A method for preparing tuna oil lipid-modified glyceride

Abstract

The present invention belongs to the technical field of fish oil fatty acid processing, and relates to a method for preparing tuna oil lipid modified glyceride. The method includes adding lipase and phosphate buffer solution to fish oil, enzymolysis under stirring to obtain fish oil hydrolyzate, adding anhydrous ethanol to the fish oil hydrolyzate to terminate the enzymolysis reaction, then adding potassium hydroxide solution to neutralize free fatty acids, adding adsorbent and centrifuging to extract oil phase, using a small amount of n-hexane extraction, rotary evaporation, nitrogen blowing to obtain glyceride rich in ω-3 polyunsaturated fatty acids. The enrichment method provided by the present invention can significantly increase the content of ω-3 polyunsaturated fatty acids, reduce the content of saturated fatty acids, and simultaneously produce monoglyceride and diglyceride, reducing the use of organic solvents and pollution to the environment. The present invention provides a green, pollution-free, low-cost, and simple-to-operate method for the enrichment of ω-3 polyunsaturated fatty acids in fish oil and the preparation of functional lipids.

Description

Technical Field

[0001] The invention belongs to the technical field of fish oil glyceride processing and relates to a method for preparing tuna oil lipid-modified glyceride. Background Art

[0002] Deep-sea fish oil is rich in ω-3 polyunsaturated fatty acids (ω-3 PUFAs). ω-3 PUFAs refer to a class of fatty acids with two or more unsaturated double bonds in the molecule, with the carbon atom at the methyl end at the ω-1 position and an unsaturated double bond at the ω-3 position. ω-3 PUFAs mainly include α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). ω-3 PUFAs have been proven to have anti-inflammatory and immune-enhancing functions, help reduce sports fatigue and recovery from fatigue, assist in improving memory and regulating blood lipids, but the content of ω-3 PUFAs in natural fish oil generally does not exceed 30%, which limits the application of fish oil in functional foods and medicine.

[0003] Methods for increasing the content of ω-3 PUFAs in fish oil include low-temperature crystallization, urea embedding, silver nitrate complexation, etc. These methods often use organic solvents such as ether, acetone, acetonitrile, methanol, etc. to extract fatty acids. Although the extraction efficiency is high, the residues of organic reagents during the extraction process may cause pollution to the final product. Chemical alkali refining is usually used to remove unstable fatty acids in oils and fats in oil and fats production, but this method has the disadvantages of high energy efficiency, waste of raw materials and low economic benefits.

[0004] Fish oil contains glycerides, which can be divided into monoglycerides, diglycerides and triglycerides according to their types. Triglycerides are the most abundant lipids in the human body and can help provide energy. However, long-term consumption of foods rich in triglycerides can lead to increased triglycerides in the body, which can lead to liver damage and hyperlipidemia. Increased triglycerides may also cause increased transaminase, pancreatitis, fatty liver, etc. Diaglyceride oil has important health benefits such as reducing visceral fat, inhibiting weight gain, and lowering blood lipids.

[0005] Therefore, it is of great significance to explore a preparation process for enriching ω-3 PUFAs in fish oil and producing modified glycerides while producing diglycerides without using or with a small amount of organic solvents. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for preparing tuna oil lipid-modified glyceride, which comprises: enzymolyzing fish oil with lipase, neutralizing free fatty acids in the enzymolysis product and then adding an adsorbent, then preliminarily separating the oil by centrifugation, and then extracting the oil with a small amount of n-hexane to further purify, rotary evaporate, and nitrogen blow to obtain glyceride rich in ω-3 polyunsaturated fatty acids. The enrichment method provided by the present invention can significantly increase the content of ω-3 polyunsaturated fatty acids, and the residual solvent content in the prepared glyceride does not increase. The present invention provides a green, pollution-free, low-cost, and simple-to-operate method for enriching ω-3 polyunsaturated fatty acids in fish oil and increasing the content of functional lipids.

[0007] To achieve the technical purpose of the present invention, on the one hand, the present invention provides a method for preparing tuna oil lipid-modified glyceride, comprising:

[0008] S1. Fish oil hydrolysis: Tuna oil was placed in a round-bottom flask, and 5 wt% of immobilized lipase ROL or DF 15 and a phosphate buffer solution with a pH value of 6.0-8.0 were added respectively. The mixture was placed on a magnetic temperature-controlled stirrer at a speed of 100-500 rpm and enzymatic hydrolysis at 20-60°C to obtain fish oil hydrolyzate.

[0009] S2, glyceride extraction: Add 3mL of anhydrous ethanol to the fish oil hydrolyzate of S1 to terminate the reaction, neutralize the free fatty acids with 0.5mol / L KOH solution, add the adsorbent, stir and mix well, and let stand for 10 minutes. Centrifuge at 2000~10000rpm for 5~25min, absorb the upper oil layer, extract with 1~3 times the volume of the oil phase with n-hexane, absorb the supernatant and rotary evaporate at 50℃. If the oil sample is not clear after rotary evaporation, repeat the extraction and rotary evaporation until the oil sample is clear. Blow nitrogen into the oil sample after rotary evaporation, weigh it, and seal it at 4℃.

[0010] Furthermore, in a method for preparing tuna oil lipid-modified glyceride provided by the present invention, the ratio of the added volume of the phosphate buffer solution to the added mass of the fish oil is 1:1, and the ω-3 polyunsaturated fatty acids include eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid. The added volume of the potassium hydroxide solution is determined by first sampling and titrating and calculating the alkali solution required to neutralize the unit weight of the hydrolyzate, calculating the theoretical amount of the required alkali solution according to the weight of the hydrolyzate, and adding the hydrolyzate at 100-110% of the theoretical amount.

[0011] Preferably, in the method for preparing tuna oil lipid-modified glyceride provided by the present invention, when the lipase is immobilized lipase ROL, the pH of the phosphate buffer solution is 7.5, the rotation speed is 300 rpm, the enzymatic hydrolysis temperature is 40°C, and the enzymatic hydrolysis time is 8 hours. When the lipase is immobilized lipase DF 15, the pH of the phosphate buffer solution is 8.0, the rotation speed is 300 rpm, the enzymatic hydrolysis temperature is 40°C, and the enzymatic hydrolysis time is 2 to 5 hours.

[0012] Furthermore, in a method for preparing tuna oil lipid-modified glyceride provided by the present invention, the adsorbent is any one of silicon dioxide, oil silica gel, activated carbon and zeolite powder, and the ratio of the amount of the adsorbent added to the mass of the fish oil is 1:50 to 250. Preferably, the adsorbent is oil silica gel, and the ratio of the amount of the adsorbent added to the mass of the fish oil is 1:150; centrifugation is performed at 6000 rpm for 5 minutes in S2.

[0013] On the other hand, the present invention claims the use of the above-mentioned method for preparing tuna oil lipid-modified glyceride in preparing glyceride rich in ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids include eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid.

[0014] In addition, the present invention claims the use of the above method for preparing tuna oil lipid-modified glyceride in preparing glyceride rich in monoglyceride and diglyceride.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0016] The present invention obtains a process with better effect of preparing tuna oil lipid-modified glyceride by hydrolysis of immobilized lipase ROL by optimizing process conditions, specifically, the enzymolysis temperature is 40 DEG C, the enzymolysis pH is 7.5, the enzymolysis speed is 300 rpm, the oil silica gel: fish oil is 1:150, the centrifugal speed is 6000 rpm, and the centrifugation time is 5 min; under the optimal process conditions, the fish oil hydrolysis degree is 37.76±1.00%, the RSD is 3.60%, and the data repeatability is good; the glyceride yield is 46.89±0.60%, the RSD is 1.27%, and the data repeatability is good. The optimal process for preparing tuna oil lipid-modified glycerides by hydrolysis of immobilized lipase DF 15 is: enzymatic hydrolysis temperature 40℃, enzymatic hydrolysis pH 8.0, enzymatic hydrolysis time 2-5h, enzymatic hydrolysis speed 300rpm, oil silica gel: fish oil 1:150, centrifugal speed 6000rpm, and centrifugal time 5min; under the optimal process conditions, the fish oil hydrolysis degree was 35.27±0.91%, and the glyceride yield was 62.99±2.01%.

[0017] The method for preparing tuna oil lipid-modified glyceride provided by the present invention can significantly increase the content of ω-3 polyunsaturated fatty acids and eicosapentaenoic acid and docosapentaenoic acid. The present invention analyzes the composition and content of the main fatty acids in tuna oil and glyceride and finds that the content of ω-3 PUFAs in the glyceride modified by ROL enzyme method is 46.22%, and the content of ω-3 PUFAs in the glyceride modified by DF15 enzyme method is 43.80%, which is significantly different from the content of ω-3 PUFAs in tuna oil of 35.13%, and the content of polyunsaturated fatty acids in the glyceride prepared by the present invention is significantly increased.

[0018] The method provided by the present invention can significantly increase the content of monoglyceride and diglyceride in tuna oil. Triglyceride accounts for the largest proportion in tuna oil, and the total content of monoglyceride and diglyceride accounts for less than 1%. After hydrolysis treatment by the method of the present invention, the content of monoglyceride and diglyceride is significantly increased, and the total content is 14.45-19.32%, and the content of triglyceride is significantly reduced.

[0019] The method for preparing tuna oil lipid-modified glyceride provided by the present invention has the advantages of being green and pollution-free, low in cost, and simple to operate. The anisidine value in the fatty acid prepared by the present invention is not much different from the anisidine value in the tuna oil before enzymatic hydrolysis, indicating that the enrichment method of the present invention does not cause solvent residue, reduces the use of organic reagents in fish oil extraction, and reduces pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention.

[0021] Figure 1 The effect of different enzymatic hydrolysis temperatures on the hydrolysis degree of fish oil.

[0022] Figure 2 The effect of different enzymatic hydrolysis pH on the hydrolysis degree of fish oil.

[0023] Figure 3 The effect of different enzymatic hydrolysis speeds on the hydrolysis degree of fish oil.

[0024] Figure 4 The effect of different adsorbents on the yield of glyceride.

[0025] Figure 5 The effect of different mass ratios of oil silica gel and fish oil on the yield of glyceride.

[0026] Figure 6 The effect of different centrifugal speeds on the glyceride yield.

[0027] Figure 7 The effect of different centrifugation times on the glyceride yield.

[0028] Figure 8 This is a Fourier infrared image of tuna oil and glycerides. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0030] Tuna oil was donated by Zhoushan Xinnuojia Bioengineering Co., Ltd.

[0031] Immobilized lipase ROL (Rhizopus oryzae lipase) was donated by Weifang Kangdian Biotechnology Co., Ltd.

[0032] Immobilized lipase DF 15 (Lipase DF “Amano” 15) was provided by Jiangsu Amano Enzyme Preparation Co., Ltd.

[0033] B932R silica was donated by Qingdao Borite Silicon Energy Technology Co., Ltd.

[0034] R92 Grease Silicone, donated by PQ Company.

[0035] Zeolite powder and activated carbon were purchased from Guangzhou Keming Reagent Co., Ltd.

[0036] Anhydrous ether, n-hexane, methanol, toluene, and acetyl chloride were purchased from Guangzhou Chemical Reagent Factory.

[0037] Example 1

[0038] This embodiment provides a method for preparing tuna oil lipid-modified glyceride, which specifically comprises the following steps:

[0039] S1. Fish oil hydrolysis: 5 g of tuna oil was placed in a round-bottom flask, and 5% of the mass of tuna oil immobilized lipase ROL and 5 mL of phosphate buffer solution with a pH value of 7.5 (prepared by 0.2 mol / L Na2HPO4 and 0.3 mol / L NaH2PO4) were added respectively, and the mixture was placed on a magnetic temperature-controlled stirrer (purchased from Shenzhen Broad Jingke Biotechnology Co., Ltd.) and enzymatically hydrolyzed at 200 rpm and 50°C for 8 h to obtain fish oil hydrolyzate.

[0040] S2. Determination of hydrolysis degree: Weigh 0.2g of fish oil hydrolyzate, add 5mL of anhydrous ethanol-ether (the volume ratio of ethanol to ether is 1:2) solution, 1-2 drops (1g / L) of phenolphthalein indicator, mix well, and titrate with 0.1mol / L NaOH solution. The titration endpoint is when the reaction solution turns slightly red and does not fade within 30s. The calculation formula of hydrolysis degree (HD) is as follows:

[0041]

[0042] In the formula, C is the concentration of NaOH solution (mol / L); V is the amount of NaOH solution used (mL); m is the mass of fish oil hydrolysate (g); 0.25 is the proportion of oil phase in the hydrolysate; 187 is the saponification value of tuna oil.

[0043] S3, glyceride extraction: add 3mL of anhydrous ethanol to the fish oil hydrolysate of S1 to terminate the reaction, take a sample to determine the free fatty acid content in the hydrolysate, calculate the amount of potassium hydroxide solution to be added, then add a calculated amount of 100~110% potassium hydroxide solution to neutralize the free fatty acids, neutralize the free fatty acids with 0.5mol / L KOH solution (containing 30vol% anhydrous ethanol), add 1% of the fish oil mass ratio of the adsorbent and stir thoroughly, and ultrasonically treat for 10min. Centrifuge at 6000rpm for 5min, absorb the upper oil layer, extract with 1~3 times the volume of the oil phase with n-hexane, absorb the supernatant and rotary evaporate at 50℃. If the oil sample is not clear after rotary evaporation, repeat the extraction and rotary evaporation until the oil sample is clear. Nitrogen blow the oil sample after rotary evaporation, weigh it, and seal it at 4℃. The calculation formula for glyceride yield is as follows:

[0044]

[0045] In the formula, m1 is the mass of glyceride (g); m is the mass of fish oil weighed (g).

[0046] Example 2

[0047] This example provides optimization of the glyceride preparation process, specifically including single factor experiment and orthogonal experiment.

[0048] 1. Single factor test

[0049] (1) Single factor experiment of enzymatic hydrolysis temperature: the enzymatic hydrolysis temperatures were 20, 30, 40, 50, and 60°C.

[0050] The preparation method of glyceride was the same as that in Example 1, and the effects of different enzymatic hydrolysis temperatures on the degree of hydrolysis of fish oil were compared ( Figure 1), in the range of 20~50℃, the degree of fish oil hydrolysis was positively correlated with the hydrolysis temperature, and then the degree of fish oil hydrolysis decreased with the increase of the hydrolysis temperature. The highest degree of hydrolysis was 30.56% at 50℃. Based on the test results, three levels of hydrolysis temperature, 40℃, 50℃ and 60℃, were selected for subsequent orthogonal tests.

[0051] (2) Single factor test of enzymatic hydrolysis pH: the enzymatic hydrolysis pH were 6.0, 6.5, 7.0, 7.5, and 8.0, respectively.

[0052] The preparation method of glyceride was the same as that in Example 1, and the effect of different enzymatic hydrolysis pH on the hydrolysis degree of fish oil was compared ( Figure 2 ), the degree of fish oil hydrolysis was the highest at pH 7.5, which was 26.64%. Based on the test results, three levels of pH 7.0, 7.5 and 8.0 were selected for subsequent orthogonal tests.

[0053] (3) Single factor test of enzymatic hydrolysis speed, the enzymatic hydrolysis speeds were 100, 200, 300, 400, and 500 rpm respectively.

[0054] The preparation method of glyceride is the same as that of Example 1, and the effect of different enzymatic hydrolysis speeds on the hydrolysis degree of fish oil is compared ( Figure 3 ), the fish oil hydrolysis degree was the highest at 200rpm, which was 30.56%. This may be because the contact time between the enzyme and the fish oil was too short when the speed was too high, which was not suitable for reaction. Based on the test results, three levels of enzymatic hydrolysis speed of 100, 200 and 300rpm were selected for subsequent orthogonal experiments.

[0055] (4) Single factor test of adsorbents, including oil silica, silica (B932R silica), activated carbon (edible oil adsorption activated carbon) and zeolite powder.

[0056] The preparation method of glyceride is the same as that of Example 1, and the yield of glyceride obtained by different types of adsorbents is compared ( Figure 4 ) found that the glyceride yield using oil silica gel as an adsorbent was higher, and the yield was significantly higher than that of other adsorbents. This may be because oil silica gel promotes the formation of free fatty acid salts from the free fatty acids obtained by enzymatic hydrolysis of tuna oil by adsorbing polar impurities (soaps, phospholipids and metal ions, etc.) produced during oil hydrolysis and neutralization, forming more precipitation under centrifugal conditions, resulting in better glyceride separation and higher glyceride yield.

[0057] (5) Single factor test on the mass ratio of adsorbent oil silica gel to fish oil. The mass ratios of oil silica gel to fish oil were 1:50, 1:100, 1:150, 1:200 and 1:250 respectively.

[0058] Adding an appropriate amount of adsorbent during the extraction of fish oil can enhance the adsorption capacity of impurities, thereby improving the removal effect of impurities. However, adding too much adsorbent may have side effects, and the adsorption of fish oil on the adsorbent is reversible, and there will be a certain degree of fish oil loss during the adsorption process. Therefore, it is very important to determine the optimal mass ratio of adsorbent to fish oil for the preparation of glyceride. The preparation method of glyceride is the same as that of Example 1, and the glyceride yields obtained by different mass ratios of oil silica gel to fish oil are compared ( Figure 5 ) found that the glyceride yield was the highest at 42.1% when the ratio of oil and silicone: fish oil was 1:150. The yield decreased significantly when the ratio was increased or decreased. Based on the test results, three levels of oil and silicone: fish oil were selected for subsequent orthogonal tests, namely 1:100, 1:150, and 1:200.

[0059] (6) Single factor test of centrifugal speed, the centrifugal speeds were 2000, 4000, 6000, 8000 and 10000 rpm respectively.

[0060] The preparation method of glyceride is the same as that of Example 1, and the glyceride yields obtained at different centrifugal speeds are compared ( Figure 6 ) found that when the centrifugal speed was 4000rpm, the glyceride yield was the highest, 46.73%. As the speed increased, the yield decreased significantly. Appropriate centrifugal conditions are conducive to the release and separation of glycerolipids. Based on the experimental results, three levels of centrifugal speeds of 2000, 4000 and 6000rpm were selected for subsequent orthogonal experiments.

[0061] (7) Single factor test of centrifugation time: the centrifugation times were 5, 10, 15, 20, and 25 min, respectively.

[0062] The preparation method of glyceride is the same as that of Example 1, and the glyceride yields obtained by different centrifugation times are compared ( Figure 7 ) found that when the centrifugation time was 10 minutes, the glyceride yield was the highest, which was 48.23%. Based on the experimental results, three levels of centrifugation time of 5, 10 and 15 minutes were selected for subsequent orthogonal experiments.

[0063] 2. Orthogonal test

[0064] (1) According to the orthogonal factor level table (Table 1), the effects of enzymatic hydrolysis temperature, enzymatic hydrolysis pH and enzymatic hydrolysis speed on the hydrolysis degree of fish oil were determined by orthogonal test. Based on the above orthogonal test determination, according to the orthogonal factor level table (Table 2), the effects of the mass ratio of oil silica gel to fish oil, centrifugal speed and centrifugal time on the glyceride yield were determined by orthogonal test. SPSS 27 and Minitab 20 software were used for data analysis, and Origin 2022 was used for plotting. Each group of data was repeated 3 times, and the test results were expressed as x±s. P<0.05 indicated significant differences. The statistical results are shown in Table 3. The statistical results were subjected to variance analysis, and the results are shown in Tables 4 and 5. The optimal process of significance analysis and range analysis was verified, and the results are shown in Table 6.

[0065] Table 1: Orthogonal factor level table with fish oil hydrolysis degree as index

[0066]

[0067] Table 2: Orthogonal factor level table with glyceride yield as an indicator

[0068]

[0069] Table 3: Orthogonal test results

[0070]

[0071] Table 4: ANOVA with fish oil hydrolysis degree as an indicator

[0072]

[0073] Table 5: ANOVA with glyceride yield as an indicator

[0074]

[0075] Table 6: Verification test results

[0076]

[0077] As shown in Table 3, the order of influence on the degree of fish oil hydrolysis is: enzymatic temperature > enzymatic speed > enzymatic pH, and the process conditions with the best degree of fish oil hydrolysis are A1B2C3. From the significant difference analysis, it can be seen that the result of experiment 2 is the best, and the degree of fish oil hydrolysis is 35.48±0.52, which is significantly different from other experimental groups (P<0.05), that is, the best process experiment of orthogonal experiment is A1B2C2, which is inconsistent with the range analysis. After verification tests, it was found that under the conditions of the optimal process A1B2C3 (specifically, the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis pH was 7.5, and the enzymatic hydrolysis speed was 300rpm) in the range analysis, the degree of fish oil hydrolysis was 37.76±1.00%, the RSD was 3.60%, and the data repeatability was good; under the conditions of the optimal process A1B2C2 (specifically, the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis pH was 7.5, and the enzymatic hydrolysis speed was 200rpm) in the significant difference analysis, the degree of fish oil hydrolysis was 35.29±0.85%. It can be seen that the optimal process is A1B2C3. As shown in Table 4, the enzymatic hydrolysis temperature has a significant effect on the degree of fish oil hydrolysis.

[0078] As shown in Table 3, the order of influence on glyceride yield is: centrifugation time > centrifugation speed > oil silica gel: fish oil, and the process condition with the best glyceride yield is A2B3C1. From the significant difference analysis, it can be seen that the results of group 6 are the most significant (P<0.05), that is, the orthogonal test of the best process test is the same as the range analysis. Therefore, under the optimal process A2B1C1 (oil silica gel: fish oil is 1:150, centrifugation speed is 6000rpm, centrifugation time is 5min), the glyceride yield is 46.89±0.60%, and the data repeatability is good; as shown in Table 5, the ratio of oil silica gel to fish oil has a significant effect on the glyceride yield.

[0079] 3. This example further determined the optimal process conditions for enzymatic hydrolysis of tuna oil with immobilized lipase DF 15 through single factor experiment and orthogonal experiment. It was found that the fish oil hydrolysis degree was high at 35.27±0.91% and the glyceride yield was 62.99±2.01% when the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis pH was 8.0, the enzymatic hydrolysis time was 2-5h and the enzymatic hydrolysis speed was 300rpm.

[0080] Example 3

[0081] This example provides composition analysis of tuna oil and glycerides prepared by hydrolyzing tuna oil with ROL lipase of the present invention.

[0082] The preparation method of glyceride is referred to Example 1, and the optimal process conditions obtained by orthogonal test in Example 2 are used for preparation. The lipid composition of tuna oil and glyceride obtained by enzymatic hydrolysis of different immobilized lipases is shown in Table 7, the main fatty acid composition and content results are shown in Table 8, the physical and chemical indicators of tuna oil and glyceride are shown in Table 9, and the Fourier transform infrared images of tuna oil and glyceride are shown in Table Figure 8As shown in Figure 1. SFA is saturated fatty acid, MUFA is linolenic acid, and PUFA is polyunsaturated fatty acid.

[0083] Table 7: Lipid composition of tuna oil and glycerides

[0084]

[0085] Table 8: Composition and content of major fatty acids in tuna oil and glycerides

[0086]

[0087] Table 9: Physical and chemical indicators of tuna oil and glycerides

[0088]

[0089] As shown in Table 7, triglycerides accounted for the largest proportion in tuna oil, and the total content of monoglycerides and diglycerides accounted for less than 1%. After hydrolysis by immobilized lipase ROL or DF 15, the content of monoglycerides and diglycerides increased significantly, with a total content of 14.45-19.32%, and the content of triglycerides decreased significantly. This may be due to the selective hydrolysis of sn-1,3 fatty acids by immobilized lipase ROL or DF 15, which led to the formation of monoglycerides and enriched the fatty acids at the sn-2 position.

[0090] As shown in Table 8, tuna oil contains 21.3% palmitic acid and 19.2% oleic acid, which are not significantly different from the content in glycerides. The content of ω-3 PUFAs in glycerides modified by ROL enzyme method is 46.22%, and the content of ω-3 PUFAs in glycerides modified by DF 15 enzyme method is 43.80%, which is significantly different from the ω-3 PUFAs content in tuna oil of 35.13% (P<0.01). After lipase hydrolysis, the content of DHA and the ratio of DHA to EPA in tuna oil increased significantly compared with tuna oil, and the content of polyunsaturated fatty acids increased significantly. It shows that lipase hydrolysis can effectively enrich DHA and EPA and can effectively supplement ω-3 PUFAs.

[0091] As shown in Table 9, the anisidine value indicates the residual amount of amine solvents in the oil. The difference between the anisidine values ​​before and after enzymatic hydrolysis is small. It can be seen that the residual glyceride solvent extracted by the process of the present invention is small and is not much different from that of unhydrolyzed tuna oil.

[0092] Depend on Figure 8It can be seen that tuna oil does not have an obvious absorption peak at the first hydroxyl peak, but glycerol esters do. This may be because tuna oil mainly exists in the form of triglycerides before hydrolysis, and the content of monoglycerides and diglycerides increases after hydrolysis, resulting in an increase in hydroxyl groups. -1 The characteristic peak at is caused by the asymmetric vibration of saturated alkane methyl groups, reflecting the chain length and molecular weight of oil fatty acids. The signal intensity of other peaks of tuna oil is significantly greater than that of glycerol esters. This may be because lipase shears the fatty acids on the glycerol skeleton during the hydrolysis of fish oil, destroying the ether bond and replacing one or two fatty acids containing carbon-carbon double bonds, methylene and carbonyl functional groups in glycerol esters with hydroxyl groups, resulting in a weakened signal intensity.

[0093] The embodiments described above are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A method for preparing tuna oil lipid-modified glyceride, characterized in that: include: S1, adding lipase and phosphate buffer solution to fish oil, and performing enzymolysis under stirring to obtain fish oil hydrolyzate; S2, adding anhydrous ethanol to the fish oil hydrolyzate to terminate the enzymatic hydrolysis reaction, then adding potassium hydroxide solution to neutralize free fatty acids, adding an adsorbent and stirring to mix, standing for 10 minutes, then centrifuging to separate the upper oil layer, adding n-hexane to extract the oil layer, rotary evaporation, and then nitrogen blowing to obtain the glyceride rich in ω-3 polyunsaturated fatty acids; The lipase is immobilized lipase ROL or immobilized lipase DF 15, and the amount of the lipase added is 5wt% of the amount of the fish oil added; The adsorbent is grease silica gel; The added volume of n-hexane is 1 to 3 times of the oil layer; The ω-3 polyunsaturated fatty acids include eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid; When the lipase is immobilized lipase ROL, the pH of the phosphate buffer solution is 7.5, the rotation speed in the stirring condition is 300 rpm, the enzymatic hydrolysis temperature is 40° C., and the enzymatic hydrolysis time is 8 h; When the lipase is immobilized lipase DF 15, the pH of the phosphate buffer solution is 8.0, the rotation speed in the stirring condition is 300 rpm, the enzymatic hydrolysis temperature is 40° C., and the enzymatic hydrolysis time is 2 to 5 h; The ratio of the amount of the adsorbent added to the mass of the fish oil is 1:150; The centrifugal speed is 6000 rpm and the centrifugal time is 5 min.

2. A method for preparing tuna oil lipid-modified glyceride according to claim 1, characterized in that: The ratio of the added volume of the anhydrous ethanol to the added mass of the fish oil is 3:5; The concentration of the potassium hydroxide solution is 0.5 mol / L, and the potassium hydroxide solution contains 30 vol% of anhydrous ethanol; The added volume of the potassium hydroxide solution is 100-110% of the theoretical value of the potassium hydroxide solution required to neutralize the fish oil hydrolyzate.

3. A method for preparing tuna oil lipid-modified glyceride according to claim 1, characterized in that: The ratio of the added volume of the phosphate buffer solution to the added mass of the fish oil is 1:1; The pH of the phosphate buffer solution is 6.0-8.0, and is prepared by mixing 0.2 mol / L Na2HPO4 and 0.3 mol / L NaH2PO4.

4. Use of the method for preparing tuna oil lipid-modified glyceride according to any one of claims 1 to 3 in preparing glyceride rich in ω-3 polyunsaturated fatty acids; The ω-3 polyunsaturated fatty acids include eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid.

5. Use of the method for preparing tuna oil lipid-modified glyceride according to any one of claims 1 to 3 in preparing glyceride rich in monoglyceride and diglyceride.

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