A method for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme

Abstract

The present invention provides a method for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling liquid enzyme. The present invention uses a cationic surfactant to modify a hydrophobic resin, increases the adsorption capacity of the hydrophobic resin to gossypol pigment, makes the gossypol pigment in the product more easily adsorbed on resin particles, reduces the residual amount of gossypol pigment in the product, improves product quality, and the modified hydrophobic resin can avoid the adsorption of liquid enzyme at the same time, and almost no loss of liquid enzyme is caused, and then the hydrophobic resin is washed by using the first to the third washing solution, and the pigment and the cationic surfactant on the hydrophobic resin can be effectively removed, so that the hydrophobic resin can be used as an immobilized carrier of liquid enzyme, and the immobilization of liquid enzyme is realized, and then the recycling of liquid enzyme is realized.

Description

Technical Field

[0001] The invention relates to the technical field of biodiesel, and in particular to a method for preparing fatty acid esters by using cottonseed oil and liquid enzyme as raw materials and recovering and reusing the liquid enzyme. Background Art

[0002] Fatty acid methyl ester (biodiesel) is a clean oxygen-containing fuel that is renewable, biodegradable, produces fewer pollutants than petroleum diesel, and has almost no greenhouse effect. It is an environmentally friendly fuel that can achieve combustion characteristics that are almost the same as those of petroleum diesel. Biodiesel has a high flash point, is easy to store, transport and use, and has better safety. According to statistics, biodiesel has become the fastest growing alternative to petroleum diesel fuel.

[0003] At present, the main factor restricting the development of biodiesel is the insufficient supply of raw oil. Cottonseed oil contains harmful ingredients (such as gossypol) and is not eaten. my country is a major cotton producer in the world. Choosing cottonseed oil as raw material makes full use of existing resources and reduces costs compared to edible oil. Cottonpol is a yellow polyphenolic hydroxybisnaphthaldehyde compound. Cottonpol is a colored and toxic substance unique to cottonseed. It mainly exists in the roots, stems, leaves and seeds of the mallow plant cotton. The highest content is in cottonseed kernels, accounting for 0.7%-4.8% of cottonseed. Refined cottonseed oil is refined and impurity-removed by equipment to remove toxic substances such as gossypol and can be eaten by humans. However, there is still a large amount of free gossypol in raw cottonseed oil. When raw cottonseed is pressed for oil, most of the gossypol moves into the oil, and the content in the oil can reach 1% to 1.3%. After producing fatty acid methyl ester using cottonseed oil as raw material, the glycerol layer contains a large amount of methanol. Cottonpol itself is insoluble in glycerol but soluble in methanol, so most of the free cottonpol is concentrated in the lower glycerol mixture, and a small amount remains in the fatty acid methyl ester. Cottonpol is not only toxic, but also causes the color of fatty acid methyl ester and glycerol as finished products to deepen, thus affecting their selling prices. Therefore, it is necessary to remove the cottonpol in the product.

[0004] At present, the research on using enzyme catalysts to replace chemical catalysts (acids or alkalis) to prepare biodiesel has been very extensive. Enzymatic production of biodiesel can overcome some disadvantages of chemical methods, such as high energy demand, high requirements for raw material purity (such as anhydrous short-chain alcohols and low-acid oils), complex product recovery and purification, and the need for sewage treatment. However, the main disadvantage of using lipase is the high cost of biocatalysts. Compared with liquid free lipase, immobilized enzyme has high stability, high selectivity and long half-life. The most critical point is that after being fixed on a carrier, it is conducive to the separation and recovery of lipase, which can reduce production costs. Since most of the liquid enzyme is dispersed in the glycerol and water mixture in the lower layer after generating fatty acid methyl esters with oils and short-chain alcohols, it is difficult to further purify and recover it. Summary of the invention

[0005] The invention aims to provide a method for preparing fatty acid esters by using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme. The method not only realizes the recycling of the liquid enzyme, but also has good quality of the prepared fatty acid esters and glycerin.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, comprising:

[0008] Step 1, reacting cottonseed oil with a first alcohol in the presence of a liquid enzyme and water, and standing and stratifying after the reaction to obtain an upper layer material and a lower layer material;

[0009] Step 2, washing the upper layer material with a sodium carbonate aqueous solution, and dehydrating and dealcoholizing to obtain fatty acid esters after washing;

[0010] Step 3, filtering the lower layer material, decolorizing and adsorbing it with a hydrophobic resin modified by a cationic surfactant to obtain a decolorized material, and washing the decolorized and adsorbed hydrophobic resin with a first washing liquid and water, wherein the first washing liquid is selected from an alkaline solution and / or a salt solution;

[0011] Step 4, washing the hydrophobic resin in the above step 3 with a second washing liquid and a third washing liquid in sequence, wherein the second washing liquid comprises a second alcohol and / or water, and the third washing liquid comprises a phosphate buffer solution;

[0012] Step 5, contacting the decolorized material with the hydrophobic resin washed in the above step 4 to obtain immobilized lipase, and collecting the residual liquid material;

[0013] Step 6: removing alcohol and water from the residual liquid material to obtain glycerol.

[0014] Since cottonseed oil contains a large amount of gossypol, using it to prepare fatty acid esters will result in residual gossypol in the product. The present invention can effectively remove the residual gossypol in the fatty acid esters and reduce the color number of the fatty acid esters by washing with a sodium carbonate aqueous solution.

[0015] For the cottony phenol residue in the glycerol by-product, the present invention modifies the hydrophobic resin by using a cationic surfactant, increases the adsorption capacity of the hydrophobic resin to the cottony phenol pigment, makes the pigment in the material more easily adsorbed on the resin particles, and the glycerol obtained is light in color and of higher quality. The modified hydrophobic resin can also avoid the adsorption of liquid enzymes at the same time, and the loss of liquid enzymes is hardly caused, and then the hydrophobic resin is washed by using the first to the third washing solution, and the pigment and the cationic surfactant on the hydrophobic resin can be effectively removed, so that the hydrophobic resin can be used as the immobilization carrier of liquid enzymes, and the immobilization of liquid enzymes is realized, and then the recycling of liquid enzymes is realized.

[0016] Preferably, the alkaline solution comprises sodium hydroxide solution.

[0017] Preferably, the salt solution includes one or more of sodium chloride solution, sodium carbonate solution, potassium chloride solution and potassium carbonate solution.

[0018] In some embodiments, the first washing solution is a mixture of an alkaline solution and a salt solution.

[0019] Furthermore, the alkaline solution and the salt solution are mixed in a mass ratio of 1:(0.7-1).

[0020] Preferably, the mass concentration of the alkaline solution is 0.1-0.4%, more preferably 0.2-0.35%.

[0021] Preferably, the mass concentration of the salt solution is 3-6%, more preferably 4-5%.

[0022] Preferably, the cationic surfactant is a quaternary ammonium salt type cationic surfactant.

[0023] More preferably, the cationic surfactant is selected from one or more of alkyl trimethyl ammonium salts and dialkyl dimethyl ammonium salts.

[0024] In some embodiments, the cationic surfactant is cetyltrimethylammonium chloride.

[0025] Preferably, the preparation method of the hydrophobic resin modified by a cationic surfactant comprises: contacting the hydrophobic resin with a cationic surfactant aqueous solution with a mass concentration of 3 to 8%, and performing cyclic adsorption, wherein the mass volume ratio of the hydrophobic resin to the cationic surfactant aqueous solution is 1 g: 5 to 10 mL.

[0026] In some embodiments, the preparation method comprises filling a packed bed reactor with a hydrophobic resin, introducing the cationic surfactant aqueous solution, and performing cyclic adsorption.

[0027] Preferably, the preparation method further comprises a step of pretreating the hydrophobic resin before the hydrophobic resin contacts the cationic surfactant aqueous solution, wherein the pretreatment comprises washing the hydrophobic resin with a mixture of n-hexane and isopropanol, and then washing with ethanol and drying.

[0028] More preferably, in the mixed liquid, the mass ratio of n-hexane to isopropanol is 1:(0.5-1.5).

[0029] More preferably, the mass ratio of the hydrophobic resin to the mixed liquid is 1:(1-3).

[0030] Preferably, the mass ratio of the hydrophobic resin to the ethanol is 1:(1-2).

[0031] Further preferably, the hydrophobic resin is rinsed several times with ethanol.

[0032] Preferably, the hydrophobic resin is polypropylene resin.

[0033] Polypropylene resin has strong adsorption properties for organic matter, and it also has the advantages of acid and alkali resistance, high temperature resistance, and large porosity. It can not only adsorb cotton phenol pigment in the product, but also serve as an immobilized carrier for lipase. In the presence of a hydrophobic surface, the enzyme is adsorbed on it and fixed as an open structure with the active center completely exposed, achieving the effect of purifying, immobilizing and overactivating the lipase. Using polypropylene resin as both a decolorizing adsorbent and an immobilized carrier simplifies the operation steps and reduces production costs.

[0034] In some embodiments, the polypropylene resin has a diameter of 455 to 550 μm and a pore size of 15 to 50 nm.

[0035] Preferably, in step 2, the mass concentration of the sodium carbonate aqueous solution is 5 to 8%, more preferably 6 to 7%.

[0036] Further preferably, in step 2, the mass ratio of the sodium carbonate aqueous solution to the cottonseed oil is (5-10):100.

[0037] Preferably, in step 1, the reaction system further comprises an aqueous sodium carbonate solution, which accounts for 0.1-0.6% of the mass of the cottonseed oil, and further 0.3-0.5%. The addition of the aqueous sodium carbonate solution can adjust the pH value of the system while ensuring the activity of the lipase, thereby facilitating the reaction, and can also reduce the residual cottonpol in the fatty acid ester, thereby improving the quality of the fatty acid ester.

[0038] Preferably, in step 1, the liquid enzyme accounts for 0.1-0.5% of the mass of the cottonseed oil.

[0039] In some embodiments, the liquid enzyme is lipase.

[0040] Preferably, in step 1, water accounts for 1 to 3% by mass of the cottonseed oil, and further 1.5 to 2.5% by mass.

[0041] Preferably, in step 1, the first alcohol accounts for 25-50% by mass of the cottonseed oil, and further 30-40% by mass.

[0042] Preferably, the reaction temperature is controlled at 30-50°C.

[0043] Preferably, the first alcohol is selected from alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propanol, and the like.

[0044] Preferably, the second alcohol is ethanol.

[0045] Preferably, the pH of the third washing liquid is 7-8.

[0046] Further preferably, the third washing solution is a sodium phosphate or potassium phosphate buffered saline solution.

[0047] Preferably, the step 5 further comprises drying the decolorized material with acetone after contacting the decolorized material with the hydrophobic resin.

[0048] Further preferably, the volume mass ratio of the acetone to the hydrophobic resin is 6 to 16 mL / g.

[0049] Preferably, the step 6 specifically comprises removing alcohol and water from the residual liquid material at a vacuum degree of -0.075 to -0.95 MPa and a temperature of 125 to 135°C.

[0050] Preferably, the dehydration and dealcoholization in step 2 are carried out at a vacuum degree of -0.085 to -0.098 MPa and a temperature of 105 to 115°C.

[0051] Preferably, the method further comprises using the immobilized lipase for preparing biodiesel.

[0052] Further preferably, the raw material oil for preparing biodiesel includes one or more of soybean oil, waste cooking oil, palm oil, and castor oil.

[0053] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0054] The present invention uses a cationic surfactant to modify the hydrophobic resin, thereby increasing the adsorption capacity of the hydrophobic resin on the gossypol pigment, making the gossypol pigment in the product more easily adsorbed on the resin particles, reducing the residual amount of the gossypol pigment in the product, and improving the product quality;

[0055] At the same time, the modified hydrophobic resin can avoid the adsorption of liquid enzymes and hardly cause the loss of liquid enzymes. By washing the hydrophobic resin with the first to third washing solutions, the pigments and cationic surfactants on the hydrophobic resin can be effectively removed, so that the hydrophobic resin can be used as an immobilization carrier of the liquid enzyme to achieve the immobilization of the liquid enzyme and further realize the recovery and reuse of the liquid enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a schematic diagram of a process of preparing biodiesel using cottonseed oil and liquid enzyme as raw materials and recovering and reusing the liquid enzyme according to the present invention;

[0057] Figure 2 The figure is a schematic diagram of the process of using the immobilized lipase of the present invention to prepare biodiesel. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0059] Unless otherwise specified, the raw materials and equipment used in the following examples and comparative examples are all commercially available products.

[0060] Cottonseed oil was purchased from Linqing, Shandong; Novus Enversa Transform 2.0 lipase was purchased from Novus Company; polypropylene particles were purchased from Membrane GmbH (Germany), model Accurel MP 1000.

[0061] Example 1

[0062] like Figure 1 and Figure 2 As shown, a method for preparing biodiesel using cottonseed oil and liquid enzyme as raw materials and recovering and reusing the liquid enzyme comprises the following steps:

[0063] (1) Weigh 300 g of cottonseed oil, add 0.35% (w / w) of Noweixin EnversaTransform 2.0 lipase based on the total mass of cottonseed oil, add 2% (w / w) water based on the total mass of cottonseed oil and 0.4% (w / w) of a dilute sodium carbonate aqueous solution with a mass concentration of 12%, heat to 40°C and slowly add 35% (w / w) methanol based on the total mass of cottonseed oil dropwise using a peristaltic pump under stirring, react for 20 hours, and allow to stand for stratification to obtain 311.5 g of an upper layer of crude alcohol-containing ester and 98.8 g of a lower layer of material (glycerol, lipase, water, methanol, pigment and a small amount of suspended impurities, i.e., material ①), and a small amount of methanol will be lost due to volatilization during the reaction.

[0064] (2) Add 8% (w / w) dilute sodium carbonate aqueous solution (mass concentration 6.2%) based on the total mass of cottonseed oil to the upper alcohol-containing crude ester, stir and wash thoroughly, and then let stand until stratification. Repeat the washing three times, then dehydrate and dealcoholize the crude ester under reduced pressure, and use the platinum-cobalt colorimetric method to determine that the color number of the methyl ester is 50.

[0065] (3) Weigh 25 g of polypropylene resin particles, wash them with 50 g of detergent (n-hexane and isopropanol mixed in a mass ratio of 1:1), filter out the polypropylene resin particles after washing, rinse them three times with 37.5 g of anhydrous ethanol, and vacuum dry them to obtain the washed polypropylene resin carrier.

[0066] (4) The washed polypropylene resin was filled into a packed bed reactor, and 175 mL of a surfactant aqueous solution (hexadecyltrimethylammonium chloride, 5% by mass) was introduced. After sufficient cyclic adsorption, material ① was filtered using a 10 μm filter cloth and then introduced into the resin at a flow rate of 0.8 mL / min. The cycle was repeated several times to obtain material ②. After decolorization and adsorption, the resin was washed several times with 65 mL of a mixed solution (0.2% by mass NaOH aqueous solution and 5% by mass NaCl aqueous solution, with a mass ratio of 1:0.75) and then washed with deionized water to separate the pigment from the resin.

[0067] (5) washing the microporous polymer resin with a mixture of ethanol and water in a mass ratio of 1:1 and water at a flow rate of 5.5 mL / min at 50-60° C. for 1 h. After washing, washing the microporous polymer resin with a phosphate buffer solution with a pH range of 7.5, while cooling the system to 25° C.;

[0068] (6) Material ② was introduced into a packed bed reactor at a flow rate of 5 mL / min, circulated for 80 min, and then 200 mL of acetone was introduced to dry the reactor to obtain immobilized lipase.

[0069] (7) After the lipase is fully immobilized, the residual liquid material is a mixture of glycerol, water, and methanol. The relative vacuum degree is -0.95 MPa, the maximum temperature is 125°C, and it is maintained for 25 min to remove methanol and water to obtain 11.6 g of glycerol product with a slightly yellow appearance.

[0070] (8) Weigh 300 g of soybean oil and 105 g of methanol, add 2.5% of water based on the total mass of soybean oil to the soybean oil, and introduce methanol into the raw material at a flow rate of 3.16 mL / min for mixing. At the same time, the raw material enters the packed bed reactor loaded with immobilized lipase at a flow rate of 12 mL / min, and the reaction is circulated at 45° C. for 16 h. After the reaction is completed, the lower layer of the material is separated and the dealcoholization test shows that the yield of methyl ester (i.e., fatty acid methyl ester) is 91.7%.

[0071] Example 2

[0072] (1) Weigh 300 g of cottonseed oil, add 0.35% (w / w) of Novus EnversaTransform 2.0 lipase based on the total mass of cottonseed oil, add 2% (w / w) water based on the total mass of cottonseed oil and 0.4% (w / w) of a 12% dilute sodium carbonate aqueous solution, heat to 40°C, and slowly add 35% (w / w) methanol based on the total mass of cottonseed oil dropwise using a peristaltic pump under stirring. After reacting for 20 hours, stand and separate to obtain 309.6 g of an upper layer of crude alcohol ester and 99.3 g of a lower layer of material (which is glycerol, lipase, water, methanol, pigment and a small amount of suspended impurities, i.e., material ①).

[0073] (2) Add 8% (w / w) dilute sodium carbonate aqueous solution (mass concentration 6.2%) based on the total mass of cottonseed oil to the upper alcohol-containing crude ester, stir and wash thoroughly, and then let stand until stratification. Repeat the washing three times, then dehydrate and dealcoholize the crude ester under reduced pressure, and use the platinum-cobalt colorimetric method to determine that the color number of the methyl ester is 50.

[0074] (3) Weigh 25 g of polypropylene resin particles, wash them with 50 g of detergent (n-hexane and isopropanol mixed in a mass ratio of 1:1), filter out the polypropylene resin particles after washing, rinse them three times with 37.5 g of anhydrous ethanol, and vacuum dry them to obtain the washed polypropylene resin carrier.

[0075] (4) The washed polypropylene resin is filled in a packed bed reactor, and 175 mL of an aqueous surfactant solution (hexadecyltrimethylammonium chloride, 5% by mass) is introduced. After sufficient cyclic adsorption, material ① is filtered using a 10 μm filter cloth and then introduced into the resin at a flow rate of 0.8 mL / min. The cycle is repeated several times to obtain material ②. After decolorization and adsorption, the resin is washed several times with 65 mL of a mixed solution (0.2% by mass concentration of NaOH aqueous solution and 5% by mass concentration of Na2CO3 aqueous solution, with a mass ratio of 1:0.75) and then washed with deionized water to separate the pigment from the resin.

[0076] (5) The microporous polymer resin was washed with a mixture of ethanol and water in a mass ratio of 1:1 and water at a flow rate of 5.5 mL / min for 1 h. After washing, the microporous polymer resin was washed with a phosphate buffer solution in the pH range of 7.5, and the system was cooled to 25° C.

[0077] (6) Material ② was introduced into a packed bed reactor at a flow rate of 5 mL / min, circulated for 80 min, and then 200 mL of acetone was introduced to dry the reactor to obtain immobilized lipase.

[0078] (7) After the lipase is fully immobilized, the residual liquid material is a mixture of glycerol, water, and methanol. The relative vacuum degree is -0.95 MPa, the maximum temperature is 125°C, and it is maintained for 25 min to remove methanol and water to obtain 9.88 g of glycerol product with a slightly yellow appearance.

[0079] (8) Weigh 300 g of soybean oil and 105 g of methanol, add 2.5% of water based on the total mass of soybean oil to the soybean oil, and introduce methanol into the raw material at a flow rate of 3.16 mL / min to mix. At the same time, the raw material enters the packed bed reactor loaded with immobilized lipase at a flow rate of 12 mL / min, and the reaction is circulated at 45° C. for 16 h. After the reaction is completed, the lower layer of the material is separated and the methyl ester yield is 90.4% after dealcoholization test.

[0080] This batch of immobilized enzyme packed bed reactors was used to catalyze the production of fatty acid methyl esters from soybean oil for multiple experiments. The methyl ester yield was still maintained above 88% after the fifth reaction.

[0081] Example 3

[0082] (1) Weigh 300 g of cottonseed oil, add 0.35% (w / w) of Novus EnversaTransform 2.0 lipase based on the total mass of cottonseed oil, add 2% (w / w) water based on the total mass of cottonseed oil and 0.4% (w / w) of a 12% dilute sodium carbonate aqueous solution, heat to 40°C, and slowly add 35% (w / w) methanol based on the total mass of cottonseed oil dropwise using a peristaltic pump under stirring. After reacting for 20 hours, stand and separate to obtain 309.4 g of an upper layer of crude alcohol-containing ester and 98.8 g of a lower layer of material (which is glycerol, lipase, water, methanol, pigment and a small amount of suspended impurities, i.e., material ①).

[0083] (2) Add 8% (w / w) dilute sodium carbonate aqueous solution (mass concentration 6.2%) based on the total mass of cottonseed oil to the upper alcohol-containing crude ester, stir and wash thoroughly, and then let stand until stratification. Repeat the washing three times, then dehydrate and dealcoholize the crude ester under reduced pressure, and use the platinum-cobalt colorimetric method to determine that the color number of the methyl ester is 50.

[0084] (3) Weigh 25 g of polypropylene resin particles, wash them with 50 g of detergent (n-hexane and isopropanol mixed in a mass ratio of 1:1), filter out the polypropylene resin particles after washing, rinse them three times with 37.5 g of anhydrous ethanol, and vacuum dry them to obtain the washed polypropylene resin carrier.

[0085] (4) The washed polypropylene resin is filled in a packed bed reactor, and 175 ml of a surfactant aqueous solution (hexadecyltrimethylammonium chloride, mass concentration 5%) is introduced. After sufficient cyclic adsorption, material ① is filtered using a 10 μm filter cloth and then introduced into the resin at a flow rate of 0.8 mL / min. The cycle is repeated several times to obtain material ②. After decolorization and adsorption, the resin is washed several times with 65 mL of a mixed solution (NaOH aqueous solution with a mass concentration of 0.35% and KCl aqueous solution with a mass concentration of 4.5%, with a mass ratio of 1:0.75) and then washed with deionized water to separate the pigment from the resin.

[0086] (5) The microporous polymer resin was washed with a mixture of ethanol and water in a mass ratio of 1:1 and water at a flow rate of 5.5 mL / min for 1 h. After washing, the microporous polymer resin was washed with a phosphate buffer solution in the pH range of 7.5, and the system was cooled to 25° C.

[0087] (6) Material ② was introduced into a packed bed reactor at a flow rate of 5 mL / min, circulated for 80 min, and then 200 mL of acetone was introduced to dry the mixture to obtain immobilized lipase.

[0088] (7) After the lipase is fully immobilized, the residual liquid material is a mixture of glycerol, water, and methanol. The relative vacuum degree is -0.95 MPa, the maximum temperature is 125°C, and it is maintained for 25 min to remove methanol and water to obtain 10.7 g of glycerol product with a slightly yellow appearance.

[0089] (8) Weigh 300 g of soybean oil and 105 g of methanol, add 2.5% of water based on the total mass of soybean oil to the soybean oil, and introduce methanol into the raw material at a flow rate of 3.16 mL / min to mix. At the same time, the raw material enters the packed bed reactor loaded with immobilized lipase at a flow rate of 12 mL / min, and the reaction is circulated at 45° C. for 16 h. After the reaction is completed, the lower layer of the material is separated and the methyl ester yield is 90.8% after dealcoholization test.

[0090] This batch of immobilized enzyme packed bed reactors was used to catalyze the production of fatty acid methyl esters from soybean oil for multiple experiments. The methyl ester yield was still maintained above 88% after the sixth reaction.

[0091] Example 4

[0092] (1) Weigh 300 g of cottonseed oil, add 0.35% (w / w) of Novus EnversaTransform 2.0 lipase based on the total mass of the cottonseed oil, add 2% (w / w) water based on the total mass of the cottonseed oil and 0.4% (w / w) of a 12% dilute sodium carbonate aqueous solution, heat to 40°C, and slowly add 35% (w / w) methanol based on the total mass of the cottonseed oil dropwise using a peristaltic pump under stirring. After reacting for 20 hours, stand and separate to obtain 308.5 g of an upper layer of crude alcohol-containing ester and 99.0 g of a lower layer of material (which is glycerol, lipase, water, methanol, pigment, and a small amount of suspended impurities, i.e., material ①).

[0093] (2) Add 8% (w / w) dilute sodium carbonate aqueous solution (concentration 6.2%) based on the total mass of cottonseed oil to the upper alcohol-containing crude ester, stir and wash thoroughly, and then stand to separate. Repeat the washing three times, then dehydrate and dealcoholize the crude ester under reduced pressure. Use platinum-cobalt colorimetry to determine that the color number of the methyl ester is 50.

[0094] (3) Weigh 25 g of polypropylene resin particles, wash them with 50 g of detergent (n-hexane and isopropanol mixed in a mass ratio of 1:1), filter out the polypropylene resin particles after washing, rinse them three times with 37.5 g of anhydrous ethanol, and vacuum dry them to obtain the washed polypropylene resin carrier.

[0095] (4) The washed polypropylene resin is filled in a packed bed reactor, and 175 ml of a surfactant aqueous solution (hexadecyltrimethylammonium chloride, mass concentration 5%) is introduced. After sufficient cyclic adsorption, material ① is filtered using a 10 μm filter cloth and then introduced into the resin at a flow rate of 0.8 mL / min. The cycle is repeated several times to obtain material ②. After decolorization and adsorption, the resin is washed several times with 65 mL of a mixed solution (a KOH aqueous solution with a mass concentration of 0.35% and a KCl aqueous solution with a mass concentration of 4.5%, with a mass ratio of 1:0.75), and then washed with deionized water to separate the pigment from the resin.

[0096] (5) The microporous polymer resin was washed with a mixture of ethanol and water in a mass ratio of 1:1 and water at a flow rate of 5.5 mL / min for 1 h. After the washing was completed, the microporous polymer resin was washed with a phosphate buffer solution in the pH range of 7.5, and the system was cooled to 25° C.

[0097] (6) Material ② was introduced into the resin reactor at a flow rate of 5 mL / min, circulated for 80 min, and then 200 mL of acetone was introduced to dry the mixture to obtain immobilized lipase.

[0098] (7) After the lipase is fully immobilized, the residual liquid material is a mixture of glycerol, water, and methanol. The relative vacuum degree is -0.95 MPa, the maximum temperature is 125°C, and it is maintained for 25 min to remove methanol and water to obtain 11.6 g of glycerol product with a slightly yellow appearance.

[0099] (8) Weigh 300 g of soybean oil and 105 g of methanol, add 2.5% of water based on the total mass of soybean oil to the soybean oil, and introduce methanol into the raw material at a flow rate of 3.16 mL / min to mix. At the same time, the raw material enters the packed bed reactor loaded with immobilized lipase at a flow rate of 12 mL / min, and the reaction is circulated at 45° C. for 16 h. After the reaction is completed, the lower layer of the material is separated and the methyl ester yield is 86.2% after dealcoholization test.

[0100] Example 5

[0101] The method is substantially the same as the above-mentioned Example 1, with the main difference being that the washing liquid used after the decolorization and adsorption in step (4) is 65 mL of a 0.2% NaOH aqueous solution.

[0102] The immobilized lipase prepared in this example was used to prepare methyl ester, and the yield of methyl ester was 88.3%.

[0103] Example 6

[0104] The method is substantially the same as the above-mentioned Example 1, with the main difference being that the washing liquid used after the decolorization and adsorption in step (4) is 65 mL of a 5% NaCl aqueous solution.

[0105] The immobilized lipase prepared in this example was used to prepare methyl ester, and the yield of methyl ester was 86.1%.

[0106] Comparative Example 1

[0107] (1) 300 g of cottonseed oil was weighed, 0.35% (w / w) of Noweixin EnversaTransform 2.0 lipase based on the total mass of the cottonseed oil was added, 2% (w / w) of water based on the total mass of the cottonseed oil and 0.4% (w / w) of a 12% dilute sodium carbonate aqueous solution were added, and the temperature was raised to 40° C., and 35% (w / w) of methanol based on the total mass of the cottonseed oil was slowly added dropwise using a peristaltic pump under stirring. After the reaction was completed, the mixture was allowed to stand and separate to obtain 308.1 g of an upper layer of crude alcohol-containing ester and 101.2 g of a lower layer of material (which was glycerol, lipase, water, methanol, pigment and a small amount of suspended impurities, i.e., mixed ①).

[0108] (2) Add 8% (w / w) dilute sodium carbonate aqueous solution (mass concentration 6.2%) based on the total mass of cottonseed oil to the upper alcohol-containing crude ester, stir and wash thoroughly, and then let stand until stratification. Repeat the washing three times, then dehydrate and dealcoholize the crude ester under reduced pressure, and use the platinum-cobalt colorimetric method to determine that the color number of the methyl ester is 50.

[0109] (3) Weigh 25 g of polypropylene resin particles, wash them with 50 g of detergent (n-hexane and isopropanol mixed in a mass ratio of 1:1), filter out the polypropylene resin particles after washing, rinse them three times with 37.5 g of anhydrous ethanol, and vacuum dry them to obtain the washed polypropylene resin carrier.

[0110] (4) The washed polypropylene resin is filled in a packed bed reactor, and 175 mL of an aqueous surfactant solution (hexadecyltrimethylammonium chloride, 5% by mass concentration) is introduced. After sufficient cyclic adsorption, material ① is filtered using a 10 μm filter cloth and then introduced into the resin at a flow rate of 0.8 mL / min. The reaction is repeated several times to obtain material ②. After decolorization and adsorption, the material is washed with deionized water to separate the pigment from the resin.

[0111] (5) The microporous polymer resin was washed with a mixture of ethanol and water in a mass ratio of 1:1 and water at a flow rate of 5.5 mL / min for 1 h. After the washing was completed, the microporous polymer resin was washed with a phosphate buffer solution in the pH range of 7.5, and the system was cooled to 25° C.

[0112] (6) Material ② was introduced into a packed bed reactor at a flow rate of 5 mL / min, circulated for 80 min, and then 200 mL of acetone was introduced to dry the mixture to obtain immobilized lipase.

[0113] (7) After the lipase is fully immobilized, the residual liquid material is a mixture of glycerol, water, and methanol. The vacuum degree is -0.95 MPa, the maximum temperature is 125°C, and it is maintained for 25 min to remove methanol and water to obtain 9.68 g of glycerol product with a slightly yellow appearance.

[0114] (8) Weigh 300 g of soybean oil and 105 g of methanol, add 2.5% of water based on the total mass of soybean oil to the soybean oil, and introduce methanol into the raw material at a flow rate of 3.16 mL / min to mix. At the same time, the raw material enters the packed bed reactor loaded with immobilized lipase at a flow rate of 12 mL / min, and the reaction is circulated at 45° C. for 16 h. After the reaction is completed, the lower layer of the material is separated and the methyl ester yield is 84.4% after dealcoholization test.

[0115] Comparative Example 2

[0116] The method is substantially the same as Example 1, except that step (4) and step (5) are not performed, but the washed polypropylene resin is filled into a packed bed reactor, and then material ① is filtered using a 10 μm filter cloth and passed into the packed bed reactor at a flow rate of 5 mL / min, circulated for 80 min, and 200 mL of acetone is passed into the reactor for drying to obtain immobilized lipase.

[0117] This comparative example obtained 11.75 g of glycerol product, which had an orange-red appearance.

[0118] The immobilized lipase prepared in this comparative example was used to prepare methyl ester, and the yield of methyl ester was 78.4%.

[0119] Comparative Example 3

[0120] The method is substantially the same as the above-mentioned Example 1, with the main difference being that the surfactant used is an anionic surfactant alkyl sulfonate.

[0121] This comparative example obtained 10.11 g of glycerol product, which had an orange appearance.

[0122] The immobilized lipase prepared in this comparative example was used to prepare methyl ester, and the yield of methyl ester was 90.3%.

[0123] Comparative Example 1

[0124] (1) Weigh 300 g of cottonseed oil, add 0.35% (w / w) of Novus EnversaTransform 2.0 lipase based on the total mass of cottonseed oil, add 2% (w / w) of water based on the total mass of cottonseed oil, heat to 40°C, and slowly add 35% (w / w) of methanol based on the total mass of cottonseed oil dropwise using a peristaltic pump under stirring. After reacting for 20 hours, stand and separate to obtain 311.3 g of crude alcohol-containing ester in the upper layer and 92.5 g of material in the lower layer (glycerol, lipase, water, methanol and a small amount of suspended impurities).

[0125] (2) The crude ester was dehydrated and dealcoholized under reduced pressure, and the color number of the methyl ester was determined to be 70 using the potassium dichromate colorimetric method.

[0126] (3) The lower layer of materials, including glycerol, lipase, water, methanol and a small amount of suspended impurities, was filtered to separate the impurities. The relative vacuum degree was -0.95 MPa and the maximum temperature was 125 °C. The temperature was maintained for 25 min to remove methanol and water, and 13.3 g of crude glycerol product was obtained, which was orange-red in appearance.

[0127] Comparative Example 2

[0128] Weigh 200g of soybean oil into a three-necked flask, add 2.5% water based on the total mass of soybean oil and 0.25% Novus Enversa Transform 2.0 lipase, weigh 60g of methanol, and pass methanol into the raw materials at a flow rate of 1.36mL / min to mix, react at 45°C for 16h, and after the reaction, separate the lower layer of the material and the methyl ester yield of the dealcoholization test is 84.5%.

[0129] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, It is characterized in that The method comprises: Step 1, reacting cottonseed oil with a first alcohol in the presence of a liquid enzyme and water, and standing and stratifying after the reaction to obtain an upper layer material and a lower layer material, wherein the first alcohol is methanol and the liquid enzyme is lipase; Step 2, washing the upper layer material with a sodium carbonate aqueous solution, and dehydrating and dealcoholizing to obtain fatty acid esters after washing; Step 3, filtering the lower layer material, decolorizing and adsorbing it with a hydrophobic resin modified by a cationic surfactant to obtain a decolorized material, and washing the decolorized and adsorbed hydrophobic resin with a first washing liquid and water, wherein the first washing liquid is selected from an alkaline solution and / or a salt solution, and the hydrophobic resin is a polypropylene resin; Step 4, washing the hydrophobic resin in the above step 3 with a second washing liquid and a third washing liquid in sequence, wherein the second washing liquid comprises a second alcohol and / or water, the second alcohol is ethanol, and the third washing liquid comprises a phosphate buffer solution; Step 5, contacting the decolorized material with the hydrophobic resin washed in the above step 4 to obtain immobilized lipase, and collecting the residual liquid material; Step 6: removing alcohol and water from the residual liquid material to obtain glycerol.

2. The method according to claim 1 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, It is characterized in that The alkaline solution includes a sodium hydroxide solution, and the salt solution includes one or more of a sodium chloride solution, a sodium carbonate solution, a potassium chloride solution, and a potassium carbonate solution.

3. The method according to claim 2 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, It is characterized in that The mass concentration of the alkaline solution is 0.1-0.4%; and / or, The mass concentration of the salt solution is 3-6%.

4. The method according to claim 1 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, It is characterized in that The cationic surfactant is a quaternary ammonium salt type cationic surfactant.

5. The method according to claim 4 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, It is characterized in that The cationic surfactant is selected from one or more of alkyl trimethyl ammonium salts and dialkyl dimethyl ammonium salts.

6. The method according to any one of claims 1, 4 and 5, wherein the method uses cottonseed oil and liquid enzyme as raw materials to prepare fatty acid esters and recycle the liquid enzyme. It is characterized in that The preparation method of the hydrophobic resin modified by a cationic surfactant comprises: contacting the hydrophobic resin with a cationic surfactant aqueous solution with a mass concentration of 3-8%, and performing cyclic adsorption, wherein the mass volume ratio of the hydrophobic resin to the cationic surfactant aqueous solution is 1g:5-10mL.

7. The method according to claim 6 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme, It is characterized in that The preparation method further comprises the step of pre-treating the hydrophobic resin before the hydrophobic resin contacts the cationic surfactant aqueous solution, wherein the pre-treatment comprises washing the hydrophobic resin with a mixture of n-hexane and isopropanol, then washing with ethanol and drying.

8. The method according to claim 1 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recovering and reusing the liquid enzyme, It is characterized in that In the step 2, the mass concentration of the sodium carbonate aqueous solution is 5-8%.

9. The method according to claim 1 for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recovering and reusing the liquid enzyme, It is characterized in that In step 1, the reaction system further comprises an aqueous sodium carbonate solution, wherein the aqueous sodium carbonate solution accounts for 0.1 to 0.6% of the mass of the cottonseed oil; and / or, The liquid enzyme accounts for 0.1-0.5% of the mass of the cottonseed oil; and / or, The water accounts for 1-3% of the mass of the cottonseed oil; and / or, The first alcohol accounts for 25-50% of the mass of the cottonseed oil; and / or, Control the reaction temperature at 30~50℃.

10. The method for preparing fatty acid esters using cottonseed oil and liquid enzyme as raw materials and recycling the liquid enzyme according to claim 1, It is characterized in that The method also includes using the immobilized lipase to produce biodiesel.

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