阅读说明：本技术 一种富集深海鱼油中epa和dha的方法 (Method for enriching EPA and DHA in deep sea fish oil ) 是由 曹茜 廖琳萌 包清彬 袁永俊 刘庆庆 潘亚瑜 于 2020-05-22 设计创作，主要内容包括：本发明提供了一种富集深海鱼油中EPA和DHA的方法,包括如下步骤:(1)将sn-1,3位选择性脂肪酶溶解在Tris-HCl缓冲液中,再加入酰基迁移促进剂二氧化硅颗粒；(2)添加深海鱼油,反应过程中不断磁力搅拌；(3)添加氢氧化钾溶液和正己烷,充分震荡后离心,转移收集上层溶液并利用氮吹去除正己烷,除去正己烷后的余液即为富含EPA和DHA的脂质。其优点在于,在利用脂肪酶与酰基迁移促进剂二氧化硅的协同催化后,鱼油中EPA的富集得到显著改善,甚至在达到最高含量后不再下降而进入平台期,由于DHA较难被脂肪酶水解,酶催化富集DHA一般存在平台期,因此,当EPA保持高含量的时间延长之后,就非常容易选择到合适的反应时间,使得EPA和DHA实现同步富集。(The invention provides a method for enriching EPA and DHA in deep sea fish oil, which comprises the following steps of (1) dissolving sn-1,3 site selective lipase in Tris-HCl buffer solution, and then adding acyl migration promoter silica particles; (2) adding deep sea fish oil, and continuously stirring by magnetic force in the reaction process; (3) adding potassium hydroxide solution and n-hexane, shaking fully, centrifuging, transferring and collecting upper layer solution, removing n-hexane by nitrogen blowing, and obtaining residual liquid which is lipid rich in EPA and DHA after n-hexane is removed. The method has the advantages that after the synergistic catalysis of the lipase and the acyl migration promoter silicon dioxide is utilized, the enrichment of EPA in the fish oil is obviously improved, even the EPA does not decline any more after reaching the highest content and enters the plateau phase, and as DHA is difficult to be hydrolyzed by the lipase, the DHA enriched by the enzyme catalysis generally exists in the plateau phase, so that after the time for keeping the high content of the EPA is prolonged, the proper reaction time is very easy to select, and the EPA and the DHA are synchronously enriched.)

1. A method for enriching EPA and DHA in deep sea fish oil is characterized by comprising the following steps:

(1) dissolving sn-1, 3-site selective lipase in Tris-HCl buffer solution, adding acyl migration promoter silica particles, and magnetically stirring to obtain a dispersion containing lipase and silica;

(2) adding deep sea fish oil into the dispersion prepared in the step (1), putting the mixture of the catalyst and the substrate into a reaction vessel, filling nitrogen into the reaction vessel, sealing, and continuously stirring by magnetic force in the reaction process;

(3) after the reaction is finished, adding a potassium hydroxide solution and n-hexane into the reaction solution, fully shaking, centrifuging, transferring and collecting an upper layer solution, removing the n-hexane by using nitrogen blowing, and obtaining a residual liquid which is the lipid rich in EPA and DHA after the n-hexane is removed.

2. The method for enriching EPA and DHA in deep sea fish oil according to claim 1, wherein the sn-1,3 position selective lipase in the step (1) is Rhizopus oryzae lipase.

3. The method for enriching EPA and DHA in deep sea fish oil according to claim 1, wherein in step (1), the sn-1,3 site selective lipase is 3 parts by weight, the concentration of Tris-HCl buffer is 0.2mol/L, the pH is 7.0, the parts by weight is 50, the parts by weight of silica is 5, the particle size of silica is 1nm to 100 μm, and the magnetic stirring time is 5 min.

4. The method for enriching EPA and DHA in deep sea fish oil according to claim 1, wherein the weight part of the deep sea fish oil in the step (2) is 100, the reaction temperature is 40 ℃, and the reaction time is 1-10 h.

5. The method for enriching EPA and DHA in deep sea fish oil according to claim 1, wherein the concentration of the potassium hydroxide solution in step (3) is 2mol/L, the weight part of the potassium hydroxide solution is 400, the weight part of n-hexane is 300, and the centrifugation condition is 5000g for 30 min.

Technical Field

The invention belongs to the technical field of methods for enriching specific fatty acid in grease, and particularly relates to a method for enriching EPA and DHA in deep sea fish oil.

Background

Many studies have confirmed that two kinds of omega-3 long-chain polyunsaturated fatty acids EPA and DHA have many benefits for human health, and as the market for functional foods and health products related thereto is continuously expanding, there is an increasing demand for oils rich in EPA and DHA. However, the quality and quantity of deep sea fish oil, which is a main source of EPA and DHA, are drastically reduced, and thus the enrichment of EPA and DHA in deep sea fish oil is receiving attention. Among the enrichment methods, lipase catalysis is the most promising method because of mild conditions and environmental friendliness. The lipase is generally selective for fatty acid, and is not easy to hydrolyze long-chain polyunsaturated fatty acid EPA and DHA to keep on a glycerol skeleton, while other (short-chain and less unsaturated bond) fatty acid is hydrolyzed, so that the EPA and DHA in the glycerol ester is enriched. Most lipases have sn-1,3 position selectivity due to the fact that the steric hindrance of sn-2 position of glyceride is larger than that of sn-1,3 position, but the position distribution of EPA and DHA in fish oil is significantly different, EPA is mainly located at sn-1,3 position and DHA is mainly located at sn-2 position, which makes the risk of EPA being hydrolyzed larger than DHA; on the other hand, EPA itself is more easily enzymatically hydrolyzed than DHA because EPA has a shorter carbon chain than DHA and a smaller number of unsaturated bonds than DHA. The two reasons cause that the difficulty of enriching the EPA is greater than that of the DHA, so that the EPA and the DHA are not enriched synchronously, when the DHA is enriched to the maximum concentration, the content of the EPA starts to be reduced even lower than the concentration before enrichment, or in order to make the content of the EPA considerable, the reaction can be stopped in advance so that the DHA cannot reach the maximum concentration. Therefore, a new method is needed to improve the problems of the prior art in enzymatically enriching EPA and DHA.

Disclosure of Invention

In order to solve the technical problem that EPA and DHA in deep sea fish oil are difficult to be enriched at high concentration at the same time in the prior art, the invention provides a method for enriching EPA and DHA in deep sea fish oil, which aims to improve the enrichment condition of EPA and synchronize the enrichment of EPA and DHA, thereby simultaneously obtaining high-concentration EPA and DHA.

In order to achieve the purpose, the technical scheme disclosed by the invention is as follows: the invention provides a method for enriching EPA and DHA in deep sea fish oil, which comprises the following steps:

(1) dissolving sn-1, 3-site selective lipase in Tris-HCl buffer solution, adding acyl migration promoter silica particles, and magnetically stirring to obtain a dispersion containing lipase and silica;

(2) adding deep sea fish oil into the dispersion prepared in the step (1), putting the mixture of the catalyst and the substrate into a reaction vessel, filling nitrogen into the reaction vessel, sealing, and continuously stirring by magnetic force in the reaction process;

(3) after the reaction is finished, adding a potassium hydroxide solution and n-hexane into the reaction solution, fully shaking, centrifuging, transferring and collecting an upper layer solution, removing the n-hexane by using nitrogen blowing, and obtaining a residual liquid which is the lipid rich in EPA and DHA after the n-hexane is removed.

The EPA has relatively low sn-2 site content in deep sea fish oil and has large enrichment potential. The Sn-1, 3-site lipase can generate a primary product 1, 2-diglyceride and a secondary product 2-monoglyceride after hydrolyzing triglyceride, if an acyl migration promoter exists in the system, the two products are respectively isomerized into 1, 3-diglyceride and 1-monoglyceride, and then can be continuously hydrolyzed by the Sn-1, 3-site selective lipase, so that other (short-chain and few unsaturated bonds) fatty acids originally positioned at the Sn-2 site can be hydrolyzed, and the enrichment potential of the Sn-2 site EPA is favorably realized. The method utilizes silicon dioxide to achieve the purpose of promoting the acyl migration at the sn-2 position.

Further, the sn-1,3 position selective lipase in the step (1) is rhizopus oryzae lipase.

Further, in the step (1), the sn-1, 3-position selective lipase is 3 parts by weight, the concentration of a Tris-HCl buffer solution is 0.2mol/L, the pH value is 7.0, the weight part is 50, the weight part of silicon dioxide is 5, the particle size of the silicon dioxide is 1nm-100 mu m, and the magnetic stirring time is 5 min.

Further, the weight portion of the deep sea fish oil in the step (2) is 100, the reaction temperature is 40 ℃, and the reaction time is 1-10 h.

Further, in the step (3), the concentration of the potassium hydroxide solution is 2mol/L, the weight part of the potassium hydroxide solution is 400, the weight part of n-hexane is 300, and the centrifugation condition is 5000g for 30 min.

The invention has the beneficial effects that: the method creatively combines the two phenomena that the hydrolysate of the sn-1,3 position selective lipase is 1, 2-diglyceride and 2-monoglyceride and the sn-2 position acyl of glyceride is easy to migrate, and utilizes the concerted catalysis of the enzyme and the acyl migration promoter to improve the problems existing when the sn-1,3 position selective lipase is used for enriching EPA and DHA in the deep sea fish oil. After the synergistic catalysis of the enzyme and the acyl migration accelerator silicon dioxide is utilized, the enrichment of EPA in fish oil is obviously improved, even the EPA does not drop after reaching the highest content and enters a platform stage, and because DHA is difficult to be hydrolyzed by lipase, the DHA enriched by the enzyme catalysis generally exists in the platform stage, so that after the time of keeping high content of EPA is prolonged, proper reaction time is very easy to select, and the EPA and DHA are synchronously enriched.

Drawings

FIG. 1 shows the EPA content change when Rhizopus oryzae lipase is synergistically catalyzed by 45 μm silica;

FIG. 2 shows the EPA content change when Rhizopus oryzae lipase catalyzes independently;

FIG. 3 is a graph showing the change in the ratio of EPA to DHA in Rhizopus Oryzae Lipase (ROL) alone or in combination with 45 μm silica;

FIG. 4 shows the EPA content change of Rhizopus oryzae lipase in combination with 10-20nm silica;

FIG. 5 shows the variation of the EPA to DHA ratio of Rhizopus Oryzae Lipase (ROL) alone or in combination with 10-20nm silica.

Detailed Description

The present invention is described in further detail below with reference to specific examples.