A method for preparing fatty acids from acidulated oil

Abstract

This invention discloses a method for preparing fatty acids from acidified oil, belonging to the field of biochemical engineering. The method includes preparing an immobilized lipase and enzymatic hydrolysis; the preparation of the immobilized lipase includes preparing a carrier and immobilization; the preparation of the carrier involves placing magnetic Fe... 3 O 4 The granules, anhydrous ethanol, deionized water, and ammonia were mixed, stirred at room temperature, and ultrasonically vibrated. Then, they were mixed with tetraethyl orthosilicate, stirred at room temperature, collected by magnetic decantation, and washed to obtain Fe. 3 O 4 @SiO 2­ Particles; Fe 3 O 4 @SiO 2­ Granules, hexadecyltrimethylammonium bromide, and deionized water are mixed and stirred. Calcium nitrate tetrahydrate is added and stirred. The pH value is adjusted to 9.5-10. Diammonium hydrogen phosphate aqueous solution is added dropwise and the pH value is stabilized at 9.5-10. The mixture is stirred and collected by magnetic decantation. The method of this invention can simultaneously achieve high hydrolysis rate and good reusability, and the hydrolysis rate is less affected by the acid value of acidified oil.

Description

A method for preparing fatty acids from acidified oil Technical Field

[0001] This invention relates to the field of biochemical engineering, and more specifically to a method for preparing fatty acids from acidified oil. Background Technology

[0002] Fatty acids are a class of carboxylic acids composed of an alkyl chain and a single carboxyl group attached to one end. They are commonly used in food, cosmetics, pharmaceuticals, and industrial chemicals—fields closely related to human life. Acidified oil refers to the mixture obtained after acidifying soapstock. Acidified oil contains various components such as glycerides, phospholipids, fatty acids, and pigments. Due to its high content of free fatty acids and relatively low production cost, acidified oil is considered a good raw material for fatty acid production. Large-scale production of fatty acids from acidified oil generally employs saponification-acidification, high-pressure continuous hydrolysis, and medium-pressure intermittent hydrolysis methods. Among them, the saponification-acidification method involves using caustic soda to saponify neutral fats, generating soap base, followed by acid hydrolysis with sulfuric acid to produce fatty acids and sodium sulfate. Sodium sulfate is then removed by water washing to obtain fatty acids. This method has high acid and alkali consumption, high energy consumption, and serious wastewater pollution, and has been largely phased out. The high-pressure continuous hydrolysis method utilizes the single-tower counter-current principle, allowing the oil to gradually decompose into fatty acids and glycerol as it rises within the hydrolysis tower. Glycerol is separated and discharged from the bottom of the tower, while fatty acids are discharged from the upper separation zone. This method has high requirements for equipment and production conditions, and requires a large amount of water and steam, resulting in a low concentration of sweet glycerol water, reducing the economic viability of glycerol recovery, and generating a large amount of organic wastewater, which is not conducive to energy conservation and emission reduction. The medium-pressure intermittent hydrolysis method uses water to decompose oils into fatty acids and glycerol at medium pressure and high temperature. This method has poor hydrolysis efficiency, long production time, and high energy consumption.

[0003] Enzymatic hydrolysis utilizes the catalytic action of lipases to hydrolyze oils into fatty acids. The lipases used (EC 3.1.1.3) are primarily derived from microorganisms (including molds, yeasts, bacteria, and fungi), and can efficiently catalyze oil hydrolysis at the oil-water interface. Lipases from different sources all possess a characteristic "α / β hydrolase fold" domain, consisting of an outer α-helix "cap" and a β-sheet "center." This domain forms the substrate-binding region and the catalytic active site of the lipase. The substrate-binding region, or catalytic region "pocket," determines the substrate and stereoselectivity of the lipase. The catalytic active site is composed of the typical nucleophilic triplet of lipases: "serine-histidine-aspartic acid" or "serine-histidine-glutamate." The catalytic active site is usually covered by an amphiphilic peptide chain "cap," exhibiting an "interfacial activation" phenomenon. When lipase is in an aqueous phase, the "cap" exhibits a closed conformation, separating the active site of the lipase from the external medium. In this state, the lipase is inactive. When the lipase is at the water-oil interface, the "cap" changes to an open conformation, exposing the catalytic active site and further transforming it into an activated form, thus enabling the catalytic reaction. Enzymatic hydrolysis offers advantages such as low pollution and low energy consumption. Furthermore, the high specificity of enzymes ensures the high efficiency and selectivity of the hydrolysis reaction, thus attracting increasing attention.

[0004] When commercially available free lipases are directly applied to the preparation of fatty acids, it has been found that free lipases suffer from poor stability and tolerance, as well as poor reusability. Furthermore, as water-soluble protein molecules, lipases are difficult to effectively separate from the hydrolysis system after the hydrolysis reaction. Immobilized lipases can be converted into water-insoluble enzyme preparations that still possess enzymatic activity through physical or chemical treatment, enabling the reusability of lipases. Therefore, immobilized lipases are typically used when preparing fatty acids from acidified oils. In its research and development, the applicant used immobilized lipases provided by Novozymes and Qingdao Weilan Biotechnology Co., Ltd., but found the following problems in both:

[0005] Problem 1. Low hydrolysis rate or poor reusability. Analysis revealed that this problem is caused by the presence of a large amount of acidic impurities and pigments in the acidified oil, as well as the impact of the immobilization process on lipase activity. As described in Zhang Pingbo et al., *China Oils and Fats*, October 2024, the presence of acidic impurities and pigments in acidified oils can affect the catalytic effect of immobilized lipases. Similarly, in the study of glutaraldehyde-immobilized lipase catalyzing the hydrolysis of vegetable oils to synthesize fatty acids, as described in Fan Xiulin's master's thesis, June 2023, methods used in the preparation of immobilized lipases include covalent bonding, adsorption cross-linking, and adsorption. Both covalent bonding and adsorption cross-linking methods use glutaraldehyde as a cross-linking agent. Glutaraldehyde reacts with lipase via a Schiff base reaction, which negatively impacts the lipase's structure, leading to distortion of its secondary structure and reduced activity. Adsorption methods immobilize lipases on a hydrophobic carrier surface through hydrophobic interactions. However, these interactions are weak physical interactions, resulting in poor reusability. Therefore, it is difficult to simultaneously achieve high hydrolysis rates and good reusability for immobilized lipases.

[0006] Question 2. The hydrolysis rate is greatly affected by the acid value of the acidified oil. The applicant prepared fatty acids using acidified soybean oil, acidified cottonseed oil, and acidified rice bran oil with different acid values ​​as raw materials. It was found that as the acid value increased, the change in acid value of the hydrolysate decreased, indicating that the hydrolysis rate decreased as the acid value of the raw material increased. Subsequently, to verify the above issue, the applicant used edible soybean oil with an acid value of 0.1 mg KOH / g as raw material and carried out enzymatic hydrolysis using immobilized lipases provided by Novozymes and Qingdao Blue Ocean Biotechnology Co., Ltd. It was found that the acid value of the hydrolysate was much higher than that of the fatty acids prepared from acidified oil. Analysis revealed that the problem stems from the significant pH-dependent activity of lipases and the product inhibition issues associated with fatty acid hydrolysis. For instance, as described in the study of magnetic Fe3O4 immobilized lipase catalyzing the synthesis of structural phospholipids (Cheng Ke, Master's Thesis, Dalian University of Technology, June 2022), free lipases exhibit poor pH stability, while immobilized lipases demonstrate superior pH stability. This indicates that immobilization reduces the impact of pH on lipase activity. Furthermore, the immobilization method itself influences lipase pH stability; poor immobilization results in lipase activity being highly susceptible to the acid value of acidified oils. Additionally, as discussed in Liang Liang et al., "Characteristics of Lipase Catalytic Hydrolysis of Intradermal Oils and Its Degreasing Effect," Leather Science and Engineering, April 2013, lipases also exhibit product inhibition during catalytic hydrolysis. To address this issue, the applicant attempted to pretreat the acidified oil before enzymatic hydrolysis by adjusting the pH to 5-5.5 and increasing the amount of water used during enzymatic hydrolysis. However, the effect was limited. Analysis showed that pretreatment could not solve the product inhibition problem. Furthermore, as described in Zhang Pingbo et al., *Chinese Oils and Fats*, October 2024, there is an optimal water-oil ratio during enzymatic hydrolysis. When this optimal water-oil ratio is exceeded, the hydrolysis rate decreases. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing fatty acids from acidified oil. This method can simultaneously achieve high hydrolysis rate and good reusability, and the hydrolysis rate is less affected by the acid value of the acidified oil.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing fatty acids from acidified oil includes preparing an immobilized lipase and enzymatic hydrolysis;

[0010] The preparation of immobilized lipase includes the preparation of a carrier and immobilization;

[0011] The preparation of the carrier involves adding magnetic Fe3O4 particles, anhydrous ethanol, a first portion of deionized water, and a first portion of ammonia water to a reaction vessel. The stirring speed is adjusted to 200-300 r / min, and the mixture is stirred at room temperature for 10-30 min. The mixture is then transferred to an ultrasonic oscillator for ultrasonic vibration, transferred back to the reaction vessel, and the stirring speed is adjusted to 200-300 r / min. Tetraethyl orthosilicate is added, and the mixture is stirred at room temperature for 25-30 h. The mixture is collected by magnetic decyl precipitation and washed 2-4 times each with deionized water and anhydrous ethanol to obtain Fe3O4@SiO2 particles. All the obtained Fe3O4@SiO2 particles and hexadecyltrimethyl... Ammonium bromide and the second part of deionized water were added to the reactor. The temperature of the reactor was adjusted to 30-35℃, the stirring speed was adjusted to 200-300 r / min, and the mixture was stirred for 30-60 min. Calcium nitrate tetrahydrate was added and stirred for 30-60 min. The second part of ammonia was added to adjust the pH value to 9.5-10. Diammonium hydrogen phosphate aqueous solution was added dropwise. During the dropwise addition, the second part of ammonia was added simultaneously to stabilize the pH value to 9.5-10. After the dropwise addition was completed, stirring was continued for 1-1.5 h. The mixture was collected by magnetic decantation, washed 2-3 times with deionized water, transferred to a vacuum drying oven, and dried at 60-70℃ to obtain the carrier.

[0012] In the preparation of the carrier, the ratio of magnetic Fe3O4 particles, anhydrous ethanol, first part of deionized water, first part of ammonia water, and tetraethyl orthosilicate is 20-25g:1000-1500mL:80-110mL:50-65mL:40-50mL.

[0013] The ratio of magnetic Fe3O4 particles, hexadecyltrimethylammonium bromide, second part of deionized water, calcium nitrate tetrahydrate, and diammonium hydrogen phosphate aqueous solution is 20-25g:3-3.5g:1500-2000mL:9-10g:300-350mL;

[0014] The dropping rate of the diammonium hydrogen phosphate aqueous solution is 10-15 mL / min;

[0015] The average particle size of the magnetic Fe3O4 particles is 500 nm.

[0016] The mass concentration of the first portion of ammonia solution is 25%.

[0017] The mass concentration of the second portion of ammonia solution is 2-3%;

[0018] The mass concentration of the diammonium hydrogen phosphate aqueous solution is 1%;

[0019] The frequency of the ultrasonic oscillation is 20-30kHz, and the duration is 10-20min;

[0020] For the immobilization process, lipase, sodium dodecyl sulfate, and the first phosphate buffer solution were added to a reaction vessel. The stirring speed was adjusted to 200-300 rpm, and the mixture was stirred at room temperature for 20-40 minutes. The mixture was then transferred to a refrigeration unit, and the temperature was adjusted to 4°C. After standing for 10-15 hours, the supernatant was added to the reaction vessel, followed by the addition of the carrier. The temperature of the reaction vessel was adjusted to 30-40°C, and the stirring speed was adjusted to 50-100 rpm. The mixture was stirred for 10-12 hours, and the mixture was collected by magnetic decantation to obtain the immobilized carrier. Polyvinylpyrrolidone, chitosan, and an aqueous acetic acid solution were then added to the reaction vessel, and the stirring speed was adjusted... Stir at 50-100 rpm for 20-40 min at room temperature, add the obtained immobilized carrier, stir for 2-3 h, collect by magnetic decantation to obtain the chitosan-coated carrier; add the second part of phosphate buffer solution and glutaraldehyde aqueous solution to the reactor, adjust the stirring speed to 50-100 rpm, stir at room temperature for 10-30 min, add the obtained chitosan-coated carrier, adjust the temperature of the reactor to 30-40℃, add ammonia to adjust the pH to 8-8.5, stir for 4-5 h, collect by magnetic decantation, wash 2-3 times with deionized water, and dry at room temperature to obtain the immobilized lipase;

[0021] In the immobilization process, the ratio of lipase, sodium dodecyl sulfate, the first phosphate buffer solution, and the carrier is 5-7 mL: 5-8 g: 5000-8000 mL: 20-25 g.

[0022] The ratio of carrier, polyvinylpyrrolidone, chitosan, acetic acid aqueous solution, second phosphate buffer solution, and glutaraldehyde aqueous solution is 20-25g:2-2.5g:9-10g:1500-2000mL:1000-1500mL:100-120mL;

[0023] The lipase is in liquid form with an enzyme activity of 5000 U / mL. It is derived from Aspergillus niger and purchased from Qingdao Weilan Biotechnology Co., Ltd.

[0024] The chitosan has a degree of deacetylation of 85% and a number-average molecular weight of 1 million.

[0025] The polyvinylpyrrolidone is polyvinylpyrrolidone K30;

[0026] The mass concentration of the acetic acid aqueous solution is 2%;

[0027] The phosphate buffer solutions used all had a molar concentration of 0.1 mol / L and a pH of 7.0.

[0028] The mass concentration of the glutaraldehyde aqueous solution is 25%.

[0029] The mass concentration of the ammonia solution is 2-3%;

[0030] The enzymatic hydrolysis involves washing the acidified oil with water to obtain washed acidified oil; adding the washed acidified oil and deionized water into a reaction vessel, adjusting the temperature of the reaction vessel to 40-45℃, adjusting the stirring speed to 150-200 r / min, adding immobilized lipase, stirring for 24-30 h, collecting the immobilized lipase by magnetic decantation, taking the oil phase after standing, washing the oil phase with water to obtain fatty acids;

[0031] In the enzymatic hydrolysis, the ratio of acidified oil after water washing to deionized water is 200-250g:400-450mL;

[0032] The mass ratio of acidified oil after water washing to immobilized lipase is 200-250:20-30.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The method for preparing fatty acids from acidified oil of the present invention can simultaneously achieve high hydrolysis rate and good reusability, and the hydrolysis rate is less affected by the acid value of the acidified oil. When treating acidified oil with an acid value of 101.13 mg KOH / g and a saponification value of 166.42 mg KOH / g, under the conditions of a water-oil ratio of 400-450 mL: 200-250 g, an immobilized lipase content of 10-12% of the acidified oil mass fraction, and a catalytic hydrolysis time of 24-30 h, the hydrolysis rate can reach 97.35-100%, and the hydrolysis activity retention rate of the immobilized lipase after 5 reuses can reach 70.85-72.41%. When treating acidified oil with an acid value of 142.59 mg KOH / g and a saponification value of 181.72 mg KOH / g... When acidified oil has a KOH content of 1g, and the water-to-oil ratio is 400-450mL:200-250g, the immobilized lipase accounts for 10-12% of the mass fraction of the acidified oil, and the catalytic hydrolysis time is 24-30h, the hydrolysis rate can reach 95.45-100%. Detailed Implementation

[0035] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0036] The lipases used in Examples 1-2 and Comparative Examples 1-5 were taken from the same fermentation batch.

[0037] The room temperature in Examples 1-2 and Comparative Examples 1-5 was 22±2℃.

[0038] Example 1

[0039] A method for preparing fatty acids from acidified oil is as follows:

[0040] Step 1. Preparation of immobilized lipase:

[0041] Step (1). Preparation of the carrier: 20g of magnetic Fe3O4 particles, 1000mL of anhydrous ethanol, 80mL of deionized water and 50mL of the first portion of ammonia water were added to the reaction vessel. The stirring speed was adjusted to 200r / min and stirred at room temperature for 10min. The mixture was then transferred to an ultrasonic oscillator, the frequency was adjusted to 20kHz, and ultrasonic oscillation was performed for 10min. The mixture was then transferred back to the reaction vessel, the stirring speed was adjusted to 200r / min, and 40mL of tetraethyl orthosilicate was added. The mixture was stirred at room temperature for 25h, collected by magnetic decantation, and washed twice each with deionized water and anhydrous ethanol to obtain Fe3O4@SiO2 particles. All the obtained Fe3O4@SiO2 particles were then collected. IO2 particles, 3g of cetyltrimethylammonium bromide, and 1500mL of deionized water were added to a reaction vessel. The temperature of the reaction vessel was adjusted to 30℃, the stirring speed was adjusted to 200r / min, and the mixture was stirred for 30min. Then, 9g of calcium nitrate tetrahydrate was added, and the mixture was stirred for 30min. A second portion of ammonia water was added to adjust the pH value to 9.5. 300mL of diammonium hydrogen phosphate aqueous solution was added dropwise at a rate of 10mL / min. During the dropwise addition, a second portion of ammonia water was added simultaneously to stabilize the pH value at 9.5. After the dropwise addition was completed, the mixture was stirred for 1h. The mixture was collected by magnetic decantation, washed twice with deionized water, transferred to a vacuum drying oven, and dried at 60℃ to obtain the carrier.

[0042] The average particle size of the magnetic Fe3O4 particles is 500 nm.

[0043] The mass concentration of the first portion of ammonia solution is 25%.

[0044] The mass concentration of the second portion of ammonia solution is 2%;

[0045] The mass concentration of the diammonium hydrogen phosphate aqueous solution is 1%;

[0046] Step (2). Immobilization: Add 5 mL of lipase, 5 g of sodium dodecyl sulfate and 5000 mL of phosphate buffer solution to the reaction vessel, adjust the stirring speed to 200 r / min, stir at room temperature for 20 min, transfer to a refrigeration device, adjust the temperature of the refrigeration device to 4℃, let stand for 10 h, take the supernatant and add it to the reaction vessel, add 20 g of carrier, adjust the temperature of the reaction vessel to 30℃, adjust the stirring speed to 50 r / min, stir for 10 h, collect by magnetic decantation to obtain the immobilized carrier; add 2 g of polyvinylpyrrolidone, 9 g of chitosan and 1500 mL of acetic acid aqueous solution. The mixture was added to a reaction vessel, and the stirring speed was adjusted to 50 r / min. The mixture was stirred at room temperature for 20 min, and the obtained immobilized carrier was added. The mixture was stirred for 2 h, and the mixture was collected by magnetic decantation to obtain the chitosan-coated carrier. 1000 mL of phosphate buffer solution and 100 mL of glutaraldehyde aqueous solution were added to the reaction vessel, and the stirring speed was adjusted to 50 r / min. The mixture was stirred at room temperature for 10 min, and the obtained chitosan-coated carrier was added. The temperature of the reaction vessel was adjusted to 30 °C, and ammonia was added to adjust the pH to 8. The mixture was stirred for 4 h, and the mixture was collected by magnetic decantation. The mixture was washed twice with deionized water and dried at room temperature to obtain the immobilized lipase.

[0047] The lipase is in liquid form with an enzyme activity of 5000 U / mL. It is derived from Aspergillus niger and purchased from Qingdao Weilan Biotechnology Co., Ltd.

[0048] The chitosan has a degree of deacetylation of 85% and a number-average molecular weight of 1 million.

[0049] The polyvinylpyrrolidone is polyvinylpyrrolidone K30;

[0050] The mass concentration of the acetic acid aqueous solution is 2%;

[0051] The phosphate buffer solutions used all had a molar concentration of 0.1 mol / L and a pH of 7.0.

[0052] The mass concentration of the glutaraldehyde aqueous solution is 25%.

[0053] The mass concentration of the ammonia solution is 2%;

[0054] Step 2. Enzymatic hydrolysis: After washing the acidified oil with water, the acidified oil after washing with water is obtained; 200g of the acidified oil after washing with water and 400mL of deionized water are added to the reaction vessel, the temperature of the reaction vessel is adjusted to 40℃, the stirring speed is adjusted to 150r / min, 20g of immobilized lipase is added, and the mixture is stirred for 24h. The immobilized lipase is collected by magnetic decantation, and after standing, the oil phase is taken and washed with water to obtain fatty acids.

[0055] The acidified oil is soybean oil acidified oil, with an acid value of 101.13 mg KOH / g and a saponification value of 166.42 mg KOH / g.

[0056] Example 2

[0057] A method for preparing fatty acids from acidified oil is as follows:

[0058] Step 1. Preparation of immobilized lipase:

[0059] Step (1). Preparation of the carrier: 25g of magnetic Fe3O4 particles, 1500mL of anhydrous ethanol, 110mL of deionized water and 65mL of the first portion of ammonia water were added to the reaction vessel. The stirring speed was adjusted to 300r / min and stirred at room temperature for 30min. The mixture was then transferred to an ultrasonic oscillator, the frequency was adjusted to 30kHz, and ultrasonic oscillation was performed for 20min. The mixture was then transferred back to the reaction vessel, the stirring speed was adjusted to 300r / min, and 50mL of tetraethyl orthosilicate was added. The mixture was stirred at room temperature for 30h, collected by magnetic decantation, and washed 4 times each with deionized water and anhydrous ethanol to obtain Fe3O4@SiO2 particles. All the obtained Fe3O4@Si particles were then collected. O2 particles, 3.5g cetyltrimethylammonium bromide, and 2000mL deionized water were added to a reaction vessel. The temperature of the reaction vessel was adjusted to 35℃, the stirring speed was adjusted to 300r / min, and the mixture was stirred for 60min. Then, 10g calcium nitrate tetrahydrate was added, and the mixture was stirred for 60min. A second portion of ammonia water was added to adjust the pH value to 10. 350mL of diammonium hydrogen phosphate aqueous solution was added dropwise at a rate of 15mL / min. During the dropwise addition, a second portion of ammonia water was added simultaneously to stabilize the pH value at 10. After the dropwise addition was completed, the mixture was stirred for 1.5h. The mixture was collected by magnetic decantation, washed three times with deionized water, transferred to a vacuum drying oven, and dried at 70℃ to obtain the carrier.

[0060] The average particle size of the magnetic Fe3O4 particles is 500 nm.

[0061] The mass concentration of the first portion of ammonia solution is 25%.

[0062] The mass concentration of the second portion of ammonia solution is 2%;

[0063] The mass concentration of the diammonium hydrogen phosphate aqueous solution is 1%;

[0064] Step (2). Immobilization: 7 mL of lipase, 8 g of sodium dodecyl sulfate and 8000 mL of phosphate buffer solution were added to the reaction vessel. The stirring speed was adjusted to 300 r / min and stirred at room temperature for 40 min. The mixture was then transferred to a refrigeration unit and the temperature of the refrigeration unit was adjusted to 4℃. After standing for 15 h, the supernatant was added to the reaction vessel, along with 25 g of carrier. The temperature of the reaction vessel was adjusted to 40℃, the stirring speed was adjusted to 100 r / min, and the mixture was stirred for 12 h. The mixture was collected by magnetic decantation to obtain the immobilized carrier. 2.5 g of polyvinylpyrrolidone, 10 g of chitosan and 2000 mL of acetic acid aqueous solution were added. The mixture was added to a reaction vessel, and the stirring speed was adjusted to 100 r / min. The mixture was stirred at room temperature for 40 min, and the obtained immobilized support was added. The mixture was stirred for 3 h, and the mixture was collected by magnetic decantation to obtain the chitosan-coated support. 1500 mL of phosphate buffer solution and 120 mL of glutaraldehyde aqueous solution were added to the reaction vessel, and the stirring speed was adjusted to 100 r / min. The mixture was stirred at room temperature for 30 min, and the obtained chitosan-coated support was added. The temperature of the reaction vessel was adjusted to 40 °C, and ammonia was added to adjust the pH to 8.5. The mixture was stirred for 5 h, and the mixture was collected by magnetic decantation. The mixture was washed three times with deionized water and dried at room temperature to obtain the immobilized lipase.

[0065] The lipase is in liquid form with an enzyme activity of 5000 U / mL. It is derived from Aspergillus niger and purchased from Qingdao Weilan Biotechnology Co., Ltd.

[0066] The chitosan has a degree of deacetylation of 85% and a number-average molecular weight of 1 million.

[0067] The polyvinylpyrrolidone is polyvinylpyrrolidone K30;

[0068] The mass concentration of the acetic acid aqueous solution is 2%;

[0069] The phosphate buffer solutions used all had a molar concentration of 0.1 mol / L and a pH of 7.0.

[0070] The mass concentration of the glutaraldehyde aqueous solution is 25%.

[0071] The mass concentration of the ammonia solution is 2%;

[0072] Step 2. Enzymatic hydrolysis: After washing the acidified oil with water, the acidified oil after washing with water is obtained; 250g of the acidified oil after washing with water and 450mL of deionized water are added to the reaction vessel, the temperature of the reaction vessel is adjusted to 45℃, the stirring speed is adjusted to 200r / min, 30g of immobilized lipase is added, and the mixture is stirred for 30h. The immobilized lipase is collected by magnetic decantation, and after standing, the oil phase is taken and washed with water to obtain fatty acids.

[0073] The acidified oil is soybean oil acidified oil, with an acid value of 101.13 mg KOH / g and a saponification value of 166.42 mg KOH / g.

[0074] Comparative Example 1

[0075] The difference between this comparative example and Example 1 is that in step 1. Preparation of immobilized lipase, step (1) does not involve the preparation of SiO2 in the carrier preparation process; that is, step (1). The preparation of the carrier is changed to:

[0076] 20g of magnetic Fe3O4 particles, 3g of hexadecyltrimethylammonium bromide, and 1500mL of deionized water were added to a reaction vessel. The temperature of the reaction vessel was adjusted to 30℃, the stirring speed was adjusted to 200r / min, and the mixture was stirred for 30min. 9g of calcium nitrate tetrahydrate was added, and the mixture was stirred for 30min. Ammonia water was added to adjust the pH value to 9.5. 300mL of diammonium hydrogen phosphate aqueous solution was added dropwise at a rate of 10mL / min. During the dropwise addition, ammonia water was added simultaneously to stabilize the pH value at 9.5. After the dropwise addition was completed, the mixture was stirred for 1h. The mixture was collected by magnetic decantation, washed twice with deionized water, transferred to a vacuum drying oven, and dried at 60℃ to obtain the carrier.

[0077] The average particle size of the magnetic Fe3O4 particles is 500 nm.

[0078] The mass concentration of the ammonia solution is 2%;

[0079] The mass concentration of the diammonium hydrogen phosphate aqueous solution is 1%.

[0080] All other steps and parameters are the same as in Example 1.

[0081] Comparative Example 2

[0082] The difference between this comparative example and Example 1 is that: in step 1. the preparation of immobilized lipase (1), cetyltrimethylammonium bromide is not added to the carrier.

[0083] All other steps and parameters are the same as in Example 1.

[0084] Comparative Example 3

[0085] The difference between this comparative example and Example 1 is that in step 1. Preparation of immobilized lipase (1), hydroxyapatite is not prepared in the preparation of the carrier; that is, step (1). Preparation of the carrier is changed to:

[0086] 20g of magnetic Fe3O4 particles, 1000mL of anhydrous ethanol, 80mL of ammonia, and 50mL of ammonia were added to a reaction vessel. The stirring speed was adjusted to 200r / min, and the mixture was stirred at room temperature for 10min. The mixture was then transferred to an ultrasonic oscillator, the frequency was adjusted to 20kHz, and the mixture was ultrasonically oscillated for 10min. The mixture was then transferred back to the reaction vessel, the stirring speed was adjusted to 200r / min, and 40mL of tetraethyl orthosilicate was added. The mixture was stirred at room temperature for 25h, and the particles were collected by magnetic decantation. The particles were washed twice each with deionized water and anhydrous ethanol to obtain Fe3O4@SiO2 particles. All the obtained Fe3O4@SiO2 particles, 3g of hexadecyltrimethylammonium bromide, and 1500mL of deionized water were added to a reaction vessel. The temperature of the reaction vessel was adjusted to 30℃, the stirring speed was adjusted to 200r / min, and the mixture was stirred for 30min. The particles were collected by magnetic decantation, washed twice with deionized water, and then transferred to a vacuum drying oven, the temperature was adjusted to 60℃, and the mixture was dried to obtain the support.

[0087] The average particle size of the magnetic Fe3O4 particles is 500 nm.

[0088] The mass concentration of the ammonia water is 25%.

[0089] All other steps and parameters are the same as in Example 1.

[0090] Comparative Example 4

[0091] The difference between this comparative example and Example 1 is that in step 1. Preparation of immobilized lipase (2). Sodium dodecyl sulfate is not added during immobilization.

[0092] All other steps and parameters are the same as in Example 1.

[0093] Comparative Example 5

[0094] The difference between this comparative example and Example 1 is that: in step 1, step (2) of preparing immobilized lipase, polyvinylpyrrolidone is not added during immobilization.

[0095] All other steps and parameters are the same as in Example 1.

[0096] Experimental Example 1

[0097] The acid values ​​of the fatty acids prepared in Examples 1-2 and Comparative Examples 1-5 were tested, and then the hydrolysis rate was calculated according to the method described in Zhang Pingbo et al., *China Oils and Fats*, October 2024. The calculation results are shown in Table 1.

[0098] Table 1

[0099]

[0100] As can be seen from the results in Table 1, the hydrolysis rate of Example 1 is higher than that of Comparative Examples 1-5.

[0101] Analysis revealed that, compared to Example 1, Comparative Example 1 did not coat the surface of the magnetic Fe3O4 particles with silica. Instead, it directly coated the surface of the magnetic Fe3O4 particles with hydroxyapatite containing hexadecyltrimethylammonium bromide. Silica can combine with hydroxyapatite to form a coating layer with a higher specific surface area. Moreover, silica can also act as an intermediary, improving the binding force between hydroxyapatite and the magnetic Fe3O4 particles through the adsorption and fixation of calcium ions, thereby adsorbing more hydroxyapatite and further increasing the binding area of ​​the carrier for lipase. Therefore, the amount and binding force of the carrier for lipase in Comparative Example 1 are inferior to those in Example 1.

[0102] Compared with Example 1, Comparative Example 2 did not add hexadecyltrimethylammonium bromide in the preparation of hydroxyapatite. As a result, when immobilizing lipase, immobilization was mainly achieved through the interaction between the hydrophilic and hydrophobic groups of sodium dodecyl sulfate and the carrier and lipase. The electrostatic interaction between sodium dodecyl sulfate and hexadecyltrimethylammonium bromide was lacking, resulting in the carrier in Comparative Example 2 having a lower binding amount and binding force to lipase than the carrier in Example 1.

[0103] Compared with Example 1, Comparative Example 3 only adsorbed a layer of hexadecyltrimethylammonium bromide on the surface of silica without coating hydroxyapatite. As a result, the binding area of ​​the carrier for lipase in Comparative Example 3 was smaller than that in Example 1. In addition, due to the interaction between calcium ions in hydroxyapatite and fatty acids, hydroxyapatite can dynamically adsorb fatty acids and reduce the product inhibition caused by fatty acids. Therefore, compared with Example 1, the product inhibition effect in Comparative Example 3 was stronger, which further resulted in the catalytic hydrolysis of lipase in Comparative Example 3 being weaker than that of lipase in Example 1.

[0104] Compared with Example 1, Comparative Example 4 did not add sodium dodecyl sulfate to pretreat the lipase during immobilization. Firstly, sodium dodecyl sulfate can improve the enzyme activity of lipase. Secondly, sodium dodecyl sulfate can improve the binding amount and binding force between lipase and carrier through electrostatic force with hexadecyltrimethylammonium bromide. Therefore, the binding amount and binding force of the carrier to lipase in Comparative Example 4 are worse than those of the carrier in Example 1.

[0105] Compared with Example 1, Comparative Example 5 did not include polyvinylpyrrolidone in the immobilization process. Firstly, polyvinylpyrrolidone can crosslink with chitosan, regulating the crosslinking network structure and further enhancing the protective effect on lipase. Secondly, polyvinylpyrrolidone can form an association with sodium dodecyl sulfate, reducing the crosslinking of glutaraldehyde on lipase through the steric hindrance of the association, thus reducing the influence of glutaraldehyde on lipase activity. Thirdly, it can adsorb acidic impurities and pigments, reducing their influence on lipase. Therefore, the immobilized lipase in Comparative Example 5 has lower activity than the immobilized lipase in Example 1.

[0106] Experimental Example 2

[0107] In step 2 of Examples 1-2 and Comparative Examples 1-5, the acidified oil in enzymatic hydrolysis was replaced by rice bran acidified oil with an acid value of 142.59 mg KOH / g and a saponification value of 181.72 mg KOH / g. Fatty acids were then prepared according to the methods for preparing fatty acids from acidified oil as described in Examples 1-2 and Comparative Examples 1-5. The acid value of the prepared fatty acids was then tested, and the hydrolysis rate was calculated. The calculation results are shown in Table 2.

[0108] Table 2

[0109]

[0110] Comparing the results in Table 2 and Table 1, it can be seen that when treating acidified oil with a higher acid value, the reduction in hydrolysis rate in Example 1 is much lower than the reduction in hydrolysis rate in Comparative Examples 2-3.

[0111] Analysis revealed that, compared to Example 1, Comparative Example 2 did not include hexadecyltrimethylammonium bromide in the preparation of hydroxyapatite. Hexadecyltrimethylammonium bromide can synergistically enhance the dynamic adsorption of fatty acids with hydroxyapatite, thereby reducing the product inhibition effect. Therefore, when treating acidified oils with higher acid values, the reduction in hydrolysis rate in Comparative Example 2 is much greater than the reduction in hydrolysis rate in Example 1.

[0112] Compared with Example 1, Comparative Example 3 only adsorbed a layer of hexadecyltrimethylammonium bromide on the surface of silica, without coating hydroxyapatite. The calcium ions in hydroxyapatite can dynamically adsorb fatty acids and reduce the product inhibition caused by fatty acids. Therefore, when treating acidified oil with higher acid value, the reduction in hydrolysis rate of Comparative Example 3 is much greater than the reduction in hydrolysis rate of Example 1.

[0113] Experimental Example 3

[0114] Following the steps of Example 1-2 and Comparative Examples 1-5 for preparing fatty acids from acidified oil, specifically step 1, preparing immobilized lipase, the immobilized lipase was reused 5 times. In each reuse, the same batch of soybean oil acidified oil with an acid value of 101.13 mg KOH / g and a saponification value of 166.42 mg KOH / g was used. Then, following the method described in [Zhang Pingbo et al., China Oils and Fats, October 2024], the hydrolytic activity retention rate of the immobilized lipase in the 5th use relative to the immobilized lipase in the 1st use was calculated. The results are shown in Table 3.

[0115] Table 3

[0116]

[0117] As can be seen from the results in Table 3, the reusability results of Example 1 are higher than those of Comparative Examples 1-5.

[0118] Analysis of Example 1 shows that, compared to Example 1, Comparative Examples 1, 2, and 4 all exhibit poorer binding affinity of the carrier to the lipase. Therefore, during the reuse of the immobilized lipase, the lipase is prone to loss. Comparative Example 5, compared to Example 1, shows poorer protection of the lipase by the outer chitosan cross-linked network and lacks the protective effect of the associative network formed by polyvinylpyrrolidone and sodium dodecyl sulfate. Therefore, during the reuse of the immobilized lipase, the lipase is also prone to loss.

[0119] Compared with Example 1, Comparative Example 3 only adsorbed a layer of hexadecyltrimethylammonium bromide on the surface of silica without coating hydroxyapatite. The hydroxyapatite and silica have different pore sizes, which further immobilizes the lipase and reduces the loss of lipase. Therefore, the reusability result of Comparative Example 3 is lower than that of Example 1.

Claims

1. A method for preparing fatty acids from acidified oil, characterized in that, The method includes preparing immobilized lipase and enzymatic hydrolysis; the preparation of immobilized lipase includes preparing a carrier and immobilization; the preparation of the carrier involves mixing magnetic Fe3O4 particles, anhydrous ethanol, a first portion of deionized water, and ammonia, stirring at room temperature, ultrasonically vibrating, mixing with tetraethyl orthosilicate, stirring at room temperature, collecting by magnetic decantation, washing, and obtaining Fe3O4@SiO2 particles; mixing all the obtained Fe3O4@SiO2 particles, hexadecyltrimethylammonium bromide, and a second portion of deionized water, stirring at 30-35°C, adding calcium nitrate tetrahydrate, stirring, and adjusting the pH to 9.5-1. Add diammonium hydrogen phosphate aqueous solution dropwise, stabilizing the pH value to 9.5-10 during the dropwise addition. Stir, collect by magnetic decantation, wash, and dry to obtain the carrier. In the preparation of the carrier, the ratio of magnetic Fe3O4 particles, anhydrous ethanol, the first part of deionized water, ammonia water, and tetraethyl orthosilicate is 20-25g:1000-1500mL:80-110mL:50-65mL:40-50mL; the ratio of magnetic Fe3O4 particles, hexadecyltrimethylammonium bromide, the second part of deionized water, calcium nitrate tetrahydrate, and diammonium hydrogen phosphate aqueous solution is 20-25g:3-3.5g:1 500-2000mL: 9-10g: 300-350mL; For the immobilization, lipase, sodium dodecyl sulfate, and the first phosphate buffer solution are mixed, stirred at room temperature, allowed to stand at 4°C, and the supernatant is mixed with the carrier, stirred at 30-40°C, and collected by magnetic decantation to obtain the immobilized carrier; polyvinylpyrrolidone, chitosan, and acetic acid aqueous solution are mixed, stirred at room temperature, the immobilized carrier is added, stirred, and collected by magnetic decantation to obtain the chitosan-coated carrier; the second phosphate buffer solution and glutaraldehyde aqueous solution are mixed, stirred at room temperature, the chitosan-coated carrier is added, and the pH is adjusted... The mixture is heated to H 8-8.5, stirred at 30-40℃, collected by magnetic decantation, washed, and dried to obtain immobilized lipase. In the immobilization process, the ratio of lipase, sodium dodecyl sulfate, the first phosphate buffer solution, and the carrier is 5-7 mL: 5-8 g: 5000-8000 mL: 20-25 g; the ratio of carrier, polyvinylpyrrolidone, chitosan, acetic acid aqueous solution, the second phosphate buffer solution, and glutaraldehyde aqueous solution is 20-25 g: 2-2.5 g: 9-10 g: 1500-2000 mL: 1000-1500 mL: 100-120 mL.

2. The method for preparing fatty acids from acidified oil according to claim 1, characterized in that, In the preparation of the carrier, the dropping rate of the diammonium hydrogen phosphate aqueous solution is 10-15 mL / min; the average particle size of the magnetic Fe3O4 particles is 500 nm; the mass concentration of the ammonia water is 25%; the mass concentration of the diammonium hydrogen phosphate aqueous solution is 1%; and the frequency of the ultrasonic oscillation is 20-30 kHz, and the time is 10-20 min.

3. The method for preparing fatty acids from acidified oil according to claim 1, characterized in that, In the immobilization process, the lipase is a liquid formulation with an enzyme activity of 5000 U / mL, derived from Aspergillus niger and purchased from Qingdao Weilan Biotechnology Co., Ltd.; the chitosan has a degree of deacetylation of 85% and a number-average molecular weight of 1 million; the polyvinylpyrrolidone is polyvinylpyrrolidone K30; the acetic acid aqueous solution has a mass concentration of 2%; the phosphate buffer solution used has a molar concentration of 0.1 mol / L and a pH of 7.0; and the glutaraldehyde aqueous solution has a mass concentration of 24-26%.

4. The method for preparing fatty acids from acidified oil according to claim 1, characterized in that, The enzymatic hydrolysis involves mixing the acidified oil after water washing, deionized water, and immobilized lipase, stirring at 40-45℃ for 24-30 hours, collecting the immobilized lipase by magnetic decantation, taking the oil phase after standing, and washing the oil phase with water to obtain fatty acids.

5. The method for preparing fatty acids from acidified oil according to claim 4, characterized in that, In the enzymatic hydrolysis, the ratio of acidified oil after washing to deionized water is 200-250g:400-450mL; the mass ratio of acidified oil after washing to immobilized lipase is 200-250:20-30.

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