One-pot enzymatic cascade degumming and deacidifying method for grease and application of one-pot enzymatic cascade degumming and deacidifying method

Abstract

The invention belongs to the technical field of food lipid and processing, particularly discloses a one-pot enzymatic cascade degumming and deacidification method for grease and application, and particularly relates to a method for performing cascade degumming and deacidification on multiple kinds of grease, generating functional lipid, improving the quality of the grease and maintaining the flavor of the grease. According to the method, phospholipase is immobilized through the porous carrier and is successfully applied to enzymatic cascade degumming and deacidification, and degumming, deacidification or cascade degumming and deacidification can be carried out on different raw materials according to requirements. Moreover, the prepared grease is rich in functional lipid components, the flavor is efficiently reserved, the oxidation stability is improved, the nutritional quality is improved, the immobilized carrier is cheap and easy to obtain, the immobilized enzyme can be repeatedly used for multiple times and is remarkably superior to free enzyme, and the industrial application potential is huge.

Description

Technical Field

[0001] The invention belongs to the technical field of oil modification and healthy food deep processing, and discloses a preparation method of an immobilized enzyme with simultaneous edible oil degumming and deacidification functions and a one-pot enzymatic cascade degumming and deacidification method for oils. Background Art

[0002] When different edible oils are processed through traditional processes such as pressing and leaching, they often contain colloidal substances such as phospholipids (PL). In particular, the content of phospholipids is relatively high, usually between 100 mg / kg and 800 mg / kg, which can cause problems such as aftertaste and discoloration of the oil. Similarly, during the extraction, processing and storage of oils, triglycerides are easily hydrolyzed and a large amount of free fatty acids are generated, resulting in an excessively high acid value of the oil and a peculiar smell. Rancid oils are not only inedible, but also produce harmful substances, disrupting the normal metabolism of the human body and potentially causing a variety of health problems. Therefore, oil refining is a key link in removing impurities and free fatty acids from oils and improving their quality. It is also an urgent need for oil processing and efficient utilization of oil resources in my country.

[0003] At present, grease is mainly based on chemical method and physical method for degumming and deacidification refining method, and there are problems such as incomplete removal, high pollution, serious waste, many by-products, and difficulty in separation. In contrast, enzymatic refining has the advantages of mild reaction conditions, few by-products, and strong selectivity and has attracted much attention. A kind of process for optimizing the degumming of high acid value rice oil is disclosed in CN118516177A, but this method uses free enzyme and cannot be reused and the operation steps are cumbersome and cannot complete the degumming and deacidification cascade reaction simultaneously. In general, free enzyme activity is low, poor stability, and is difficult to reuse, while immobilized enzyme can not only be repeatedly used, but also can show good stability and catalytic activity. Currently common immobilized enzyme varieties are more, but all can not meet the degumming and deacidification cascade reaction. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an immobilized enzyme for the preparation and application of edible oil degumming and deacidification functions to address the deficiencies of the existing technology. The immobilized enzyme can meet the needs of edible oil degumming, deacidification, or cascade deacidification and degumming, and has the characteristics of high catalytic efficiency, simple operation, and easy product separation.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first object of the present invention is to provide a method for preparing an immobilized enzyme having both edible oil degumming and deacidification functions, comprising the following steps:

[0007] (1) Hydrophobic modification of the mesoporous carrier: The mesoporous carrier and the hydrophobic silane coupling agent are added to a n-hexane solution, ultrasonically dispersed, and the mixture is placed in a constant temperature shaker for reaction. After the reaction is completed, the hydrophobic mesoporous carrier is obtained by filtering, washing, and drying.

[0008] (2) Preparation of immobilized phospholipase: The free enzyme was immobilized on the prepared hydrophobic mesoporous carrier by physical adsorption to obtain immobilized phospholipase.

[0009] It should be noted that degumming involves the conversion of non-hydratable phospholipids in crude oil into hydrated phospholipids through the immobilized phospholipase, generating hydrated phospholipids. Hydrated phospholipids are insoluble in oil and can therefore be separated from the oil by centrifugation and removed, achieving the purpose of degumming. Deacidification, on the other hand, involves the esterification of free fatty acids in the oil with added glycerol by the immobilized phospholipase, generating functional oils such as diacylglycerol, thereby removing the free fatty acids.

[0010] Furthermore, the molar ratio of the hydrophobic silane coupling agent to the mesoporous carrier is 1:30-1:100, the ultrasonic dispersion time is 5-15 minutes, the constant temperature shaking reaction time is 1.5-3 hours, the anhydrous ethanol washing is 3-6 times, and the drying time is 12-24 hours.

[0011] Moreover, the mesoporous carrier is one or more combinations of ordered mesoporous SiO2 materials, hollow mesoporous SiO2 materials, mesoporous molecular sieves, macroporous resins, and mesoporous silica gel; the hydrophobic silane coupling agent is one or more combinations of ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, polymethylhydrogensiloxane, butyltriethoxysilane, octyltriethoxysilane, dodecyltriethoxysilane, and octadecyltriethoxysilane.

[0012] The hydrophobic mesoporous support material prepared by the above method has a size of 2-200 μm and a pore volume of 0.8-2 cm 3 / g, with a specific surface area of 800-1200m 2 / g, the mesopore diameter is 3-10nm, and the contact angle after hydrophobic modification is 110-130°.

[0013] Alternatively, the specific process for preparing immobilized phospholipase by physical adsorption is as follows:

[0014] Adding a phospholipase solution to a phosphate buffer for dilution, mixing the diluted enzyme solution with a hydrophobic carrier for immobilization to obtain an immobilized phospholipase; wherein the pH value of the phospholipase solution is 5.0-10.0, the dilution ratio is 2-10 times; the immobilization time is 0.5-2 hours, the incubation temperature is 20-30° C.; and the pH value of the phosphate buffer is 5.0-10.0;

[0015] The phospholipase is one or more combinations of phospholipase A1 from Escherichia coli, phospholipase A1 from Streptomyces alboflavin, phospholipase A1 from bumblebee venom, phospholipase A1 from the outer membrane of Salmonella typhi, and phospholipase A1 from Aspergillus niger.

[0016] The immobilized phospholipase prepared by the above method has a solid loading capacity of 50-250 mg / g, a solid loading rate of 15-35%, a thermal stability of 2-4 times that of the free enzyme, a higher stability than the free enzyme in common solvents such as methanol, ethanol, acetonitrile, acetone, ether and petroleum ether, an activity 1-3 times higher than that of the free enzyme, and a pH stability 1-2.5 times higher than that of the free enzyme.

[0017] A second object of the present invention is to provide an application of an immobilized enzyme having both edible oil degumming and deacidification functions, the method specifically comprising the following steps:

[0018] (1) Enzymatic degumming of edible oil: After heating the raw oil, add citric acid solution, cool it, add sodium hydroxide solution to adjust the pH value, add the immobilized enzyme prepared by the above method and distilled water, and carry out degumming reaction in a constant temperature water bath. After the reaction is completed, separate the solid and liquid by centrifugation. The solid product obtained is a mixture of immobilized phospholipase and phospholipids, and the resulting solution is the oil after degumming;

[0019] (2) Enzymatic deacidification of edible oil: the raw oil and acyl acceptor are mixed, and then the immobilized phospholipase is added to carry out esterification reaction in a constant temperature water bath; after the reaction is completed, the solid and liquid are separated, and the obtained solid product is the immobilized phospholipase, and the obtained solution is the oil after deacidification;

[0020] (3) Cascade degumming and deacidification of edible oil: After the edible oil is treated in step (1), the mixture of immobilized enzyme and phospholipid is removed by centrifugation, and then the deacidification of the edible oil is carried out in step (2) to achieve cascade enzymatic degumming and deacidification of the edible oil.

[0021] Furthermore, when the degummed crude oil is heated to 70-90° C., the concentration of citric acid buffer added is 30%-50% (w / w), and the added amount accounts for 0.02-0.05% of the mass of the undegummed crude oil. The concentration of NaOH added is 1-5% (w / w), the pH value is adjusted to 4-6, the amount of immobilized phospholipase added is 2-5%, the amount of distilled water added is 0-1%, the constant temperature reaction temperature is 15-45° C., and the reaction time is 1.5-3.5 hours.

[0022] The raw oil is one or a combination of rapeseed oil, linseed oil, soybean oil, corn oil, peanut oil, sesame oil, rice bran oil, camellia oil, plant oil, algae oil rich in polyunsaturated fatty acids, and fish oil.

[0023] Furthermore, the molar ratio of free fatty acids to acyl acceptors in the raw oil is 1:1-1:8; the amount of the immobilized phospholipase added is 2-5% of the substrate mass; the temperature of the constant temperature water bath is 25-45° C.; and the esterification reaction time is 3-6 hours.

[0024] The acyl acceptor is one or more combinations of monoglyceride, glycerol, D-isoascorbic acid, sterol ester, sterol, and short-chain monohydric alcohol.

[0025] The quality of edible oil after degumming by the above method is as follows:

[0026] The phospholipid content is 1-10 mg / kg, the degumming rate reaches 90-98%, the peroxide value is 0.10-0.15 g / 100 g, the oxidation induction time is 2-4 h, and the flavor characteristics remain basically unchanged.

[0027] The quality of edible oil after the above cascade degumming and deacidification is as follows:

[0028] The phospholipid content is 1-13.5 mg / kg, the degumming rate reaches 90-98%, the acid value is 1-2.5 mg KOH / g, the mass percentage of the generated diglyceride in the oil product is 5-30%, the moisture and volatile matter contents are less than 0.02%, and the peroxide value is less than 0.1 g / 100 g.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention utilizes simple process conditions to perform hydrophobic modification on the mesoporous carrier, and the preparation process is simple, the raw materials are economical and readily available, the time consumption is short, the yield is high, and it is easy to scale up.

[0031] 2. The present invention uses a hydrophobic mesoporous carrier to immobilize free phospholipase, and its activity, stability and number of reuses are all higher than those of the free enzyme; and the size of the carrier can be adjusted according to actual conditions, with a wide range of applications and diverse scenarios.

[0032] 3. The present invention discloses an immobilized enzyme that has the functions of degumming and deacidifying edible oil. Different raw materials can be selected according to needs for degumming, deacidification or degumming and deacidification cascade reaction. The process conditions are simple and efficient, and the quality of the crude product is controllable, meeting the needs of different application scenarios.

[0033] 4. The present invention has mild reaction conditions, short reaction time and simple separation throughout the entire process; compared with traditional chemical and physical methods, it does not produce by-products, large amounts of wastewater and secondary waste, effectively saving costs, and the immobilized enzyme can be regenerated and reused multiple times, with great potential for industrial application.

[0034] 5. The immobilized phospholipase prepared by the present invention has a thermal stability 2-4 times that of the free enzyme, a higher stability in common solvents such as methanol, ethanol, acetonitrile, acetone, ether and petroleum ether than the free enzyme, an activity 1-3 times higher than the free enzyme, and a pH stability 1-2.5 times higher than the free enzyme. After repeated use for 7 times, the activity still remains above 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0036] Figure 1 (a) and (b) are SEM and TEM images of the mesoporous material MCM-41 in Example 1, respectively. Figure 1 (c) is the contact angle between the hydrophobic material and water in Example 1, Figure 1 (d) is the thermogravimetric value of the mesoporous material before and after enzyme immobilization in Example 1, Figure 1 (e) and (f) are the nitrogen adsorption-desorption curves of the mesoporous material before and after enzyme immobilization in Example 1.

[0037] Figure 2 The figure is a gas chromatogram of the ARA oil composition in Example 1; wherein FFA represents free fatty acids, MAG represents monoglyceride, DAG represents diglyceride, and TAG represents triglyceride.

[0038] Figure 3 The stability comparison chart of the free enzyme and the immobilized enzyme in Example 1 when incubated for the same time at different temperatures, pH values and solvents is shown.

[0039] Figure 4 This is the relative activity of the immobilized enzyme in Example 8 after 7 reuses.

[0040] Figure 5 (a) and (b) are diagrams showing the effects of the immobilized enzyme in Example 1 on degumming and deacidification of different oils. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] The term "embodiment" is used herein specifically to describe any embodiment as "exemplary," and should not be construed as superior or preferable to other embodiments. Performance indicators in the embodiments of this application were tested using conventional testing methods in the art, unless otherwise specified. It should be understood that the terms used in this application are intended solely to describe specific implementations and are not intended to limit the disclosure herein.

[0043] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0044] In order to better illustrate the content of this application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that this application can be implemented without certain specific details. In the examples, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of this application.

[0045] Under the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of this application.

[0046] The invention discloses the preparation and application of an immobilized enzyme with the functions of degumming and deacidifying edible oil, and specifically discloses a one-pot enzymatic cascade degumming and deacidification method for oils and fats.

[0047] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content are also considered to fall within the scope of protection of the present invention.

[0048] It should be noted that the composition of oils and fats degummed and deacidified by phospholipase immobilized on hydrophobic mesoporous materials is mainly determined by gas chromatography, and the gas chromatography detection conditions are as follows:

[0049] The chromatographic column was a DB-5HT (15 m × 0.320 mm, 0.10 μm) with an inlet temperature of 380°C, an injection volume of 1 μL, and a split ratio of 50:1. High-purity helium was used as the carrier gas at a flow rate of 2 mL / min. Hydrogen flow was 32 mL / min, and air flow was 200 mL / min. The detector was an FID at 380°C. The oven temperature program was as follows: initial temperature at 170°C, hold for 2 min, then increase the temperature at 5°C / min to 380°C and hold for 6 min.

[0050] The detection of flavor substances in degummed and deacidified oils and fats catalyzed by phospholipase immobilized on hydrophobic mesoporous materials is mainly carried out by gas chromatography-mass spectrometry. The detection conditions of gas chromatography-mass spectrometry are as follows:

[0051] Chromatographic condition 1: DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm); temperature program: 40 °C for 2 min, increase to 200 °C at 4 °C / min, hold for 2 min, and increase to 230 °C at 5 °C / min; helium flow rate of 1.5 mL / min, and injection port temperature of 250 °C.

[0052] Chromatographic condition 2: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), temperature program: 40°C for 2 min, increase to 200°C at 4°C / min, hold for 2 min, and increase to 230°C at 5°C / min; helium flow rate of 1.5 mL / min, injection mode of splitless flow.

[0053] Mass spectrometry conditions: electron energy 70 eV; ion source temperature 230°C; transfer line temperature 250°C; quadrupole temperature 150°C; mass scan range m / z 40-350.

[0054] Qualitative analysis: GC-MS raw data were matched with the NIST17 spectral library for qualitative analysis, and standards and retention time indices were used for comparison.

[0055] Example 1

[0056] A method for enzymatic degumming and deacidification of ARA algae oil, comprising the following steps:

[0057] (1) Hydrophobic modification of the mesoporous support: 1.0 g of MCM-41 sample and 50 mM C8 were added to 10 mL of n-hexane, mixed well, and then ultrasonicated for 10 min. The mixture was then placed in a thermostatic shaker at 30°C and allowed to react at 200 rpm for 1.5 h. Finally, the reaction mixture was filtered, washed at least three times with ethanol, and dried at 60°C for 24 h to obtain the final MCM-41-C8. After hydrophobic modification, the contact angle was 117°.

[0058] (2) Preparation of immobilized enzyme: 3 g of MCM-41-C8 was added to 100 mL of a 2-fold diluted enzyme solution of phospholipase A1 (PLA1) from Aspergillus niger (diluent: 0.1 mol / mL, pH 7.0 PBS), and the mixture was shaken at 220 rpm at 30°C for 40 min. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain PLA1@MCM-41-C8. The immobilized amount could reach 190.8 mg / g, and the immobilized efficiency was 18.2%.

[0059] (3) Enzymatic degumming of ARA algae oil: ARA algae oil crude oil (45.0 g) was heated to 70°C, and then 0.2 mL of citric acid solution (45%, w / w) was added. The mixture was vigorously stirred at 10,000 rpm for 1 min. The solution was cooled to 45°C, and NaOH solution (4%, w / w) was added to adjust the pH of the reaction solution to 5.8. PLA1@MCM-41-C8 (0.2%, w / w) was added, and the reaction temperature was maintained at 35°C. The enzymatic degumming was carried out for 3 h. The reaction contents were centrifuged (4863×g, 10 min, 4°C). The supernatant was the degummed oil, and the bottom was a mixture of phospholipids and immobilized enzyme.

[0060] (4) Enzymatic deacidification of ARA algae oil: The degummed ARA algae oil was mixed with glycerol at a molar ratio of 1:4 and then 5% Molecular sieve, mixed evenly, added 3% immobilized enzyme and carried out enzymatic deacidification at 35℃.

[0061] It was determined that the phospholipid content of ARA algae oil after enzymatic degumming and deacidification was 13.1 mg / kg, and the degumming rate was 95.9%; the acid value was 1.1 mgKOH / g, the ARA retention rate was 98%, the DAG content was 12.7%, and the peroxide value was 0.04 g / 100 g. The flavor substances of ARA algae oil before and after degumming and deacidification were mainly alcohols and aldehydes. After degumming and deacidification, the content of alcohols was 15025.1 μg / kg, accounting for 58.5% of the total flavor substances, and the content of aldehydes was 5146.1 μg / kg, accounting for 20.4% of the total flavor substances.

[0062] Example 2

[0063] A method for enzymatic degumming and deacidification of rapeseed oil, specifically comprising the following steps:

[0064] Step (1) is the same as in Example 1

[0065] (2) Preparation of immobilized enzyme: Phospholipase A1 (PLA1) from Aspergillus niger was dissolved in phosphate buffer (50 mM, pH = 7) to prepare an enzyme solution of 90 mg / mL. Then, hydrophobic mesoporous silica modified with n-octylsilane was added to a ground-mouth conical flask at a ratio of 10 mg / mL to the enzyme solution. After reacting on a shaker (200 rpm, 30°C) for 40 minutes, the solution was taken out and centrifuged. The precipitate after centrifugation was placed in a freeze dryer and freeze-dried to obtain PLA1@SG211-C8. The immobilized amount could reach 150.1 mg / g, and the immobilized rate was 15.2%.

[0066] (3) Enzymatic degumming of rapeseed oil: Rapeseed oil with a phospholipid content of 300.6 mg / kg was heated to 70°C, and then 0.2 mL of citric acid solution (45%, w / w) was added. The mixture was vigorously stirred at 10,000 rpm for 1 min. The solution was cooled to 45°C, and NaOH solution (4%, w / w) was added to adjust the pH of the reaction solution to 5.8. PLA1@SG211-C8 (0.2%, w / w) was added, and the reaction temperature was maintained at 35°C. Enzymatic degumming was performed for 3 h. The reaction contents were centrifuged (4863×g, 10 min, 4°C). The supernatant was the degummed rapeseed oil, and the bottom was a mixture of phospholipids and immobilized enzyme.

[0067] (4) Enzymatic deacidification of rapeseed oil: The rapeseed oil treated in step (3) has an acid value of 5.68 mgKOH / g. The rapeseed oil and glycerol are mixed at a molar ratio of 1:4 and 5% (w / w) of Molecular sieves were mixed evenly and then 3% (w / w) immobilized enzyme PLA1@SG211-C8 was added for enzymatic deacidification at 35°C. After the reaction, the collected liquid was rapeseed oil and the solid was the immobilized enzyme.

[0068] The collected rapeseed oil is a mixture of degummed and deacidified rapeseed oil and diglyceride. The rapeseed oil product has a phospholipid content of 3.8 mg / kg, an acid value of 1.5 mg KOH / g, and a diglyceride content of 6.8%.

[0069] Example 3

[0070] A method for enzymatic degumming and deacidification of ARA oil, comprising the following steps:

[0071] (1) Hydrophobic modification of mesoporous materials: 1.0 g of HMSS sample and 60 mM C8 were added to 10 mL of n-hexane, mixed well, and then treated with ultrasound for 10 min. The mixture was then placed in a thermostatic shaker at 30°C and allowed to react at 200 rpm for 1.5 h. Finally, the reactant was filtered, washed with ethanol at least three times, and dried at 60°C for 24 h to obtain the final HMSS-C8. After hydrophobic modification, the contact angle was 124°.

[0072] Steps (2) (3) (4) are the same as in Example 1

[0073] The results showed that the phospholipid content of the ARA algae oil was 7.1 mg / kg, the acid value was 0.98 mgKOH / g, the ARA retention rate was 98%, the DAG content was 14.1%, and the peroxide value was 0.02 g / 100 g.

[0074] Example 4

[0075] A method for enzymatic degumming and deacidification of rice bran oil specifically comprises the following steps:

[0076] (1) Hydrophobic modification of mesoporous materials: 2.0 g of OMS sample, 100 mM C8, and 15 μL of triethylamine were added to 10 mL of toluene. To achieve uniform dispersion, the mixture was ultrasonicated for 5 min. The mixture was then placed in a reactor and reacted at 130°C for 20 h. Finally, the reactants were filtered, washed with ethanol at least three times, and dried at 60°C for 18 h to obtain the final OMS-C8. After hydrophobic modification, the contact angle was 135°.

[0077] Step (2) is the same as in Example 1

[0078] (3) Enzymatic degumming of rice bran oil: Rice bran oil with a phospholipid content of 142.4 mg / kg was heated to 70°C, and then 0.2 mL of citric acid solution (45%, w / w) was added. 0.2 mL of pure water was added and the mixture was vigorously stirred at 10,000 rpm for 1 min. The solution was cooled to 45°C and a NaOH solution (4%, w / w) was added to adjust the pH of the reaction solution to 5.8. PLA1@OMS-C8 (0.2%, w / w) was added and the reaction temperature was maintained at 35°C. Enzymatic degumming was performed for 3 h. The reacted contents were centrifuged (4863×g, 10 min, 4°C). The supernatant was rice bran oil, and the bottom was a mixture of phospholipids and immobilized enzyme.

[0079] (4) Enzymatic deacidification of rice bran oil: The rice bran oil treated in step (3) has an acid value of 19.2 mgKOH / g. The rice bran oil is mixed with glycerol at a molar ratio of 1:3 and 5% (w / w) of Molecular sieves were mixed evenly and then 5% (w / w) immobilized enzyme PLA1@OMS-C8 was added for enzymatic deacidification at 35°C. After the reaction, the collected liquid was rice bran oil and the solid was immobilized enzyme PLA1@OMS-C8.

[0080] The rice bran oil was determined to have a phospholipid content of 1.4 mg / kg, an acid value of 1.5 mgKOH / g, a DAG content of 24.5%, and a peroxide value of 0.02 g / 100 g.

[0081] Example 5

[0082] A method for enzymatic deacidification of tea seed oil, comprising the following steps:

[0083] Step (1) is the same as in Example 4

[0084] Step (2) is the same as in Example 2

[0085] (3) Enzymatic deacidification of tea seed oil: tea seed oil with an acid value of 5.8 mgKOH / g was mixed with glycerol at a molar ratio of 1:5 and then 5% (w / w) of Molecular sieves were mixed evenly and then 5% (w / w) immobilized enzyme PLA1@OMS-C8 was added for enzymatic deacidification at 35°C. After the reaction, the liquid collected was tea seed oil and the solid was immobilized enzyme PLA1@OMS-C8.

[0086] The results show that the acid value of the tea seed oil is 1.0 mgKOH / g, the DAG content is 7.5%, and the peroxide value is 0.02 g / 100 g.

[0087] Example 6

[0088] A method for enzymatic degumming of peanut oil specifically comprises the following steps:

[0089] (1) Hydrophobic modification of mesoporous materials: 1.0 g of MCM-49 sample and 50 mM C8 were added to 10 mL of n-hexane, mixed well, and then ultrasonicated for 10 min. The mixture was then placed in a thermostatic shaker at 30°C and allowed to react at 200 rpm for 1.5 h. Finally, the reactant was filtered, washed at least three times with ethanol, and dried at 60°C for 24 h to obtain the final MCM-49-C8. After hydrophobic modification, the contact angle was 120°.

[0090] Step (2) is the same as in Example 1

[0091] (3) Enzymatic degumming of peanut oil: Peanut oil with a phospholipid content of 277.6 mg / kg was heated to 70°C, and then 0.2 mL of citric acid solution (45%, w / w) was added. 0.2 mL of pure water was added and the mixture was vigorously stirred at 10,000 rpm for 1 min. The solution was cooled to 45°C and the pH of the reaction solution was adjusted to 5.8 by adding NaOH solution (4%, w / w). PLA1@OMS-C8 (0.2%, w / w) was added and the reaction temperature was maintained at 35°C. Enzymatic degumming was performed for 3 h. The contents after the reaction were centrifuged (4863×g, 10 min, 4°C). The supernatant was peanut oil, and the bottom was a mixture of phospholipids and immobilized enzyme.

[0092] The peanut oil was determined to have a phospholipid content of 12.4 mg / kg, an acid value of 1.9 mgKOH / g, a DAG content of 2.5%, and a peroxide value of 0.02 g / 100 g.

[0093] Example 7

[0094] A method for enzymatic degumming and deacidification of soybean oil, comprising the following steps:

[0095] Steps (1) and (2) are the same as in Example 1

[0096] (3) Enzymatic degumming of soybean oil: Soybean oil with a phospholipid content of 304.1 mg / kg was heated to 70°C, and then 0.2 mL of citric acid solution (45%, w / w) was added. 0.2 mL of pure water was added and the mixture was vigorously stirred at 10,000 rpm for 1 min. The solution was cooled to 45°C and the pH of the reaction solution was adjusted to 5.8 by adding NaOH solution (4%, w / w). PLA1@MCM-41-C8 (0.2%, w / w) was added and the reaction temperature was maintained at 35°C. Enzymatic degumming was performed for 3 h. The reaction contents were centrifuged (4863×g, 10 min, 4°C). The supernatant was soybean oil, and the bottom was a mixture of phospholipids and immobilized enzyme.

[0097] (4) Enzymatic deacidification of soybean oil: Soybean oil with an acid value of 8.2 mgKOH / g was mixed with glycerol at a molar ratio of 1:4 and then 5% (w / w) Molecular sieves were mixed evenly and then 5% (w / w) immobilized enzyme PLA1@MCM-41-C8 was added for enzymatic deacidification at 35°C. After the reaction, the collected liquid was soybean oil and the solid was immobilized enzyme PLA1@MCM-41-C8.

[0098] The soybean oil was determined to have a phospholipid content of 3.0 mg / kg, an acid value of 2.5 mgKOH / g, a DAG content of 11.4%, and a peroxide value of 0.03 g / 100 g.

[0099] Example 8

[0100] A method for enzymatic degumming of walnut oil specifically comprises the following steps:

[0101] Steps (1) and (2) are the same as in Example 1

[0102] (3) Enzymatic degumming of walnut oil: Walnut oil with a phospholipid content of 155.9 mg / kg was heated to 70°C, and then 0.2 mL of citric acid solution (45%, w / w) was added. 0.2 mL of pure water was added and the mixture was vigorously stirred at 10,000 rpm for 1 min. The solution was cooled to 45°C and the pH of the reaction solution was adjusted to 5.8 by adding NaOH solution (4%, w / w). PLA1@MCM-41-C8 (0.2%, w / w) was added and the reaction temperature was maintained at 35°C. Enzymatic degumming was performed for 3 h. The contents after the reaction were centrifuged (4863×g, 10 min, 4°C). The supernatant was walnut oil, and the bottom was a mixture of phospholipids and immobilized enzyme.

[0103] The results show that the walnut oil has a phospholipid content of 10.09 mg / kg, an acid value of 1.88 mgKOH / g, a DAG content of 1.5%, and a peroxide value of 0.02 g / 100 g.

[0104] Example 9

[0105] A method for enzymatic degumming and deacidification of ARA algae oil, comprising the following steps:

[0106] Steps (1) (2) (3) (4) are the same as in Example 1

[0107] (5) Reuse of immobilized enzyme: Collect the immobilized enzyme from step (3) (4) and continue with step (3) (4) to achieve the purpose of reuse of the immobilized enzyme.

[0108] After testing, it was found that the activity of the immobilized enzyme remained above 80% after being reused 7 times, which can achieve a good degumming and deacidification effect.

[0109] Example 10

[0110] A method for enzymatic degumming and deacidification of ARA algae oil, comprising the following steps:

[0111] (1) Pretreatment of mesoporous materials: The resin LXTE-1000 was washed three times with deionized water to remove dust and other impurities; then it was acid-washed with 4% HCl solution under stirring for 2 h, and then alkaline-washed with 4% NaOH solution for 2 h; finally, it was washed with deionized water until neutral and stored in deionized water for later use.

[0112] (2) Preparation of immobilized enzyme: The cleaned resin LXTE-1000 was drained of water, and 10 g of LXTE-1000 was added to 100 mL of a 2-fold diluted PLA1 enzyme solution (diluent: 0.1 mol / mL, pH 7.0 PBS). The mixture was shaken at 220 rpm at 30°C for 40 min. The precipitate after centrifugation was dried in a cold air drying oven for 24 h to obtain PLA1@LXTE-1000. The immobilized amount could reach 100 mg / g, and the immobilized efficiency was 14.5%.

[0113] Steps (3) and (4) are the same as in Example 1

[0114] It was determined that the phospholipid content of the ARA algae oil was 15.1 mg / kg, the acid value was 1.89 mgKOH / g, the DAG content was 11.2%, the ARA retention rate was 96%, the peroxide value was 0.04 g / 100 g, the oxidation induction time was 3.8 h, the flavor substances were mainly alcohols and aldehydes, and the flavor substance content was reduced.

[0115] Example 11

[0116] A method for enzymatic degumming and deacidification of ARA algae oil, comprising the following steps:

[0117] Steps (1) (2) (3) (4) are the same as in Example 9;

[0118] (5) Repeated practicality of immobilized enzyme: Collect the immobilized enzyme PLA1@LXTE-1000 from steps (3) and (4) and continue with steps (3) and (4) to achieve the purpose of repeated use of the immobilized enzyme.

[0119] After testing, it was found that the activity of the immobilized enzyme remained above 80% after being reused 5 times, which can achieve a good degumming and deacidification effect.

[0120] Table 1 Summary of Degumming and Deacidification Effects in Examples

[0121]

[0122] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a hydrophobically modified mesoporous carrier, characterized in that: The hydrophobically modified mesoporous carrier is obtained by adding a mesoporous carrier and a hydrophobic silane coupling agent into n-hexane, performing ultrasonic dispersion, placing the mixture in a constant temperature shaker for shaking reaction, filtering, washing and drying.

2. The method for preparing a hydrophobically modified mesoporous carrier according to claim 1, wherein: The mesoporous carrier is one or more combinations of ordered mesoporous SiO2 material, hollow mesoporous SiO2 material, mesoporous molecular sieve, macroporous resin, and mesoporous silica gel; the hydrophobic silane coupling agent is one or more combinations of ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, polymethylhydrogensiloxane, butyltriethoxysilane, octyltriethoxysilane, dodecyltriethoxysilane, and octadecyltriethoxysilane.

3. The method for preparing a hydrophobically modified mesoporous carrier according to claim 1, wherein: The molar ratio of the hydrophobic silane coupling agent to the mesoporous carrier is 1:30-1:100, the ultrasonic dispersion time is 5-15 minutes, the shaking reaction time is 1.5-3 hours, and the drying time is 12-24 hours.

4. A method for preparing an immobilized enzyme having both edible oil degumming and deacidification functions, characterized in that: The method specifically comprises the following steps: Free phospholipase is immobilized on the hydrophobic mesoporous carrier prepared by the method as claimed in claim 1 by physical adsorption to obtain immobilized phospholipase.

5. The method for preparing an immobilized enzyme having the functions of degumming and deacidifying edible oil according to claim 4, characterized in that: The specific process for preparing immobilized phospholipase by physical adsorption is as follows: The phospholipase solution is added to a phosphate buffer solution for dilution, and the diluted enzyme solution is mixed with a hydrophobic carrier for immobilization to obtain an immobilized phospholipase; wherein, The enzyme solution concentration is 10-100 mg / mL; the ratio of the hydrophobic carrier mass to the enzyme solution volume is 1:20-1:100 (g / mL); the immobilization time is 0.5-2 hours, the incubation temperature is 20-40°C; the pH value of the phosphate buffer is 5.0-10.0; The phospholipase is one or more combinations of phospholipase A1 from Escherichia coli, phospholipase A1 from Streptomyces alboflavin, phospholipase A1 from bumblebee venom, phospholipase A1 from the outer membrane of Salmonella typhi, and phospholipase A1 from Aspergillus niger.

6. An application of an immobilized enzyme having both edible oil degumming and deacidification functions, characterized in that: The specific steps include: (1) Enzymatic degumming of edible oil: After heating the raw oil, add citric acid solution, cool it, add sodium hydroxide solution to adjust the pH value, add the immobilized phospholipase prepared by the method as described in claim 4 and distilled water, and carry out degumming reaction in a constant temperature water bath. After the reaction is completed, separate the solid and liquid by centrifugation. The obtained solid product is a mixture of immobilized phospholipase and phospholipids, and the obtained solution is the oil after degumming; (2) Enzymatic deacidification of edible oil: The raw oil and acyl acceptor are mixed, and then the immobilized phospholipase prepared by the method as described in claim 4 is added to carry out esterification reaction in a constant temperature water bath; after the reaction is completed, the solid and liquid are separated, and the obtained solid product is the immobilized phospholipase, and the obtained solution is the oil after deacidification; (3) Cascade degumming and deacidification of edible oil: After the edible oil is treated in step (1), the mixture of immobilized enzyme and phospholipid is removed by centrifugation, and then the deacidification of the edible oil is carried out in step (2) to achieve cascade enzymatic degumming and deacidification of the edible oil.

7. The use according to claim 6, characterized in that In step (1), when heated to 70-90° C., the concentration of the added citric acid buffer is 30%-50% (w / w), the amount of the added citric acid buffer accounts for 0.02-0.05% of the mass of the undegummed crude oil, the concentration of the added NaOH is 1-5% (w / w), the pH value is adjusted to 4-6, the amount of the added immobilized phospholipase is 2-5%, the amount of the added distilled water is 0-1%, the isothermal reaction temperature is 15-45° C., and the reaction time is 1.5-3.5 hours; The raw oil is one or a combination of vegetable oils such as rapeseed oil, linseed oil, soybean oil, corn oil, peanut oil, sesame oil, rice bran oil, camellia oil, and algae oil and fish oil rich in polyunsaturated fatty acids.

8. The use according to claim 6, characterized in that In step (3), the molar ratio of free fatty acids to acyl acceptors in the feed oil is 1:1-1:8; the amount of immobilized phospholipase added is 2-5% of the substrate mass; the temperature of the constant temperature water bath is 25-45° C.; and the esterification reaction time is 3-6 hours; The acyl acceptor is one or more combinations of monoglyceride, glycerol, D-isoascorbic acid, sterol ester, sterol, and short-chain monohydric alcohol.

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