Method for extracting rice bran oil based on ultrasonic coupling enzymolysis technology

Abstract

The invention relates to the field of vegetable oil extraction, in particular to a method for extracting rice bran oil from rice bran by an ultrasonic coupling composite enzymolysis technology, and provides a novel method for preparing the rice bran oil. The invention discloses a method for preparing rice bran oil through an ultrasonic coupling enzyme method. The method comprises the steps of mixed rice bran pretreatment, ultrasonic coupling enzymolysis reaction and lipid extraction. Wherein the ultrasonic coupling enzymolysis reaction comprises the following steps: adding water into the rice bran, uniformly mixing, adjusting the pH value of the system, adding quantitative plant extraction enzyme, putting into an ultrasonic instrument, setting specific temperature, ultrasonic power and reaction time for enzymolysis, and finally terminating the reaction for extraction. An ultrasonic coupling enzyme method adopted by the invention is combined with biological enzymolysis and physical ultrasonic action, and a brand new path is opened up for efficiently preparing oil from the rice bran.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant oil extraction, in particular to a method for extracting rice bran oil from rice bran by ultrasonic coupling complex enzymatic hydrolysis technology, which provides a new method for the preparation of rice bran oil. BACKGROUND

[0002] Traditional rice bran oil extraction mainly adopts pressing method, leaching method and supercritical fluid extraction method and subcritical water extraction method developed in recent years. The pressing method is a conventional extraction technology, which has simple equipment, convenient operation, but relatively low oil yield, and easy to produce more residual oil; the leaching method has high oil yield, but has problems such as using a large amount of organic solvent, harsh reaction conditions, high energy consumption, producing toxic and harmful substances, and serious environmental pollution. With the popularization of the concept of green and environmentally friendly production, supercritical fluid extraction method as a green extraction technology is applied to oil extraction, which can better preserve active ingredients at low temperature extraction, but requires high temperature and high pressure device, consumes a large amount of supercritical fluid, has high cost, and is difficult to be large-scale industrialized production; subcritical water extraction has poor selectivity, which easily dissolves non-target components, increases the difficulty and cost of separation and purification, and the high temperature and high pressure environment may cause the degradation of heat-sensitive components, affecting the product quality. Compared with the above, ultrasonic extraction as a green extraction method has obvious advantages, which does not require high temperature and high pressure equipment, has low equipment cost and energy consumption, and is simple to operate, and can effectively destroy the dense network structure formed by lignin, cellulose and hemicellulose, so as to improve the diffusion efficiency of enzyme molecules due to the tight structure of cell wall. In summary, it is of great significance to develop a simple, efficient, green and safe preparation method of high-quality rice bran oil to expand the market of rice bran oil and break through the bottleneck of rice bran oil development. SUMMARY

[0003] The problem to be solved by the present application is to provide a method for extracting rice bran oil based on ultrasonic coupling enzymatic hydrolysis technology.

[0004] In order to solve the above problems, the present application provides a method for extracting rice bran oil, comprising the following steps: 1) dehulling fresh rice, collecting husks, pretreating the husks to obtain rice bran powder (the pretreated husks can be stored at-20℃ for later use); 2) mixing the rice bran powder with water, adjusting the pH of the system to 5±0.2 (using a conventional acetic acid-sodium acetate buffer solution to adjust the pH of the system), and adding plant extraction enzyme for enzymatic extraction (placing in an ultrasonic instrument, setting specific temperature, ultrasonic power and reaction time for enzymatic extraction); After the set enzymatic extraction time is reached, the enzymatic reaction is terminated, and the rice bran oil is obtained by lipid extraction.

[0005] As the improvement of the extraction method of the rice bran oil of the present application: the pretreatment is to crush the bran (pass through a 40-60 mesh sieve), 95±5℃ water bath for 10±1 min, and after cooling (cool to ≤ extraction temperature), the rice bran powder is obtained.

[0006] As a further improvement of the extraction method of the rice bran oil of the present application, in step 2): The solid-liquid ratio of rice bran powder: water (sterile water) = 1 g / 5.5-6.5 mL; The enzyme addition amount of plant extraction enzyme is 1.8-2.2%; The extraction temperature is 45±2℃; The extraction time is 4±0.5 h.

[0007] Note: The enzyme addition amount of plant extraction enzyme is the enzyme mass / rice bran powder mass.

[0008] As a further improvement of the extraction method of the rice bran oil of the present application, in step 2): The ultrasonic power is 175±10 W, and the frequency is 40 Hz (5 g of rice bran powder).

[0009] As a further improvement of the extraction method of the rice bran oil of the present application, in step 2): The solid-liquid ratio of rice bran powder: water = 1 g / 6 mL; The enzyme addition amount of plant extraction enzyme is 2%.

[0010] As a further improvement of the extraction method of the rice bran oil of the present application, in step 2): The method for terminating the enzyme hydrolysis reaction is to quickly place in a water bath at 95±5℃ for heating for 10 min.

[0011] As a further improvement of the extraction method of the rice bran oil of the present application, in step 2): The lipid extraction is to add n-hexane to the obtained product after terminating the enzyme hydrolysis reaction, fully shake and mix, then centrifuge (6000 r / min), take the supernatant, and remove the n-hexane.

[0012] As a further improvement of the extraction method of the rice bran oil of the present application, 1±0.2 mL of n-hexane is used for every 1 g of rice bran powder.

[0013] The present application also simultaneously provides the rice bran oil prepared by any of the above methods.

[0014] The rice bran oil obtained by the present application is mainly composed of palmitic acid, oleic acid, linoleic acid, etc., and the unsaturated fatty acid accounts for 65.84% of the total fatty acid.

[0015] This invention overcomes the shortcomings of existing rice bran oil preparation processes by providing a method for preparing rice bran oil using an ultrasound-coupled enzymatic method. The method includes mixed rice bran pretreatment, ultrasound-coupled enzymatic hydrolysis, and lipid extraction. Specifically, the ultrasound-coupled enzymatic hydrolysis involves adding water to the rice bran and mixing thoroughly, adjusting the pH of the system, adding a measured amount of plant-derived enzyme, placing the mixture in an ultrasound instrument, setting specific temperature, ultrasound power, and reaction time for enzymatic hydrolysis, and finally terminating the reaction for extraction.

[0016] This invention has the following technical advantages: The ultrasound-coupled enzyme method employed in this invention, combining biological enzymatic hydrolysis with physical ultrasound, opens up a completely new pathway for the efficient extraction of oil from rice bran. On one hand, based on the cell wall structure of rice bran, plant-extracted enzymes (plant-extracted complex enzymes, including cellulase, pectinase, xylanase, and glucanase) are selected to specifically recognize and hydrolyze the cell wall, promoting the release of intracellular oils and other contents. On the other hand, when ultrasound acts on the rice bran system, it generates a large number of cavitation bubbles, and the resulting shear force further disrupts the cell wall integrity, significantly increasing the enzyme's contact area and improving the oil yield. Compared to traditional single-enzyme or ultrasound methods, the ultrasound-coupled enzyme method has lower energy consumption, milder reaction conditions, higher oil yield, and effectively preserves nutrients, providing a theoretical basis and technical support for the high-value utilization of rice bran oil. Attached Figure Description

[0017] Figure 1 Scanning electron microscopy (SEM) development of rice bran materials under different treatment conditions; Figure 1 middle: A~B: Phenotypes of rice bran inside and on the surface under untreated conditions (bar=40 μm); C~D: Internal and surface phenotypes of rice bran under optimal conventional enzymatic hydrolysis (bar=40 μm); E~F: Internal and surface phenotypes of rice bran under optimal ultrasonic treatment conditions (bar=40 μm); G~H: Internal and surface phenotypes of rice bran under optimal ultrasonic-coupled enzymatic hydrolysis conditions (bar=40 μm). Detailed Implementation

[0018] To more fully explain the implementation of the present invention, the following are implementation examples of a process for preparing rice bran oil using an ultrasonic-coupled enzyme method. The described embodiments are merely some, not all, embodiments of the present invention, and these embodiments are for illustrative purposes only and not for limiting the scope of the present invention.

[0019] The rice can be supplied by the China National Rice Research Institute; the plant-extracted enzymes (plant-extracted compound enzymes) can be purchased from Ningxia Heshibi Biotechnology Co., Ltd.; the other products and reagents are available on the market.

[0020] Example 1: Rice bran pretreatment Fresh rice is threshed to remove the grains and collect the bran. The bran is then pulverized until it can pass through a 40-60 mesh sieve, and subjected to a 95°C water bath for 10 minutes. After cooling to room temperature, the resulting product is named rice bran powder (pre-treated rice bran powder). The rice bran powder can be directly used for subsequent rice bran oil extraction, or it can be stored at -20°C for later use.

[0021] Example 2: Ultrasonic-coupled enzyme extraction of rice bran oil Accurately weigh 5 g of rice bran powder (obtained in Example 1) and place it in a 50 mL centrifuge tube. Add sterile water at a material-to-liquid ratio of 1:6 (rice bran powder: sterile water, g / mL). Adjust the pH of the system to 5.0 using a conventional acetate-sodium acetate buffer solution. Add plant extract enzyme equivalent to 2% of the weight of the rice bran powder. Place the centrifuge tube in an ultrasonic instrument and react for 4 h at 45°C, 40 Hz, and 175 W. After the reaction is complete, immediately place the tube in a 95°C water bath for 10 min to terminate the reaction.

[0022] Add 5 mL of n-hexane to the obtained reaction product, shake thoroughly to mix, and centrifuge at 6000 × g for 10 min. Repeat this operation twice and combine the supernatants obtained from the two operations. Remove the solvent (n-hexane) by nitrogen stripping.

[0023] Comparative Example 1: Ultrasonic Extraction of Rice Bran Oil Accurately weigh 5 g of rice bran powder (obtained in Example 1) and place it in a 50 mL centrifuge tube. Add sterile water at a ratio of 1:6 (rice bran powder: sterile water, g / mL), and adjust the pH of the system to 5.0 using a conventional acetate-sodium acetate buffer solution. Place the centrifuge tube in an ultrasonic instrument and react at 45°C, 40 Hz, and 175 W for 4 h. After the reaction is complete, quickly place the tube in a 95°C water bath for 10 min to terminate the reaction.

[0024] Add 5 mL of n-hexane to the obtained reaction product, shake thoroughly to mix, and centrifuge at 6000 × g for 10 min. Repeat this operation twice and combine the supernatants. Remove the solvent by nitrogen stripping.

[0025] Comparative Example 2: Extraction of rice bran oil by plant enzyme hydrolysis Accurately weigh 5 g of rice bran powder (obtained in Example 1) and place it in a 50 mL centrifuge tube. Add sterile water at a material-to-liquid ratio of 1:6 (rice bran powder: sterile water, g / mL). Adjust the pH of the system to 5.0 using an acetate-sodium acetate buffer solution. Add plant extract enzyme equivalent to 2% of the material weight. Place the centrifuge tube in a water bath and react at 45°C for 4 h. After the reaction is complete, immediately heat in a 95°C water bath for 10 min. Add 5 mL of n-hexane to the reaction system, shake thoroughly to mix, and centrifuge at 6000 × g for 10 min. Repeat this operation twice and combine the supernatants. Remove the solvent by nitrogen stripping.

[0026] Example 4: Determination of rice bran oil yield .

[0027] Example 5: Scanning Electron Microscopy Microstructure Analysis The microstructure of rice bran samples treated with un-enzymatic hydrolysis, conventional enzymatic hydrolysis, ultrasonication, and ultrasonic-coupled enzyme treatment was analyzed using scanning electron microscopy. The results are as follows: Figure 1 As shown, the enzymatic hydrolysis group showed an increase in the surface voids of the rice bran material, while the ultrasonic treatment group showed an increase in surface cracks. Ultrasonic coupled enzyme treatment can further destroy the material structure and promote the release of oil.

[0028] Example 6: Lipid Composition Analysis Weigh 60.0 mg of the rice bran oil sample prepared above (Example 2, Comparative Example 1, Comparative Example 2), dissolve it in 4.0 mL of isooctane, add 200 μL of 2 mol / L potassium hydroxide-methanol solution, react for 10 min, then add 1.0 g of sodium bisulfate, shake to mix, let stand to separate the layers, use a 1 mL syringe to take the upper n-hexane layer, filter it through a lipid-soluble filter membrane into a 2 mL brown sample vial, and use it for instrumentation.

[0029] After methyl esterification, the fatty acid composition of the sample was analyzed by gas chromatography using an HP-5 capillary column (30 m × 0.32 mm × 0.25 μm), with N2 (purity >99.99%) as the carrier gas and a flow rate of 1 mL / min. The sample injection volume was 1 μL, with a split ratio of 1:100. The injection port and detector temperatures were set to 270℃ and 280℃, respectively. The temperature program was as follows: initial column temperature 100℃, held for 10 min; increased to 180℃ at 10℃ / min, held for 6 min; continued to increase to 200℃ at 1℃ / min, held for 20 min; then increased to 230℃ at 4℃ / min, held for 10.5 min. The TIC chromatogram of the sample was compared with that of the fatty acid methyl ester mixed standard to determine the peak time of each fatty acid in the sample, and the percentage content of each fatty acid in rice bran was calculated by peak area normalization.

[0030] The results of the above measurements are shown in Table 1: Table 1

[0031] As shown in Table 1 above, the rice bran oil extracted by ultrasonic-coupled plant-based enzyme hydrolysis achieved an oil yield of 10.21%, which is higher than that obtained by ultrasonic extraction and plant-based enzyme hydrolysis. Gas chromatography analysis revealed that the rice bran oil mainly consists of palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2), and also contains fatty acids such as myristic acid (C14:0), stearic acid (C18:0), arachidic acid (C20:0), and α-linolenic acid (C18:3n3). Calculations showed that unsaturated fatty acids accounted for 65.84% of the total fatty acid composition in the rice bran oil obtained by ultrasonic-coupled enzyme extraction, with oleic acid, linoleic acid, and α-linolenic acid accounting for 31.45%, 28.99%, and 5.40%, respectively. Comparison with the fatty acid composition of rice bran oil extracted by conventional enzymatic hydrolysis showed no significant difference in the composition and content of each fatty acid. In summary, this invention utilizes the synergistic effect of ultrasonic extraction and enzymatic hydrolysis to extract rice bran oil, which can effectively improve the oil yield of rice bran oil. Furthermore, the ultrasonic-coupled enzyme extraction method does not have a substantial impact on the composition and content of fatty acids in rice bran oil.

[0032] The embodiments in this specification are written in a progressive manner, with each embodiment focusing on the differences from other embodiments. Identical or similar content can be referred to interchangeably. Furthermore, it should be noted that the above-listed examples are only some specific embodiments of the present invention. Obviously, the scope of protection of the present invention is not limited to the above embodiments, and many modifications exist. All equivalent modifications directly derived or reasonably conceived by those skilled in the art based on the disclosure of this invention should be included within the scope of protection of this invention.

Claims

1. A method for extracting rice bran oil, characterized by The method comprises the following steps: 1) dehulling fresh rice, collecting the husks, pretreating the husks to obtain rice bran powder; 2) mixing the rice bran powder with water, adjusting the pH of the system to 5±0.2, and adding a plant extraction enzyme for enzymatic extraction; After the set enzymatic extraction time is reached, the enzymatic reaction is terminated, and the rice bran oil is obtained through lipid extraction.

2. The extraction method of rice bran oil according to claim 1, characterized by: The pretreatment is to crush the husks, then water bath at 95±5℃ for 10±1 min, and after cooling, the rice bran powder is obtained.

3. The extraction method of the rice bran oil according to claim 1 or 2, characterized by In the step 2): The solid-liquid ratio of rice bran powder:water is 1 g / 5.5~6.5 mL; The enzyme addition amount of the plant extraction enzyme is 1.8~2.2%; The extraction temperature is 45±2℃; The extraction time is 4±0.5 h.

4. The extraction method of the rice bran oil according to claim 3, characterized by In the step 2): The ultrasonic power is 175±10 W.

5. The rice bran oil extraction method according to claim 4, characterized by In the step 2): The solid-liquid ratio of rice bran powder:water is 1 g / 6 mL; The enzyme addition amount of the plant extraction enzyme is 2%.

6. The rice bran oil extraction method according to any one of claims 1 to 5, characterized by In the step 2): The method for terminating the enzymatic reaction is to quickly heat in a water bath at 95±5℃ for 10 min.

7. The rice bran oil extraction method according to claim 6, characterized by In the step 2): The lipid extraction is to add n-hexane to the obtained product after the enzymatic reaction is terminated, mix uniformly, centrifuge, take the supernatant, and remove the n-hexane.

8. The rice bran oil extraction method according to claim 7, characterized by In the step 2): 1±0.2 mL of n-hexane is used for every 1 g of rice bran powder.

9. The rice bran oil prepared by the method of any one of claims 1~8.

10. The rice bran oil according to claim 9, characterized by, The rice bran oil mainly comprises palmitic acid, oleic acid, and linoleic acid, and the content of unsaturated fatty acids in the total fatty acids is high.

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