Method for preparing sn-2 site fat rich in alpha-linolenic acid structure based on enzyme method

Abstract

The invention discloses a method for preparing sn-2 site fat rich in alpha-linolenic acid structure based on an enzymatic method, and relates to the technical field of biology, and the method comprises the following steps: (1) enzymatic acidolysis: mixing an alpha-linolenic acid product (the purity is greater than or equal to 70%), walnut oil and deionized water according to a ratio of 1: 1-5: 1 (mol / mol), and placing in a spiral test tube with a plug; adding 1 to 20 percent (w / w) of lipase Novozym 435 into the test tube; after the reactants are fully mixed, carrying out nitrogen charging treatment on the test tube to remove oxygen, and then tightly sealing the test tube; placing the test tube on a shaking table, setting the rotating speed to be 200r / min, and reacting at the temperature of 35-75 DEG C for 8-48 hours to obtain an acidolysis product. And (2) molecular distillation purification: putting the enzymatic acidolysis product into a molecular distillation instrument, and carrying out molecular distillation on the enzymatic acidolysis product to purify the crude product of the acidolysis product. The invention provides the preparation method of the structural grease, which has the advantages of high access rate, easiness in product purification and lower cost.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a structured lipid rich in alpha-linolenic acid at the sn-2 position based on an enzyme method. Background Art

[0002] Structured lipids are a new type of functional lipids with broad application prospects and research value. In the food industry: structured lipids can be used as a substitute for low-fat foods, with the effects of regulating blood lipids, lowering cholesterol, and preventing cardiovascular diseases. In addition, structured lipids can also be used as functional additives to improve the texture and taste of food. In the pharmaceutical industry: structured lipids have good biocompatibility and biodegradability, and can be used as drug carriers or controlled-release agents to improve the bioavailability of drugs and reduce toxic side effects. In the cosmetics industry: structured lipids have good antioxidant and moisturizing properties, and can be used in skin care products, lipsticks, and lip balms; in the fuel industry: structured lipids can be used as raw materials for biodiesel, which has the advantages of environmental protection and renewability compared to traditional petroleum fuels.

[0003] Structural lipids are composed of different compositions or distributions of fatty acids in the triglyceride backbone, which can be obtained through chemical, enzymatic or genetic modification. Triglycerides have three fatty acid binding sites (sn-1, sn-2 and sn-3), among which the fatty acids at the sn-2 position are easier for the human body to absorb, that is, the fatty acid composition and content at this site can affect the physiological metabolism of the human body.

[0004] The main methods for preparing structural lipids are: (1) Chemical transesterification. As a mature technology in the field of oil modification, chemical transesterification is known for its low cost, perfect process flow and convenience of large-scale production. However, this technology also faces significant limitations: the randomness of the reaction process makes it difficult to accurately control the product composition and it is difficult to meet the production requirements of lipids with specific physiological functions. (2) Direct esterification. The advantage of using this technology is that the reaction must be carried out once, and under the corresponding substrate ratio environment, there are relatively few by-products, and the product separation operation is simple. However, the disadvantage of this method is that the raw materials need to be prepared separately, so the cost is relatively high. (3) Transesterification. This reaction involves the exchange of internal and external glycerol molecules. The operation is simple and flexible. Different oils can be used directly or partially hydrolyzed first. It is worth mentioning that the overall production cost is greatly reduced due to the low cost and easy availability of raw materials. However, this reaction has the problem of low yield, and the content of monoglyceride and diglyceride in the product is relatively high, which brings certain challenges to the subsequent separation work. (4) Glycerol hydrolysis. This method is suitable for the preparation of monoglyceride and diglyceride. (4) Acid hydrolysis: This process cleverly combines the hydrolysis of biological enzymes with the subsequent esterification reaction to achieve directional adjustment of the molecular structure of oils. The raw oils for this method are widely available, with low production costs, and the excess free fatty acids can be easily removed by molecular short-range distillation.

[0005] Therefore, in order to solve the above technical problems, it is necessary to provide a new technical solution to solve this technical problem, especially a method for preparing sn-2 position rich α-linolenic acid structured lipids based on enzymatic method. Summary of the invention

[0006] The purpose of the present invention is to solve the technical problems of high cost, low access rate and inconvenient separation and purification of structural lipids in existing methods, and provide a method for preparing sn-2 position rich α-linolenic acid structural lipids based on enzyme method.

[0007] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing a sn-2 position-rich α-linolenic acid structural lipid based on an enzymatic method, comprising the following steps:

[0008] (1) Enzymatic acid hydrolysis

[0009] S11. Mix the α-linolenic acid product and walnut oil in a ratio of 1:1-5:1 (mol / mol) and deionized water in a ratio of 1%, and place in a screw-stoppered test tube;

[0010] S12. 1-20% (w / w) lipase Novozym 435 was added to the test tube;

[0011] S13. The reactants are thoroughly mixed, the test tube is filled with nitrogen to exclude oxygen, and then the test tube is tightly sealed;

[0012] S14. Place the test tube on a shaker, set the speed to 200 r / min, and react at a temperature of 35-75°C for 8-48 hours to obtain an acid hydrolysis product;

[0013] (2) Molecular distillation purification

[0014] S21. placing the product of enzymatic acid hydrolysis in a molecular distillation apparatus, setting the parameter conditions of the molecular distillation apparatus; and performing molecular distillation on the product of enzymatic acid hydrolysis to purify the crude product of acid hydrolysis.

[0015] Preferably, the parameter conditions of the molecular distillation apparatus are: preheating 80°C, feed 2mL / min, wall temperature 180°C, scraper 100r / min, condensed water 25°C, and pressure 1Pa.

[0016] Preferably, in step (1), the molar ratio of α-linolenic acid to walnut oil is 3:1, the amount of lipase Novozym 435 is 10%, the reaction temperature is 55° C., and the reaction time is 24 h.

[0017] Preferably, in step (1), the molar ratio of α-linolenic acid to walnut oil is 2.65:1, the dosage of lipase Novozym 435 is 7.65%, the reaction temperature is 55° C., and the reaction time is 38 h.

[0018] Preferably, the α-linolenic acid content in the α-linolenic acid product is ≥ 70%.

[0019] The beneficial effects of the present invention are:

[0020] The invention uses an enzymatic acid hydrolysis method to prepare a lipid structure rich in α-linolenic acid at the sn-2 position, and the total accession rate of α-linolenic acid and the accession rate at the sn-2 position can reach 20.46% and 10.33%, respectively, and the product accession rate is high. At the same time, the yield reaches more than 70%, and the product yield is high. After molecular distillation purification, the acid value of the product is reduced from 88.7 mg KOH / g to 0.43 mg KOH / g, and the triglyceride (TAG) content in the product reaches more than 90%, which is convenient for purification and has a high purity. 75.35% of the prepared triglyceride products contain α-linolenic acid. At the same time, the lipase can be reused more than 10 times and still maintain a high activity, thereby reducing the production cost.

[0021] The product prepared by the present invention has an acid value and a peroxide value lower than the national standard level while maintaining the basic level of crude oil after the enzymatic reaction, and has no significant changes in nutritional indicators and functional group changes, and has good thermal stability. 75.35% of the prepared triglyceride products contain α-linolenic acid, have good thermal stability, can be used as a substitute for low-fat food, and have the effects of regulating blood lipids, lowering cholesterol, and preventing cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The enzymatic acid hydrolysis conditions of the present invention are screened Figure 1 .

[0023] Figure 2 The enzymatic acid hydrolysis conditions screening of the present invention Figure 2 .

[0024] Figure 3 The enzymatic acid hydrolysis conditions screening of the present invention Figure 3 .

[0025] Figure 4 The enzymatic acid hydrolysis conditions screening of the present invention Figure 4 .

[0026] Figure 5 It is a response surface diagram of the total access rate of ALA affected by the interaction of factors in the present invention.

[0027] Figure 6 It is a response surface diagram of the interaction of factors affecting the sn-2 access rate of ALA in the present invention.

[0028] Figure 7 It is a schematic diagram of the enzyme activity of the lipase of the present invention after multiple tests.

[0029] Figure 8 It is a schematic diagram comparing the product of the present invention and crude oil.

[0030] Fig. 9 It is a schematic diagram of the thermal stability analysis structure of the product of the present invention.

[0031] Fig.10 It is a comparative schematic diagram of infrared analysis of the products of the present invention. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] Professional term explanation: Triglycerides have three fatty acid binding sites (sn-1, sn-2 and sn-3), among which the fatty acid at sn-2 is easier for the human body to absorb. The sn-2 in this specification refers to the second fatty acid binding site of triglycerides. Walnut oil (WO), alpha-linolenic acid (ALA), sn-2 rich alpha-linolenic acid structural lipid (ALA-SL).

[0036] The Chinese meaning of the English abbreviations: L (lauric acid), MA (myristic acid), PA (palmitic acid), SA (stearic acid), OA (oleic acid), LA (linoleic acid), ALA (α-linolenic acid).

[0037] 1. Embodiment:

[0038] (I) Preparation of structural lipids

[0039] Enzymatic acid hydrolysis: Mix α-linolenic acid product (purity ≥ 70%), walnut oil in a ratio of 1:1-5:1 (mol / mol) and deionized water at a ratio of 1%, and place in a spiral stopper test tube. Subsequently, add 1-20% (w / w) of lipase Novozym 435 to the test tube. After ensuring that all reactants are fully mixed, the test tube is filled with nitrogen to exclude oxygen, and then the test tube is tightly sealed. Next, the test tube is placed on a shaker, the speed is set to 200r / min, and the acid hydrolysis product can be obtained after 8-48h of reaction at a temperature of 35-75°C. This process aims to produce the target product by acidolysis of walnut oil and α-linolenic acid (ALA) through enzyme catalysis.

[0040] Taking into account the acid value and yield of the product, the product of enzymatic acid hydrolysis was placed in a molecular distillation apparatus, and the parameter conditions of the molecular distillation apparatus were set: preheating 80°C, feed 2mL / min, wall temperature 180°C, scraper 100r / min, condensed water 25°C, pressure 1Pa, and molecular distillation was performed to purify the crude product rich in α-linolenic acid structural lipids (ALA-SL) at the sn-2 position, and a secondary heavy phase was obtained through secondary molecular distillation.

[0041] In some embodiments, in step (1), the molar ratio of α-linolenic acid to walnut oil is 3:1, the amount of lipase Novozym435 is 10%, the reaction temperature is 55° C., and the reaction time is 24 h.

[0042] In a more preferred embodiment, in step (1), the molar ratio of α-linolenic acid to walnut oil is 2.65:1, the amount of lipase Novozym 435 is 7.65%, the reaction temperature is 55° C., and the reaction time is 38 h.

[0043] The comparative experiment of optimizing experimental parameter conditions is as follows:

[0044] (II) Preliminary screening of conditions for enzymatic acid hydrolysis

[0045] (1) Substrate molar ratio

[0046] The enzymatic reaction was carried out at five gradient molar ratios of α-linolenic acid (ALA) / walnut oil (mol / mol) substrate, namely 1:1, 2:1, 3:1, 4:1, and 5:1. The other reaction conditions were: 1% water content in the system, 10% enzyme loading, 55°C reaction temperature, and 24h reaction time.

[0047] like Figure 1 As shown, when the ratio of α-linolenic acid (ALA) / walnut oil is 3:1, the total accession rate and sn-2 accession rate of α-linolenic acid (ALA) reach 14.87% and 5.57% respectively. When the substrate ratio exceeds 3:1, the increase in the content of α-linolenic acid (ALA) in walnut oil is not obvious, that is, the substrate molar ratio is determined to be 2:1, 3:1, and 4:1 for optimization experiments.

[0048] (2) Enzyme loading

[0049] The enzymatic reaction was carried out at five gradient enzyme loadings (w / w) of 1, 5, 10, 15, and 20%, respectively. Other reaction conditions were: system water content of 1%, substrate molar ratio of 3:1, reaction temperature of 55°C, and reaction time of 24h.

[0050] like Figure 2As shown in the figure, when Novozym 435 is used, the total access rate of α-linolenic acid (ALA) increases with the increase of enzyme dosage, but when the enzyme dosage is greater than 10%, the total access rate of α-linolenic acid (ALA) decreases. Since the water content of the system does not increase with the increase of enzyme dosage, the water content in the system is insufficient for the enzyme to exert its maximum activity, thereby reducing the access rate. That is, the enzyme loading of 5%, 10%, and 15% was determined for optimization experiments.

[0051] (3) Reaction temperature

[0052] The enzymatic reaction was carried out at five gradient temperatures of 35°C, 45°C, 55°C, 65°C and 75°C, respectively. Other reaction conditions were: system water content 1%, substrate molar ratio 3:1, enzyme loading 10% and reaction time 24h.

[0053] like Figure 3 As shown in the figure, when the temperature rises from 35°C to 55°C, the total accession rate and sn-2 accession rate of α-linolenic acid (ALA) gradually increase, and the accession rates are 13.8% and 2.31%, respectively. When the temperature reaches 55°C, the accession degree of α-linolenic acid (ALA) is the largest. However, when the temperature continues to rise, the accession degree decreases slightly, which may be due to the inactivation of some enzymes, reducing the catalytic efficiency. It can be seen that the optimal temperature for Novozym 435 to catalyze this acidolysis reaction is 45-65°C. That is, the reaction temperatures of 45°C, 55°C, and 65°C were determined for optimization experiments.

[0054] (4) Response time

[0055] The enzymatic reaction was carried out at five gradient temperatures of 8, 10, 12, 24, 36 and 48 h, respectively. Other reaction conditions were: 1% water content in the system, 3:1 substrate molar ratio, 10% enzyme loading and 55°C reaction temperature.

[0056] like Figure 4 As shown, in the first 8 to 36 hours, the total accession rate of α-linolenic acid (ALA) increased from 7.59% to 16.42%, and the accession rate at the sn-2 position increased from 0.92% to 5.72%. After 36 hours, the accession rate of α-linolenic acid (ALA) did not increase significantly, indicating that the best time for Novozym 435 to catalyze this acidolysis reaction is 24 to 48 hours. That is, the reaction time of 24 hours, 36 hours, and 48 hours was determined for optimization experiments.

[0057] (III) Experimental condition parameters of enzymatic acid hydrolysis process response surface optimization

[0058] The influencing parameters of the response surface optimization experiment were selected from the results of the initial screening of conditions for the enzymatic acid hydrolysis reaction. Using the above results, the following experiments were carried out.

[0059] The substrate molar ratio (A), enzyme loading (B), reaction temperature (C) and reaction time (D) were selected as influencing factors, and the total accession rate of α-linolenic acid (ALA) and the sn-2 accession rate of α-linolenic acid (ALA) were used as response values. The response surface optimization experiment with four factors and three levels was designed using DesignExpert 8.0. In order to analyze and optimize the influence of process parameters on the response variables, the RSM design included 29 groups of randomized experiments. The experiment was carried out randomly, and the experimental values ​​were averaged. The total accession rate and sn-2 accession rate were measured by GB5009.168-2016 National Food Safety Standard for the Determination of Fatty Acid Content in Food and GB / T24894-2010 for the Determination of 2-position of Triglycerides in Animal and Plant Oils and Fats, respectively. The response surface optimization design scheme and results are shown in Table 1.

[0060] Table 1 Response surface optimization design scheme and results

[0061]

[0062]

[0063] With A, B, C, and D as independent variables and Y1 and Y2 as response values, the optimized fitting quadratic equations of the total access rate of ALA and the sn-2 access rate of ALA were obtained through analysis with Design Expert 8.0.6 software:

[0064] Y1(%)=20.40+2.36A+2.59B+0.9308C+0.4475D+0.5150AB+0.1000AC-0.2700AD+0.7275BC-0.0075BD-0.8450CD-2.10A2-3.37B2-2.08C2-1.10D2

[0065] Y2(%)=10.59+0.9867A+1.19B+0.4117C+0.3467D+0.3475AB+0.4825AC-0.0 650AD-0.0950BC+0.2875BD+0.0275CD-0.5077A2-1.31B2-1.42C2-0.6952D2

[0066] Table 2 Regression analysis

[0067]

[0068]

[0069] Note: P<0.05 indicates statistical significance; P<0.01 indicates relatively significant statistics; P<0.001 indicates extremely significant statistics

[0070] In line with the expected results, all the first-order coefficients are positive, which means that each factor has a positive direct impact on the results; while the second-order coefficients are all negative, indicating that the impact of each factor on the results has a nonlinear decreasing effect. At the same time, the cross-term coefficients vary in positive and negative, revealing that when the factors work together, they may produce both synergistic and offsetting effects, that is, there are complex interactions between the factors. Figure 5 and Figure 6 As shown in the figure, the 3D surface plot generated by the response surface model can well explain the relationship between reaction parameters and response values. The maximum total ALA accession rate and sn-2 position accession rate appear in the range of higher substrate ratio (2.00-3.00) and higher reaction temperature (50.0-60.0℃).

[0071] Figure 5 (a) substrate molar ratio and enzyme loading, (b) substrate molar ratio and reaction temperature, (c) substrate molar ratio and reaction time, (d) enzyme loading and reaction temperature, (e) enzyme loading and reaction time, (f) reaction temperature and reaction time. Figure 6 (a) substrate molar ratio and enzyme loading, (b) substrate molar ratio and reaction temperature, (c) substrate molar ratio and reaction time, (d) enzyme loading and reaction temperature, (e) enzyme loading and reaction time, (f) reaction temperature and reaction time.

[0072] based on Figure 5 and Figure 6 Based on the 3D surface data analysis, we derived a set of finely optimized production process parameters, including: the substrate molar ratio was set to a precise ratio of 2.65:1, the enzyme addition was fine-tuned to 7.65% to maximize efficiency, the reaction temperature was controlled at 55°C to maintain optimal enzyme activity, and the reaction time was optimized to 38h to ensure complete reaction. Under this optimized parameter framework, we used a predictive model to estimate the overall accession rate of ALA and its accession rate specific to the sn-2 position, which reached the expected 20.58% and 10.47%, respectively.

[0073] (IV) Figure 5 and Figure 6 Verification of optimized parameters obtained in experiments

[0074] In order to verify the effectiveness of the optimization strategy and the accuracy of the prediction model, we designed and executed a set of experiments in strict accordance with the above steps and the above optimization conditions. The experimental results show that the measured value of the overall access rate of α-linolenic acid (ALA) is 20.46%, while the specific access rate of α-linolenic acid (ALA) at the sn-2 position is 10.33%. These two data are highly consistent with the model prediction value, with very small deviations, which strongly proves the accuracy and reliability of the established response surface methodology (RSM) model. This verification process not only confirms the scientific nature of the optimization conditions, but also provides a solid theoretical basis and data support for subsequent process amplification and production applications. It can be seen that the experimental results are very consistent with the predicted results.

[0075] Through the above, preliminary screening is carried out through experiments, and the best experimental parameter data is obtained after optimization calculation through response surface. After verification, it is basically consistent with the actual conclusion, so that an experimental method with high output rate is obtained through optimized experimental condition ratio.

[0076] 2. Experimental analysis of repeated utilization of lipase, an important component of the present invention

[0077] like Figure 7 As shown, the immobilized lipase Novozym 435 in the reaction system was treated by washing and filtering with petroleum ether 2-3 times and drying in a fume hood at a constant temperature, then recovered and directly applied to subsequent multiple test batches, and the stability and reusability of its catalytic activity were deeply evaluated. Figure 7 As shown, in the continuous multiple rounds of reactions using the optimized process conditions of the present invention, the immobilized enzyme Novozym 435 still has durability and catalytic efficiency. Specifically, the immobilized enzyme can be reused at least more than ten times without significant performance degradation. Even in the eighth reaction, the total access rate of α-linolenic acid (ALA) is still as high as 17.80%. Compared with the initial reaction efficiency, this value shows that its catalytic activity can still be maintained at a high level of about 80%. The lipase used in the method of the present invention can be recycled and reused multiple times, thereby further reducing the cost of the present invention.

[0078] 3. Product distillation purity and product yield analysis

[0079] As can be seen from Table 3, before distillation, the sample had a high content of free fatty acids (FFA) and also contained a small amount of monoacylglycerol (MAG) and diacylglycerol (DAG). However, after distillation, a significant decrease in the content of FFA was observed, while the content of MAG and DAG also decreased. It is worth noting that the content of triglycerides (TAG) increased significantly, and its relative content reached 93.4%. This change indicates that the distillation process effectively purified TAG and reduced the content of other impurities, thereby improving the quality of the sample.

[0080] Table 3 Comparison of purity before and after molecular distillation

[0081] MAG (%) DAG(%) TAG(%) FFA(%) Before molecular distillation <![CDATA[5.60±1.71 a ]]> <![CDATA[1.95±0.13 a ]]> <![CDATA[45.6±1.71 b ]]> <![CDATA[46.8±1.81 a ]]> After molecular distillation <![CDATA[3.40±0.95 a ]]> <![CDATA[1.82±0.15 a ]]> <![CDATA[93.4±0.95 a ]]> <![CDATA[1.38±0.03 b ]]>

[0082] The sn-2 position-rich α-linolenic acid structural lipids (ALA-SL) were purified by molecular distillation, and the type composition of the products was analyzed by ultra-performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS).

[0083] Table 4 TAG type composition of ALA-SL products after molecular distillation

[0084]

[0085]

[0086] The specific triglyceride (TAG) structure type results are shown in Table 4. A total of 33 types of triglycerides (TAG) were identified, of which 75.35% contained α-linolenic acid (ALA). In this study, OA-ALA-ALA was the most abundant, accounting for 12.26%. Therefore, the content of triglycerides (TAG) rich in α-linolenic acid (ALA) in the product of the present invention is high, and the product prepared by this experimental method has a high yield.

[0087] 4. Analysis of the physical and chemical properties of the product of the present invention

[0088] (1) Physical and chemical indicators of the product

[0089] like Figure 8 As shown, the color of ALA-SL becomes darker and exhibits a more intense yellow color compared to the crude, walnut oil.

[0090] As shown in Table 5, compared with WO (0.03 g / 100 g), the peroxide value of ALA-SL increased to 0.04 g / 100 g, which may be due to the increase in temperature during the extraction process, which led to the generation of peroxides and hydroperoxides, but the peroxide value of ALA-SL was still lower than the edible walnut oil requirement (0.16 g / 100 g) specified in GB / T22327-2019 "National Standard Walnut Oil of the People's Republic of China". The p-anisidine values ​​of ALA-SL and WO were 3.97 and 1.50, respectively.

[0091] Table 5 Basic physical and chemical indicators of ALA-SL and WO

[0092]

[0093]

[0094] Note: Different letters in the same line indicate significant differences (P<0.05), WO is walnut oil, ALA-SL is sn-2 α-linolenic acid glyceride

[0095] (2) Thermal stability

[0096] Differential scanning calorimeter (DSC) is an important analytical instrument used to evaluate the quality of fats and vegetable oils. The peaks of the DSC curve have corresponding physical and chemical processes. Differential scanning calorimeter can reveal a variety of thermodynamic and kinetic properties of samples, such as melting enthalpy, melting point, crystallization rate and other parameters. The Asn-2 position α-linolenic acid structured lipid (ALA-SL) oil sample was analyzed to obtain the DSC melting curve diagram, as shown in Fig. 9 As shown, the melting peak endothermic temperature of the sn-2 position α-linolenic acid-rich structural lipid (ALA-SL) is 103°C, which is greater than 100°C, indicating that the thermal stability of the oil is good.

[0097] (3) Nutritional indicators

[0098] Table 6 compares the fatty acid composition and content of sn-2 α-linolenic acid-rich structural lipids (ALA-SL) and walnut oil (WO). The highest relative content of fatty acids in WO is linoleic acid, followed by α-linolenic acid. Obviously, the highest relative content of α-linolenic acid in sn-2 α-linolenic acid-rich structural lipids (ALA-SL) is because sn-2 α-linolenic acid-rich structural lipids (ALA-SL) have α-linolenic acid grafted compared to walnut oil crude oil (WO). There is no significant difference in the content of polyunsaturated fatty acids and saturated fatty acids between α-linolenic acid structural lipids (ALA-SL) and walnut oil crude oil (WO) (P>0.05). This indicates that enzymatic acid hydrolysis has no significant effect on the content of polyunsaturated fatty acids and saturated fatty acids in sn-2 α-linolenic acid-rich structural lipids (ALA-SL) compared to walnut oil crude oil (WO) (P>0.05).

[0099] Table 6 Fatty acid composition and content of ALA-SL and WO

[0100]

[0101] The results of tocopherol determination of sn-2 α-linolenic acid-rich structural lipids (ALA-SL) and walnut oil crude oil (WO) are shown in Table 7. Enzymatic acid hydrolysis had no significant effect on the γ-tocopherol and δ-tocopherol contents of WO (P>0.05), resulting in a decrease in the α-tocopherol content from 72.50 μg / g to 62.36 μg / g. The reason for this phenomenon is that enzymatic acid hydrolysis causes the degradation of α-tocopherol in WO and a decrease in its content. The content of sterols did not change significantly during the enzymatic acid hydrolysis process (P>0.05).

[0102] Table 7 Lipid concomitant contents of ALA-SL and WO

[0103] ALA-SL WO δ-Tocopherol (μg / g) <![CDATA[113.36±2.95 a ]]> <![CDATA[114.79±0.43 a ]]> γ-Tocopherol (μg / g) <![CDATA[368.57±0.38 a ]]> <![CDATA[371.47±1.63 a ]]> α-Tocopherol (μg / g) <![CDATA[62.36±2.73 b ]]> <![CDATA[72.50±1.20 a ]]> Total tocopherols (μg / g) <![CDATA[544.29±6.06 b ]]> <![CDATA[558.77±3.26 a ]]> Stigmasterol (μg / g) <![CDATA[115.57±1.47 a ]]> <![CDATA[118.37±1.37 a ]]> β-Sitosterol (μg / g) <![CDATA[0.34±0.01 a ]]> <![CDATA[0.31±0.02 a <!-- 9 -->]]> Campesterol (μg / g) <![CDATA[115.92±1.47 a ]]> <![CDATA[113.55±1.63 a ]]>

[0104] (4) Infrared spectroscopy

[0105] 1465cm-1 is the shear vibration absorption peak of aliphatic -CH2, 1654cm-1 corresponds to the vibration absorption of C=O and C=C; 2867cm-1 and 2948cm-1 represent the asymmetric and symmetric stretching vibrations of -CH2 and -CH3 in aliphatic respectively, 3006cm-1 is the -CH stretching vibration absorption peak of cis-HC=CH-, and 3485cm-1 is the absorption peak of OH-π bond.

[0106] like Fig.10 As shown, there is no obvious difference in the stretching vibration of the main characteristic peaks of sn-2 position α-linolenic acid-rich structural lipids (ALA-SL) and walnut oil (WO), indicating that enzymatic acid hydrolysis did not lead to significant changes in the characteristic functional group structure of WO.

[0107] The method provided by the present invention uses an enzymatic acid hydrolysis method to prepare a structured lipid rich in α-linolenic acid at the sn-2 position, and the total accession rate of α-linolenic acid and the accession rate at the sn-2 position can reach 20.46% and 10.33%. After molecular distillation purification, the acid value of the product is reduced from 88.7 mg KOH / g to 0.43 mg KOH / g. The yield of the structured lipid product is 41.3%, and the yield is 81.6%. At the same time, the lipase can be reused more than 10 times and still maintain a high activity.

[0108] On the prepared products: after the enzymatic reaction, the structured fat products can maintain the basic level of crude oil, and the acid value and peroxide value are still lower than the national standard level. There is no significant change in nutritional indicators and functional groups, and it has good thermal stability.

Claims

1. A method for preparing a sn-2-position α-linolenic acid-rich structural lipid based on an enzymatic method, characterized in that: The following steps are involved: (1) Enzymatic acid hydrolysis S11. Mix the α-linolenic acid product and walnut oil in a ratio of 1:1-5:1 (mol / mol) and deionized water in a ratio of 1%, and place in a screw-stoppered test tube; S12. 1-20% (w / w) lipase Novozym 435 was added to the test tube; S13. The reactants are thoroughly mixed, the test tube is filled with nitrogen to exclude oxygen, and then the test tube is tightly sealed; S14. Place the test tube on a shaker, set the speed to 200 r / min, and react at a temperature of 35-75°C for 8-48 hours to obtain an acid hydrolysis product; (2) Molecular distillation purification S21. placing the product of enzymatic acid hydrolysis in a molecular distillation apparatus, setting the parameter conditions of the molecular distillation apparatus; and performing molecular distillation on the product of enzymatic acid hydrolysis to purify the crude product of acid hydrolysis.

2. The method according to claim 1, characterized in that: The parameter conditions of the molecular distillation apparatus are: preheating 80°C, feed 2mL / min, wall temperature 180°C, scraper 100r / min, condensed water 25°C, and pressure 1Pa.

3. The method according to claim 2, characterized in that: In the step (1), the molar ratio of α-linolenic acid to walnut oil is 3:1, the amount of lipase used is 10%, the reaction temperature is 55° C., and the reaction time is 24 h.

4. The method according to claim 2, characterized in that: In the step (1), the molar ratio of α-linolenic acid to walnut oil is 2.65:1, the amount of lipase used is 7.65%, the reaction temperature is 55° C., and the reaction time is 38 hours.

5. The method according to claim 2, characterized in that: The α-linolenic acid content in the α-linolenic acid product is ≥70%.

Images