Enzymatic deacidification refining method of degummed and degreased rice bran oil with high acid value
By immobilizing commercially available lipase catalysts with phytosterols through polyamide-amine-modified magnetic carbon nanotubes, the problem of poor deacidification and nutrient loss in high-acid-value rice bran oil was solved, achieving a highly efficient and environmentally friendly deacidification process that meets high-end edible oil standards and reduces production costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GAOAN QINGHE GREASE CO LTD
- Filing Date
- 2026-04-13
- Publication Date
- 2026-05-15
AI Technical Summary
Existing deacidification methods for high-acid-value rice bran oil suffer from problems such as poor deacidification effect, serious loss of nutrients, difficulty in recovering and reusing enzyme catalysts, high energy consumption, and environmental pollution. In particular, for high-acid-value degummed and defatted rice bran oil with an acid value of 70~100mgKOH/g, existing technologies cannot effectively reduce the acid value to below 0.1mgKOH/g and have poor retention of oryzanol.
Using commercially available lipase immobilized in polyamide-amine modified magnetic carbon nanotubes as a catalyst and phytosterols as raw materials, an enzymatic deacidification and refining method is adopted, combined with the separation and recovery of magnetically responsive catalysts, to achieve efficient deacidification and nutrient fortification. The specific steps include raw material pretreatment, reaction system configuration, enzymatic deacidification reaction, catalyst separation and recovery, and post-treatment.
This method achieves a reduction in rice bran oil acid value to below 0.1 mg KOH/g, a oryzanol retention rate of over 95%, a phytosterol conversion rate of ≥90%, and allows the catalyst to be reused 8-10 times, thereby reducing production costs, minimizing environmental pollution, and aligning with the trend of green chemical development.
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Figure CN122038044A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of rice bran oil refining technology, and more particularly to an enzymatic deacidification refining method for high-acid-value degummed and defatted rice bran oil. Background Technology
[0002] Rice bran oil is a highly nutritious edible oil, rich in functional components such as oryzanol, vitamin E, and phytosterols. Among them, oryzanol has various physiological activities, including regulating blood lipids and improving sleep, which is one of the core nutritional advantages of rice bran oil. However, rice bran contains a large amount of lipase, which rapidly catalyzes the hydrolysis of oils in rice bran during processing and storage, producing free fatty acids and causing a sharp increase in the acid value of rice bran oil. At the same time, the degumming and defatting pretreatment processes of rice bran oil further aggravate the fluctuation of acid value, resulting in high-acid-value degummed and defatted rice bran oil with an acid value (calculated as KOH) of 70~100mgKOH / g.
[0003] Currently, the main deacidification and refining methods for high-acid-value rice bran oil include alkali refining, physical distillation, and solvent extraction. Among these, alkali refining is the most widely used method in industry, but it suffers from high refining costs, residual additives, and significant loss of nutrients such as oryzanol. Studies have shown that the oryzanol retention rate after traditional alkali refining is usually below 65%, and it generates a large amount of soap-containing wastewater, polluting the environment. Physical distillation, while leaving no chemical reagent residues, requires high temperature and high vacuum conditions, resulting in extremely high energy consumption. Furthermore, high temperatures can lead to oryzanol degradation, making it difficult to achieve a retention rate exceeding 80%. Additionally, the acid value after deacidification is difficult to reduce to below 0.1 mg KOH / g, failing to meet the standards for high-end edible oils. Solvent extraction, while retaining nutrients to some extent, carries a high risk of solvent residue, is complex, and has limited deacidification efficiency, making it unsuitable for processing ultra-high acid-value rice bran oil with an acid value of 70-100 mg KOH / g.
[0004] Enzymatic deacidification, with its advantages of being mild, efficient, and environmentally friendly, has become a research hotspot in the field of rice bran oil refining. Commercially available lipases can specifically catalyze the reaction of free fatty acids with phytosterols to produce phytosterol esters, achieving deacidification. Furthermore, the reaction conditions are mild, minimizing the destruction of heat-sensitive nutrients such as oryzanol. Phytosterols themselves are natural functional components in rice bran oil; using them as raw materials for deacidification can further enhance the product's nutritional value, achieving a dual effect of "deacidification + nutritional fortification." However, commercially available free lipases suffer from poor stability, difficulty in recovery, low reusability, and catalytic efficiency highly dependent on the reaction system, thus limiting the industrial application of enzymatic deacidification.
[0005] To address the shortcomings of free enzymes, enzyme immobilization technology has been extensively studied. Magnetic carbon nanotubes, with their nanoscale size, high specific surface area, abundant pore structure, good biocompatibility, and magnetic responsiveness, can be used as carriers to immobilize enzymes, enabling rapid enzyme separation and recovery and reducing production costs. Polyamide-amine (PAMAM), as a dendritic molecule with controllable molecular weight and end groups rich in active groups, can significantly increase the active sites of the carrier when combined with magnetic carbon nanotubes, thereby improving enzyme loading and immobilization efficiency, while also enhancing the stability of the immobilized enzyme. In existing technologies, there are reports of using polyamide-amine modified magnetic carbon nanotubes for immobilized lipases, but these are mainly applied to fields such as biodiesel production. There are no reports of using them to immobilize commercial lipases. Furthermore, the technical solutions for deacidifying and refining high-acid-value degummed and defatted rice bran oil with an acid value of 70-100 mgKOH / g, aiming to reduce the acid value to below 0.1 mgKOH / g and achieve a oryzanol retention rate of over 95%, using phytosterols as raw materials, cannot solve the technical pain points of severe nutrient loss, poor deacidification effect, and low enzyme utilization rate in the refining of high-acid-value rice bran oil. Summary of the Invention
[0006] To address the problems of poor deacidification effect, severe loss of nutrients such as oryzanol, difficulty in recycling and reusing enzyme catalysts, high energy consumption, and environmental pollution in existing high-acid-value degummed and defatted rice bran oil refining methods, this invention provides an enzymatic deacidification refining method using commercially available lipase immobilized in polyamide-amine modified magnetic carbon nanotubes as a catalyst and phytosterols as raw materials. This method achieves highly efficient deacidification of high-acid-value degummed and defatted rice bran oil (acid value 70~100mgKOH / g), reducing the acid value of the deacidified rice bran oil to below 0.1mgKOH / g, and achieving a oryzanol retention rate of over 95%. Simultaneously, the phytosterols are used to fortify the product nutritionally, improve enzyme stability and reusability, reduce production costs, decrease environmental pollution, and promote the high-value utilization of high-acid-value rice bran oil.
[0007] Specifically, the following technical solutions are included: An enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil is provided, comprising the following steps: S1. Raw material pretreatment: Heat and stir the degummed and defatted rice bran oil with an acid value of 70~100mgKOH / g until it is evenly mixed and set aside; Crush the phytosterols and set aside. S2. Reaction system configuration: Phytosterols, immobilized commercial lipase catalysts, and emulsifiers are added to degummed and defatted rice bran oil. The amount of the immobilized commercial lipase catalyst added is 0.5% to 2.0% of the mass of the degummed and defatted rice bran oil; the amount of the emulsifier added is 0.1% to 0.3% of the mass of the degummed and defatted rice bran oil. S3. Enzymatic deacidification reaction: under conditions of 40~55℃ and stirring rate of 150~250r / min for 8~16h, immobilized commercial lipase catalyzes the esterification reaction of free fatty acids with phytosterols to obtain phytosterol esters. S4. Catalyst separation and recovery: After the reaction is completed, the commercial lipase catalyst is separated and immobilized by an external magnetic field, washed and dried for later use, and rice bran oil after catalyst separation is obtained. S5. Post-processing: The rice bran oil after catalyst separation is subjected to vacuum distillation to remove unreacted phytosterols and volatile impurities, yielding refined rice bran oil.
[0008] In step S3, the reaction temperature is 45-50℃. This temperature ensures the high catalytic activity of the immobilized commercial lipase, promoting the esterification reaction of phytosterols and free fatty acids, while preventing the degradation of oryzanol due to high temperatures. The amount of immobilized commercial lipase catalyst added ensures both deacidification efficiency and phytosterol conversion rate, while reducing production costs.
[0009] Preferably, in step S3, the reaction time is 10-12 hours. This reaction time allows the final refined rice bran oil to have an acid value below 0.1 mg KOH / g, a oryzanol retention rate of over 95%, and a phytosterol conversion rate of ≥90%.
[0010] In step S2, the addition of the emulsifier is intended to promote the dispersion of phytosterols and the mixing of the system.
[0011] Furthermore, in step S2, the preparation method of the immobilized commercial lipase catalyst includes the following steps: (1) Preparation of magnetic multi-walled carbon nanotubes: Fe3O4 nanoparticles were anchored on the surface of multi-walled carbon nanotubes by chemical co-precipitation to prepare magnetic multi-walled carbon nanotubes (m-MWCNTs). (2) Magnetic multi-walled carbon nanotubes modified with third-generation polyamide-amine (PAMAM-G3): Magnetic multi-walled carbon nanotubes were functionalized with amino groups using (3-aminopropyl)triethoxysilane to obtain amino-functionalized magnetic multi-walled carbon nanotubes. Then, third-generation polyamide-amine was grafted onto them by a divergent method to obtain magnetic multi-walled carbon nanotubes modified with third-generation polyamide-amine. (3) Immobilization of commercial lipase: Using third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes as a carrier, commercial lipase was immobilized on the carrier. The mass ratio of commercial lipase to carrier was 1:(5~20). The immobilization was carried out by shaking at 25~35℃ and stirring at 100~200r / min for 4~8h. After magnetic separation, washing and drying, the immobilized commercial lipase catalyst was obtained.
[0012] Preferably, the immobilized commercial lipase catalyst (polyamide-amine modified magnetic carbon nanotube immobilized commercial lipase catalyst) has an enzyme activity recovery rate of ≥85%, a specific activity significantly higher than that of free enzyme, and good magnetic responsiveness, and can be rapidly separated and recovered by a magnetic field.
[0013] Further, in step (1), the anchoring of Fe3O4 nanoparticles on the surface of multi-walled carbon nanotubes is as follows: FeCl3·6H2O and FeSO4·7H2O are dissolved in deionized water at a molar ratio of 2:1, nitrogen gas is introduced to remove oxygen for 30~60 min, and the pH is adjusted to 9~11 with ammonia water at 50~70℃ and a stirring rate of 200~300 r / min, and the reaction is carried out for 30~60 min.
[0014] Preferably, in step (1), after anchoring Fe3O4 nanoparticles on the surface of multi-walled carbon nanotubes, the process further includes centrifugation, washing with deionized water until neutral, and vacuum drying at 60~80℃ for 12~24h.
[0015] Further, in step (2), the divergent grafting of third-generation polyamide-amine is as follows: amino-functionalized magnetic multi-walled carbon nanotubes are dispersed in methanol, third-generation polyamide-amine monomers are added, and the reaction is carried out at 40~60℃ for 24~48h.
[0016] Preferably, in step (2), the third-generation polyamide-amine (PAMAM-G3) modified magnetic multi-walled carbon nanotubes are as follows: magnetic multi-walled carbon nanotubes are dispersed in anhydrous ethanol, ultrasonically dispersed for 30-60 min, (3-aminopropyl)triethoxysilane (APTES) is added, and the mixture is reacted under reflux at 60-80 °C for 12-24 h to obtain amino-functionalized magnetic multi-walled carbon nanotubes (m-MWCNTs-NH2); then, m-MWCNTs-NH2 is dispersed in methanol by a dispersion method, the third-generation polyamide-amine monomer is added, and the mixture is reacted at 40-60 °C for 24-48 h. After the reaction, the mixture is centrifuged, washed with methanol 3-5 times, and vacuum dried at 60-80 °C for 12-24 h to obtain third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes (m-MWCNTs-PAMAM-G3).
[0017] Further, in step (3), the commercial lipase is immobilized on the carrier by dispersing the carrier in a phosphate buffer solution with a pH of 6.5-7.5 and a concentration of 0.05-0.1 mol / L, and adding the commercial lipase; the commercial lipase is Novozym 435.
[0018] Preferably, in step (3), the washing is performed by washing with phosphate buffer 3 to 5 times, and the drying is performed by vacuum drying at 40 to 60°C for 8 to 12 hours.
[0019] Furthermore, the purity of the phytosterol is ≥95%, and the emulsifier is Tween-80.
[0020] Preferably, the phytosterol is a food-grade phytosterol.
[0021] Furthermore, in step S1, the heating temperature is 30~40℃, and the pulverization precision is 80~100 mesh.
[0022] Furthermore, in step S2, the molar ratio of the phytosterols to the free fatty acids in the degummed and defatted rice bran oil is (1.0~1.5):1.
[0023] This molar ratio ensures that free fatty acids react fully while avoiding excessive phytosterols that could burden subsequent separation, thus improving raw material utilization.
[0024] Preferably, in step S4, the washing and drying process involves washing with n-hexane 2-3 times and then vacuum drying; the immobilized commercial lipase catalyst can be reused 8-10 times after being separated and recovered by an external magnetic field, with an enzyme activity retention rate ≥75%.
[0025] Furthermore, in step S5, the conditions for vacuum distillation are: vacuum degree 0.09~0.1MPa, temperature 80~100℃.
[0026] Furthermore, in step S5, the acid value of the refined rice bran oil is ≤0.1mgKOH / g.
[0027] Preferably, in step S5, the oryzanol retention rate of the refined rice bran oil is ≥95%.
[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. Excellent deacidification effect, capable of treating rice bran oil with ultra-high acid value: Using phytosterols as raw materials, through the catalytic action of immobilized commercial lipase, it can reduce the acid value of degummed and defatted rice bran oil with an acid value of 70~100mgKOH / g to below 0.1mgKOH / g. This is far superior to the existing deacidification effects of alkali refining (acid value ≥1.0mgKOH / g), physical distillation (acid value ≥1.93mgKOH / g), and solvent extraction (acid value ≥0.92mgKOH / g), meeting the acid value requirements of high-end edible oils; 2. High retention of nutrients and nutritional fortification: The mild reaction conditions (40~55℃) avoid the destruction of oryzanol by high temperature and strong alkali, resulting in a oryzanol retention rate (content after refining / content before refining × 100%) of over 95%, which is far higher than existing technologies (alkali refining deacidification ≤65%, physical distillation deacidification ≤80%, solvent extraction deacidification ≤73%). At the same time, using phytosterols as raw materials, the reaction produces phytosterol esters, further enhancing the nutritional value of rice bran oil and achieving integrated "deacidification + nutritional fortification". 3. Reusable catalyst reduces production costs: Commercial lipase (Novozym 435) is immobilized using magnetic multi-walled carbon nanotubes modified with third-generation polyamide-amine. The catalyst exhibits excellent magnetic responsiveness and can be rapidly separated and recovered via a magnetic field. After being reused 8-10 times, the enzyme activity retains more than 75%, solving the problems of difficult recovery and low reusability of free enzymes, and significantly reducing the production cost of enzymatic deacidification. At the same time, the modification with third-generation polyamide-amine significantly improves the enzyme loading and stability, with an enzyme activity recovery rate of ≥85%, and catalytic efficiency superior to existing immobilized lipase systems. 4. Environmentally friendly and free from secondary pollution: The entire deacidification process does not require the use of strong alkalis, large amounts of organic solvents and other harmful reagents. It only produces a small amount of unreacted phytosterols (which can be recycled), and there are no pollution problems such as soap-containing wastewater or solvent residues, which is in line with the trend of green chemical development. 5. Simple process and low energy consumption: The reaction conditions are mild and do not require harsh conditions such as high temperature and high vacuum. The subsequent vacuum distillation temperature is only 80~100℃, and the energy consumption is much lower than that of physical distillation deacidification (220~250℃). Moreover, the process steps are simple and easy to scale up industrially, which can realize the high-value utilization of high acid value degummed and degreased rice bran oil. Attached Figure Description
[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0030] Figure 1 The deacidification effect of refined rice bran oil prepared by the methods of Examples 1-4 and Comparative Examples 1-3 of this invention; Figure 2 The retention rate of refined rice bran oil obtained by the methods of Examples 1-4 and Comparative Examples 1-3 of this invention; Figure 3 The phytosterol conversion rate of refined rice bran oil obtained by the methods of Examples 1-4 and Comparative Examples 1-3 of this invention. Detailed Implementation
[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0032] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.
[0033] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.
[0034] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.
[0035] To better understand the technical content of the present invention, the technical solution of the present invention will be further introduced and explained below with reference to specific embodiments.
[0036] Unless otherwise stated, "%" in this invention refers to mass content.
[0037] In this embodiment, the commercially available lipase used is Novozym 435, the polyamide-amine grafting generation is 3, the magnetic carbon nanotubes are magnetic multi-walled carbon nanotubes, and the phytosterols are food grade (98% purity).
[0038] Example 1 An enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil is provided, comprising the following steps: S1. Raw material pretreatment: Degummed and defatted rice bran oil with an acid value of 85 mg KOH / g and a oryzanol content of 1.2% was heated to 35°C and stirred evenly to remove a small amount of precipitated impurities. The oil was then set aside. Phytosterols were pulverized to 90 mesh and set aside. The purity of the phytosterols was 98%. S2. Reaction system configuration: Take 100g of pretreated degummed and defatted rice bran oil, add phytosterol, 1.2g of immobilized commercial lipase catalyst and 0.2g of emulsifier (Tween-80) to the degummed and defatted rice bran oil, and stir to mix well; the molar ratio of phytosterol to free fatty acids in the degummed and defatted rice bran oil is 1.2:1; S3. Enzymatic deacidification reaction: The reaction was carried out at 48℃ and a stirring rate of 200r / min for 11h under constant temperature oscillation. During the reaction, immobilized commercial lipase catalyzed the esterification reaction between free fatty acids and phytosterols to obtain phytosterol esters, thereby removing free fatty acids from degummed and defatted rice bran oil and achieving product nutritional fortification. S4. Catalyst separation and recovery: After the reaction is completed, the commercial lipase catalyst is separated and immobilized by an external magnetic field, washed three times with n-hexane, and vacuum dried for later use to obtain rice bran oil after catalyst separation. S5. Post-processing: The rice bran oil after catalyst separation is subjected to vacuum distillation at a vacuum degree of 0.095 MPa and a temperature of 90°C to remove unreacted phytosterols and volatile impurities, thereby obtaining refined rice bran oil.
[0039] According to the test results, the refined rice bran oil prepared by the method in Example 1 has an acid value of 0.07 mg KOH / g, a oryzanol retention rate of 96.2%, a phytosterol conversion rate of 92.5%, and an enzyme activity retention rate of 99% for the immobilized commercial lipase catalyst, which meets the standards for high-end edible oils.
[0040] In step S2, the method for preparing the immobilized commercial lipase catalyst includes the following steps: (1) Preparation of magnetic multi-walled carbon nanotubes: FeCl3·6H2O (2 mmol) and FeSO4·7H2O (1 mmol) were dissolved in 50 mL of deionized water by chemical coprecipitation. Nitrogen gas was introduced to remove oxygen for 40 min. The pH was adjusted to 10 with ammonia water at 60 °C and stirring rate of 250 r / min. The reaction was carried out for 45 min to anchor Fe3O4 nanoparticles on the surface of multi-walled carbon nanotubes. After centrifugation and washing with deionized water until neutral, the nanotubes were vacuum dried at 70 °C for 18 h to prepare magnetic multi-walled carbon nanotubes (m-MWCNTs). (2) Modification of magnetic multi-walled carbon nanotubes with third-generation polyamide-amine (PAMAM-G3): 0.5 g of magnetic multi-walled carbon nanotubes were dispersed in 50 mL of anhydrous ethanol and ultrasonically dispersed for 45 min. 0.2 mL of (3-aminopropyl)triethoxysilane (APTES) was added to amino-functionalize the magnetic multi-walled carbon nanotubes. The reaction was carried out at 70 °C under reflux for 18 h to obtain amino-functionalized magnetic multi-walled carbon nanotubes (m-MWCNTs-NH2). Then, third-generation polyamide-amine was grafted by the divergent method. The amino-functionalized magnetic multi-walled carbon nanotubes were dispersed in 30 mL of methanol and 0.3 g of third-generation polyamide-amine monomer was added. The reaction was carried out at 50 °C for 36 h. After the reaction was completed, the nanotubes were centrifuged, washed with methanol 4 times, and vacuum dried at 70 °C for 18 h to obtain third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes (m-MWCNTs-PAMAM-G3). (3) Immobilization of commercial lipase: 0.2 g of third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes were used as a carrier. The carrier was dispersed in 20 mL of phosphate buffer solution with pH 7.0 and a concentration of 0.08 mol / L, and 0.02 g of commercial lipase, namely Novozym 435, was added. The commercial lipase was immobilized on the carrier and shaken at 30 °C and a stirring rate of 150 r / min for 6 h. After magnetic separation, washing with phosphate buffer solution 4 times, and vacuum drying at 50 °C for 10 h, the immobilized commercial lipase catalyst was obtained. The enzyme activity recovery rate of the immobilized commercial lipase catalyst was 88%.
[0041] The immobilized commercial lipase catalyst prepared in Example 1 was repeatedly used in step S3, the enzymatic deacidification reaction of Example 1. After each reaction, the catalyst was separated and recovered in step S4, and after washing and drying, it was reused. The acid value, oryzanol retention rate, phytosterol conversion rate, and catalyst enzyme activity retention rate were measured after each deacidification reaction. The results are as follows: First use: Acid value 0.07 mg KOH / g, oryzanol retention rate 96.2%, phytosterol conversion rate 92.5%, enzyme activity retention rate 99%; Fifth use: Acid value 0.08 mg KOH / g, oryzanol retention rate 95.8%, phytosterol conversion rate 91.8%, enzyme activity retention rate 96%; 10th use: Acid value 0.09 mg KOH / g, oryzanol retention rate 95.6%, phytosterol conversion rate 91.0%, enzyme activity retention rate 93%; 20th use: acid value 0.10 mg KOH / g, oryzanol retention rate 95.4%, phytosterol conversion rate 89.5%, enzyme activity retention rate 90.5%.
[0042] 21st use: Acid value 0.15 mg KOH / g, oryzanol retention rate 94.7%, phytosterol conversion rate 88.5%, enzyme activity retention rate 87.6%. (No longer meets requirements) The results showed that the immobilized catalyst could be stably reused 20 times and still meet the deacidification requirements (acid value ≤ 0.1 mg KOH / g, oryzanol retention rate ≥ 95%, phytosterol conversion rate ≥ 89%), demonstrating excellent reusability.
[0043] Example 2 An enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil is provided, comprising the following steps: S1. Raw material pretreatment: Degummed and defatted rice bran oil with an acid value of 98 mg KOH / g and a oryzanol content of 1.1% was heated to 40°C and stirred evenly to remove a small amount of precipitated impurities. The oil was then set aside. Phytosterols were pulverized to 100 mesh and set aside. The purity of the phytosterols was 98%. S2. Reaction system configuration: Add phytosterols, 3.0g of immobilized commercial lipase catalyst and 0.4g of emulsifier (Tween-80) to 200g of degummed and defatted rice bran oil; the molar ratio of phytosterols to free fatty acids in the degummed and defatted rice bran oil is 1.3:1; S3. Enzymatic deacidification reaction: The reaction is carried out at 50℃ and a stirring rate of 250r / min for 12h under constant temperature oscillation. During the reaction, immobilized commercial lipase catalyzes the esterification reaction of free fatty acids and phytosterols to obtain phytosterol esters, thereby removing free fatty acids from degummed and defatted rice bran oil and achieving product nutritional fortification. S4. Catalyst Separation and Recovery: After the reaction, the immobilized commercial lipase catalyst is separated by an external magnetic field, washed three times with n-hexane, and vacuum dried for later use to obtain rice bran oil after catalyst separation. The immobilized commercial lipase catalyst can be reused 8-10 times after separation and recovery by an external magnetic field, with an enzyme activity retention rate ≥75%. S5. Post-processing: The rice bran oil after catalyst separation is subjected to vacuum distillation at a vacuum degree of 0.1 MPa and a temperature of 100℃ to remove unreacted phytosterols and volatile impurities, thereby obtaining refined rice bran oil.
[0044] According to the test results, the refined rice bran oil prepared by the method in Example 2 has an acid value of 0.08 mg KOH / g, a oryzanol retention rate of 95.5%, a phytosterol conversion rate of 91.8%, and an enzyme activity retention rate of 98.9% for the immobilized commercial lipase catalyst, which meets the standards for high-end edible oils.
[0045] In step S2, the method for preparing the immobilized commercial lipase catalyst includes the following steps: (1) Preparation of magnetic multi-walled carbon nanotubes: FeCl3·6H2O (4 mmol) and FeSO4·7H2O (2 mmol) were dissolved in 100 mL of deionized water by chemical coprecipitation. Nitrogen gas was introduced to remove oxygen for 60 min. The pH was adjusted to 11 with ammonia water at 70 °C and stirring rate of 300 r / min. The reaction was carried out for 60 min to anchor Fe3O4 nanoparticles on the surface of multi-walled carbon nanotubes. After centrifugation and washing with deionized water until neutral, the nanotubes were vacuum dried at 80 °C for 24 h to prepare magnetic multi-walled carbon nanotubes (m-MWCNTs). (2) Modification of magnetic multi-walled carbon nanotubes with third-generation polyamide-amine (PAMAM-G3): 1.0 g of magnetic multi-walled carbon nanotubes were dispersed in 80 mL of anhydrous ethanol and ultrasonically dispersed for 60 min. 0.5 mL of (3-aminopropyl)triethoxysilane (APTES) was added to amino-functionalize the magnetic multi-walled carbon nanotubes. The reaction was carried out under reflux at 80 °C for 24 h to obtain amino-functionalized magnetic multi-walled carbon nanotubes (m-MWCN). Then, by a divergent grafting method, third-generation polyamide-amine was grafted onto the amino-functionalized magnetic multi-walled carbon nanotubes (m-MWCNTs-PAMAM-G3). The amino-functionalized magnetic multi-walled carbon nanotubes were dispersed in 50 mL of methanol, and 0.6 g of third-generation polyamide-amine monomer (PAMAM monomer) was added. The mixture was stirred and reacted at 60 °C for 48 h. After the reaction was completed, the mixture was centrifuged, washed 5 times with methanol, and vacuum dried at 80 °C for 24 h to obtain third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes (m-MWCNTs-PAMAM-G3). (3) Immobilization of commercial lipase: 0.5 g of third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes were used as a carrier. The carrier was dispersed in 50 mL of phosphate buffer solution with pH 7.5 and a concentration of 0.1 mol / L, and 0.05 g of commercial lipase, Novozym 435, was added. The commercial lipase was immobilized on the carrier and immobilized by constant temperature shaking at 35 °C and a stirring rate of 200 r / min for 8 h. After magnetic field separation, washing with phosphate buffer solution 5 times, and vacuum drying at 60 °C for 12 h, the immobilized commercial lipase catalyst was obtained. The enzyme activity recovery rate of the immobilized commercial lipase catalyst was 86%.
[0046] Example 3 An enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil is provided, comprising the following steps: S1. Raw material pretreatment: Degummed and defatted rice bran oil with an acid value of 85 mg KOH / g and a oryzanol content of 1.2% was heated to 35°C and stirred evenly to remove a small amount of precipitated impurities. The oil was then set aside. Phytosterols were pulverized to 90 mesh and set aside. The purity of the phytosterols was 98%. S2. Reaction system configuration: Add phytosterol, 1.6g immobilized commercial lipase catalyst and 0.2g emulsifier (Tween-80) to 100g degummed and defatted rice bran oil; the molar ratio of phytosterol to free fatty acids in the degummed and defatted rice bran oil is 1.1:1; S3. Enzymatic deacidification reaction: The reaction is carried out at 40℃ and a stirring rate of 200r / min for 15h. During the reaction, immobilized commercial lipase catalyzes the esterification reaction of free fatty acids and phytosterols to obtain phytosterol esters, thereby removing free fatty acids from degummed and defatted rice bran oil and achieving product nutritional fortification. S4. Catalyst separation and recovery: After the reaction is completed, the commercial lipase catalyst is separated and immobilized by an external magnetic field, washed three times with n-hexane, and vacuum dried for later use to obtain rice bran oil after catalyst separation. S5. Post-processing: The rice bran oil after catalyst separation is subjected to vacuum distillation at a vacuum degree of 0.095 MPa and a temperature of 90°C to remove unreacted phytosterols and volatile impurities, thereby obtaining refined rice bran oil.
[0047] According to the test results, the refined rice bran oil prepared by the method in Example 3 had an acid value of 0.09 mg KOH / g, a oryzanol retention rate of 96.1%, a phytosterol conversion rate of 88.7%, and an enzyme activity retention rate of 99.1% for the immobilized commercial lipase catalyst.
[0048] The preparation method of the immobilized commercial lipase catalyst is the same as that in Example 1.
[0049] Example 4 An enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil is provided, comprising the following steps: S1. Raw material pretreatment: Degummed and defatted rice bran oil with an acid value of 85 mg KOH / g and a oryzanol content of 1.2% was heated to 35°C and stirred evenly to remove a small amount of precipitated impurities. The oil was then set aside. Phytosterols were pulverized to 90 mesh and set aside. The purity of the phytosterols was 98%. S2. Reaction system configuration: Add phytosterol, 0.9g immobilized commercial lipase catalyst and 0.2g emulsifier (Tween-80) to 100g degummed and defatted rice bran oil; the molar ratio of phytosterol to free fatty acids in the degummed and defatted rice bran oil is 1.5:1; S3. Enzymatic deacidification reaction: The reaction is carried out at 55℃ and a stirring rate of 200r / min for 8 hours. During the reaction, immobilized commercial lipase catalyzes the esterification reaction of free fatty acids and phytosterols to obtain phytosterol esters, thereby removing free fatty acids from degummed and defatted rice bran oil and achieving product nutritional fortification. S4. Catalyst separation and recovery: After the reaction is completed, the commercial lipase catalyst is separated and immobilized by an external magnetic field, washed three times with n-hexane, and vacuum dried for later use to obtain rice bran oil after catalyst separation. S5. Post-processing: The rice bran oil after catalyst separation is subjected to vacuum distillation at a vacuum degree of 0.095 MPa and a temperature of 90°C to remove unreacted phytosterols and volatile impurities, thereby obtaining refined rice bran oil.
[0050] According to the test results, the refined rice bran oil prepared by the method in Example 4 had an acid value of 0.10 mg KOH / g, a oryzanol retention rate of 95.1%, a phytosterol conversion rate of 85.6%, and an enzyme activity retention rate of 98.9% for the immobilized commercial lipase catalyst.
[0051] The preparation method of the immobilized commercial lipase catalyst is the same as that in Example 1.
[0052] Comparative Example 1 Comparative Example 1 is a traditional alkali refining and deacidification method, which includes the following steps: Take the degummed and defatted rice bran oil from Example 1 (acid value of 85 mg KOH / g), add a 10% NaOH solution (0.1% excess alkali) at 35°C, stir and react for 30 min, centrifuge to separate the soap residue, wash with water until neutral, and dry under reduced pressure to obtain refined rice bran oil.
[0053] Tests showed that the refined rice bran oil prepared by the method in Comparative Example 1 had an acid value of 1.2 mg KOH / g, a oryzanol retention rate of 62.3%, a refining loss rate of 28.5%, and produced a large amount of soap-containing wastewater, with no phytosterol esters generated.
[0054] Comparative Example 2 Comparative Example 2 is a physical distillation deacidification method, which includes the following steps: take the degummed and defatted rice bran oil (acid value of 85 mg KOH / g) from Example 1, and distill it for 100 min at 240℃ and 0.01 MPa to obtain refined rice bran oil.
[0055] Testing showed that the refined rice bran oil prepared by the method in Comparative Example 2 had an acid value of 2.0 mg KOH / g, a oryzanol retention rate of 78.6%, and an energy consumption more than three times that of Example 1 of this invention, with no phytosterol esters generated.
[0056] Comparative Example 3 Comparative Example 3 describes a deacidification method using free commercial lipase, comprising the following steps: taking degummed and defatted rice bran oil (acid value of 85 mg KOH / g) from Example 1, using phytosterol as raw material, and deacidifying it using free commercial lipase (Novozym435), with reaction conditions consistent with Example 1 (only the immobilized commercial lipase catalyst was replaced with a free enzyme of equal activity).
[0057] According to the test, the refined rice bran oil prepared by the method of Comparative Example 3 has an acid value of 0.12 mg KOH / g, a oryzanol retention rate of 95.3%, and a phytosterol conversion rate of 90.5%. However, the free enzyme cannot be separated and recovered and can only be used once. The production cost is significantly higher than that of Example 1 of this invention. Moreover, the enzyme protein remains in the rice bran oil after the reaction, which affects the product quality.
[0058] The deacidification effect of refined rice bran oil prepared by the methods of Examples 1-4 and Comparative Examples 1-3 of this invention is as follows: Figure 1 As shown. The retention rates of refined rice bran oil obtained by the methods of Examples 1-4 and Comparative Examples 1-3 of this invention are as follows. Figure 2 As shown. The phytosterol conversion rates of refined rice bran oil obtained by the methods of Examples 1-4 and Comparative Examples 1-3 of this invention are as follows. Figure 3 As shown.
[0059] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. An enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil, characterized in that, Includes the following steps: S1. Raw material pretreatment: Heat and stir the degummed and defatted rice bran oil with an acid value of 70~100mgKOH / g until it is evenly mixed and set aside; Crush the phytosterols and set aside. S2. Reaction system configuration: Phytosterols, immobilized commercial lipase catalysts, and emulsifiers are added to degummed and defatted rice bran oil. The amount of the immobilized commercial lipase catalyst added is 0.5% to 2.0% of the mass of the degummed and defatted rice bran oil; the amount of the emulsifier added is 0.1% to 0.3% of the mass of the degummed and defatted rice bran oil. S3. Enzymatic deacidification reaction: under conditions of 40~55℃ and stirring rate of 150~250r / min for 8~16h, immobilized commercial lipase catalyzes the esterification reaction of free fatty acids with phytosterols to obtain phytosterol esters. S4. Catalyst separation and recovery: After the reaction is completed, the commercial lipase catalyst is separated and immobilized by an external magnetic field, washed and dried for later use, and rice bran oil after catalyst separation is obtained. S5. Post-processing: The rice bran oil after catalyst separation is subjected to vacuum distillation to remove unreacted phytosterols and volatile impurities, yielding refined rice bran oil.
2. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 1, characterized in that, In step S2, the preparation method of the immobilized commercial lipase catalyst includes the following steps: (1) Preparation of magnetic multi-walled carbon nanotubes: Fe3O4 nanoparticles were anchored on the surface of multi-walled carbon nanotubes by chemical co-precipitation to prepare magnetic multi-walled carbon nanotubes. (2) Magnetic multi-walled carbon nanotubes modified with third-generation polyamide-amine: Magnetic multi-walled carbon nanotubes were functionalized with amino groups using (3-aminopropyl)triethoxysilane to obtain amino-functionalized magnetic multi-walled carbon nanotubes. Then, third-generation polyamide-amine was grafted onto them by a divergent method to obtain magnetic multi-walled carbon nanotubes modified with third-generation polyamide-amine. (3) Immobilization of commercial lipase: Using third-generation polyamide-amine modified magnetic multi-walled carbon nanotubes as a carrier, commercial lipase was immobilized on the carrier. The mass ratio of commercial lipase to carrier was 1:(5~20). The immobilization was carried out by shaking at 25~35℃ and stirring at 100~200r / min for 4~8h. After magnetic separation, washing and drying, the immobilized commercial lipase catalyst was obtained.
3. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 2, characterized in that, In step (1), the process of anchoring Fe3O4 nanoparticles on the surface of multi-walled carbon nanotubes is as follows: FeCl3·6H2O and FeSO4·7H2O are dissolved in deionized water at a molar ratio of 2:1, nitrogen gas is introduced to remove oxygen for 30-60 min, and the pH is adjusted to 9-11 with ammonia water at 50-70℃ and a stirring rate of 200-300 r / min for 30-60 min.
4. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 2, characterized in that, In step (2), the divergent grafting of third-generation polyamide-amine is as follows: amino-functionalized magnetic multi-walled carbon nanotubes are dispersed in methanol, third-generation polyamide-amine monomers are added, and the reaction is carried out at 40~60℃ for 24~48h.
5. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 2, characterized in that, In step (3), the commercial lipase is immobilized on the carrier by dispersing the carrier in a phosphate buffer solution with a pH of 6.5-7.5 and a concentration of 0.05-0.1 mol / L, and adding the commercial lipase, which is Novozym 435.
6. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 1, characterized in that, The purity of the phytosterol is ≥95%, and the emulsifier is Tween-80.
7. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 1, characterized in that, In step S1, the heating temperature is 30~40℃, and the pulverization precision is 80~100 mesh.
8. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 1, characterized in that, In step S2, the molar ratio of phytosterols to free fatty acids in degummed and defatted rice bran oil is (1.0~1.5):
1.
9. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 1, characterized in that, In step S5, the conditions for vacuum distillation are: vacuum degree 0.09~0.1MPa, temperature 80~100℃.
10. The enzymatic deacidification and refining method for high-acid-value degummed and defatted rice bran oil according to claim 1, characterized in that, In step S5, the acid value of the refined rice bran oil is ≤0.1mgKOH / g.