Preparation method of camellia oleosa seed oil with high oxidation resistance

Abstract

The invention relates to a preparation method of camellia oleosa seed oil with high oxidation resistance, and belongs to the technical field of camellia oleosa seed oil. The preparation method comprises the following steps: mixing camellia seed powder with distilled water, carrying out ultrasonic treatment, preheating, adding immobilized compound enzyme, mixing, stirring at a constant temperature, and drying to obtain an intermediate; pressing the intermediate in a double-screw press; the preparation method of the immobilized compound enzyme comprises the following steps: preparing composite particles from chitosan, amino acid and cellulose, adding an auxiliary agent and an aromatic aldehyde solution to prepare a porous carrier, and immobilizing the compound enzyme by the porous carrier. According to the method, after the camellia seeds are subjected to ultrasonic treatment, compound enzyme can permeate into a plant tissue structure more easily, the extraction rate of grease is increased, in addition, chitosan, cellulose and amino acid are compounded to form a composite carrier, and the composite carrier and syringaldehyde form a Schiff base structure, so that the heat stability and pH tolerance of the immobilized enzyme are improved; the activity of the compound enzyme is improved and extraction of camellia oleosa seed oil is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of camellia oil and relates to a method for preparing camellia oil with high antioxidant properties. Background Art

[0002] After the tea seeds are harvested, they go through a series of pretreatment steps, including drying, and then through a pressing process to obtain tea seed crude oil. Subsequently, the crude oil undergoes a refining process to finally obtain finished tea seed oil. As an advanced oil extraction technology, the water enzyme method is centered on using water as a solvent, and on the basis of mechanical crushing, using enzymes (such as proteases, cell wall degrading enzymes, complex enzymes, etc.) that can degrade macromolecular complexes such as lipoproteins, lipopolysaccharides, and cell walls in oil cells to destroy their tissue structure. By utilizing the difference in oil-water density and the difference in the affinity of each component to oil and water, effective separation of oil and protein is achieved.

[0003] As a biocatalyst, enzymes are widely used, especially in the fields of food processing, medicine and fine chemicals. Their high selectivity, mild catalytic reaction conditions and pollution-free characteristics make them a key factor in the above industries. Among the many enzyme application studies, immobilized enzymes are particularly eye-catching. As one of the core contents of enzyme engineering, they are of great significance for improving the utilization efficiency of enzymes and extending their service life. In the process of aqueous enzymatic extraction of camellia seeds, since free enzymes are usually soluble in water and have poor stability, a large amount of enzymes need to be added, and the separation process after the use of enzymes is complicated, which easily leads to resource waste. By immobilizing enzymes on various carriers, convenient separation and reuse of enzymes can be achieved. However, the performance of immobilized enzymes is significantly affected by the characteristics of the carrier materials used and the immobilization method. Natural polysaccharides, as a low-cost, renewable resource widely present in nature, have become the focus of research on immobilized enzyme carrier materials due to their good degradability, easy modification and environmental friendliness. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing camellia oil with high antioxidant property. The present invention is more conducive to the penetration of composite enzyme into plant tissue structure after the camellia seeds are subjected to ultrasonic treatment, thereby improving the extraction rate of oil. In addition, the present invention forms a composite carrier by compounding chitosan, cellulose and amino acids, so that the Schiff base structure formed by the composite carrier and syringaldehyde is improved, thereby improving the thermal stability and pH tolerance of the immobilized enzyme, improving the activity of the composite enzyme, and promoting the extraction of camellia oil.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for preparing highly antioxidant camellia oil comprises the following steps:

[0007] Step 1: After mixing camellia seed powder and distilled water and subjecting them to ultrasonic treatment, the mixture is preheated, and then the immobilized complex enzyme is added, mixed, stirred at a constant temperature, and dried at a low temperature to obtain an intermediate;

[0008] Step 2: placing the intermediate in a twin-screw press for pressing;

[0009] As a preferred technical solution of the present invention, in step 1, the particle size of the camellia seed powder is 20 meshes; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.0-5.4:0.30-0.32.

[0010] As a preferred technical solution of the present invention, in step one, the oil tea seed powder is obtained by picking, drying, shelling, seed shelling, magnetic separation and sieving the oil tea seeds.

[0011] As a preferred technical solution of the present invention, in step one, the ultrasonic treatment is performed at a frequency of 400-600W for 30-40min; the preheating temperature is 35-40°C; the constant temperature stirring is performed at a temperature of 40-50°C and a speed of 200-300r / min for 30-36h; the low-temperature drying is performed at a temperature of 60°C to control the moisture content of the tea seeds to 8-10%.

[0012] As a preferred technical solution of the present invention, in step 2, the pressing temperature is room temperature.

[0013] As a preferred technical solution of the present invention, in the process of extracting camellia oil by ultrasonic-assisted water-enzyme method, ultrasonic treatment can destroy the structure of plant cell walls through its cavitation effect, enhance the permeability of plant tissues, and thus promote the rapid release of oils, improve the extraction efficiency of oils, and shorten the processing cycle. Compared with the simple application of water-enzyme method, the ultrasonic-water-enzyme method not only significantly improves the extraction rate of oils in plant oil extraction, but also improves the quality of extracted oils, making the quality indicators of camellia oil surpass traditional solvent extraction technology. This method helps to retain more bioactive substances and nutrients, thereby producing camellia oil with better quality.

[0014] The present invention discloses a method for preparing the immobilized complex enzyme, comprising the following steps:

[0015] S1, ultrasonically mixing chitosan and acetic acid solution, adding amino acid and cellulose, stirring and mixing, heating and grinding, cooling to room temperature and freeze-drying to obtain composite particles;

[0016] S2, stirring the composite particles, the additive and the aromatic aldehyde solution at low temperature, centrifuging to remove the supernatant, placing in an acidic solution for ultrasonic treatment, filtering, washing and vacuum drying to obtain a porous carrier;

[0017] S3. Place the porous carrier in the composite enzyme and sodium acetate buffer solution, stir and mix, let stand, filter, and wash to obtain the product.

[0018] As a preferred technical solution of the present invention, in step S1, the amino acid is cysteine; the mass fraction of the acetic acid solution is 5%; the ultrasonic mixing is ultrasonication at a frequency of 1200-1400W for 40-60min; the heated grinding is grinding at a temperature of 50-60°C and a speed of 200-300r / min for 30-45min; in step S1, under the action of ultrasound, chitosan is degraded to form a small molecular structure, thereby increasing its solubility and increasing the number of free amino groups.

[0019] As a preferred technical solution of the present invention, in step S1, the mass ratio of chitosan, cellulose, amino acid and acetic acid solution is 4.2-4.6:1.0-1.2:0.3-0.5:80-90.

[0020] As a preferred technical solution of the present invention, the present invention increases the porosity of the porous carrier and the surface area of ​​the porous carrier by adding cellulose with higher specific surface area and stability on the basis of chitosan, so as to make the contact and combination with the complex enzyme closer; the amino group and thiol group of cysteine ​​can also complex metal ions, so that the complex enzyme is protected from inhibition by metal ions.

[0021] As a preferred technical solution of the present invention, in step S2, the mass ratio of the composite particles, the additive, the aromatic aldehyde solution and the acidic solution is 10-12: 0.12-0.14: 22-26: 30-35.

[0022] As a preferred technical solution of the present invention, in step S2, the acidic solution is a hydrochloric acid solution with a mass fraction of 4%; the aromatic aldehyde solution is a mixture of syringaldehyde and ethanol in a mass ratio of 4.2-5.0:80-90; the low-temperature stirring is stirring at a temperature of 20-25°C for 3-4h; the auxiliary agent is sodium bicarbonate; the ultrasonic treatment is ultrasonic treatment at a frequency of 200-300W for 40-60min; the washing is washing to neutrality; and the vacuum drying is drying at a temperature of 80°C to constant weight.

[0023] As a preferred technical solution of the present invention, in step S3, the usage ratio of the composite carrier, the composite enzyme and the sodium acetate buffer solution is 1.0-1.2 g: 32-36 U: 30 mL.

[0024] As a preferred technical solution of the present invention, the sodium acetate buffer solution is prepared by mixing 5.4 g of sodium acetate and 50 mL of deionized water, adjusting the pH to 4.6 with glacial acetic acid, and then diluting the mixture to 100 mL with deionized water.

[0025] As a preferred technical solution of the present invention, in step S3, the complex enzyme is composed of cellulase, protease and pectinase mixed in a mass ratio of 4-6:20-30:5-8; the stirring and mixing is stirring at a temperature of 20-25°C for 30-40 minutes; the standing time is 3-4 hours; and the washing is washing with sodium acetate buffer.

[0026] Beneficial effects of the present invention:

[0027] The invention is more conducive to the penetration of the composite enzyme into the plant tissue structure by ultrasonic treatment of the oil tea seeds, thereby improving the oil extraction rate. In addition, the invention forms a composite carrier by compounding chitosan, cellulose and amino acids, so that the Schiff base structure formed by the composite carrier and syringaldehyde is improved, thereby improving the thermal stability and pH tolerance of the immobilized enzyme, improving the activity of the composite enzyme, and promoting the extraction of oil tea seed oil. DETAILED DESCRIPTION

[0028] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0029] Example 1

[0030] A method for preparing highly antioxidant camellia oil comprises the following steps:

[0031] Step 1: After mixing 20-mesh camellia seed powder and distilled water, ultrasonicate at a frequency of 400W for 30 minutes, preheat at 35°C, add immobilized complex enzyme and mix, stir at a constant temperature of 40°C and 200r / min for 30 hours, control the moisture content of camellia seeds to 8% at 60°C, and obtain an intermediate; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.0:0.30;

[0032] Step 2: Place the intermediate in a twin-screw press for pressing at room temperature.

[0033] Wherein, the preparation method of the immobilized complex enzyme comprises the following steps:

[0034] S1. After chitosan and 5% mass fraction acetic acid solution were ultrasonicated at a frequency of 1200W for 40 minutes, cysteine ​​and cellulose were added and stirred for 10 minutes, and then ground at 50°C and 200r / min for 30 minutes, and then cooled to room temperature and freeze-dried to obtain composite particles; the mass ratio of chitosan, cellulose, cysteine ​​and acetic acid solution was 4.2:1.0:0.3:80;

[0035] S2. Stir the composite particles, sodium bicarbonate and aromatic aldehyde solution at 20°C for 3h, centrifuge to remove the supernatant, place in a 4% by mass hydrochloric acid solution for ultrasonic treatment, filter, wash and vacuum dry to obtain a porous carrier; the mass ratio of the composite particles, sodium bicarbonate, aromatic aldehyde solution and hydrochloric acid solution is 10:0.12:22:30; the aromatic aldehyde solution is a mixture of syringaldehyde and ethanol in a mass ratio of 4.2:80; the ultrasonic treatment is ultrasonic treatment at a frequency of 200W for 40min; the washing is washing to neutrality; the vacuum drying is drying at 80°C to constant weight;

[0036] S3. Place the porous carrier in a composite enzyme and sodium acetate buffer solution, stir for 30 minutes at 20°C, let stand for 3 hours, filter, and wash with sodium acetate buffer to obtain; the dosage ratio of the composite carrier, composite enzyme and sodium acetate buffer solution is 1.0g:32U:30mL; the composite enzyme is a mixture of pectinase, cellulase and protease in a mass ratio of 5:4:20.

[0037] Example 2

[0038] A method for preparing highly antioxidant camellia oil comprises the following steps:

[0039] Step 1: After mixing 20-mesh camellia seed powder and distilled water, ultrasonicate at a frequency of 450W for 32 minutes, preheat to 36°C, add immobilized complex enzyme and mix, stir at a constant temperature of 42°C and 220r / min for 32 hours, control the moisture content of camellia seeds to 8% at 60°C, and obtain an intermediate; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.1:0.31;

[0040] Step 2: Place the intermediate in a twin-screw press for pressing at room temperature.

[0041] Wherein, the preparation method of the immobilized complex enzyme comprises the following steps:

[0042] S1. After chitosan and 5% acetic acid solution were ultrasonicated at a frequency of 1250W for 45 minutes, cysteine ​​and cellulose were added and stirred for 10 minutes, and then ground at a temperature of 52°C and a speed of 220r / min for 35 minutes, and then cooled to room temperature and freeze-dried to obtain composite particles; the mass ratio of chitosan, cellulose, cysteine ​​and acetic acid solution was 4.3:1.05:0.35:82;

[0043] S2. Stir the composite particles, sodium bicarbonate and aromatic aldehyde solution at 21°C for 3.5h, centrifuge to remove the supernatant, place in a 4% by mass hydrochloric acid solution for ultrasonic treatment, filter, wash and vacuum dry to obtain a porous carrier; the mass ratio of the composite particles, sodium bicarbonate, aromatic aldehyde solution and hydrochloric acid solution is 10.5:0.125:23:31; the aromatic aldehyde solution is a mixture of syringaldehyde and ethanol in a mass ratio of 4.4:82; the ultrasonic treatment is ultrasonic treatment at a frequency of 220W for 45min; the washing is washing to neutrality; the vacuum drying is drying at 80°C to constant weight;

[0044] S3. Place the porous carrier in a composite enzyme and sodium acetate buffer solution, stir for 32 minutes at 21°C, let stand for 3.5 hours, filter, and wash with sodium acetate buffer to obtain; the dosage ratio of the composite carrier, composite enzyme and sodium acetate buffer solution is 1.05g:33U:30mL; the composite enzyme is a mixture of pectinase, cellulase and protease in a mass ratio of 5.8:4.5:22.

[0045] Example 3

[0046] A method for preparing highly antioxidant camellia oil comprises the following steps:

[0047] Step 1: After mixing 20-mesh camellia seed powder and distilled water, ultrasonicate at a frequency of 500W for 35 minutes, preheat to 38°C, add immobilized complex enzyme and mix, stir at a constant temperature of 45°C and 250r / min for 33 hours, control the moisture content of camellia seeds to 9% at 60°C, and obtain an intermediate; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.2:0.31;

[0048] Step 2: Place the intermediate in a twin-screw press for pressing at room temperature.

[0049] Wherein, the preparation method of the immobilized complex enzyme comprises the following steps:

[0050] S1. After chitosan and 5% mass fraction acetic acid solution were ultrasonicated at a frequency of 1300W for 50 minutes, cysteine ​​and cellulose were added and stirred for 10 minutes, and then ground at 55°C and 250r / min for 38 minutes, and then cooled to room temperature and freeze-dried to obtain composite particles; the mass ratio of chitosan, cellulose, cysteine ​​and acetic acid solution was 4.4:1.1:0.4:85;

[0051] S2. Stir the composite particles, sodium bicarbonate and aromatic aldehyde solution at 22°C for 3.5h, centrifuge to remove the supernatant, place in a 4% by mass hydrochloric acid solution for ultrasonic treatment, filter, wash and vacuum dry to obtain a porous carrier; the mass ratio of the composite particles, sodium bicarbonate, aromatic aldehyde solution and hydrochloric acid solution is 11:0.13:24:32; the aromatic aldehyde solution is a mixture of syringaldehyde and ethanol in a mass ratio of 4.6:85; the ultrasonic treatment is ultrasonic treatment at a frequency of 250W for 50min; the washing is washing to neutrality; the vacuum drying is drying at 80°C to constant weight;

[0052] S3. Place the porous carrier in a composite enzyme and sodium acetate buffer solution, stir for 35 minutes at 22°C, let stand for 3.5 hours, filter, and wash with sodium acetate buffer to obtain the composite carrier, the composite enzyme and sodium acetate buffer solution in a dosage ratio of 1.1 g: 34 U: 30 mL; the composite enzyme is a mixture of pectinase, cellulase and protease in a mass ratio of 6.5: 5: 25.

[0053] Example 4

[0054] A method for preparing highly antioxidant camellia oil comprises the following steps:

[0055] Step 1: After mixing 20-mesh camellia seed powder and distilled water, ultrasonicate at a frequency of 550W for 38 minutes, preheat to 39°C, add immobilized complex enzyme and mix, stir at a constant temperature of 48°C and 270r / min for 34 hours, control the moisture content of camellia seed to 9% at 60°C, and obtain an intermediate; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.3:0.31;

[0056] Step 2: Place the intermediate in a twin-screw press for pressing at room temperature.

[0057] Wherein, the preparation method of the immobilized complex enzyme comprises the following steps:

[0058] S1. After chitosan and 5% mass fraction acetic acid solution were ultrasonicated at a frequency of 1350W for 55 minutes, cysteine ​​and cellulose were added and stirred for 10 minutes, and then ground at a temperature of 58°C and a speed of 270r / min for 40 minutes, and then cooled to room temperature and freeze-dried to obtain composite particles; the mass ratio of chitosan, cellulose, cysteine ​​and acetic acid solution was 4.5:1.15:0.45:88;

[0059] S2. Stir the composite particles, sodium bicarbonate and aromatic aldehyde solution at 24°C for 3.5h, centrifuge to remove the supernatant, place in a 4% by mass hydrochloric acid solution for ultrasonic treatment, filter, wash and vacuum dry to obtain a porous carrier; the mass ratio of the composite particles, sodium bicarbonate, aromatic aldehyde solution and hydrochloric acid solution is 11.5:0.135:25:34; the aromatic aldehyde solution is a mixture of syringaldehyde and ethanol in a mass ratio of 4.8:88; the ultrasonic treatment is ultrasonic treatment at a frequency of 270W for 55min; the washing is washing to neutrality; the vacuum drying is drying at 80°C to constant weight;

[0060] S3. Place the porous carrier in a composite enzyme and sodium acetate buffer solution, stir for 38 minutes at 24°C, let stand for 3.5 hours, filter, and wash with sodium acetate buffer to obtain; the dosage ratio of the composite carrier, composite enzyme and sodium acetate buffer solution is 1.15g:35U:30mL; the composite enzyme is composed of pectinase, cellulase and protease mixed in a mass ratio of 7:5.5:28.

[0061] Example 5

[0062] A method for preparing highly antioxidant camellia oil comprises the following steps:

[0063] Step 1: After mixing 20-mesh camellia seed powder and distilled water, ultrasonicate at a frequency of 600W for 40 minutes, preheat at 40°C, add immobilized complex enzyme and mix, stir at a constant temperature of 50°C and a speed of 300r / min for 36 hours, and control the moisture content of the camellia seeds to 10% at a temperature of 60°C to obtain an intermediate; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.4:0.32;

[0064] Step 2: Place the intermediate in a twin-screw press for pressing at room temperature.

[0065] Wherein, the preparation method of the immobilized complex enzyme comprises the following steps:

[0066] S1. After chitosan and 5% mass fraction acetic acid solution were ultrasonicated at a frequency of 1400W for 60 minutes, cysteine ​​and cellulose were added and stirred for 10 minutes, and then ground at 60°C and 300r / min for 45 minutes, and then cooled to room temperature and freeze-dried to obtain composite particles; the mass ratio of chitosan, cellulose, cysteine ​​and acetic acid solution was 4.6:1.2:0.5:90;

[0067] S2. Stir the composite particles, sodium bicarbonate and aromatic aldehyde solution at 25°C for 4 hours, centrifuge to remove the supernatant, place in a 4% by mass hydrochloric acid solution for ultrasonic treatment, filter, wash and vacuum dry to obtain a porous carrier; the mass ratio of the composite particles, sodium bicarbonate, aromatic aldehyde solution and hydrochloric acid solution is 12:0.14:26:35; the aromatic aldehyde solution is a mixture of syringaldehyde and ethanol in a mass ratio of 5.0:90; the ultrasonic treatment is ultrasonic treatment at a frequency of 300W for 60 minutes; the washing is washing to neutrality; the vacuum drying is drying at 80°C to constant weight;

[0068] S3. Place the porous carrier in a composite enzyme and sodium acetate buffer solution, stir for 40 minutes at 25°C, let stand for 4 hours, filter, and wash with sodium acetate buffer to obtain the composite carrier, the composite enzyme and sodium acetate buffer solution in a dosage ratio of 1.2g:36U:30mL; the composite enzyme is a mixture of pectinase, cellulase and protease in a mass ratio of 8:6:30.

[0069] Comparative Examples 1-3

[0070] Compared with Example 1, the difference between Comparative Examples 1-3 is that the amounts of chitosan, cellulose and cysteine ​​are as shown in Table 1, and the other components, preparation steps and parameters are the same.

[0071] Table 1 (Unit: parts by weight)

[0072] Chitosan Cellulose Cysteine Example 1 4.2 1.0 0.3 Comparative Example 1 0 3.1 2.4 Comparative Example 2 4.7 0 0.8 Comparative Example 3 4.3 1.2 0

[0073] Comparative Example 4

[0074] Compared with Example 1, Comparative Example 4 is different in that alanine replaces cysteine, and the other components, preparation steps and parameters are the same.

[0075] Comparative Example 5

[0076] Compared with Example 1, the difference of Comparative Example 5 is that step S1;

[0077] Step S1, chitosan, 5% mass fraction acetic acid solution, cysteine ​​and cellulose are stirred and mixed for 50 minutes, ground at 50°C and 200 r / min for 30 minutes, cooled to room temperature and freeze-dried to obtain composite particles; the remaining components, preparation steps and parameters are the same.

[0078] Comparative Example 6

[0079] Compared with Example 1, Comparative Example 6 is different in that sodium bicarbonate is not used, and the remaining components, preparation steps and parameters are the same.

[0080] Comparative Example 7

[0081] Compared with Example 1, the difference in Comparative Example 7 is that the aromatic aldehyde solution is prepared by mixing syringaldehyde and ethanol in a mass ratio of 6:80, and the other components, preparation steps and parameters are the same.

[0082] Comparative Example 8

[0083] Compared with Example 1, Comparative Example 8 is different in that no syringaldehyde is used, and the remaining components, preparation steps and parameters are the same.

[0084] Comparative Example 9

[0085] Compared with Example 1, Comparative Example 9 is different in that ultrasonic treatment is not performed in step 1, and the remaining components, preparation steps and parameters are the same.

[0086] The camellia oils of Examples 1-5 and Comparative Examples 1-9 were subjected to DPPH free radical scavenging tests respectively, and the test results are shown in Table 2.

[0087] DPPH free radical scavenging test:

[0088] In a 10mL colorimetric tube, 4.0mL of DPPH solution and the sample to be tested (Example / Comparative Example) were added in sequence, and then anhydrous ethanol was added to the scale. After mixing, the absorbance (A) was measured at a wavelength of 517nm using a 1cm colorimetric dish immediately. The absorbance was recorded as Ai. The absorbance was measured after being placed at room temperature and protected from light for 30min, and recorded as Aj. The blank control was only added with DPPH ethanol solution, and its absorbance was recorded as Ac. The DPPH free radical scavenging rate K (%) was calculated according to the following formula; the experiment was repeated 3 times and the average value was taken. The test results are shown in Table 2.

[0089] K=[1-(Ai-Aj) / Ac]*100%;

[0090] Table 2

[0091] DPPH free radical scavenging rate (%) Example 1 96.1 Example 2 95.8 Example 3 95.4 Example 4 95.5 Example 5 95.7 Comparative Example 1 76.7 Comparative Example 2 86.3 Comparative Example 3 84.5 Comparative Example 4 89.3 Comparative Example 5 81.4 Comparative Example 6 78.2 Comparative Example 7 88.3 Comparative Example 8 84.8 Comparative Example 9 82.4

[0092] From the test results in Table 2, it can be seen that compared with Comparative Examples 1-9, the DPPH free radical scavenging ability of the rapeseed oil prepared in Examples 1-5 is significantly better than that in Comparative Examples 1-9.

[0093] Compared with Comparative Examples 1-9, Examples 1-5 and Table 2 show that this is because the present invention uses ultrasonic treatment to destroy the cell wall of Camellia oleifera seeds, accelerates the release of oil, and produces more oxidation products, which promotes the dissolution of bioactive components in the oil, thereby increasing the oil content and obtaining better antioxidant properties; and after ultrasonic treatment, it is more conducive to the penetration of the composite enzyme into the plant tissue structure, thereby improving the oil extraction rate. In addition, the present invention forms a composite carrier with an interpenetrating network structure by compounding chitosan, cellulose and cysteine, so that the cross-linking group is more abundant, the pore structure and specific surface area are increased, and the free amino acid is increased by compounding chitosan and cysteine. The number of bases increases, which increases the Schiff base structure formed with syringaldehyde, and combines with sodium bicarbonate to expand the pores, further enriching the pore structure of the porous carrier, improving the immobilization effect of the complex enzyme, and making it more thermally stable and pH tolerant. The cellulose skeleton contains a large number of hydroxyl groups combined with chitosan and cysteine ​​carboxyl groups, which promote the interaction with syringaldehyde through hydrogen bonds, increase the stability of the porous carrier, and form an interactive force with the complex enzyme, making it more tightly bound to the complex enzyme. The introduction of amino and thiol groups into the porous carrier can also complex metal ions, protect the complex enzyme from metal ion inhibition, improve the activity of the complex enzyme, and promote the extraction of camellia oil.

[0094] In Comparative Example 7, due to the excessive syringaldehyde, the amino groups of the porous carrier were cross-linked, so that the porosity of the porous carrier surface was reduced, thereby affecting the immobilization effect and activity of the complex enzyme.

[0095] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing highly antioxidant camellia oil, characterized in that: The preparation method comprises the following steps: Step 1: After mixing camellia seed powder and distilled water and subjecting them to ultrasonic treatment, the mixture is preheated, and then the immobilized complex enzyme is added, mixed, stirred at a constant temperature, and dried at a low temperature to obtain an intermediate; Step 2: placing the intermediate in a twin-screw press for pressing; Wherein, the preparation method of the immobilized complex enzyme comprises the following steps: S1, ultrasonically mixing chitosan and acetic acid solution, adding amino acid and cellulose, stirring and mixing, heating and grinding, cooling to room temperature and freeze-drying to obtain composite particles; S2, stirring the composite particles, the additive and the aromatic aldehyde solution at low temperature, centrifuging to remove the supernatant, placing in an acidic solution for ultrasonic treatment, filtering, washing and vacuum drying to obtain a porous carrier; S3. Place the porous carrier in the composite enzyme and sodium acetate buffer solution, stir and mix, let stand, filter, and wash to obtain the product.

2. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step 1, the particle size of the camellia seed powder is 20 meshes; the mass ratio of the camellia seed powder, distilled water and immobilized complex enzyme is 1:5.0-5.4:0.30-0.

32.

3. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step one, the ultrasonic treatment is performed at a frequency of 400-600W for 30-40 minutes; the preheating temperature is 35-40°C; the constant temperature stirring is performed at a temperature of 40-50°C and a speed of 200-300r / min for 30-36h; the low-temperature drying is performed at a temperature of 60°C to control the moisture content of the tea seeds to 8-10%.

4. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step 2, the pressing temperature is room temperature.

5. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step S1, the amino acid is cysteine; the mass fraction of the acetic acid solution is 5%; the ultrasonic mixing is performed at a frequency of 1200-1400 W for 40-60 min; and the heated grinding is performed at a temperature of 50-60° C. and a rotation speed of 200-300 r / min for 30-45 min.

6. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step S1, the mass ratio of chitosan, cellulose, amino acid and acetic acid solution is 4.2-4.6:1.0-1.2:0.3-0.5:80-90.

7. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step S2, the mass ratio of the composite particles, the additive, the aromatic aldehyde solution and the acidic solution is 10-12: 0.12-0.14: 22-26: 30-35.

8. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step S2, the acidic solution is a 4% by mass hydrochloric acid solution; the aromatic aldehyde solution is prepared by mixing syringaldehyde and ethanol in a mass ratio of 4.2-5.0:80-90; the low-temperature stirring is stirring at a temperature of 20-25°C for 3-4h; the auxiliary agent is sodium bicarbonate; the ultrasonic treatment is ultrasonic treatment at a frequency of 200-300W for 40-60min; the washing is washing to neutrality; and the vacuum drying is drying at a temperature of 80°C to constant weight.

9. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step S3, the usage ratio of the composite carrier, the composite enzyme and the sodium acetate buffer solution is 1.0-1.2 g: 32-36 U: 30 mL.

10. The method for preparing a highly antioxidant camellia oil according to claim 1, characterized in that: In step S3, the complex enzyme is prepared by mixing cellulase, protease and pectinase in a mass ratio of 4-6:20-30:5-8; the stirring and mixing is performed at a temperature of 20-25°C for 30-40 minutes; the standing time is 3-4 hours; and the washing is performed by washing with sodium acetate buffer.