Method for synergistically promoting enzymatic degumming of rapeseed oil by phospholipase c and phospholipase a2

Abstract

The application discloses a method for synergistically promoting enzymatic degumming of rapeseed oil by phospholipase C and phospholipase A2, and comprises the following steps: S1, acidizing crude rapeseed oil; S2, adjusting the pH value of the acidized rapeseed oil; S3, adding phospholipase C, phospholipase A2, calcium chloride and deionized water into the system after adjusting the pH value, mixing uniformly, and then performing an enzymatic degumming reaction under a preset reaction temperature by means of ultrasonic assistance; and S4, performing enzyme inactivation treatment and centrifugal separation to obtain degummed rapeseed oil. The method adopts a phospholipase C and phospholipase A2 complex enzyme system, utilizes the catalytic complementarity of the two enzymes to different phospholipid components, breaks through the bottleneck that single enzyme cannot completely degum high-phosphorus rapeseed oil, and combines an ultrasonic auxiliary technology, so that the mass transfer efficiency of the enzyme and the substrate is strengthened through an ultrasonic cavitation effect, the degumming time is shortened, an efficient, economical and green new degumming solution is provided for the rapeseed oil refining industry, and the method has significant technical innovation and industrial application value.

Description

Technical Field

[0001] This invention relates to the field of oil processing, and specifically to a method for promoting enzymatic degumming of rapeseed oil by synergistic use of phospholipase C and phospholipase A2. Background Technology

[0002] Degumming is the first and crucial step in the refining process of edible vegetable oils. Its primary purpose is to remove colloidal impurities, mainly phospholipids, from the crude oil. Efficient degumming technology is of significant practical importance to the oil and fat industry, especially for oils with high non-hydrated phospholipid content, such as rapeseed oil, where degumming is more challenging and requires more stringent technical standards.

[0003] Currently, the traditional degumming methods widely used in industry include hydration degumming and acid degumming. However, both methods have inherent drawbacks: hydration degumming is difficult to effectively remove non-hydrated phospholipids from crude oil, resulting in high residual phosphorus content in the degummed oil, which is difficult to meet high-standard refining requirements; while acid degumming has a certain removal effect on non-hydrated phospholipids, it requires the use of a large amount of chemical reagents, which not only increases the burden of wastewater treatment, but also the high temperature and high acid environment can easily damage the quality of oils, which is not in line with the trend of green and environmentally friendly industrial development.

[0004] To address these issues, enzymatic degumming technology has emerged. This technology utilizes the site specificity of specific phospholipases to hydrolyze difficult-to-remove phospholipids (such as non-hydrated phospholipids) in crude oil into more hydrophilic lysophospholipids or diglycerides, thereby achieving degumming through hydration separation. Compared to traditional processes, enzymatic degumming offers advantages such as mild reaction conditions, high specificity, and fewer side reactions.

[0005] Despite the significant potential of enzymatic degumming, current technologies still face numerous bottlenecks. On one hand, existing research largely focuses on optimizing processes using single phospholipases (such as phospholipase A1, A2, and C). However, crude oil contains complex phospholipid compositions, making it difficult for a single enzyme system to simultaneously and efficiently hydrolyze phospholipids with different structures, thus limiting degumming efficiency and limiting its adaptability to crude oils of varying qualities. On the other hand, while some researchers have attempted to use dual-enzyme or multi-enzyme degumming methods, the differences in optimal reaction conditions (such as pH, temperature, and metal ion requirements) among different enzymes often lead to mutual inhibition of enzyme activity or difficulty in coordinating reaction conditions, resulting in minimal improvement in degumming efficiency or even adverse effects. Furthermore, enzymatic degumming generally suffers from long reaction times and low production efficiency, which to some extent restricts its large-scale industrial application.

[0006] Therefore, how to construct a degumming process that can fully leverage the synergistic effect of different phospholipases and possess efficient mass transfer characteristics to improve degumming efficiency and oil quality remains a pressing technical challenge in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a method for promoting enzymatic degumming of rapeseed oil by synergistic use of phospholipase C and phospholipase A2, so as to overcome the technical problems existing in the prior art when using a single phospholipase or a combination of phospholipases for degumming.

[0008] To achieve this objective, the present invention adopts the following technical solution: A method for enzymatic degumming of rapeseed oil using the synergistic effect of phospholipase C and phospholipase A2 includes the following steps: S1. Heat crude rapeseed oil to the first temperature, add acid solution and shear and mix to carry out acidification treatment; S2. Cool the acidified rapeseed oil to the second temperature, and then adjust the pH of the mixture to a range suitable for enzyme reaction; S3. Add phospholipase C, phospholipase A2, calcium chloride and deionized water to the pH-adjusted system, and form a mixed system by shear dispersion. Perform enzymatic degumming reaction with ultrasonic assistance at the preset reaction temperature. S4. The reaction system is heated to a third temperature for enzyme inactivation treatment, and then degummed rapeseed oil is obtained by centrifugation.

[0009] In one optional embodiment, in step S1, the first temperature is 60-70°C; the acid solution is a citric acid solution with a mass concentration of 45%.

[0010] In one optional embodiment, in step S1, the shearing speed is 8000-15000 rpm and the time is 1-2 min.

[0011] In one alternative embodiment, in step S2, the second temperature is 50–55°C; and the pH value is 6.0.

[0012] In an optional embodiment, in step S3, the enzyme activity ratio of phospholipase C to phospholipase A2 is 1:1.

[0013] In one alternative embodiment, in step S3, the amount of deionized water added is 4% of the mass of crude rapeseed oil.

[0014] In one alternative implementation, the ultrasonic power is 70% in step S3.

[0015] In one optional embodiment, in step S3, the reaction temperature is 45°C and the time is 60–90 min.

[0016] In an optional embodiment, in step S3, the mixing system further includes calcium chloride, wherein the amount of calcium chloride added is 0.04% of the mass of crude rapeseed oil.

[0017] In one optional embodiment, in step S4, the third temperature is 80-90°C, and the inactivation treatment time is 15-30 minutes.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention addresses the complex phospholipid composition of rapeseed oil by combining phospholipase C (PLC) and phospholipase A2 (PLA2). Utilizing the specificity of PLC for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and the highly efficient hydrolysis capability of PLA2 for phosphatidylinositol (PI) and phosphatidic acid (PA), a catalytic system covering all phospholipid components is constructed. Through systematic screening and optimization of single-factor process conditions, the optimal reaction window for the synergistic effect of the two enzymes is successfully identified, overcoming the technical bottleneck of single phospholipases being unable to adapt to the degumming requirements of crude oils of different qualities due to substrate specificity limitations. Rapeseed oil treated with this invention can have its phosphorus content stably reduced to below 10 mg / kg, significantly better than the degumming effect of single enzymatic methods, meeting or even exceeding the feed requirements of advanced refining processes.

[0019] 2. This invention couples ultrasonic-assisted technology, utilizing the microjets and shock waves generated by ultrasonic cavitation effect to effectively reduce the oil-water interfacial tension, greatly increasing the collision probability and mass transfer rate between the enzyme and the substrate at the interface. Under the premise of ensuring enzyme activity stability, it significantly shortens the degumming reaction time, improves production efficiency, and lays a feasible foundation for industrial application.

[0020] 3. The process conditions of this invention are mild, which maximizes the preservation of natural active ingredients in rapeseed oil while achieving efficient degumming. After accelerated storage at 70℃ for 7 days, the retention of four tocopherols in the oil is significantly higher than that of untreated rapeseed oil and rapeseed oil degummed by a single enzymatic method, indicating that this method causes less damage to natural nutrients. In addition, the degummed rapeseed oil exhibits excellent storage stability, with an acid value below 0.28 mg / g and a peroxide value below 0.23 mg / g, which are far below the national quality standard limits, effectively delaying oxidative rancidity of the oil during storage.

[0021] 4. This invention achieves reduced chemical reagent usage and water conservation through a dual-enzyme synergistic combined with ultrasound-assisted coupling process, avoiding the environmental burden of traditional acid degumming. While improving degumming efficiency and oil quality, it also has economic and environmental benefits, providing a new, efficient, economical, and green degumming solution for the rapeseed oil refining industry. It has significant technological innovation and broad prospects for industrial application. Attached Figure Description

[0022] Figure 1 The graph shows the changes in α-tocopherol content in degummed rapeseed oil treated with different degumming processes during storage.

[0023] Figure 2 The graph shows the changes in β-tocopherol content in degummed rapeseed oil treated with different degumming processes during storage.

[0024] Figure 3 The graph shows the changes in γ-tocopherol content in degummed rapeseed oil treated with different degumming processes during storage.

[0025] Figure 4 The graph shows the changes in δ-tocopherol content in degummed rapeseed oil treated with different degumming processes during storage.

[0026] Figure 5 The graph shows the changes in acid value of degummed rapeseed oil treated with different degumming processes during storage.

[0027] Figure 6 The graph shows the changes in peroxide value of degummed rapeseed oil treated with different degumming processes during storage. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0029] Example 1 100g of crude rapeseed oil (containing 250ppm phosphorus) was heated to 65℃ in a water bath, and then 0.2ml of 45% citric acid solution was added. The mixture was sheared at 10000 rpm for 1 min and then stirred for 20 min to carry out the acidification reaction. The acidified sample was then cooled from 65℃ to 50℃. The pH of the above sample was adjusted to 6.0 using a 1 mol / L sodium hydroxide solution. Then, 4 g of preheated deionized water, 10 g of phospholipase C (PLC, Shanghai Huashang Xiangyang Biotechnology Co., Ltd., 200 U / g), 0.2 mL of phospholipase A2 (PLA2, Shanghai Huashang Xiangyang Biotechnology Co., Ltd., 10,000 U / mL) and 4 g of calcium chloride were added sequentially. The resulting mixture was first dispersed at 10,000 rpm for 1 min, and then ultrasonically reacted at 70% ultrasonic power and 45℃ for 60 min. After the reaction was completed, the enzyme was inactivated at 85℃ for 15 min, and the degummed rapeseed oil was obtained after centrifugation for 10 min.

[0030] Example 2 100g of crude rapeseed oil (phosphorus content 250ppm) was heated to 70℃ in a water bath, and then 0.2ml of 45% citric acid solution was added. The mixture was sheared at 8000 rpm for 2 min and then stirred for 25 min for acidification. The acidified sample was then cooled from 65℃ to 55℃. The pH of the sample was adjusted to 6.0 with 1mol / L sodium hydroxide solution. Then, 4g of preheated deionized water, 10g of phospholipase C (PLC, 200 U / g), 0.2mL of phospholipase A2 (PLA2, 10,000 U / mL), and 4g of calcium chloride were added sequentially. The resulting mixture was first dispersed at 10000 rpm for 1 min, and then ultrasonically reacted at 70% power and 45℃ for 60 min. After the reaction, the enzyme was inactivated at 90℃ for 15 min, and the degummed rapeseed oil was obtained after centrifugation for 10 min.

[0031] Example 3 100g of crude rapeseed oil (phosphorus content 250ppm) was heated to 65℃ in a water bath, and then 0.2ml of 45% citric acid solution was added. The mixture was sheared at 15000 rpm for 1 min and then stirred for 15 min for acidification. The acidified sample was then cooled from 65℃ to 55℃. The pH of the sample was adjusted to 6.0 with 1mol / L sodium hydroxide solution. Then, 4g of preheated deionized water, 10g of phospholipase C (PLC, 200 U / g), 0.2mL of phospholipase A2 (PLA2, 10,000 U / mL), and 4g of calcium chloride were added sequentially. The resulting mixture was first dispersed at 10000 rpm for 1 min, and then sonicated at 70% power and 45℃ for 60 min. After the reaction, the enzyme was inactivated at 80℃ for 20 min, and the degummed rapeseed oil was obtained after centrifugation for 10 min.

[0032] Comparative Example 1 The difference between this comparative example and Example 1 is that the degumming reaction under ultrasonic conditions is replaced by a degumming reaction under stirring conditions; otherwise, it is the same as Example 1.

[0033] Comparative Example 2 The difference between this comparative example and Example 1 is that phospholipase A2 is not added; otherwise, they are the same as in Example 1.

[0034] Comparative Example 3 The difference between this comparative example and Example 1 is that phospholipase C is not added; otherwise, they are the same as in Example 1.

[0035] Comparative Example 4 The difference between this comparative example and Example 1 is that phospholipase A2 is replaced with phospholipase A1, otherwise it is the same as Example 1.

[0036] Comparative Example 5 The difference between this comparative example and Example 1 is that phospholipase A2 is replaced with phospholipase B, otherwise it is the same as Example 1.

[0037] Comparative Example 6 The difference between this comparative example and Example 1 is that phospholipase C is replaced with phospholipase D, otherwise it is the same as Example 1.

[0038] I. Study on Single Factors in the Synergistic Degumming Process of Phospholipase C and Phospholipase A2 (a) Screening based on moisture content Using phosphorus content as an indicator, the effect of different moisture contents in the degumming reaction system on the degumming effect of rapeseed oil was investigated. With other factors and the preparation process kept constant, different amounts of deionized water were added to achieve moisture contents of 3%, 4%, and 5% in the degumming reaction system, respectively. The results are shown in Table 1.

[0039] Table 1. Effect of different water contents in the degumming reaction system on the phosphorus content in the degummed oil.

[0040] The results showed that a water content of 4% in the degumming reaction system had a better effect on the removal of phospholipids, which could reduce the phosphorus content in the degummed oil to 7 mg / kg. When the water content was lower or higher than this value, the phosphorus content in the degummed oil was greater than 10 mg / kg. Therefore, the water content of the degumming reaction system should be controlled at 4%.

[0041] (ii) pH screening The effect of different pH values ​​in the degumming reaction system on the degumming effect of rapeseed oil was investigated using phosphorus content as an indicator. With other factors and the preparation process kept constant, the pH of the degumming reaction system was adjusted to 5.0, 6.0, and 7.0, respectively. The results are shown in Table 2.

[0042] Table 2. Effect of different pH values ​​in the degumming reaction system on the phosphorus content in the degummed oil.

[0043] The results showed that the degumming reaction system had a better effect on phospholipid removal when the pH was 6.0, which reduced the phosphorus content in the degummed oil to below 8 mg / kg. When the pH was lower or higher than this value, the phosphorus content in the degummed oil was greater than 15 mg / kg. Therefore, the pH of the degumming reaction system should be controlled at 6.0.

[0044] (III) Screening of reaction temperature Using phosphorus content as an indicator, the effect of different reaction temperatures in the degumming reaction system on the degumming effect of rapeseed oil was investigated. With other factors and preparation process kept constant, the temperature during the degumming reaction was adjusted to 40℃, 45℃, and 50℃, respectively. The results are shown in Table 3.

[0045] Table 3. Effect of different reaction temperatures during degumming on phosphorus content in the degummed oil.

[0046] The results showed that the removal effect of phospholipids was better when the temperature in the degumming reaction system was 45℃, which reduced the phosphorus content in the degummed oil to below 8mg / kg. When the temperature was lower or higher than this value, the phosphorus content in the degummed oil was greater than 8mg / kg. Therefore, the temperature of the degumming reaction system should be controlled at 45℃.

[0047] (iv) Screening of ultrasonic power Using phosphorus content as an indicator, the effect of different ultrasonic powers on the degumming effect of rapeseed oil was investigated. With other factors and the preparation process kept constant, the ultrasonic power was adjusted to 60%, 70%, and 80%, respectively. The results are shown in Table 4.

[0048] Table 4. Effect of different ultrasonic powers during the degumming reaction on the phosphorus content in the degummed oil.

[0049] The results showed that the ultrasonic power at 70% during the degumming reaction was more effective in removing phospholipids, reducing the phosphorus content in the degummed oil to below 5 mg / kg. When the ultrasonic power was lower or higher than this value, the phosphorus content in the degummed oil was greater than 5 mg / kg. Therefore, the ultrasonic power during the degumming reaction should be controlled at 70%.

[0050] (v) Screening of ultrasound time Using phosphorus content as an indicator, the effect of different ultrasonic times during the degumming reaction on the degumming effect of rapeseed oil was investigated. With other factors and preparation process kept constant, the ultrasonic time was adjusted to 45 min, 60 min, and 75 min, respectively. The results are shown in Table 5.

[0051] Table 5. Effect of different ultrasonic times during degumming reaction on phosphorus content in the degummed oil.

[0052] The results showed that the ultrasonic time of the degumming reaction reached 60 min, which achieved the best effect on the removal of phospholipids and reduced the phosphorus content in the degummed oil to below 4 mg / kg. Therefore, the ultrasonic time of the degumming reaction should be controlled at 60 min.

[0053] (vi) Screening of calcium chloride addition amount Using phosphorus content as an indicator, the effect of different amounts of calcium chloride in the degumming reaction system on the degumming effect of rapeseed oil was investigated. With other factors and preparation process kept constant, the amount of calcium chloride added (relative to the mass of acidified rapeseed oil) was adjusted to 0.03%, 0.04%, and 0.05%, respectively. The results are shown in Table 6.

[0054] Table 6. Effect of different amounts of calcium chloride added in the degumming reaction system on the phosphorus content in the degummed oil.

[0055] The results showed that the removal effect of phospholipids was optimal when the amount of calcium chloride added to the degumming reaction system was 0.04%, which reduced the phosphorus content in the degummed oil to below 5 mg / kg. Therefore, the amount of calcium chloride added to the degumming reaction system should be controlled at 0.04%.

[0056] (vii) Screening of different phospholipase C to phospholipase A2 activity ratios Using phosphorus content as an indicator, the effect of different enzyme activity ratios of phospholipase C and phospholipase A2 on the degumming effect of rapeseed oil was investigated. With other factors and preparation process kept constant, the enzyme activity ratio of phospholipase C to phospholipase A2 was adjusted to 2:1, 1:1, and 1:2, respectively. The results are shown in Table 7.

[0057] Table 7. Effect of different enzyme activity ratios in the degumming reaction system on the phosphorus content in the degummed oil.

[0058] The results showed that the optimal ratio of phospholipase C to phospholipase A2 activity in the degumming reaction system was 1:1, which reduced the phosphorus content in the degummed oil to below 8 mg / kg. When the ratio was lower or higher, the phosphorus content would exceed 10 mg / kg. Therefore, the ratio of phospholipase C to phospholipase A2 activity in the degumming reaction system should be controlled at 1:1.

[0059] In summary, in the process of synergistic degumming of rapeseed oil by phospholipase C and phospholipase A2, the optimal parameters for degumming were: water content of 4%, pH of 6.0, reaction temperature of 45℃, ultrasonic amplitude of 70%, ultrasonic time of 60 min, calcium chloride addition of 0.04%, and phospholipase C to phospholipase A2 enzyme activity ratio of 1:1. These screening results indicate that in the rapeseed oil degumming process using the synergistic effect of phospholipase C and phospholipase A2, by optimizing and controlling key process parameters such as system water content, pH value, reaction temperature, ultrasonic power, ultrasonic time, calcium chloride addition, and the ratio of the two enzyme activities, the synergistic effect of these factors can significantly improve degumming efficiency, reducing the phosphorus content of degummed rapeseed oil to below 10 mg / kg. This optimized process demonstrates a highly efficient and stable degumming effect.

[0060] II. Testing the effect of different degumming processes on the phosphorus content of rapeseed oil after degumming The degummed rapeseed oils obtained in Examples 1-3 and Comparative Examples 1-6 were used as oil samples to be tested, and their phosphorus content was determined.

[0061] 10 g of the oil sample to be tested and 0.5 g of zinc oxide were weighed and placed in a crucible. After the sample was completely carbonized, it was transferred to a muffle furnace and ashed at 550 °C for 2 h. After ashing, the ash was dissolved in 10 mL of 1:1 (v / v) hydrochloric acid solution and heated to a gentle boil for 5 min. The resulting mixture was filtered and diluted to 100 mL. 10 mL of the sample solution was taken and neutralized with 50% (w / v) potassium hydroxide solution, and then 1:1 (v / v) hydrochloric acid solution was slowly added dropwise until the precipitate was completely dissolved. Subsequently, 8 mL of 0.015% hydrazine sulfate solution and 2 mL of disodium molybdate dilute sulfuric acid solution (w / v) were added. After mixing, the mixture was heated in a boiling water bath for 10 min and then cooled to room temperature. The reaction solution was appropriately diluted and its absorbance was measured at a wavelength of 650 nm. Each experiment was performed in triplicate and the average value was taken. The results are shown in Table 8 below.

[0062] Table 8. Effects of different degumming processes on phosphorus content in rapeseed oil after degumming.

[0063] The results showed that after degumming using the degumming processes of Examples 1-3, the phosphorus content in rapeseed oil could be reduced to below 6 mg / kg. Specifically, the phosphorus content in rapeseed oil obtained in Example 1 was reduced to 2.89 ± 0.63 mg / kg, demonstrating excellent degumming effect. When stirring was used instead of ultrasound (Comparative Example 1), the phosphorus content in the resulting rapeseed oil was 7.54 ± 0.93 mg / kg, more than double that of Example 1. When a single enzyme was used for degumming (Comparative Examples 2 and 3), the phosphorus content... The obtained rapeseed oils contained phosphorus of 14.83±2.04 mg / kg and 8.79±0.60 mg / kg, respectively, which were more than five times and more than three times higher than those in Example 1. When one of the enzymes (phospholipase C (PLC) or phospholipase A2 (PLA2)) was replaced with other enzymes (Comparative Examples 4-6), the phosphorus content in the degummed rapeseed oils was 8.36±0.54, 13.65±0.31, and 10.84±1.21, respectively, which was much higher than that in Example 1. This indicates that only a specific combination of degumming processes (ultrasound + compound enzymes (phospholipase C and phospholipase A2)) can achieve excellent degumming effects in rapeseed oil. The absence of one enzyme or the alteration of another will reduce the degumming effect of the rapeseed oil.

[0064] III. Tests on the Effects of Different Degumming Processes on Tocopherol Content in Rapeseed Oil During Storage The degummed rapeseed oils obtained in Example 1 and Comparative Examples 1-3 were used as test samples. Under accelerated storage conditions of 70°C for seven days, the contents of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol were determined.

[0065] 1.0 g of oil sample was weighed for analysis. The oil sample was dissolved in chromatographic grade n-hexane and diluted to a final volume in a 10 mL brown volumetric flask. After shaking well, the solution was passed through a 0.22 μm nylon membrane and then separated by high-performance liquid chromatography (HPLC). Instrument and separation conditions: LC 20AT HPLC system (Tsushima Corporation, Japan) equipped with an SPD-20A UV detector; silica gel column (5 µm, 250 × 4.6 mm; Jiangsu Hanbang Technology Co., Ltd., China); n-hexane / isopropanol (98.5:1.5, v / v) as the mobile phase, flow rate 1.0 mL / min; injection volume 20 µL, column temperature 30℃, detection wavelength 295 nm. Qualitative and quantitative analysis was performed using the external standard method. The changes in the content of the four tocopherols during storage are shown below. Figure 1-4 As shown. Among them, Figure 1 The graph shows the change in α-tocopherol content during 7 days of storage. Figure 2 The graph shows the change in β-tocopherol content during 7 days of storage. Figure 3 The graph shows the change in γ-tocopherol content during 7 days of storage. Figure 4This is a graph showing the change in δ-tocopherol content during 7 days of storage.

[0066] from Figure 1-4 It can be seen that the tocopherol content of rapeseed oil degummed by enzymatic methods is slightly lower than that of crude oil in the early stage of storage. With increasing storage time, the content of all four tocopherols decreases. However, the tocopherol content of the four different enzymatically degummed rapeseed oils is higher than that of crude oil with increasing storage time. The tocopherol content of the four tocopherols in the ultrasonic combined enzymatic degumming group (α-tocopherol: 56.62 μg / ml - 45.69 μg / ml; β-tocopherol: 13.26 μg / ml - 7.74 μg / ml; γ-tocopherol: 114.94 μg / ml - 88.95 μg / ml; σ-tocopherol: 32.63 μg / ml - 21.22 μg / ml) remained relatively stable throughout the storage process.

[0067] The results show that although the environmental factors of ultrasonic cavitation may cause loss of tocopherols in the early stage of storage, the rapeseed oil treated by the ultrasonic compound enzymatic degumming method of the present invention can significantly increase the content of tocopherols during storage.

[0068] IV. Tests on the Effect of Different Degumming Processes on the Acid Value of Rapeseed Oil During Storage The degummed rapeseed oils obtained in Example 1 and Comparative Examples 1-3 were used as test samples. Under accelerated storage conditions of 70°C for seven days, the acid value content of the samples was determined.

[0069] Weigh 5 g of the oil sample to be tested into an Erlenmeyer flask, add 50 mL of neutral ethanol, heat to boiling and maintain for 5 minutes. Then add 1 mL of phenolphthalein indicator and titrate to the endpoint with 0.1 mol / L potassium hydroxide standard solution. Calculate the acid value using the following formula:

[0070] Where V is the titration volume (mL) of 0.1 N potassium hydroxide standard solution, N is the concentration of potassium hydroxide (0.1 N), and W is the mass of the oil sample (g).

[0071] The results are as follows Figure 5 As shown, from Figure 5The results show that on day 0, the AV values ​​of rapeseed oils degummed using all four different enzymatic methods were higher than those of untreated rapeseed oil. The AV values ​​of each group increased with prolonged storage time. Furthermore, after day 2, the AV value of crude rapeseed oil began to rise significantly, and the AV values ​​of all four treatments were lower than those of untreated rapeseed oil. Throughout the entire storage period, the AV value of the ultrasonic compound enzymatic degumming group increased more slowly, consistently remaining below 1 mg / g, indicating good storage quality. The ultrasonic phospholipase A2 degumming group and the ultrasonic phospholipase C degumming group showed poorer AV value performance.

[0072] V. Tests on the Effect of Different Degumming Processes on the Peroxide Value of Rapeseed Oil During Storage The degummed rapeseed oils obtained in Example 1 and Comparative Examples 1-3 were used as test samples. Under accelerated storage conditions of 70°C for seven days, the peroxide value was determined.

[0073] Accurately weigh 5 g of the oil sample to be tested into a 250 mL Erlenmeyer flask, add 30 mL of acetic acid-chloroform mixture (volume ratio 3:2), and shake vigorously to dissolve. Then add 1.00 mL of saturated potassium iodide solution, shake to mix, and let stand in the dark for 3 minutes. Add 100 mL of distilled water, shake thoroughly, and titrate with 0.01 mol / L sodium thiosulfate standard solution. When the yellow color of the solution disappears, add 1 mL of starch indicator and continue titrating until the blue color just disappears. Perform a blank test correction using the same method. The peroxide value is calculated using the following formula:

[0074] Where V is the volume (mL) of sodium thiosulfate standard solution consumed by the actual sample, V0 is the volume (mL) of sodium thiosulfate standard solution consumed by the blank experiment, C is the concentration of sodium thiosulfate standard solution (0.01 mol / L), and M is the mass (g) of the oil sample.

[0075] The results are as follows Figure 6 As shown, from Figure 6 The results show that on day 0, the PV value of crude oil (0.13 g / 100 g) was higher than that of the other four groups of rapeseed oil degummed by enzymatic methods. With increasing storage time, the PV value of crude oil increased significantly, consistently remaining higher than the PV values ​​of the other groups. The PV values ​​of the ultrasonic compound enzymatic degumming group remained consistently low; the stirring compound enzymatic degumming group was next; the ultrasonic phospholipase A2 degumming group and the ultrasonic phospholipase C degumming group had higher PV values, but these were still consistently lower than the PV value of crude oil. During the 7-day storage period, only the ultrasonic compound enzymatic degumming group consistently maintained a PV value below 0.20 g / 100 g, demonstrating good oxidative stability.

[0076] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0077] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0078] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for promoting enzymatic degumming of rapeseed oil by synergistically using phospholipase C and phospholipase A2, characterized in that, Includes the following steps: S1. Heat crude rapeseed oil to the first temperature, add acid solution and shear and mix to carry out acidification treatment; S2. Cool the acidified rapeseed oil to the second temperature, and then adjust the pH of the mixture to a range suitable for enzyme reaction; S3. Add phospholipase C, phospholipase A2, calcium chloride and deionized water to the pH-adjusted system, and form a mixed system by shear dispersion. Perform enzymatic degumming reaction with ultrasonic assistance at the preset reaction temperature. S4. The reaction system is heated to a third temperature for enzyme inactivation treatment, and then degummed rapeseed oil is obtained by centrifugation.

2. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect as described in claim 1, characterized in that, In step S1, the first temperature is 60-70°C; the acid solution is a citric acid solution with a mass concentration of 45%.

3. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect as described in claim 1, characterized in that, In step S1, the shearing speed is 8000-15000 rpm and the time is 1-2 min.

4. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S2, the second temperature is 50-55℃; the pH value is 6.

0.

5. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S3, the enzyme activity ratio of phospholipase C to phospholipase A2 is 1:

1.

6. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S3, the amount of deionized water added is 4% of the mass of crude rapeseed oil.

7. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S3, the ultrasonic power is 70%.

8. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S3, the reaction temperature is 45°C and the time is 60–90 min.

9. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S3, the amount of calcium chloride added is 0.04% of the mass of crude rapeseed oil.

10. The method for enzymatic degumming of rapeseed oil using phospholipase C and phospholipase A2 in synergistic effect according to claim 1, characterized in that, In step S4, the third temperature is 80-90°C, and the inactivation treatment time is 15-30 minutes.

Images