MLCT rich in medium-chain and long-chain triglyceride and preparation method thereof

Abstract

The invention provides MLCT rich in medium-chain and long-chain triglyceride and a preparation method of the MLCT, and belongs to the technical field of structural lipid preparation. According to the invention, olive oil and MCT are used as acyl donors to prepare the medium-and-long-chain lipid with a low-calorie structure, and the content of medium-and-long-chain triglyceride in the obtained product can reach 82.7%. Long-chain fatty acid is highly distributed at the Sn-2 site and reaches 81.96%, the content of oleic acid at the Sn-2 site is close to 50%, and more than 80% of oleic acid is long-chain fatty acid. The prepared MLCT can be added into food to improve nutritional ingredients of the food, can replace traditional vegetable fat, improves absorption of essential fatty acid and controls obesity, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of structured lipid preparation, and particularly relates to ML CT rich in medium and long chain triglycerides and a preparation method thereof. Background Art

[0002] The main component of traditional edible oil is long-chain triglycerides (LCTs), which are composed of long-chain fatty acids (LCFA). Excessive intake of long-chain fatty acids promotes oxidative stress and inflammation. Improving diet and limiting the intake of long-chain triglycerides can control obesity and obesity-related complications. Unlike LCFA, medium-chain fatty acids (MCFA) can be directly absorbed by the liver through the portal vein and rapidly oxidized to produce energy to reduce the accumulation of fat in various organs. However, the rapid oxidation of medium-chain fatty acids will increase the concentration of ketone bodies in the liver. Medium-chain structural lipids combine the advantages of MCFA and LCFA structural esters, which can not only avoid ketone body poisoning caused by excessive MCFA concentration, as well as oxidative stress and proinflammatory diseases caused by excessive intake of LCFA, but also provide essential fatty acids, quickly supply energy, and reduce fat accumulation, which is of great significance to human health. Therefore, how to prepare structured lipids rich in medium-chain triglycerides (MLCT) is a key that urgently needs to be studied in this field. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the object of the present invention is to provide an MLCT rich in medium and long chain triglycerides and a preparation method thereof, wherein the medium and long chain triglycerides in the prepared MLCT reach more than 80%.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a method for preparing MLCT, comprising the following steps: mixing olive oil and medium-chain triglycerides, adding immobilized lipase, reacting to obtain a crude product, and separating and purifying the crude product to obtain MLCT; the immobilized lipase is Lipozyme TLIM.

[0006] Preferably, the mass ratio of the olive oil to medium-chain triglycerides is 3:2 to 7:3.

[0007] Preferably, the addition amount of the immobilized lipase is 6-7.5 wt % based on the total mass of olive oil and medium-chain triglycerides.

[0008] Preferably, the reaction temperature is 59-60° C., and the reaction time is 2-4 hours.

[0009] Preferably, the separation and purification method comprises: sequentially performing deacidification, water removal, n-hexane extraction, and reduced pressure evaporation on the crude product.

[0010] Preferably, the deacidification method comprises: mixing the crude product with anhydrous ethanol, stirring at 35 to 37° C. for 25 to 35 minutes, and discarding the ethanol phase.

[0011] Preferably, the water removal method comprises: mixing the deacidified product with silica gel, stirring, centrifuging, and discarding the precipitate.

[0012] Preferably, the temperature of the reduced pressure evaporation treatment is 65-75° C. and the rotation speed is 90-110 rpm.

[0013] The present invention also provides MLCT prepared by the above preparation method.

[0014] The present invention also provides application of the MLCT in food processing.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The preparation method of the present invention successfully synthesized medium- and long-chain structured lipids. After optimization, the optimal process conditions for preparing medium- and long-chain low-calorie structured lipids using olive oil and MCT as raw materials were determined. Under this optimized condition, the yield of MLCTs in the crude mixture was 75.93%. Subsequently, two steps of purification were performed, and the medium- and long-chain triglycerides in the purified product could reach 82.7%. The long-chain fatty acids (from olive oil) were distributed at a higher level at the Sn-2 position, reaching 81.96%. At the Sn-2 position, the content of oleic acid was close to 50%, and more than 80% were long-chain fatty acids. The total carbon number of the newly emerged TG was mainly C 34 -C 46 .

[0017] In food processing, adding the MLCT prepared by the present invention can improve the nutritional content of food. Using the MLCT prepared by the present invention to replace traditional vegetable fats can diversify the functional food market, improve essential fatty acid absorption, control obesity, and bring health benefits to consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Effects of different lipases on MLCT content;

[0019] Figure 2 :Effects of different substrate ratios on MLCT content;

[0020] Figure 3 :The influence of different temperatures on MLCT content;

[0021] Figure 4 :The effect of different reaction time on MLCT content;

[0022] Figure 5 :Effects of different enzyme addition amounts on MLCT content;

[0023] Figure 6 : Interaction diagram between factors in the response surface;

[0024] Figure 7 : Gas chromatogram before the reaction of olive oil and MCT;

[0025] Figure 8 : Gas chromatograms after reaction of olive oil, MCT and MLCT. DETAILED DESCRIPTION

[0026] The invention provides a method for preparing MLCT, comprising the following steps: mixing olive oil and medium-chain triglycerides, adding immobilized lipase, reacting to obtain a crude product, and separating and purifying the crude product to obtain MLCT; the immobilized lipase is Lipozyme TLIM.

[0027] The present invention uses olive oil and medium-chain triglycerides as substrates (acyl donors), and in a solvent-free system, olive oil and medium-chain triglycerides are mixed in proportion and then subjected to an ester exchange reaction. The mass ratio of olive oil to medium-chain triglycerides in the present invention is 3:2 to 7:3, preferably 3.15:1.85. Preferably, the olive oil in the present invention is virgin olive oil; the fatty acids in the olive oil are mainly oleic acid and linoleic acid; the oleic acid content is 75 to 80%, and the linoleic acid content is 5 to 7%. Preferably, the medium-chain triglycerides in the present invention are mainly caprylic acid and capric acid, the caprylic acid content is 60 to 62%, and the capric acid content is 38 to 40%. The present invention does not limit the sources of olive oil and medium-chain triglycerides.

[0028] The present invention utilizes olive oil and medium-chain triglycerides to prepare MLCT, which can not only improve the thermal stability and oxidative stability of MLCT, but also provide long-chain monounsaturated fatty acids. Moreover, the prepared MLCT is easier to digest, absorb and metabolize in the human body than long-chain fatty acids, thus providing a rapid energy source for the body.

[0029] The present invention adds Lipozyme TLIM to a mixed system of olive oil and medium-chain triglycerides to catalyze transesterification reaction. Based on the total mass of the olive oil and the medium-chain triglycerides, the addition amount of the immobilized lipase is 6-7.5wt%, preferably 7-7.2wt%.

[0030] The temperature of the reaction of the present invention is 59-60°C, and the reaction time is 2-4h, preferably 3h. As an optional embodiment, the reaction of the present invention is preferably stirred under constant temperature water bath conditions, that is, the present invention adds immobilized lipase, avoids light, and stirs the reaction in a water bath at 60°C on a magnetic stirrer for 3h; after the reaction is completed, centrifuge at 4200rpm for 5min, fill the supernatant with nitrogen, and seal (to avoid oil oxidation), which is the crude product.

[0031] Preferably, the present invention purifies and separates the crude product obtained by the reaction, and the separation and purification method comprises: sequentially performing deacidification, water removal, n-hexane extraction, and reduced pressure evaporation treatment on the crude product.

[0032] The deacidification method of the present invention comprises: mixing the crude product with anhydrous ethanol, stirring at 35-37° C. for 25-35 minutes, and discarding the ethanol phase. The temperature is preferably 36° C., the stirring time is preferably 30 minutes, and the stirring is preferably stirred with a magnetic stirrer. The deacidification step of the present invention is preferably repeated 5-6 times, that is, after discarding the ethanol phase, anhydrous ethanol is added again for mixing.

[0033] The dewatering method of the present invention comprises: mixing the deacidified product with silica gel, centrifuging after stirring, and discarding the precipitate. Preferably, the mass ratio of the deacidified product to the silica gel is 15 to 20:1, more preferably 16 to 17:1. The stirring is preferably stirred with a magnetic stirrer; the stirring time is 1.5 to 2.5 hours, preferably 2 hours; the centrifugal condition is 4000 to 4500 rpm, and the centrifugation is 4 to 6 minutes; the centrifugal speed is preferably 4200 rpm, and the centrifugal time is preferably 5 minutes.

[0034] The n-hexane extraction method of the present invention comprises: adding n-hexane to the product (supernatant) after water removal, the n-hexane addition ratio is preferably 1:2 (V / V), the number of extractions is preferably 2 times, the extract is preferably contained in a brown bottle, left to stand overnight at -20°C, and filtered to obtain the extract.

[0035] The temperature of the reduced pressure evaporation treatment of the present invention is 65-75°C, and the rotation speed is 90-110rpm, the temperature is preferably 70°C, and the rotation speed is preferably 100rpm. The reduced pressure evaporation of the present invention is preferably carried out using a rotary evaporator. The sample obtained after the reduced pressure evaporation removal of the present invention is the purified MLCT. The purified MLCT of the present invention is preferably frozen at -20°C.

[0036] The present invention also provides MLCT prepared by the above preparation method. The medium and long chain triglycerides of the purified MLCT can reach 82.7%. The long chain fatty acids (from olive oil) are distributed at a higher level at the Sn-2 position, reaching 81.96%. At the Sn-2 position, the content of oleic acid is close to 50%, and more than 80% are long chain fatty acids.

[0037] The present invention also provides the use of the MLCT in food processing. The prepared MLCT can improve the nutritional components of food, replace traditional vegetable fats, improve the absorption of essential fatty acids, and control obesity.

[0038] In the following examples, unless otherwise specified, all are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0039] In a specific embodiment of the present invention, the preparation method of the physical mixed oil is as follows: olive oil and medium-chain triglycerides are mixed according to a mass ratio of 3.15:1.85. Olive oil was purchased from Yihai Kerry Grain and Oil Industry (Wuhan) Co., Ltd., and the olive oil contained 79.41% oleic acid, 6.59% linoleic acid, and 14% other fatty acids; medium-chain triglycerides (MCT) were purchased from McLean Biochemical Technology Co., Ltd. (Shanghai), with a purity of ≥98%, and MCT contained 61.33% caprylic acid and 38.67% caprylic acid. Pancreatic lipase was purchased from McLean Biochemical Technology Co., Ltd. (Shanghai). Lipozyme TLIM (275IUN / g), Lipozyme RMIM (250IUN / g), Novozyme 435 (10000PLU / g), and Lipozyme 435 (8000PLU / g) were all purchased from Novozymes Biotechnology (Shanghai) Co., Ltd. 37 fatty acid methyl ester standards were purchased from Source Leaf Biotechnology (Shanghai) Co., Ltd.

[0040] In a specific embodiment of the present invention, the magnetic stirrer is a T21-3 constant temperature magnetic stirrer, the rotation speed of the magnetic stirrer is 500 rpm, and the power is 16W.

[0041] In the specific embodiment of the present invention, the measured data were analyzed using SPSS 22.0 software version. All reactions and tests were performed in triplicate. The mean value was expressed as mean ± standard deviation, and P < 0.05 was considered to be significantly different. The response surface data were analyzed using Design-Expert V8.0.6 software.

[0042] Example 1

[0043] Optimization of conditions for MLCT preparation:

[0044] 1. Effects of different immobilized lipases on MLCT preparation

[0045] Four immobilized lipases (specific characteristics are shown in Table 1) were selected and transesterification was carried out on a magnetic stirrer. The enzyme dosage (based on the total mass of olive oil and MCT) was 6 wt%, the reaction temperature was 50 °C, the reaction time was 5 h, the olive oil: MCT ratio was 3:2 (w / w), and the total mass of olive oil and MCT was 5 g.

[0046] Table 1 Lipase source, specificity, reaction temperature, water content, color and morphology

[0047]

[0048] By catalyzing the reaction of olive oil and MCT, the yield of the generated MLCT was used as the reaction index. The results are as Figure 1 shown. The results showed that the yields of MLCT synthesized by different enzymes, from low to high, were: Novozyme435 < Lipozyme 435 < Lipozyme RMIM < Lipozyme TLIM, ranging from 47.40 wt% to 74.42 wt%. It shows that the type of lipase has an impact on the reaction yield. In terms of cost, Lipozyme 435, Novozyme 435 and Lipozyme RMIM are expensive. Therefore, Lipozyme TLIM was used as the substrate catalyst for the preparation of MLCT.

[0049] 2. Influence of different substrate ratios on the preparation of MLCT

[0050] Transesterification was carried out on a magnetic stirrer. The dosage of Lipozyme TLIM (based on the total mass of olive oil and MCT) was 6 wt%, the reaction temperature was 50 °C, the reaction time was 5 h, and the ratios of olive oil:MCT were 2:3, 1:1, 3:2, 7:3, 8:2 (w / w) respectively, and the total mass of olive oil and MCT was 5 g.

[0051] By catalyzing the reaction of olive oil and MCT, the yield of the generated MLCT was used as the reaction index. The results are as Figure 2 shown. The results showed that when the mass ratio (Olive:MCT) increased from 3:2 to 7:3, the MLCT content increased rapidly, reaching 75.47 wt%. When the mass ratio of Olive to MCT exceeded 7:3, with the increase of the olive oil content, the MLCT content decreased. The increase in the olive oil content reduced the insertion rate of medium-chain fatty acids. Therefore, the mass ratio of olive oil to MCT for the preparation of MLCT was 7:3.

[0052] 3. Influence of different reaction temperatures on the preparation of MLCT

[0053] Transesterification was carried out on a magnetic stirrer. The dosage of Lipozyme TLIM (based on the total mass of olive oil and MCT) was 6 wt%, the reaction time was 5 h, the ratio of olive oil:MCT was 3:2 (w / w), and the total mass of olive oil and MCT was 5 g. The reaction temperatures were 40 °C, 50 °C, 60 °C, 70 °C, 80 °C respectively.

[0054] By catalyzing the reaction of olive oil and MCT, the yield of the generated MLCT was used as the reaction index. The results are as Figure 3As shown. The results show that when the temperature rises from 40°C to 60°C, the MLCT content continues to rise, reaching 72.74wt%. Within a certain range, as the temperature increases, the solubility of the reactants increases, the viscosity of the mixture decreases, and the degree of transesterification between Olive and MCT becomes higher and higher. When the temperature exceeds 60°C, the MLCT yield begins to decrease as the temperature increases. After exceeding the appropriate temperature, the activity of the enzyme is affected. Therefore, the reaction temperature for preparing MLCT is 60°C.

[0055] 4. Effect of different reaction times on MLCT preparation

[0056] The transesterification was carried out on a magnetic stirrer, the amount of Lipozyme TLIM (based on the total mass of olive oil and MCT) was 6wt%, the reaction temperature was 50°C, the olive oil:MCT ratio was 3:2 (w / w), the total mass of olive oil and MCT was 5g. The reaction times were 2h, 3h, 4h, 5h, and 6h, respectively.

[0057] The MLCT yield was used as the reaction index by catalyzing the reaction between olive oil and MCT. Figure 4 As shown. The results show that in the early stage of the reaction, the substrate concentration is high and the reaction mainly proceeds in the positive direction. When the reaction lasts for 3 hours, the ester exchange gradually tends to equilibrium and the MLCT content reaches 70.51%. The MLCT content is also gradually decreasing with time. The main reason may be that the concentration of structural lipids increases, which leads to an increase in the reverse reaction rate. Therefore, 3h is selected as the optimal reaction time.

[0058] 5. Effect of different enzyme addition amounts on MLCT preparation

[0059] The ester exchange was carried out on a magnetic stirrer with a reaction temperature of 50°C, a reaction time of 5 h, an olive oil:MCT ratio of 3:2 (w / w), a total mass of olive oil and MCT of 5 g, and Lipozyme TLIM dosages (based on the total mass of olive oil and MCT) of 1 wt%, 2 wt%, 4 wt%, 6 wt%, and 8 wt%, respectively.

[0060] The MLCT yield was used as the reaction index by catalyzing the reaction between olive oil and MCT. Figure 5 As shown. The results show that as the amount of enzyme added increases, the content of MLCT gradually increases, and the content of MLCT is the highest when the enzyme addition amount is 6wt%. When the enzyme addition amount exceeds 6wt%, the content of MLCT decreases slightly, which may be due to excessive enzyme addition. Aggregated together and unable to fully contact the substrate, thereby reducing the catalytic efficiency of Lipozyme TLIM. Therefore, the best enzyme addition amount is 6wt% of the substrate weight.

[0061] 6. Response surface analysis

[0062] According to the results of the single factor test, the effects of the interaction of various factors on the MLCT yield were further studied. The substrate ratio (X1), reaction temperature (X2), and enzyme addition amount (X3) were used as independent variables, and the MLCT yield was used as the response value. The BBD response surface test was performed. The response surface was designed by the software Design Expert 8. The specific levels of the response surface test factors are shown in Table 2. The test results are shown in Table 3.

[0063] Table 2 Response surface factor levels

[0064]

[0065] Table 3 Box-Behnken matrix experimental results

[0066]

[0067] Note: The values ​​of 0.6, 0.7 and 0.8 represent the mass ratios of olive oil and MCT as 3:2, 7:3 and 4:1 respectively; the values ​​of 1, 2 and 3 in X3 represent the addition amounts of enzyme as 4wt%, 6wt% and 8wt% respectively.

[0068] The quadratic polynomial regression equation of substrate ratio (X1), reaction temperature (X2) and enzyme addition amount (X3) is as follows: Y = -117.67325 + 240.865X1 + 2.95045X2 + 24.0265X3 - 0.0375X1X2 + 4.325X1X3 - 0.25975X2X3 - 199.225X12 - 0.019072X22 - 2.20725X32 (Formula 1)

[0069] The results of variance analysis are shown in Table 4.

[0070] Table 4 Analysis of variance of the response surface four-component model

[0071] Sources of variation sum of squares Degrees of Freedom Mean Square F-number P>F Significance Model 233.09 9 25.90 272.36 <0.0001 *** A-X1 80.14 1 80.14 842.76 <0.0001 *** B-X2 10.76 1 10.76 113.21 <0.0001 *** C-X3 55.76 1 55.76 586.36 <0.0001 *** AB 5.63E-03 1 5.63E-03 0.059 0.8148 AC 0.75 1 0.75 7.87 0.0263 BC 26.99 1 26.99 283.82 <0.0001 *** A2 16.71 1 16.71 175.75 <0.0001 *** B2 15.32 1 15.32 161.07 <0.0001 *** C2 20.51 1 20.51 215.73 <0.0001 *** Residual 0.67 7 0.095 Lack of Fit 0.51 3 0.17 4.56 0.0885 Pure Error 0.15 4 0.038 Total difference 233.76 16

[0072] According to Table 4, the regression model determination coefficient R 2=0.9972, the P value of the model is <0.0001, indicating that the regression model has a very high significance, and there is a high correlation between the test value and the fitting value. The P value of lack of fitting is not significant, indicating that the model has a good degree of fitting and can better reflect the relationship between the factors and the response value (MLCT content). Therefore, the model can be used to predict the actual situation of the transesterification reaction. As can be seen from Table 4, there is a significant interaction between temperature and the amount of enzyme added. The linear and quadratic coefficients X1, X2, X3, X12, X22, and X32 have extremely significant effects. The interaction coefficient X2X3 has a significant effect. It shows that the MLCT content is affected by the substrate ratio, reaction temperature, the amount of enzyme added, and the interaction between the amount of enzyme added and the reaction temperature.

[0073] The interaction diagram between the factors in the response surface is shown in Figure 6 In the figure, (a, b) shows the interaction between reaction temperature and substrate ratio (olive oil: MCT) on MLCT yield when the enzyme addition amount is 6wt%; (c, d) shows the interaction between enzyme addition amount and substrate ratio on MLCT yield when the temperature is 60℃; (e, f) shows the interaction between enzyme addition amount and temperature on MLCT yield when the substrate ratio (olive oil: MCT) is 7:3.

[0074] Figure 6 (a, b) show that the maximum MLCT yield occurs when the substrate ratio (olive oil: MCT) is 3:2-7:3 and the temperature is 55-70°C. The interaction between temperature and substrate ratio on MLCT content is not significant. At higher temperatures, the MLCT content first increases and then decreases with the increase of olive oil content in the substrate ratio. This may be because after the olive oil content increases to a certain value, MCT is insufficient, so that the reaction yield no longer increases. As the reaction temperature increases, the MLCT content also continues to increase. While maintaining enzyme activity, higher temperatures also reduce the viscosity of the mixture and increase the solubility of the reactants. The increase in temperature increases the reaction rate and the degree of transesterification.

[0075] Figure 6 (c, d) show that the highest MLCT content appears in the range of substrate ratio 3:2-7:3 and enzyme addition amount 5wt%-8wt%. The interaction between enzyme addition amount and substrate ratio on MLCT content is not significant. When the enzyme addition amount is high, the MLCT content first increases and then decreases with the increase of olive oil content. When the enzyme addition amount is within the optimal range, the content of medium- and long-chain triglycerides increases with the increase of enzyme amount. As the enzyme amount increases, the reaction rate also increases. When the enzyme amount is added to a certain extent, the mass transfer resistance between the product and the substrate is too large, reducing the enzyme catalytic efficiency and reaction rate.

[0076] Figure 6(e, f) show that the interaction between the amount of enzyme added and the temperature has a significant effect. Higher temperatures and higher amounts of enzyme added result in higher MLCT content. Enzyme catalytic activity mainly affects the degree of transesterification and the reaction rate. Controlling the reaction temperature is very important because enzyme-catalyzed reactions are thermodynamic processes, which not only affect the catalytic activity of the enzyme but also the solubility and viscosity of the substrate and the mass transfer rate between the substrate and the product. Within a suitable temperature range, the transesterification yield increases with increasing temperature, and the increase in temperature increases the degree of transesterification and the reaction rate. Excessively high temperatures can inactivate the enzyme. An appropriate amount of enzyme will promote the reaction. If the amount of enzyme added in the reaction system is too high, aggregation will occur, increasing the mass transfer resistance between the substrate and the product, which is not conducive to the reaction.

[0077] Design Expert software was used to predict the optimal reaction conditions for the synthesis of MLCT (olive oil and MCT transesterification reaction): the mass ratio of olive oil and MCT, temperature, enzyme addition, and time were 3.15:1.85, 59.21°C, 7.2wt% (based on substrate mass), and 3h. In actual operation, the temperature was adjusted to 59-60°C. Under the predicted conditions, verification tests (three parallel runs) were carried out, and the MLCT contents were 75.80%, 75.96%, and 76.04%, respectively, with an average content of 75.93%. The model prediction value was 76.08%, and the experimental results were close to the predicted values, indicating that the experimental response surface model prediction results were effective.

[0078] Example 2

[0079] Preparation of MLCT:

[0080] A total mass of 5 g of olive oil and MCT were placed in a reaction vessel at a mass ratio of 3:2, and 7 wt% of immobilized lipase (Lipozyme TLIM) was added. The mixture was stirred in a water bath at 60°C for 3 h under light protection on a magnetic stirrer. After the reaction, the reaction system was centrifuged at 4200 rpm for 5 min, and the supernatant was filled with nitrogen, sealed, and stored at -20°C.

[0081] Example 3

[0082] Preparation of MLCT:

[0083] A total mass of 5 g of olive oil and MCT were placed in a reaction container at a mass ratio of 7:3, and 7.5 wt% of immobilized lipase (Lipozyme TLIM) was added. The mixture was stirred in a water bath at 59° C. on a magnetic stirrer under light protection for 3 h. After the reaction, the reaction system was centrifuged at 4200 rpm for 5 min, and the supernatant was the reaction product (crude product).

[0084] Separation and purification: The crude product was mixed with anhydrous ethanol in a ratio of 1:1 (V / V), stirred for 30 minutes with a magnetic stirrer at 36°C, the ethanol phase was discarded, and the extraction was repeated 6 times for deacidification. Take 30g of the deacidified sample and mix it with 1.8g of silica gel, stir it with a magnetic stirrer for 2h at room temperature, and centrifuge it at 4200rpm for 5min. The supernatant was added with n-hexane in a volume ratio of 1:2 and extracted twice. The extract was placed in a brown bottle, left to stand at -20°C overnight, and filtered with filter paper the next day. The n-hexane was removed by reduced pressure evaporation using a rotary evaporator at a temperature of 70°C and a speed of 100rpm. The purified sample was frozen at -20°C.

[0085] Example 4

[0086] Preparation of MLCT:

[0087] A total mass of 5 g of olive oil and MCT were placed in a reaction container at a mass ratio of 3.15:1.85, and 7 wt % of immobilized lipase (Lipozyme TLIM) was added. The mixture was stirred in a water bath at 60° C. on a magnetic stirrer under light protection for 3 h. After the reaction, the reaction system was centrifuged at 4200 rpm for 5 min, and the supernatant was the reaction product (crude product).

[0088] Separation and purification: The crude product was mixed with anhydrous ethanol in a ratio of 1:1 (V / V), stirred for 30 minutes with a magnetic stirrer at 36°C, the ethanol phase was discarded, and the extraction was repeated 6 times for deacidification. Take 30g of the deacidified sample and mix it with 1.8g of silica gel, stir it with a magnetic stirrer for 2h at room temperature, and centrifuge it at 4200rpm for 5min. The supernatant was added with n-hexane in a volume ratio of 1:2 and extracted twice. The extract was placed in a brown bottle, left to stand at -20°C overnight, and filtered with filter paper the next day. The n-hexane was removed by reduced pressure evaporation using a rotary evaporator at a temperature of 70°C and a speed of 100rpm. The purified sample was frozen at -20°C.

[0089] Example 5

[0090] The MLCT prepared in Example 4 was analyzed:

[0091] 1. Determination of MLCT quality indicators

[0092] The determination of color refers to GB / T 5009.37-2003; the determination of transparency, odor and taste refers to GB / T5525-2008; the determination of peroxide value refers to GB 5009.227-2023; the determination of acid value refers to GB 5009.229-2016. The specific parameter results are shown in Table 5.

[0093] Table 5 Quality indicators of MLCT prepared by olive oil transesterification

[0094] project MLCT odor Olive oil inherent smell, no peculiar smell Color Light yellow transparency Clarity and transparency Acid value / (KOH)mg / g 0.84±0.05 Peroxide value / (g / 100g) 0.04±0.01

[0095] The results showed that the prepared MLCT was clear, transparent and had no odor, and the acid value and peroxide value of the purified MLCT were lower than the limit standards in GB 15196-2015 edible oil and fat products.

[0096] 2. Analysis of fatty acid composition of MLCT triglycerides

[0097] (1) Analysis of total fatty acid composition: Weigh 0.1 g of oil sample, add 2 mL of n-heptane and shake well, then add 200 μL of NaOH-CH3OH solution and vortex for 1 min. After standing for 1 h, the supernatant was filtered through a 0.45 μm filter membrane, and 1 μL of the obtained fatty acid methyl ester solution was analyzed using a Trace GC Uitra gas chromatograph (Thermo Electron Corporation) equipped with a hydrogen flame ionization detector (FID). Fatty acids were separated on a highly polar gas chromatography column HP-88 capillary column (30 m×0.25 mm×0.2 μm, Agilent Technologies Inc., USA), with a nitrogen (carrier gas) flow rate of 1.0 mL / min, an injector temperature of 280°C, an FID temperature of 280°C, and a separation ratio of 1:40; the detector gas was 35 mL / min of hydrogen, 350 mL / min of air, and 30 mL / min of nitrogen. The initial column temperature was maintained at 90°C for 2 min, then increased to 170°C at a rate of 10°C / min, and then maintained at 170°C for 8 min. Finally, the temperature was increased to 230°C at a rate of 2.8°C / min and maintained at 230°C for 10 min. The retention time of the sample peak was compared with the retention time of 37 fatty acid methyl ester standards to determine the composition of FAs, and the relative content of each fatty acid was calculated by area normalization. MCT, olive oil, and physical mixed oil were used as comparisons.

[0098] (2) Analysis of the composition of the fatty acid at the Sn-2 position: Take an oil sample, add 2 mL of 1 mol / L Tris-HCl buffer (pH=7.6), 0.25 mL of 0.05% sodium cholate, 0.4 mL of 2.2% CaCl2 solution and 20 mg of pancreatic lipase, mix well, place in a 42°C water bath and shake for 4 min, then add 1 mL of 6 mol / L hydrochloric acid and 3 mL of ether, and then centrifuge the mixture at 4800 rpm for 3 min. Collect the upper layer and remove the ether by nitrogen evaporation. The product after pancreatic lipase hydrolysis is separated on a thin layer chromatography TLC plate for 60 to 70 min, the color development solvent is hexane / ether / acetic acid (50 / 50 / 1; V / V / V), and after color development with iodine vapor, collect the 2-monoacylglycerol (2-MAG) band, prepare 2-MAG fatty acid methyl ester, and analyze it according to the method of step (2).

[0099] The results are shown in Table 6.

[0100] Table 6 Fatty acid composition of olive oil, MCT, physical blended oil and MLCT

[0101]

[0102]

[0103] Note: Each value is the mean ± SD of three independent replicates; the values ​​with different superscripts in each row are significantly different (P<0.05); ND, not detected under this analytical condition.

[0104] The results showed that the fatty acids in medium-chain triglycerides were mainly caprylic acid (C8:0) and capric acid (C10:0), accounting for 61.33% and 38.67% respectively. Olive oil contains more unsaturated fatty acids, such as oleic acid (C18:1): 79.41%, linoleic acid (C18:2): 6.59%. Compared with the raw materials (reaction substrates), the physical mixed oil did not change the type of fatty acid composition, but its relative content changed. The fatty acids in the physical mixed oil and MLCT were mainly capric acid (C10:0), oleic acid (C18:1), palmitic acid (C16:0), caprylic acid (C8:0) and linoleic acid (C18:2). Compared with the physical mixed oil, the fatty acid composition of the transesterification product remained unchanged, and the relative content was not much different. The enzymatic reaction process is mild, and the fatty acid composition and relative content are almost the same after the transesterification reaction, because the double bonds and saturation on the fatty acids have not changed. After the transesterification reaction, the distribution of medium-chain fatty acids caprylic acid and capric acid at the Sn-2 position reached 20.21%. The distribution of long-chain fatty acids (from olive oil) at the Sn-2 position was higher, reaching 81.96%, of which oleic acid reached 49.82%.

[0105] 3. Analysis of MLCT structure and composition

[0106] Sample preparation: Dissolve the oil sample (20 mg) in n-hexane (10 mL) and filter through a 0.45 μm filter membrane for GC-MS analysis. Chromatographic conditions: DB-WAX fused silica capillary column (60 m × 250 μm × 0.25 μm), high-purity helium as carrier gas, flow rate of 1.5 mL per minute, split ratio of 50:1, injection volume of 1 μL, solvent delay of 3 min. Inlet temperature, interface temperature and column temperature were 350 ° C, 350 ° C and 100 ° C, respectively. The column temperature was maintained at 100 ° C for 1 min, then increased to 150 ° C (10 ° C / min), then increased to 270 ° C (20 ° C / min), and finally increased to 340 ° C (5 ° C / min), and maintained for 71 min. Mass spectrometry conditions: electron ionization source, electron energy of 70 eV, ion source temperature of 250 °C, scanning range of 35-1000 m / z, electron multiplier voltage of 1.444 kV.

[0107] Taking olive oil and MCT as comparison, the gas chromatogram before the reaction of olive oil and MCT is as follows: Figure 7 As shown, the gas chromatograms of olive oil, MCT and MLCT after reaction are as follows Figure 8 shown.

[0108] Table 7 TG distribution of physical mixed oil and MLCT

[0109]

[0110]

[0111] Note: C8: Caprylic acid; C 10 : capric acid; P: palmitic acid; S: stearic acid; O: oleic acid; L: linoleic acid; Ln: linolenic acid; Each value represents the mean and standard deviation (SD) of three independent replicates; ND means not identified.

[0112] The results showed that olive oil is mainly composed of triglycerides composed of fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. MCT is a triglyceride composed of caprylic acid and caprylic acid. MLCT was synthesized by transesterification of olive oil and MCT under the catalysis of 1,3-specific lipase Lipozyme TLIM. Before and after the reaction, the types and relative contents of TG molecules changed greatly. The mixed oil before the reaction contained only C 24 , C 26 , C 28 , C 30 Medium chain triglycerides and total carbon number C 50 , C 52 , C 54Long-chain triglycerides. MLCT contains four types of TG: MMM, MML, MLL and LLL. As shown in Table 7, after two-step purification, MLCT in the purified product can reach 82.7%. About 80.83% of TG in MLCT is MLCT (MML and MLL), and MML type accounts for 39.64%. The new TG that appears after the reaction is mainly C8C8O / C8C8L (C 34 ), C 10 C 10 P / C8C 10 S / C8C 10 O / C8C 10 L / C8C 10 Ln(C 36 )、C8OO / C8OL / C8LL(C 44 ), C 10 SO / C 10 OO / C 10 SL / C 10 OL / C 10 LL(C 46 ). After esterification of MCT and olive oil mixture, the content of MML and MLL type TG increased significantly, while the content of LLL and MMM type TG decreased. This indicates that the FAs in TG have been rearranged.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing MLCT, characterized in that: The method comprises the following steps: olive oil and medium chain triglyceride are mixed, immobilized lipase is added, a crude product is obtained by reaction, and the crude product is separated and purified to obtain MLC T; the immobilized lipase is Lipozyme TLIM.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the olive oil to the medium-chain triglyceride is 3:2 to 7:

3.

3. The preparation method according to claim 1, characterized in that: Based on the total weight of olive oil and medium-chain triglycerides, the added amount of the immobilized lipase is 6-7.5wt%.

4. The preparation method according to claim 1, characterized in that: The reaction temperature is 59-60° C.; the reaction time is 2-4 hours.

5. The preparation method according to claim 1, characterized in that: The separation and purification method comprises: the crude product is sequentially subjected to deacidification, water removal, n-hexane extraction, and reduced pressure evaporation treatment.

6. The preparation method according to claim 5, characterized in that: The deacidification method comprises: mixing the crude product with anhydrous ethanol, stirring at 35-37° C. for 25-35 minutes, and discarding the ethanol phase.

7. The preparation method according to claim 5, characterized in that: The dehydration method comprises: mixing the deacidified product with silica gel, stirring, centrifuging, and discarding the precipitate.

8. The preparation method according to claim 5, characterized in that: The temperature of the reduced pressure evaporation treatment is 65-75° C., and the rotation speed is 90-110 rpm.

9. MLCT prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the MLCT according to claim 9 in food processing.

Images