Preparation method of enzyme modified sea horse oil and application thereof in reducing blood lipid

Abstract

The application discloses a preparation method of enzyme modified hippocampus oil, which comprises the following steps: 1) freezing and crushing fresh inflated hippocampus; 2) adding ethanol to stir and extract, filtering, and taking lipid-containing ethanol extract; 3) performing vacuum evaporation on the lipid-containing ethanol extract to obtain inflated hippocampus oil; 4) mixing the inflated hippocampus oil, glycerol and a lipase with sn-1,3 site selectivity to perform enzymolysis; and 5) mixing the enzymolyzed inflated hippocampus oil with water, centrifuging to take the upper oil layer, and obtaining enzyme modified hippocampus oil. In the enzyme modified hippocampus oil, the content of diglyceride is more than 50%. The application further discloses application of the enzyme modified hippocampus oil in preparation of a blood lipid reducing product. The enzyme modified hippocampus oil can significantly enhance the comprehensive physiological effect of the hippocampus oil in reducing triglyceride and cholesterol.

Description

Technical Field

[0001] This invention relates to the field of marine biological agents, specifically to a method for preparing seahorse oil. Background Technology

[0002] Existing lipid-lowering products based on vegetable oil-derived diacylglycerol (DAG) have inherent limitations in their mechanism of action. They primarily rely on the unique metabolic pathway of 1,3-DAG, effectively reducing postprandial blood lipids by reducing the resynthesis of triglycerides (TG) and the assembly of chylomicrons in the intestine. However, the functional components of existing DAG oils are relatively singular, and their action is mainly concentrated on the postprandial TG-related pathway. Their comprehensive regulatory capacity on cholesterol metabolism-related processes (such as absorption, transport, and conversion) is limited, making it difficult to achieve a comprehensive improvement in blood lipid indicators. Furthermore, the body may adapt to long-term single DAG intervention through compensatory mechanisms such as upregulating endogenous lipid synthesis, causing the lipid-lowering effect to decline or plateau over time.

[0003] Therefore, there is an urgent need to develop a new type of oil raw material that can contain multiple lipid active components and can be prepared by a green and mild process, so as to enhance the comprehensive regulatory capacity and long-term application value of lipid-lowering oils.

[0004] Seahorse oil is rich in various bioactive substances. In addition to triglycerides and free fatty acids, it is characterized by high levels of glycerophospholipids, sphingomyelins, glycolipids, long-chain alkaloids, and steroidal compounds, earning it the reputation of "Southern Ginseng." The bloated seahorse, as the main seahorse species currently farmed on a large scale globally, can provide a stable and high-quality source of raw materials for seahorse oil.

[0005] In their article "Analysis of Lipids and Fatty Acid Composition of Abdominal Seahorse" (Food Research and Development, Vol. 45, No. 17, pp. 178-183, September 2024), Ning Yifei et al. pointed out that lipid components such as phospholipids, sterols, and n-3 polyunsaturated fatty acids (PUFAs) such as DHA and EPA could be detected in abdominal seahorse oil, and reported on the analysis of its lipid composition and fatty acid composition.

[0006] However, there are no reports on diglycerides derived from seahorse oil in the existing technology. Summary of the Invention

[0007] The technical problem to be solved by this invention is: addressing the issues of existing DAG oils having single functional components, relatively concentrated target sites, and insufficient comprehensive regulatory capabilities, this invention provides a method for preparing structured oils using seahorse oil as raw material under mild conditions. This method ensures that the resulting oil is rich in diglycerides while minimizing the damage or loss of natural polar lipids and steroidal components, thereby obtaining modified seahorse oil with multiple lipid active components.

[0008] Seahorse oil contains abundant free fatty acids (FFA) and triglycerides (TG). Its high FFA content and unique TG structure provide ideal reaction substrates and great potential for enzymatic modification.

[0009] Based on this characteristic, this invention uses expanded seahorse oil as raw material and prepares diglycerides rich in DHA and EPA through lipase-catalyzed hydrolysis and esterification reactions. At the same time, it retains a variety of active components such as naturally occurring glycerophospholipids, sphingomyelins, glycolipids and sterols, and finally obtains an enzymatically modified seahorse oil with multiple lipid synergistic effects.

[0010] To solve the above-mentioned technical problems, the present invention provides a method for preparing seahorse oil, using expanded seahorse oil as the base oil, and modifying its triglyceride structure by hydrolysis and esterification reaction catalyzed by lipase to prepare structured seahorse oil with increased diglyceride content; wherein, the lipase is preferably a lipase with sn-1,3 site selectivity to increase the proportion of 1,3-diacylglycerol in the product.

[0011] This invention discloses a method for preparing enzymatically modified seahorse oil, comprising the following steps:

[0012] 1) Fresh, bloated seahorse, frozen and pulverized;

[0013] 2) Add ethanol, stir and extract, filter, and take the lipid-containing ethanol extract;

[0014] 3) Vacuum evaporation of the lipid-containing ethanol extract yields bloated seahorse oil;

[0015] 4) Mix the expanded seahorse oil, glycerol, and lipase with sn-1,3 site selectivity, and enzymatically hydrolyze them;

[0016] 5) After mixing the enzymatically hydrolyzed bloated seahorse oil with water, centrifuge to obtain the upper oil layer, thus obtaining the enzymatically modified seahorse oil.

[0017] Furthermore, in the above-mentioned method for preparing enzymatically modified seahorse oil, lipases with sn-1,3 site selectivity include Candida antarcticis lipase B, Rhizopus miltiorrhiza lipase, and Thermophilus sparsely cottony lipase.

[0018] Furthermore, in the above-mentioned method for preparing enzymatically modified seahorse oil, the mass ratio of bloated seahorse oil to glycerol is 10:1.

[0019] Furthermore, in the above-mentioned method for preparing enzymatically modified seahorse oil, the amount of lipase added is 1% of the total mass of the expanded seahorse oil and glycerol.

[0020] Furthermore, in the above-mentioned method for preparing enzymatically modified seahorse oil, the enzymatic hydrolysis reaction conditions in step 4) are as follows: reaction at a temperature of 55±2℃ for 6 hours.

[0021] The present invention also discloses an enzyme-modified seahorse oil prepared according to the above-described method for preparing enzyme-modified seahorse oil.

[0022] Furthermore, the diglyceride content in the above-mentioned enzymatically modified seahorse oil is above 50%.

[0023] This invention also discloses the application of the above-mentioned enzymatically modified seahorse oil in the preparation of lipid-lowering products.

[0024] Furthermore, in the above-mentioned application of enzymatically modified seahorse oil, the lipid-lowering product is taken orally.

[0025] Furthermore, in the above-mentioned application of enzymatically modified seahorse oil, the proportion of seahorse oil diglycerides in the total food intake is 1%.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention employs hydrolysis and esterification reactions catalyzed by lipase, and achieves the following by controlling the process conditions such as the ratio of reactant solution, temperature, and reaction time: (1) increasing the content of diglycerides (DAG) in the base oil and introducing or enriching n-3 polyunsaturated fatty acids such as DHA and / or EPA; (2) relatively reducing the loss of natural components such as polar lipids and steroids in seahorse oil under mild enzymatic conditions and relatively mild post-treatment.

[0028] This invention utilizes enzymatic modification to not only reduce the content of free fatty acids and increase the content of DAG, but also to achieve a certain degree of enrichment of DHA and / or EPA within the DAG molecules, thus giving the resulting oil a combination of "structured DAG + n-3 PUFA" characteristics. Furthermore, phospholipids and sterols are retained, achieving a synergistic effect.

[0029] Existing research indicates that phospholipids and n-3 PUFAs may play a regulatory role in lipid absorption, lipid transport, and endogenous lipid synthesis. The modified oils obtained in this invention can simultaneously contain DAG, DHA / EPA, and naturally occurring phospholipids and sterols found in seahorse oil. Therefore, the combination of these multiple lipid components provides a material basis for achieving multi-pathway synergistic regulation of lipid metabolism, contributing to enhancing the comprehensive application potential of oil products in blood lipid health management.

[0030] This invention selects the bloated seahorse breed that has been commercially farmed on a large scale, ensuring the sustainability, stability and traceability of raw materials, and overcoming the industrialization bottleneck of scarce wild seahorse resources and uneven quality.

[0031] This invention provides an enzymatic preparation process using expanded seahorse oil as raw material. The modified oil obtained by this process incorporates multiple active ingredients, which can significantly enhance the comprehensive physiological effects of seahorse oil in lowering triglycerides and cholesterol through multi-pathway synergistic action, providing a new solution for developing a new generation of highly efficient and stable lipid-lowering functional oils. Attached Figure Description

[0032] Figure 1 This represents the weight measurement results of a mouse obesity model induced by diet over 6 weeks.

[0033] Figure 2 This represents the weight analysis results of a mouse obesity model induced by diet over 6 weeks.

[0034] Figure 3 The values ​​represent the body weight measurements of mice in each group during weeks 6–12 of the experiment.

[0035] Figure 4 This represents the weight analysis results of mice in each group during weeks 6 to 12 of the experiment.

[0036] Figure 5 The results represent the body fat percentage analysis of mice in each group during weeks 6–12 of the experiment.

[0037] Figure 6 The results represent the serum triglyceride analysis of mice in each group during weeks 6–12 of the experiment.

[0038] Figure 7 The results represent the serum total cholesterol analysis of mice in each group during weeks 6 to 12 of the experiment. Detailed Implementation

[0039] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0040] Example 1: Preparation of Abdominal-Expanding Seahorse Oil

[0041] Fresh, bloated seahorses were rapidly frozen in liquid nitrogen until hard and brittle, then ground into a powder using a mortar and pestle under liquid nitrogen conditions. After grinding, food-grade ethanol was added and stirred for 24 hours for extraction. After extraction, the mixture was poured into a Buchner funnel lined with medium-speed qualitative filter paper, and vacuum filtration was performed, allowing the lipid-containing ethanol extract to pass through the filter paper into a filtration flask. Solid residue was retained on the filter paper, thus achieving solid-liquid separation. The lipid-containing ethanol extract was concentrated under reduced pressure using a rotary evaporator to obtain bloated seahorse oil. The concentration conditions were: temperature 50℃, vacuum degree -0.08MPa, and rotation speed 100rpm.

[0042] Example 2: Enzymatic hydrolysis of distended seahorse oil

[0043] Swelling seahorse oil, glycerol, and lipases with sn-1,3 site selectivity were mixed. The mass ratio of swelling seahorse oil to glycerol was 10:1, and the amount of lipase added was 1% of the total mass of swelling seahorse oil and glycerol. The lipases with sn-1,3 site selectivity were complex lipases, including immobilized lipases in a mass ratio of 5:3:1: *Candida antarcticis* lipase B (Chiralzyme IM-100, Shanghai Kangdian Biotechnology Co., Ltd.), *Rhizomucormiehei* immobilized lipase (Lipozyme RM IM, Novozymes, Denmark), and *Thermomyces lanuginosus* lipase (Lipozyme TL 100 L, Novozymes, Denmark).

[0044] After mixing the expanded seahorse oil, glycerol and compound lipase, the mixture was enzymatically hydrolyzed at 55±2℃ for 6 hours to obtain the reactants.

[0045] After the reaction material was mixed with water at a mass ratio of 2:1, it was centrifuged at 8000 r / min for 15 min, and the upper oil sample was taken to obtain the enzymatic hydrolysis product of the bloated seahorse oil.

[0046] Example 3: Determination of lipid component content

[0047] Seahorse oil is naturally rich in various active ingredients such as sterols, phospholipids, and n-3 polyunsaturated fatty acids, which together provide a multi-layered material basis for its lipid-lowering effect.

[0048] The contents of each lipid component in the enzymatically hydrolyzed seahorse oil prepared in Example 2 were determined by liquid chromatography (Zhong Nanjing et al., Liquid Chromatography Analysis of Glycerides, Modern Food Science and Technology, Vol. 28, No. 1, 123-126, 2012) and HPLC-ELSD detection analysis (Zhao Xinnan et al., HPLC-ELSD Detection and Analysis of Five Phospholipids in Aquatic Products, China Fisheries Quality and Standards, Vol. 12, No. 4, 10-17, July 2022).

[0049] The measurement results are shown in Table 1.

[0050] Table 1. Content of lipid components in expanded seahorse oil before and after enzymatic hydrolysis Triglycerides diglycerides monoglycerides Free fatty acids Phospholipids lysophospholipids steroids Abdominal swelling seahorse oil 25.4% 1.08% 0.82% 43.3% 18.5% 1.2% 7.3% Enzymatic hydrolysis of seahorse oil 6.2% 56.9% 1.6% 6.8% 14.1% 5.7% 6.0%

[0051] As shown in Table 1, the content of free fatty acids in the bloated seahorse oil itself is relatively high. After enzymatic hydrolysis, the content of diglycerides and lysophospholipids increased significantly, while the content of free fatty acids decreased significantly.

[0052] Example 4: Effects of enzymatic hydrolysis of seahorse oil on blood lipids in high-fat diet-induced obese mice

[0053] Low-fat feed, high-fat feed, and oil substitute feed were all provided by Nantong Trofi Feed Technology Co., Ltd. The oil substitute feed used enzymatically modified seahorse oil obtained in Example 2 of this invention, bloated seahorse oil obtained in Example 1, and commercially available vegetable oil-derived diglycerides with corresponding diglyceride content in the enzymatically hydrolyzed bloated seahorse oil to replace part of the oil in the high-fat feed.

[0054] Six-week-old male C57BL / 6J mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After one week of acclimatization feeding, they were randomly divided into two groups according to their body weight: a low-fat group (n=6) and a high-fat group (n=24). The mice were fed a low-fat diet daily, while the high-fat group was fed a high-fat diet daily. After six weeks of feeding, the high-fat group was further randomly divided into four groups of six mice each. The feed formulations are shown in Table 2. The protein / carbohydrate / fat energy ratio of the low-fat diet was 21.03% / 67.32% / 11.65%. The protein / carbohydrate / fat energy ratio of the high-fat diet was 16.70% / 37.02% / 46.28%.

[0055] Table 2. Feed Formulation formula Low-fat feed (g / kg) High-fat feed (g / kg) Casein 200 200 sucrose 0 0 fructose 0 250 corn starch 649.98 199.98 maltodextrin 0 0 Cellulose 50 50 minerals 35 35 Vitamins 10 10 L-methionine 3 3 Choline bitartrate 2 2 TBHQ 0.02 0.02 corn oil 50 50 lard 0 200

[0056] The specific groupings are as follows.

[0057] Low-fat control group: Mice were fed a low-fat diet for 12 consecutive weeks.

[0058] High-fat model group: Mice were fed a high-fat diet for 12 consecutive weeks.

[0059] Enzyme-modified seahorse oil group: Mice were first fed a high-fat diet for 6 weeks to establish an obese mouse model; from week 7 onwards, the enzyme-modified seahorse oil obtained in Example 2 was used to replace part of the fat in the high-fat diet, and the mice were fed continuously for 6 weeks. The amount of enzyme-modified seahorse oil added was 17.6 g / kg, and based on this, the final concentration of seahorse oil diglycerides in the diet was 10 g / kg.

[0060] Obese mouse model: Mice were first fed a high-fat diet for 6 weeks to establish an obese mouse model; from week 7 onwards, the fat source in the high-fat diet was partially replaced by obese seahorse oil for 6 consecutive weeks. The amount of obese seahorse oil (unmodified) added was 17.6 g / kg, which was the same as the dosage in the enzymatically modified seahorse oil group.

[0061] Plant-derived diglycerides group: Mice were first fed a high-fat diet for 6 weeks to establish an obese mouse model. From week 7 onwards, a portion of the oil source in the high-fat diet was replaced by commercially available plant-derived diglyceride oil (Jinlongyu Fengyitang Xinqingyi Diglyceride Edible Oil, 50% DAG content, Yihai Kerry Jinlongyu Food Group Co., Ltd.), and this was continued for 6 weeks. The final addition amount of plant-derived diglycerides in the diet was 10 g / kg, consistent with the DAG addition amount in the enzymatically modified seahorse oil group.

[0062] Starting from the beginning of the experiment (week 0), the weight of the mice was measured every 2 weeks until the end of the experiment (week 12).

[0063] After the experiment, the mice were fasted for 12 hours, and their total weight was measured. The mice were then euthanized by decapitation, and the following adipose tissues were quickly separated: inguinal subcutaneous fat, epididymal fat, perirenal fat, and brown fat in the scapula. The body fat percentage of the mice was calculated based on their weight.

[0064] The method for calculating body fat percentage is as follows:

[0065] Body fat percentage = Total wet weight of collected fat ÷ Mouse body weight × 100% (Equation 1)

[0066] Blood was collected, centrifuged at 3500 rpm for 15 min, serum was separated, and stored at -80℃ for the detection of triglycerides (TG) and total cholesterol (TC).

[0067] Serum triglyceride (TG) levels were measured using a triglyceride (TG) assay kit (single-reagent GPO-PAP method) (ELISA reader and biochemical analyzer) (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., catalog number A110-1-1). All procedures were strictly performed in accordance with the kit instructions.

[0068] Total cholesterol (TC) levels were measured using a Total Cholesterol (TCH / T-CHO) Assay Kit (Single Reagent GPO-PAP Method) (ELISA reader and biochemical analyzer) (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., Catalog No. A111-1-1). All operations were strictly performed in accordance with the kit instructions.

[0069] Statistical analysis was performed using GraphPad Prism 8. Differences between two groups were analyzed using Student's t-test. Multiple comparisons among groups were performed using one-way ANOVA and Tukey's post-hoc test.

[0070] The results of weight measurement in a 6-week diet-induced obesity model of mice are shown below. Figure 1 and Figure 2 All values ​​are expressed as mean ± SD. For example... Figure 1 As shown, the weight of mice in the obesity modeling group exceeded that of mice in the low-fat control group by 22.5%. Figure 2 As shown, different letters above the data bars indicate significant differences in body weight between the two groups of mice (P < 0.0001).

[0071] The successfully modeled obese mice were randomly assigned to the high-fat model group, the enzymatically modified seahorse oil group, the bloated seahorse oil group, and the plant diglyceride group according to their body weight for subsequent experiments.

[0072] The body weight measurements of mice in each group during weeks 6–12 of the experiment are as follows: Figure 3 and Figure 4 As shown, body fat percentage, serum triglyceride, and total cholesterol levels are as follows: Figure 5 , Figure 6 and Figure 7 As shown in the figure. Different letters above the data bars indicate significant differences between groups (P<0.05), while the same letter indicates no significant differences between groups.

[0073] After 6 weeks of dietary intervention, compared with the high-fat model group, the enzymatically modified seahorse oil group showed significant reductions in body weight, body fat percentage, serum triglycerides, and total cholesterol levels. Compared with the expanded seahorse oil group and the plant diglyceride group, the enzymatically modified seahorse oil group also showed varying degrees of reduction in body weight, body fat percentage, serum triglycerides, and serum total cholesterol.

[0074] The above results demonstrate that the enzymatically modified, expanded seahorse oil provided in the application examples of this invention has a significant effect on reducing mouse body weight, body fat percentage, serum triglycerides, and serum total cholesterol, and its effect is superior to expanded seahorse oil and vegetable diglycerides. This may be because enzymatically modified expanded seahorse oil is rich in diglycerides, phospholipids, lysophospholipids, n-3 polyunsaturated fatty acids, etc., and integrates multiple active ingredients. Through multi-pathway synergistic action, it significantly enhances the comprehensive physiological efficacy of seahorse oil in reducing triglycerides and cholesterol.

Claims

1. A method for preparing enzymatically modified seahorse oil, comprising the following steps: 1) Fresh, bloated seahorse, frozen and pulverized; 2) Add ethanol, stir and extract, filter, and take the lipid-containing ethanol extract; 3) Vacuum evaporation of the lipid-containing ethanol extract yields bloated seahorse oil; 4) Mix the expanded seahorse oil, glycerol, and lipase with sn-1,3 site selectivity, and enzymatically hydrolyze them; 5) After mixing the enzymatically hydrolyzed bloated seahorse oil with water, centrifuge to obtain the upper oil layer, thus obtaining the enzymatically modified seahorse oil.

2. The method for preparing enzymatically modified seahorse oil as described in claim 1, characterized in that: In step 4), lipases with sn-1,3 site selectivity include Candida antarcticis lipase B, Rhizopus miltiorrhiza lipase, and Thermophilus sparsely cottony lipase.

3. The method for preparing enzymatically modified seahorse oil as described in claim 1, characterized in that: In step 4), the mass ratio of the expanded seahorse oil to glycerin is 10:

1.

4. The method for preparing enzymatically modified seahorse oil as described in claim 1, characterized in that: In step 4), the amount of lipase added is 1% of the total mass of the expanded seahorse oil and glycerol.

5. The method for preparing enzymatically modified seahorse oil according to any one of claims 1 to 4, characterized in that: The enzymatic hydrolysis reaction conditions for step 4) are: reaction at 55±2℃ for 6 hours.

6. Enzyme-modified seahorse oil prepared by the method according to any one of claims 1 to 5.

7. The enzymatically modified seahorse oil according to claim 6, characterized in that: The diglyceride content in enzymatically modified seahorse oil is over 50%.

8. The application of the enzymatically modified seahorse oil according to claim 6 or 7 in the preparation of lipid-lowering products.

9. The application as described in claim 8, characterized in that: The lipid-lowering product is to be taken orally.

10. The application as described in claim 9, characterized in that: Seahorse oil diglycerides account for 1% of total food intake.

Images