Method for extracting symplocos paniculata kernel oil by enzyme method

Abstract

The invention discloses a method for extracting symplocos paniculata kernel oil by an enzymic method, which mainly comprises the following steps: shelling symplocos paniculata seeds, drying, and beating into powder by using a beating crusher; weighing the symplocos paniculata kernel powder, putting the symplocos paniculata kernel powder into a conical flask, and adding a phosphate buffer solution with the pH value of 5 according to the material-liquid ratio of 1: 5; setting microwave time, temperature and power, and carrying out microwave pretreatment; performing water bath for more than 1 hour at 50-70 DEG C after microwave treatment, and then cooling to below 50 DEG C; adding a certain amount of enzyme, and oscillating at constant temperature; after the enzymolysis is finished, enzyme deactivation is performed, the mixture is transferred into a centrifugal tube to be centrifuged, and supernatant oil is the symplocos paniculata kernel oil. The symplocos paniculata kernel oil is extracted by adopting a microwave-assisted aqueous enzymatic method, the action condition is mild, the process is simple, the equipment requirement is low, and the oil is better in quality, safer, more environment-friendly and more nutritional. The method provided by the invention can provide beneficial practical guidance for subsequent industrial production, and is helpful for improving the production efficiency of enterprises for making edible oil from the symplocos paniculata oil or enterprises for producing biodiesel.

Description

Technical Field

[0001] The invention relates to separation, in particular to a method for extracting sandalwood kernel oil from sandalwood seeds. Background Art

[0002] White sandalwood ( Symplocos paniculata (Thunb.) Miq.) is an eco-economic tree species that integrates edible (oil), ornamental, medicinal and industrial uses. It is an ideal woody oil tree species suitable for the development of karst habitats. The oil content of sandalwood fruit is as high as 38.6%, and the unsaturated fatty acid content is 85%. It is an ideal edible oil and a high-quality raw material for the preparation of biodiesel. Sandalwood oil is often used in industry as a lubricant in the machinery industry and a blending agent in the ink industry. In the textile industry, it can soften wool and remove static electricity. In the chemical industry, it can be used to make soap and soap. It can be seen that sandalwood oil has great development potential and application prospects in food and industry.

[0003] Traditional vegetable oil extraction methods include pressing and extraction. Pressing methods include hot pressing and cold pressing. The high temperature conditions of hot pressing will cause polyunsaturated fatty acids to deteriorate and oxidize and become rancid, which can easily produce unpleasant odors, thus affecting the quality of the extracted oils. Cold pressing has high energy consumption and low extraction rates. The solvent extraction method has safety hazards, and organic solvents are prone to remain, which is harmful to human health. The aqueous method is an extraction method that uses water as an extractant, but the oil extraction rate is low. The aqueous enzyme oil extraction technology is based on the aqueous method and adds enzymes that can degrade plant oil cells or have a degrading effect on complexes such as lipoproteins and lipopolysaccharides, so that the oils can be easily released from the oil solids.

[0004] The research on the separation of oil and fat by water enzyme method has become a hot topic at home and abroad. Compared with the traditional oil production process, the water enzyme oil extraction process can not only separate oil and protein from oil cells at the same time, but also has simple equipment, safe operation, no solvent residue, no environmental pollution, low investment, and can remove odor components in oil. Microwave-assisted extraction uses microwave heating to increase the internal pressure of oil cells, break or dissolve cell walls and cell membranes, and release oil. Microwave-assisted water enzyme method promotes the release of oil through microwave action and enzymatic hydrolysis, thereby improving the oil extraction rate.

[0005] So far, there have been reports on the preparation of oils from many oil plants such as walnut, rapeseed, peanut, soybean, and litsea cubeba kernels, and the research on the water enzymatic extraction process and microwave-assisted water enzymatic extraction process, but there are no relevant reports on the extraction process of sandalwood kernel oil. Therefore, this experiment studied the effect of microwave pretreatment combined with single enzyme and compound enzyme on the extraction of sandalwood kernel oil, and optimized the microwave treatment conditions and enzymatic hydrolysis conditions, and determined the optimal process conditions for microwave-assisted water enzymatic extraction of sandalwood kernel oil, which will help companies that make edible oil from sandalwood oil or companies that produce biodiesel to improve production efficiency. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for extracting sandalwood kernel oil by enzyme method in view of the above defects.

[0007] The technical solution of the present invention is: a method for extracting sandalwood kernel oil by enzymatic method, the main steps of which are as follows: (1) Shell the sandalwood seeds, dry them, and grind them into powder using a crusher; (2) Weigh the sandalwood kernel powder into a conical flask and add phosphate buffer with a pH of 5 at a solid-liquid ratio of 1:5; (3) Set microwave time, temperature, and power to perform microwave pretreatment; (4) After microwave, place in a water bath at 50℃-70℃ for more than 1h, then cool to below 50℃; (5) Add a certain amount of enzyme and oscillate at a constant temperature to carry out enzymatic hydrolysis; after the enzymatic hydrolysis is completed, inactivate the enzyme at above 90°C, transfer the mixture to a centrifuge tube and centrifuge. The clear oil on the top is sandalwood kernel oil.

[0008] In the above step (3), the microwave treatment conditions are: microwave temperature 50°C, microwave time 75min, microwave power 800W.

[0009] In the above step (4), the water bath temperature is 60°C, the water bath time is 1 hour, and then cooled to 50°C.

[0010] In the above step (5), after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90°C for 20 minutes.

[0011] In the above step (5), the centrifugal speed is 10000 r / min and the centrifugal time is 15 min.

[0012] In the above step (5), the pH of the enzymatic hydrolysis is 5, the enzymatic hydrolysis time is 4 h, and the enzymatic hydrolysis temperature is 50°C.

[0013] In the above step (5), after adding the enzyme, the mixture was shaken at a constant temperature of 50°C for 4 hours; after the enzymatic hydrolysis was completed, the enzyme was inactivated at 90°C.

[0014] In the above step (5), the enzyme is at least one of protease, pectinase and cellulase.

[0015] In the above step (5), the enzyme is selected from one of protease, pectinase and cellulase, and the dosage of the enzyme is: 1.5-3.5% protease, 0.5-2.5% pectinase, and 0.25-1.5% cellulase.

[0016] In the above step (5), the enzyme is a mixture of protease, pectinase and cellulase, and the dosage of each enzyme in the mixed enzyme is: protease 1.5-3.5%, pectinase 0.5-2.5%, cellulase 0.25-1.5%.

[0017] Principle of the invention: The invention destroys the cell wall of sandalwood kernel cells and the lipoprotein and other structures in the cells to release the oil and improve the oil extraction rate. At the same time, microwave treatment is added to the enzymatic hydrolysis conditions, which is more conducive to the release of oil and improves the oil extraction rate of sandalwood kernels.

[0018] The invention adopts microwave-assisted aqueous enzyme method to extract sandalwood kernel oil, which has mild action conditions, simple process, low equipment requirements, better oil quality, and is safer, more environmentally friendly and nutritious. The method of the invention provides practical guidance for subsequent industrial production, and helps enterprises producing edible oil from sandalwood oil or enterprises producing biodiesel to improve production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the effect of protease on the oil extraction rate of sandalwood kernels; Figure 2 This is the effect of pectinase on the oil extraction rate of sandalwood kernels; Figure 3 This is the effect of cellulase on the oil extraction rate of sandalwood kernels; FIG4 is a graph showing the effect of the complex enzyme on the oil extraction rate of sandalwood kernels; Figure 5 This is a diagram showing the effects of microwave-assisted and various enzymes on the oil extraction rate of sandalwood kernels. DETAILED DESCRIPTION 1. Experimental Methods

[0020] 1. Oil extraction process After shelling the sandalwood seeds, dry them in a 60℃ oven for two hours and crush them into powder with a crusher. Weigh 3g of sandalwood kernel powder into a 250ml conical flask and add phosphate buffer with a pH of 5 at a solid-liquid ratio of 1:5. Set the microwave time, temperature, and power for microwave pretreatment. After microwave, place in a 60℃ water bath for 1h, cool to below 50℃, add a certain amount of enzyme, and oscillate at a constant temperature of 50℃ for 4 hours. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90℃ for 20min. Transfer to a centrifuge tube and centrifuge at 10000r / min for 15min. The upper clear oil is the sandalwood kernel oil.

[0021] Process flow: sandalwood seeds → shelling → crushing → microwave pretreatment → aqueous enzymatic hydrolysis → enzyme inactivation → centrifugation → supernatant oil absorption → drying → sandalwood kernel oil 2. Microwave treatment method selection Microwave treatment uses microwave heating to increase the internal pressure of oil cells, burst the cell membrane and cell wall, and release oil.

[0022] The microwave time gradient was designed as follows: 30min, 45min, 60min, 75min, 90min; the microwave power gradient was designed as follows: 400w, 500w, 600w, 700w, 800w, 850w; the microwave temperature gradient was designed as follows: 40℃, 45℃, 50℃, 55℃, 60℃. The microwave temperature with the highest oil extraction rate was determined under the conditions of 500w and 75min, as the optimal microwave temperature; the microwave time with the highest oil extraction rate was determined under the conditions of the optimal microwave temperature and 500w, as the optimal microwave time; the microwave power with the highest oil extraction rate was determined under the conditions of the optimal microwave temperature and the optimal microwave time, as the optimal microwave power.

[0023] 3. Single factor experiment of optimal addition amount of each enzyme A single factor experiment was conducted to investigate the effect of the addition of pectinase (40,000 U / g), cellulase (3000 U / g), and papain (800,000 U / g) on ​​the oil extraction rate of white sandalwood kernels. Enzymatic hydrolysis conditions: pectinase is optimal at pH 2.5-6 and temperature 50°C; cellulase is optimal at pH 4.5-6.5 and temperature 45-65°C; papain is optimal at pH 5-7 and temperature 50-60°C; the reaction conditions were set at pH 5, enzymatic hydrolysis temperature 50°C, and solid-liquid ratio 1:5 (g / ml).

[0024] The gradient of enzyme addition amount (based on the mass percentage of white sandalwood kernel) was: 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, and 3.5%.

[0025] Weigh 3g of sandalwood kernel powder into a 250ml conical flask, add the extract at a solid-liquid ratio of 1:5, and place in a water bath at 60℃ for 1 hour. Add enzymes and rotate and oscillate at 50℃ for 4 hours. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90℃ for 20min, then centrifuge at 10000r / min for 15min at room temperature, and absorb the supernatant oil. At the same time, make a blank control group to compare the oil extraction rate and determine the optimal addition amount of each enzyme. Perform three parallel experiments for each experiment and take the average.

[0026] 4. Determine the complex enzyme conditions Papain, pectinase and cellulase are combined with each other. The amount of enzyme added is based on the optimal amount of each enzyme. A combination experiment is conducted to determine the best combination. The experimental steps are the same as those for a single enzyme. Three groups of parallel experiments are performed each time to obtain the average value.

[0027] 5. Calculation of oil yield Oil extraction rate / %=(weight of sandalwood kernel oil / weight of sandalwood kernel)×% 6. Data Analysis Take the average value of the data from three parallel experiments, draw a data graph with error bars, and conduct analysis.

[0028] The results of microwave experiments conducted by other members of the same group showed that the best conditions for microwave treatment were 50℃, 75min, 800W, and 60℃ water bath for 1h after microwave treatment. 2. Results and Analysis

[0029] (1) Results of protease extraction of sandalwood kernel oil The results of protease extraction of sandalwood kernel oil are shown in Figure 1 .

[0030] Within a certain range, as the amount of protease added increases, the extraction rate of sandalwood kernel oil increases. When the amount of protease added reaches 3%, the extraction rate reaches a maximum of 23.55%. This is because the protease destroys the protein bound to phospholipids and the protein membrane encapsulated in the outer layer of the oil droplet, releasing the oil. When the amount of protease added exceeds 3%, the extraction rate decreases.

[0031] (2) Results of extracting sandalwood kernel oil with pectinase The results of extracting sandalwood kernel oil with pectinase are shown in Figure 2 Within a certain range, with the increase of enzyme amount, the extraction rate of sandalwood kernel oil increases. When the addition amount of pectinase reaches 1%, the maximum oil extraction rate is 24.4%. This is because pectinase hydrolyzes and separates pectin, freeing oil and increasing the oil extraction rate of sandalwood kernel oil. When the addition amount exceeds 1%, the extraction rate gradually decreases.

[0032] (3) Results of cellulase extraction of sandalwood kernel oil Figure 3 The results showed that within a certain range, with the increase of enzyme dosage, the extraction rate of sandalwood kernel oil increased. When the addition amount of cellulase was 0.5%, the oil extraction rate reached a maximum of 26.3%. This is because cellulase degraded the cellulose skeleton of the plant cell wall, freeing the oil, thereby increasing the oil extraction rate of sandalwood kernel oil. When the enzyme addition amount exceeded 0.5%, the extraction rate decreased and tended to be flat.

[0033] Depend on Figure 4 It can be seen that according to the optimal addition amount of each enzyme, the combined enzyme for oil extraction is cellulase and pectinase, and the oil extraction rate under this condition reaches 28.6%, which is higher than the oil yield when the three enzymes are enzymatically hydrolyzed separately. This is the result of the synergistic effect of the enzymes.

[0034] The results of the orthogonal test of composite enzymes in Table 1 show that the main factor affecting the extraction of sandalwood kernel oil is the amount of pectinase added, and the secondary factor is the amount of cellulase added. The optimal amount of composite enzyme added is 1.5% cellulase and 1% pectinase. Under this condition, the extraction rate of sandalwood kernel oil is 29.77%.

[0035] Figure 5 The results show that the oil extraction rates of the single enzyme and compound enzyme tests are higher than those of the blank control, indicating that enzyme treatment is conducive to the release of oil and can improve the oil extraction rate. As can be seen from the figure, under the condition that the enzyme conditions remain unchanged, microwave pretreatment is added before enzymolysis, the oil extraction rate of the protease and compound enzyme test groups increases, while the oil extraction rate of the pectinase and cellulase test groups is not significantly increased. Among them, the best oil extraction effect is the compound enzyme test group after microwave treatment, with an oil extraction rate of 29.77%.

[0036] From this embodiment, it can be seen that the optimal process conditions for microwave-assisted aqueous enzymatic extraction of sandalwood kernel oil are: solid-liquid ratio 1:5 (g / ml), microwave power 800W, microwave time 75min, microwave temperature 50°C, complex enzyme 1.5% cellulase and 1% pectinase, enzymatic pH 5, enzymatic time 4h, enzymatic temperature 50°C, and the yield of sandalwood kernel oil obtained under this process condition is 29.77%. The sandalwood oil extracted by microwave-assisted aqueous enzymatic method is golden, clear, and has no precipitation, and the operation is simple, which provides a certain technical reference for the industrial production of aqueous enzymatic extraction of sandalwood kernel oil.

Claims

1. A method for extracting sandalwood kernel oil by enzymatic method, the main steps are as follows: Shell the white sandalwood seeds, dry them, and grind them into powder using a crusher; Weigh the sandalwood kernel powder into a conical flask, and add phosphate buffer with a pH of 5 at a solid-liquid ratio of 1:5; Set microwave time, temperature and power to perform microwave pretreatment; After microwave, place in a water bath at 50℃-70℃ for more than 1h, then cool to below 50℃; After adding a certain amount of enzyme, the mixture is oscillated at a constant temperature to carry out enzymatic hydrolysis reaction; after the enzymatic hydrolysis is completed, the enzyme is inactivated at above 90°C, and the mixture is transferred to a centrifuge tube and centrifuged, and the upper clear oil is sandalwood kernel oil.

2. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: In step (3), the microwave treatment conditions are: microwave temperature 50°C, microwave time 75min, microwave power 800W.

3. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: In step (4), the water bath temperature is 60°C, the water bath time is 1 hour, and then cooled to 50°C.

4. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: In step (5), after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90°C for 20 minutes.

5. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: The centrifugal speed was 10000 r / min and the centrifugal time was 15 min.

6. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: After adding the enzyme, shake at a constant temperature of 50°C for 4 hours; after the enzymatic hydrolysis is completed, inactivate the enzyme at 90°C.

7. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: The enzyme is at least one of protease, pectinase and cellulase.

8. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: The enzyme is selected from one of protease, pectinase and cellulase, and the dosage of the enzyme is: 1.5-3.5% of protease, 0.5-2.5% of pectinase, and 0.25-1.5% of cellulase.

9. The method for extracting sandalwood kernel oil by enzymatic method according to claim 1, characterized in that: The enzyme is a mixture of protease, pectinase and cellulase, and the dosage of each enzyme in the mixed enzyme is: 1.5-3.5% of protease, 0.5-2.5% of pectinase, and 0.25-1.5% of cellulase.

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