CN105506008A - Preparation method for monoglyceride rich in omega-3 fatty acid - Google Patents

Abstract

The invention relates to a preparation method for monoglyceride rich in omega-3 fatty acid. The preparation method comprises the following steps: taking an organic solvent as an extraction agent to extract algae oil; adding hexane and silica gel into a chromatographic column, and then adding the algae oil into the chromatographic column to obtain triacylglycerol; mixing triacylglycerol and ethanol, and meanwhile adding immobilized lipase A-CALA and a little water for reaction to obtain a monoglyceride crude product rich in omega-3 fatty acid; mixing the monoglyceride crude product with food-grade n-hexane according to proportion, conducting extraction for 3 times, collecting an ethanol phase, and conducting rotary evaporation to remove ethanol and water to obtain purified monoglyceride rich in omega-3 fatty acid. Through the adoption of the process condition, the total content of omega-3 fatty acid in monoglyceride after enzymic catalytic reaction can reach 90% or above.

Description

A preparation method rich in omega-3 fatty acid monoglyceride

technical field

The invention relates to a preparation method of monoglyceride rich in omega-3 fatty acid.

Background technique

Monoacylglycerols (MAGs) are a class of non-polar substances with hydrophobic groups and hydrophilic groups, and their production accounts for about 50% of the total production of emulsifiers in China. As emulsifiers, MAGs have been widely used in food, pharmaceutical, cosmetic and other fields. The development of monoglycerides rich in omega-3 fatty acids as emulsifiers or additives in the food field is one of the hot directions in the field of research and development of modified oils. As we all know, omega-3 fatty acids (such as SDA (18:4n-3), EPA (20:5n-3) and DHA (22:6n-3) are essential fatty acids for the human body. Biological functions in immunity, anti-tumor, prevention of stroke and gout, etc. At present, the production technology of monoglyceride is mainly chemical catalysis and enzyme catalysis. Although chemical catalysis is currently the main production of monoglyceride with saturated fatty acids The method has the advantages of low cost and short time, but it is easy to cause the oxidation of oil at high temperature, and then produce by-products, discoloration and other phenomena. The production of monoglyceride by chemical method has the disadvantage of high melting point, which directly affects the production of monoglyceride. The application of enzyme-catalyzed preparation of monoglyceride rich in omega-3 fatty acids has the advantages of high efficiency, specificity, mild reaction conditions, less by-products, and repeated use of enzymes. It will be the preferred technology for industrial production. Europe, America, Japan, etc. Developed countries have always been in the leading position in research on oil modification and its industrial application (CamilaA.Palla et al , 2014; Aditya et al , 2014; LalitMohanNegi et al , 2014). In recent years, foreign countries have developed monoglycerides rich in omega-3 fatty acids Lipid research has entered the pilot test level, such as Alicia.R et al. (Alicia.R etal , 2012) who used Novozymes 435 enzyme alcoholysis herring liver fish oil preparation can reach 20-25% (wt% = 60-75mol %) of 2-monoglyceride, its omega-3 fatty acid content can reach 35.5%, and it is mainly used to develop functional triglycerides to replace infant milk fat. In the review paper of Feltes (FeltesM.MC etal , 2013), it was pointed out , the use of fish oil to develop monoglycerides and diglycerides rich in omega-3 fatty acids can be applied in the food field, which has broad market prospects. Subsequently, ZhengGuo’s team at Aarhus University in Denmark (Angela et al , 2016) studied the use of Novozymes 435 enzyme Catalyzed sardine fish oil and glycerol for glycerolysis reaction to prepare monoglyceride containing 31.7% (wt%) omega-3 fatty acids, which was successfully scaled up to the pilot test level. However, domestic research on this aspect is currently blank. Therefore, research and development are rich in The functional monoglyceride of omega-3 fatty acid is applied to the fields of food and pharmacy, which is of great significance.

Edible oils rich in omega-3 fatty acids are mainly fish oils and algae oils from marine microalgae. In recent years, the aquaculture industry has consumed 83% of the world's fish oil, and human direct consumption of fish oil only accounts for 6% of the total. In June 2015, FAOGlobefish announced that the price of fish oil was 2,400$/ton, which was 300$/ton higher than the price of fish oil last year; in addition, in the first half of this year, my country has imported 253,000 tons of fish oil to meet the domestic market demand. The high price of fish oil and dependence on imports directly restrict the development of functional monoglycerides rich in omega-3 fatty acids. Based on the analysis of the marine food chain, the omega-3 fatty acids enriched in marine fish mainly come from marine microalgae rich in omega-3 fatty acids. To a certain extent, it is feasible to develop functional monoglycerides with algal oil rich in omega-3 fatty acids as an alternative to fish oil; As a food additive. Therefore, the use of omega-3-rich algae oil to develop functional monoglycerides with high nutritional value can solve the oil source problem faced by this technology.

The development of functional monoglycerides in developed countries is still faced with how to screen lipases with strong specificity, high catalytic efficiency and environmental protection. Currently commonly used commercial lipases include non-selective immobilized lipases (such as Novozyme 435, CALA, etc.) and 1,3 selective enzymes (such as TLIM, RMIM, AK-20, etc.). As mentioned above, the total content of omega-3 fatty acids in monoglycerides prepared by enzymes such as Novozyme 435 is equal to or slightly higher than that of raw fish oil. Finding a lipase capable of hydrolyzing saturated fatty acids and monounsaturated fatty acids of edible oils is currently a difficult problem in the development of omega-3 lipase-rich monoglyceride technology. In 2011, Professor Xuebing Xu from Aarhus University in Denmark studied the biomarker of CALA lipase and found that the enzyme could hydrolyze 8 carbon chains and oleic acid of oil under ethanol conditions; however, they did not further use the enzyme to alcoholyze fish oil to develop its function Monoglycerides.

Contents of the invention

The purpose of the present invention is to prepare the monoglyceride rich in omega-3 fatty acid by using the algae oil rich in omega-3 fatty acid as raw material and utilizing the environment-friendly and high-efficiency enzymatic catalysis method.

The technical scheme and steps adopted for realizing the purpose of the present invention are:

1. Algae oil extraction. Weigh a certain amount of microalgae powder, use an organic solvent as an extraction agent, and use an accelerated solvent extraction device to extract algae oil. The extraction temperature is 40-125°C, the extraction time is 25-120 minutes, and the extraction times are 2-5 times. After the extraction is completed, the extract is placed in a vacuum rotary evaporator to evaporate the organic solution, and the residue obtained after evaporation is algae oil.

The organic solvent of the present invention refers to chloroform, n-hexane, ethanol or methanol.

An organic solvent is added to the microalgae powder of the present invention, and the ratio is microalgae powder (g):organic solvent (mL)=1:15-35.

2. Triacylglycerol separation. The chromatographic column is added with hexane, and 80g of silica gel is added simultaneously, and then the algal oil obtained in step 1 is added to the chromatographic column;

Use 500mL n-hexane-ether to elute the chromatographic column to separate and remove the grease of non-polar components, wherein the volume ratio of n-hexane:ether is 90:5;

Use 1000mL n-hexane-ether to elute the chromatographic column to separate and obtain triacylglycerol rich in omega-3 fatty acids, wherein the volume ratio of n-hexane:ether is 90:10;

3. Synthesis of monoglycerides rich in omega-3 fatty acids.

The above-mentioned extracted triacylglycerol is mixed with ethanol, and at the same time, immobilized lipase A-CALA and a little water are added to carry out alcoholysis reaction to obtain crude monoglyceride rich in omega-3 fatty acid. The alcoholysis reaction conditions are: the mass ratio of triacylglycerol and ethanol is 1:1~4, the reaction temperature is 25~55°C, the amount of immobilized lipase A-CALA accounts for 5~12.5% of the mass of monoglyceride, and the amount of water added accounts for The total mass of monoglyceride and ethanol is 2-9%, the reaction time is 10h-48h, and the stirring speed is 500r/min.

The immobilized lipase A-CALA was purchased from Novozymes, Denmark.

4. Purification of monoglycerides rich in omega-3 fatty acids.

The reaction product monoglyceride rich in omega-3 fatty acids obtained in step 3 is mixed with food-grade n-hexane according to the volume ratio of 1:9, after three extractions, the ethanol phase is collected, and the ethanol and water are removed by rotary evaporation to obtain Purified monoglycerides rich in omega-3 fatty acids.

Using the process conditions described in the patent of the present invention, the total content of omega-3 fatty acids in monoglyceride can reach more than 90% after the enzyme-catalyzed reaction.

detailed description

Example 1

1. Extract algae oil. Weigh 10g of Nannochloropsis algae powder, add 350mL of n-hexane:ethanol (1:2, v/v) mixture, and extract the algal oil with an accelerated solvent extraction apparatus. The extraction conditions are: temperature 75°C, extraction time 50min, Repeat the extraction 3 times. After the extraction is completed, the extract is placed in a vacuum rotary evaporator to evaporate n-hexane and ethanol, and the residue obtained after evaporation is algae oil.

2. Separation of different components of algae oil. The chromatographic column is added with hexane, and 80g of silica gel is added simultaneously, and then the algal oil obtained in step 1 is added to the chromatographic column;

Use 500mL n-hexane-ether to elute the chromatographic column to separate and remove the grease of non-polar components, wherein the volume ratio of n-hexane:ether is 90:5;

Triacylglycerol was obtained by elution of the chromatographic column with 1000 mL of n-hexane-ether, wherein the volume ratio of n-hexane:ether was 90:10.

3. Synthesis of monoglycerides rich in omega-3 fatty acids. Utilize the triacylglycerol of the algae oil extracted above to mix with ethanol, add immobilized lipase A-CALA and a little water at the same time to carry out alcoholysis reaction and prepare the monoglyceride rich in omega-3 fatty acid, the reaction condition is: triacylglycerol and The mass ratio of ethanol is 1:3, the reaction temperature is 35°C, the amount of immobilized lipase A-CALA accounts for 12.5% of the mass of triacylglycerol, the amount of water added accounts for 6% of the total mass of monoglyceride and ethanol, and the reaction time is 46h. The crude product of monoglyceride rich in omega-3 fatty acid was prepared by reaction at a stirring speed of 500 r/min.

The lipase (immobilized lipase A-CALA) was purchased from Novozymes, Denmark.

4. Purification of monoglycerides rich in omega-3 fatty acids.

The above reaction product was mixed with food-grade n-hexane (1:9, v/v), extracted three times, each extraction time was 50 minutes, and the ethanol phase was collected, and the ethanol and water were removed by rotary evaporation to obtain omega-3 fatty acid-rich monoglycerides.

Using the process conditions described in the patent of the present invention, the total content of omega-3 fatty acids in monoglyceride can reach 92% after the enzyme-catalyzed reaction.

Example 2

1. Extract algae oil. Weigh 10g of Isochrysis algae powder, add 200mL of n-hexane:methanol (1:2, v/v) mixture, and extract the algal oil with an accelerated solvent extraction apparatus. The extraction conditions are: temperature is 75°C, extraction time is 45min, Repeat the extraction 5 times. After the extraction is completed, the extract is placed in a vacuum rotary evaporator to evaporate the organic solution, and the residue obtained after evaporation is algae oil.

2. Separation of different components of algae oil. The chromatographic column is added with hexane, and 80g of silica gel is added simultaneously, and then the algal oil obtained in step 1 is added to the chromatographic column;

Use 500mL n-hexane-ether to elute the chromatographic column to remove the grease of non-polar components, wherein the volume ratio of n-hexane:ether is 90:5;

The crude product of triacylglycerol was obtained by elution of the chromatographic column with 1000 mL of n-hexane-ether, wherein the volume ratio of n-hexane:ether was 90:10.

3. Synthesis of crude triacylglycerol rich in omega-3 fatty acids. Utilize the triacylglycerol and ethanol of the algal oil extracted above to carry out the alcoholysis reaction to prepare the monoglyceride crude product rich in omega-3 fatty acids; the reaction conditions are: the mass ratio of triacylglycerol and ethanol is 1:4, and the reaction temperature is 30 ℃, the amount of immobilized lipase A-CALA accounts for 12.5% of the mass of triacylglycerol, the amount of water added accounts for 5% of the total mass of monoglyceride and ethanol, the reaction time is 40h, and the stirring speed is 500r/min. Monoglyceride rich in omega-3 fatty acids.

The lipase (immobilized lipase A-CALA) was purchased from Novozymes, Denmark.

4. Purification of monoglycerides rich in omega-3 fatty acids.

Extract the above reaction product with food-grade n-hexane (1:9, v/v) for 3 times, collect the ethanol phase, each extraction time is 50min, collect the ethanol phase, and remove ethanol and water by rotary evaporation to obtain fatty acids rich in omega-3 of monoglycerides.

Using the process conditions described in the patent of the present invention, the total content of omega-3 fatty acids in monoglyceride can reach 90% after the enzyme-catalyzed reaction.

Claims (7)

1. be rich in a preparation method for omega-fatty acid Tegin 55G, it is characterized in that:

Take a certain amount of micro-algae powder, using organic solvent as extraction agent, utilize accelerated solvent extraction to extract algae oil, after extraction terminates, extraction liquid is placed in vacuum rotary evaporator and organic dissolution is evaporated, residuum is algae oil;

Chromatographic column adds hexane, adds 80g silica gel simultaneously, and the algae oil then step 1 obtained joins in chromatographic column;

Be separated with 500mL normal hexane-ether elution chromatography post and remove the grease of non-polar component; The triacylglycerol obtaining and be rich in omega-fatty acid is separated, wherein normal hexane: the volume ratio of ether is 90:10 with 1000mL normal hexane-ether elution chromatography post;

The triacylglycerol of said extracted is mixed with ethanol, adds immobilized lipase A-CALA and little water carries out the Tegin 55G crude product that alcoholysis reaction obtains being rich in omega-fatty acid simultaneously;

The reaction product Tegin 55G crude product being rich in omega-fatty acid obtained is mixed with food grade normal hexane, through 3 extractions, collects ethanol phase, the Tegin 55G being rich in omega-fatty acid after rotary evaporation obtains purifying after removing second alcohol and water.

2. according to claim 1ly a kind ofly adopt lipase-catalyzed legal system for the method for structure glyceryl ester, it is characterized in that described extraction conditions, extraction temperature is 40 ~ 125 DEG C, extraction time 25 ~ 120min, and extraction times is 2 ~ 5 times.

3. a kind of preparation method being rich in omega-fatty acid Tegin 55G according to claim 1, is characterized in that described organic solvent refers to chloroform, normal hexane, ethanol or methyl alcohol.

4. a kind of preparation method being rich in omega-fatty acid Tegin 55G according to claim 1, it is characterized in that adding organic solvent in described micro-algae powder, its ratio is micro-algae powder (g): organic solvent (ml)=1:15 ~ 35.

5. a kind of preparation method being rich in omega-fatty acid Tegin 55G according to claim 1, is characterized in that the dose volume of normal hexane-ether in described step 2 than being 90:5.

6. a kind of preparation method being rich in omega-fatty acid Tegin 55G according to claim 1, it is characterized in that the alcoholysis described in step 3), reaction conditions is: the consumption of mass ratio 1:1 ~ 4 of triacylglycerol and ethanol, temperature of reaction 25 ~ 55 DEG C, immobilized lipase A-CALA account for Tegin 55G quality 5 ~ 12.5%, amount of water account for the total mass of Tegin 55G and ethanol 2 ~ 9%, the reaction times is 10h ~ 48h, stirring velocity 500r/min.

7. a kind of preparation method being rich in omega-fatty acid Tegin 55G according to claim 1, it is characterized in that the Tegin 55G crude product described in step 4) mixes with food grade normal hexane, is the carrying out according to Tegin 55G crude product and food grade normal hexane parts by volume ratio 1:9.