:1, while other parameters and conditions are the same as in Example 1.

[0040] Comparative Example 1 The only difference from Example 1 is that in step (2), the defatted earthworm powder and egg yolk phospholipid protein powder are enzymatically hydrolyzed, enzyme-inactivated and dried respectively, and the spray-dried products are mixed in a mass ratio of 2:1.

[0041] Comparative Example 2 The only difference from Example 1 is that the defatted earthworm powder and egg yolk phospholipid protein powder in step (2) are enzymatically hydrolyzed, enzyme-inactivated and dried to obtain the corresponding earthworm peptide and egg yolk phospholipid peptide products respectively.

[0042] Product Characterization 1. Basic indicators of the products obtained in Examples 1-3 were measured, and the results are shown in Table 1: Table 1. Proportion of protein and peptide content in earthworm egg yolk phospholipid peptides

[0043] 2. Dispersion stability test The finished products of Example 1 and Comparative Example 1 were dispersed in water at a material-to-water ratio of 5:100, and the dispersion stability was as follows. Figure 1 As shown in the results, the dispersibility of the product of Example 1 is significantly better than that of Comparative Example 1, with no obvious stratification or precipitation, thus solving the problem of unstable dispersion of traditional mixed peptides.

[0044] Application Examples Application of earthworm egg yolk phospholipid peptides in lowering blood lipids and improving memory Fifty-six SPF-grade male C57BL / 6 mice were randomly divided into four groups: a blank control group, a model group, a low-dose group (400 mg / kg·d) of Example 1, a high-dose group (800 mg / kg·d) of Example 1, a high-dose group (800 mg / kg·d) of Comparative Example 1, a high-dose group (800 mg / kg·d) of earthworm peptide (800 mg / kg·d) of Comparative Example 2, and a high-dose group (800 mg / kg·d) of egg yolk phospholipid peptide (800 mg / kg·d) of Comparative Example 2, with eight mice in each group. Except for the blank control group, the other groups were fed a 60% high-fat diet (containing 20% ​​lard and 1.25% cholesterol) to establish a hyperlipidemia model. After successful modeling, oral administration of medication was initiated. From day 1 to day 40, the blank control group and the model group were administered 0.2 ml of physiological saline by gavage. On day 38, new object recognition and Y-maze behavioral tests were performed. On day 41, all mice were fasted overnight and then sacrificed, and blood was collected for subsequent analysis.

[0045] Results Analysis 1. Measurement of mouse serum markers Table 2. Serum markers in mice

[0046] Note: Different lowercase Roman letters indicate significant differences between different groups for the same indicator. p <0.05).

[0047] 2. The results of the new object recognition experiment and the Y-maze experiment, which improved memory activity, are shown in Table 3. Table 3. Results of the new object recognition experiment and the Y-maze experiment

[0048] Note: A new object recognition index > 0 indicates that the mouse has the ability to recognize new objects; the higher the index, the stronger the recognition ability. A higher spontaneous alternation rate in the Y-maze indicates stronger spatial memory. Different lowercase Roman letters indicate significant differences between different groups for the same indicator. p <0.05).

[0049] Summary of results: (I) Conclusion on the efficacy of lowering blood lipids Compared with the blank control group, the model group showed significantly increased total cholesterol, total triglycerides, and low-density lipoprotein cholesterol, and significantly decreased high-density lipoprotein cholesterol, indicating that the hyperlipidemia mouse model was successfully established.

[0050] All treatment groups improved the blood lipid levels of hyperlipidemic mice to varying degrees. The high-dose group in Example 1 showed the best effect: compared with the model group, total cholesterol decreased by 33.30%, total triglycerides decreased by 39.81%, low-density lipoprotein cholesterol decreased by 47.15%, and high-density lipoprotein cholesterol showed a significant upward trend.

[0051] Example 1 (earthworm egg yolk phospholipid peptide prepared by targeted compound enzymatic hydrolysis) showed a better lipid-lowering effect than Comparative Example 1 (physically mixed product) and the groups using earthworm peptide or egg yolk phospholipid peptide alone (with significant differences). p The result was <0.05, confirming that the structural fusion of the two peptides achieved through the targeted compound enzymatic hydrolysis process can produce a significant synergistic effect in lowering blood lipids.

[0052] Example 1 Low-dose group (400 mg / kg) d) It can also effectively improve blood lipid indicators, showing good dose dependence, indicating that the product can play a role in lowering blood lipids within a certain dosage range.

[0053] (II) Conclusion on the efficacy of improving memory The model group mice had a negative new object recognition index and a significantly lower spontaneous alternation rate in the Y maze than the blank control group, indicating that a high-fat diet led to a significant decline in cognitive function and memory in the mice.

[0054] All treatment groups improved the cognitive function of mice: compared with the model group, the new object recognition index of the high-dose group in Example 1 turned from negative to positive and reached 35.28%, and the spontaneous alternation rate of the Y maze increased to 64.43%, which was close to the level of the blank control group, and the improvement effect was the most significant.

[0055] Example 1 showed a better memory improvement effect than Comparative Example 1 and the groups that used earthworm peptide or egg yolk phospholipid peptide alone (significant difference, p<0.05), demonstrating that earthworm peptide and egg yolk phospholipid peptide achieve functional synergy through targeted compound enzymatic hydrolysis, which can more efficiently improve the spatial learning and memory ability of mice.

[0056] Example 1 shows that the low-dose group was able to turn the new object recognition index from negative to positive, indicating that the product can start to improve memory function at a low dose. The high-dose group showed a further enhanced effect, presenting a clear dose-response relationship.

[0057] (III) Overall Conclusion The earthworm egg yolk phospholipid peptide prepared by this invention (especially Example 1 with an earthworm peptide and egg yolk phospholipid peptide mass ratio of 2:1) achieves synergistic effects of the two active ingredients through a targeted compound enzymatic hydrolysis process. Compared with physically mixed products and single-component products, it shows superior effects in lowering blood lipids and improving memory, and has high safety and good dose dependence. It provides a highly efficient and safe functional food ingredient for people with hyperlipidemia accompanied by cognitive decline.

[0058] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A type of earthworm egg yolk phospholipid peptide with lipid-lowering and memory-improving properties, characterized in that, The earthworm egg yolk phospholipid peptide was obtained by enzymatic hydrolysis of defatted earthworm powder and egg yolk phospholipid protein powder by Bacillus subtilis protease and transglutaminase.

2. The earthworm egg yolk phospholipid peptide according to claim 1, characterized in that, The earthworm egg yolk phospholipid peptide has a protein content of >70% and a peptide content of >50%.

3. The method for preparing earthworm egg yolk phospholipid peptides with lipid-lowering and memory-improving effects as described in claim 1 or 2, characterized in that, The method includes the following: Defatted earthworm powder and egg yolk phospholipid protein powder are mixed and added to an enzymatic hydrolysis tank. Water is added and stirred evenly. Then, Bacillus subtilis protease and transglutaminase are added for enzymatic hydrolysis, enzyme inactivation, concentration, and vacuum low-temperature spray drying to obtain the earthworm egg yolk phospholipid peptide product.

4. The method according to claim 1, characterized in that, The defatted earthworm powder is a commercially available product or is obtained by defatting fresh earthworms.

5. The method according to claim 1, characterized in that, The mass ratio of defatted earthworm powder to egg yolk phospholipid protein powder is 1~3:

1.

6. The method according to claim 1, characterized in that, The mass ratio of defatted earthworm powder and egg yolk phospholipid protein powder to water is 1:10~15.

7. The method according to claim 1, characterized in that, The amount of Bacillus subtilis protease added is 2-3% of the total substrate mass, and the enzyme activity is 300,000 U / g; the substrate is defatted earthworm powder and egg yolk phospholipid protein powder.

8. The method according to claim 1, characterized in that, The transglutaminase is added at a rate of 0.2-0.3% of the total substrate mass, and has an enzyme activity of 120 U / g; the substrate is defatted earthworm powder and egg yolk phospholipid protein powder.

9. The method according to claim 1, characterized in that, The enzymatic hydrolysis conditions are: 40~70℃, time 4~8h, pH 7~9.

10. The application of the earthworm egg yolk phospholipid peptide according to claim 9 in the preparation of functional foods with lipid-lowering and memory-improving effects.

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