CN108239663B - Method for hydrolyzing high-melting-point grease by enzyme method - Google Patents

Method for hydrolyzing high-melting-point grease by enzyme method Download PDF

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CN108239663B
CN108239663B CN201611207643.9A CN201611207643A CN108239663B CN 108239663 B CN108239663 B CN 108239663B CN 201611207643 A CN201611207643 A CN 201611207643A CN 108239663 B CN108239663 B CN 108239663B
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lipase
derived
weight
amount
lipozyme
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CN108239663A (en
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李磊
丛芳
郭晓峰
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Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
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Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead

Abstract

The invention relates to a method for hydrolyzing high-melting-point grease by an enzyme method. Specifically, the invention provides a preparation method of 12-hydroxystearic acid, which comprises the step of hydrolyzing hydrogenated castor oil by using lipase in the presence of a first organic solvent, a second organic solvent and water, wherein the lipase is at least two of lipase derived from thermophilic fungi, lipase derived from rhizomucor and partial glyceride lipase, the polarity of the first organic solvent is between 4 and 6, and the polarity of the second polar solvent is less than or equal to 1. The method can carry out hydrolysis reaction at low temperature by using common lipase, can obviously improve the final hydrolysis rate, and does not reduce the hydroxyl value of the obtained product.

Description

Method for hydrolyzing high-melting-point grease by enzyme method
Technical Field
The invention belongs to the field of grease processing technology, and particularly relates to a method for hydrolyzing high-melting-point grease by an enzymatic method.
Background
12-Hydroxystearic acid is an 18-carbon fatty acid containing a hydroxyl group at the 12-position and has the formula CH3(CH2)5CH(OH)(CH2)10COOH, in the form of plate or needle crystals at room temperature, with a melting point of 82-93 ℃. 12-hydroxystearic acid is an important chemical industrial material because it contains both hydroxyl and carboxyl groups, and can be used as an intermediate product in the production of plastics or as a raw material for cosmetics.
One of the main preparation methods of 12-hydroxystearic acid is to hydrolyze castor oil at high temperature and high pressure, and then hydrogenate ricinoleic acid obtained by hydrolysis to prepare 12-hydroxystearic acid, in the hydrolysis process of castor oil, hydroxyl and carboxyl in ricinoleic acid are easy to generate esterification reaction and polymerize, and the yield of 12-hydroxystearic acid can be reduced, so the method is rarely adopted. The second method is to hydrogenate castor oil to obtain hydrogenated castor oil, then saponify the hydrogenated castor oil with strong alkali and acidify the hydrogenated castor oil with strong acid to obtain 12-hydroxystearic acid. Although the method can protect hydroxyl from being damaged, the method uses excessive strong base and strong acid to generate a large amount of waste water, thereby causing environmental pollution. The enzymatic hydrolysis of hydrogenated castor oil is therefore a further development.
Some reports about the preparation of 12-hydroxystearic acid by an enzymatic method at home and abroad mainly include the following:
CN 201510313095.7 discloses a method for hydrolyzing hydrogenated castor oil, which comprises the step of carrying out three times of hydrolysis reaction on hydrogenated castor oil and deionized water according to a certain proportion in the presence of biological hydrolase to obtain 12-hydroxystearic acid. The hydrogenated castor oil, water and hydrolase in a mass ratio of 100:60-100:0.3-0.5 are mixed for each hydrolysis, and after being uniformly mixed, the mixture is hydrolyzed for three times under the stirring condition to obtain 12-hydroxystearic acid; the temperature of the hydrolysis reaction is 80-85 ℃; the time is 120-180 min. The reaction temperature of the method is above 80 ℃, and the temperature of most of hydrolyzed lipase is about 50 ℃, so that most of low-temperature lipase is eliminated, the lipase is limited to high-temperature lipase with few types, the high-temperature lipase is expensive, and the method has general significance for later-stage production operation.
JP 61139396 discloses a process for preparing 12-hydroxystearic acid by hydrolyzing hydrogenated castor oil with lipase at 75 ℃ and dissolving it in a solvent during the reaction. This method has the same disadvantage as CN 201510313095.7, i.e. it excludes most of the hydrolytic lipases.
CN 1473199A discloses a method for preparing 12-hydroxystearic acid, which mainly comprises the steps of firstly catalyzing and hydrolyzing castor oil at 15-50 ℃ by one or more lipases to form ricinoleic acid, and then hydrogenating a hydrolysis product to prepare 12-hydroxystearic acid. Although the method has low operation temperature in the hydrolysis process, the method can cause loss of hydroxyl and reduction of acid value in the later hydrogenation process, and has certain defects.
The above literature studies how to prepare 12 hydroxystearic acid under various process conditions through various processes. But has the following disadvantages:
1. when hydrogenated castor oil is used as a substrate, the hydrolysis temperature is high in the hydrolysis process, and only high-temperature lipase can be used as a catalyst;
2. when castor oil is used as a substrate, although the hydrolysis temperature is low, hydroxyl groups are lost in the later hydrogenation process, so that the product does not reach the standard.
Therefore, there is still a need in the art for a method for preparing 12-hydroxystearic acid using hydrogenated castor oil with a high melting point as a substrate, which can perform hydrolysis reaction at a low temperature using a common lipase, can significantly increase the final hydrolysis rate, and can obtain a product with no decrease in hydroxyl value.
Disclosure of Invention
The present invention provides, in a first aspect, a method for preparing 12-hydroxystearic acid, which includes a step of hydrolyzing hydrogenated castor oil using a lipase in the presence of a first organic solvent, a second organic solvent and water, wherein the lipase is at least two of a lipase derived from the genus Thermomyces (Thermomyces sp.), a lipase derived from the genus Rhizomucor (Rhizomucor sp.), and a partial glyceride lipase.
In one or more embodiments, the first organic solvent has a polarity between 4 and 6, and the second polar solvent has a polarity of 1 or less.
In one or more embodiments, the first organic solvent is selected from acetone, chloroform, or mixtures thereof.
In one or more embodiments, the second organic solvent is selected from cyclohexane, n-hexane, or mixtures thereof.
In one or more embodiments, the volume ratio of the first organic solvent to the second organic solvent is 40 to 60: 60-40.
In one or more embodiments, the volume ratio of the first organic solvent to the second organic solvent is less than or equal to 1.
In one or more embodiments, the first organic solvent is acetone, the second organic solvent is n-hexane or cyclohexane, and the volume ratio of acetone to n-hexane or cyclohexane is 40-60: 60-40.
In one or more embodiments, the volume ratio of acetone to n-hexane or cyclohexane is less than or equal to 1.
In one or more embodiments, the first organic solvent is chloroform, the second organic solvent is n-hexane, and the volume ratio of chloroform to n-hexane is 40 to 60: 60-40.
In one or more embodiments, the ratio of the total amount of organic solvent added in milliliters to the mass of hydrogenated castor oil in grams is from 1 to 8.
In one or more embodiments, the weight ratio of hydrogenated castor oil to water is 1: 2-6, preferably 1: 2 to 4.
In one or more embodiments, the partial glyceride lipase is a lipase derived from Aspergillus (Aspergillus), Penicillium (Penicillium sp.), or Malassezia (Malassezia).
In one or more embodiments, the lipase derived from Penicillium sp is a lipase derived from Penicillium camemberti (Penicillium camemberti).
In one or more embodiments, the Lipase derived from Penicillium sp is Lipase G50, a commercial enzyme Lipase G "Amano" 50.
In one or more embodiments, the partial glyceride lipase is used in an amount of 0.1 to 6.0% by weight, for example, 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 3.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 5.0% by weight of the hydrogenated castor oil.
In one or more embodiments, the lipase derived from the genus thermophilic fungus is a lipase derived from thermomyces lanuginosus.
In one or more embodiments, the lipase derived from the genus thermophilic fungus is Lipozyme TL IM.
In one or more embodiments, the lipase derived from Thermomyces is used in an amount of 0.1 to 6.0% by weight of the fat, for example 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 3.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 3.0%.
In one or more embodiments, the lipase derived from Rhizomucor sp is a lipase derived from Rhizomucor miehei (Rhizomucor miehei).
In one or more embodiments, the lipase derived from rhizomucor is Lipozyme RM IM.
In one or more embodiments, the lipase derived from Rhizomucor is used in an amount of 1.0 to 5.0% by weight, such as 1.0 to 4.0%, 1.0 to 3.0%, 1.0 to 2.0%, 2.0 to 5.0% by weight, and the like, based on the weight of the oil.
In one or more embodiments, the lipase is a lipase derived from the genus thermophilus and a partial glyceride lipase. In these embodiments, the amount of lipase derived from Thermomyces is 0.2 to 5.0%, such as 1.0 to 3.0%, and the amount of partial glyceride lipase is 0.2 to 5.0%, such as 1.0 to 3.0%, by weight of the oil.
In one or more embodiments, the lipase is a lipase derived from the genus thermophilus and a lipase derived from the genus rhizomucor. In these embodiments, the lipase derived from the genus Thermomyces is used in an amount of 1.0 to 5.0% by weight and the lipase derived from the genus Rhizomucor is used in an amount of 1.0 to 5.0% by weight based on the weight of the oil or fat.
In one or more embodiments, the lipase is a lipase derived from rhizomucor and a partial glyceride lipase. In these embodiments, the amount of lipase derived from Rhizomucor is 1.0 to 5.0%, such as 2.0 to 5.0% by weight of the oil; the amount of partial glyceride lipase is 1.0-5.0%, for example 2.0-5.0% of the weight of the oil.
In one or more embodiments, the hydrolysis is carried out at a temperature of from 35 to 55 ℃, for example, from 40 to 50 ℃.
In a second aspect, the present invention provides a method for hydrolyzing a high melting point oil or fat, comprising the step of hydrolyzing the high melting point oil or fat with a lipase in the presence of a first organic solvent, a second organic solvent and water, wherein the lipase is at least two of a lipase derived from the genus Thermomyces (Thermomyces sp.), a lipase derived from the genus Rhizomucor (Rhizomucor sp.), and a partial glyceride lipase.
In one or more embodiments, the first organic solvent has a polarity between 4 and 6, and the second organic solvent has a polarity of 1 or less.
In one or more embodiments, the first organic solvent is selected from acetone, chloroform, or mixtures thereof.
In one or more embodiments, the second organic solvent is selected from cyclohexane, n-hexane, or mixtures thereof.
In one or more embodiments, the volume ratio of the first organic solvent to the second organic solvent is 40 to 60: 60-40, preferably 40: 60-50: 50.
In one or more embodiments, the volume ratio of the first organic solvent to the second organic solvent is less than or equal to 1.
In one or more embodiments, the first organic solvent is acetone, the second organic solvent is n-hexane or cyclohexane, and the volume ratio of acetone to n-hexane or cyclohexane is 40-60: 60-40, preferably 40: 60-50: 50.
In one or more embodiments, the volume ratio of acetone to n-hexane or cyclohexane is less than or equal to 1.
In one or more embodiments, the first organic solvent is chloroform, the second organic solvent is n-hexane, and the volume ratio of chloroform to n-hexane is 40 to 60: 60-40, preferably 40: 60-50: 50.
In one or more embodiments, the ratio of the total amount of organic solvent added in milliliters to the mass of high melting point grease in grams is from 1 to 8.
In one or more embodiments, the weight ratio of high melting point grease to water is 1: 2-6, preferably 1: 2 to 4.
In one or more embodiments, the partial glyceride lipase is a lipase derived from Aspergillus (Aspergillus), Penicillium (Penicillium sp.), or Malassezia (Malassezia).
In one or more embodiments, the lipase derived from Penicillium sp is a lipase derived from Penicillium camemberti (Penicillium camemberti).
In one or more embodiments, the Lipase derived from Penicillium sp is Lipase G50, a commercial enzyme Lipase G "Amano" 50.
In one or more embodiments, the partial glyceride lipase is used in an amount of 0.1 to 6.0% by weight, for example, 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 3.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 5.0% by weight of the hydrogenated castor oil.
In one or more embodiments, the lipase derived from the genus thermophilic fungus is a lipase derived from thermomyces lanuginosus.
In one or more embodiments, the lipase derived from the genus thermophilic fungus is Lipozyme TL IM.
In one or more embodiments, the lipase derived from Thermomyces is used in an amount of 0.1 to 6.0% by weight, such as 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 3.0%, 1.0 to 2.0%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 5.0% by weight of the oil.
In one or more embodiments, the lipase derived from Rhizomucor sp is a lipase derived from Rhizomucor miehei (Rhizomucor miehei).
In one or more embodiments, the lipase derived from rhizomucor is Lipozyme RM IM.
In one or more embodiments, the lipase derived from Rhizomucor is used in an amount of 1.0 to 5.0% by weight, such as 1.0 to 4.0%, 1.0 to 3.0%, 1.0 to 2.0%, 2.0 to 5.0% by weight, and the like, based on the weight of the oil.
In one or more embodiments, the lipase is a lipase derived from the genus thermophilus and a partial glyceride lipase. In these embodiments, the amount of lipase derived from Thermomyces is 0.2 to 5.0%, such as 1.0 to 3.0%, and the amount of partial glyceride lipase is 0.2 to 5.0%, such as 1.0 to 3.0%, by weight of the oil.
In one or more embodiments, the lipase is a lipase derived from the genus thermophilus and a lipase derived from the genus rhizomucor. In these embodiments, the lipase derived from the genus Thermomyces is used in an amount of 1.0 to 5.0% by weight and the lipase derived from the genus Rhizomucor is used in an amount of 1.0 to 5.0% by weight based on the weight of the oil or fat.
In one or more embodiments, the lipase is a lipase derived from rhizomucor and a partial glyceride lipase. In these embodiments, the amount of lipase derived from Rhizomucor is 1.0 to 5.0%, such as 2.0 to 5.0% by weight of the fat; the amount of partial glyceride lipase is 1.0-5.0%, for example 2.0-5.0% of the weight of the oil.
In one or more embodiments, the hydrolysis is carried out at a temperature of from 35 to 55 ℃, for example, from 40 to 50 ℃.
In a third aspect, the present invention provides an application of a partial glyceride lipase and/or a lipase derived from a genus Thermomyces and/or a lipase derived from a genus Rhizomucor in hydrolyzing high melting point fats and oils, an application in preparing 12-hydroxystearic acid using hydrogenated castor oil as a substrate, and an application in preparing a lipase composition for hydrolyzing high melting point fats and oils or for preparing 12-hydroxystearic acid using hydrogenated castor oil as a substrate.
The third aspect of the present invention also provides a reaction mixture comprising: high-melting-point grease, a first organic solvent, a second organic solvent, water and lipase; wherein the lipase is at least two of a lipase derived from Thermomyces sp, a lipase derived from Rhizomucor sp, and a partial glyceride lipase.
In one or more embodiments, the first organic solvent has a polarity between 4 and 6 and the second organic solvent has a polarity of 1 or less.
In one or more embodiments, the first organic solvent is selected from acetone, chloroform, or mixtures thereof.
In one or more embodiments, the second organic solvent is selected from cyclohexane, n-hexane, or mixtures thereof.
In one or more embodiments, the volume ratio of the first organic solvent to the second organic solvent in the reaction mixture is from 40 to 60: 60-40, preferably 40: 60-50: 50.
In one or more embodiments, the volume ratio of the first organic solvent to the second organic solvent in the reaction mixture is less than or equal to 1.
In one or more embodiments, the first organic solvent is acetone, the second organic solvent is n-hexane or cyclohexane, and the volume ratio of acetone to n-hexane or cyclohexane is 40-60: 60-40, preferably 40: 60-50: 50.
In one or more embodiments, the volume ratio of acetone to n-hexane or cyclohexane is less than or equal to 1.
In one or more embodiments, the first organic solvent is chloroform, the second organic solvent is n-hexane, and the volume ratio of chloroform to n-hexane is 40 to 60: 60-40, preferably 40: 60-50: 50.
In one or more embodiments, the ratio of the total amount of organic solvent in milliliters to the mass of high melting point oil in grams in the reaction mixture is from 1 to 8.
In one or more embodiments, the reaction mixture further comprises 12-hydroxystearic acid.
In one or more embodiments, the weight ratio of high melting point grease to water in the mixture is 1: 2-6, preferably 1: 2 to 4.
In one or more embodiments, the partial glyceride lipase is a lipase derived from Aspergillus (Aspergillus), Penicillium (Penicillium sp.), or Malassezia (Malassezia).
In one or more embodiments, the lipase derived from Penicillium sp is a lipase derived from Penicillium camemberti (Penicillium camemberti).
In one or more embodiments, the Lipase derived from Penicillium sp is Lipase G50, a commercial enzyme Lipase G "Amano" 50.
In one or more embodiments, the partial glyceride lipase is present in an amount of 0.1 to 6.0% by weight of the high melting point oil, for example 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 2.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 3.0%.
In one or more embodiments, the lipase derived from the genus thermophilic fungus is a lipase derived from thermomyces lanuginosus.
In one or more embodiments, the lipase derived from the genus thermophilic fungus is Lipozyme TL IM.
In one or more embodiments, the lipase derived from Thermomyces is present in an amount of 0.1 to 6.0% by weight of the high melting point oil, such as 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 3.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 3.0%.
In one or more embodiments, the lipase derived from Rhizomucor sp is a lipase derived from Rhizomucor miehei (Rhizomucor miehei).
In one or more embodiments, the lipase derived from rhizomucor is Lipozyme RM IM.
In one or more embodiments, the lipase derived from Rhizomucor is present in an amount of 1.0 to 5.0%, for example, 1.0 to 4.0%, 1.0 to 3.0%, 1.0 to 2.0%, 2.0 to 5.0%, etc., based on the weight of the high melting point oil.
In one or more embodiments, the high melting point grease is a grease having a melting point above 40 ℃, preferably hydrogenated castor oil, palm stearin, or lard.
In one or more embodiments, the lipase is a lipase derived from the genus thermophilus and a partial glyceride lipase. In these embodiments, the lipase derived from the genus thermophilic fungi is present in an amount of 0.2 to 5.0%, such as 1.0 to 3.0%, and the partial glyceride lipase is present in an amount of 0.2 to 5.0%, such as 1.0 to 3.0%, by weight of the high melting point fat.
In one or more embodiments, the lipase is a lipase derived from the genus thermophilus and a lipase derived from the genus rhizomucor. In these embodiments, the content of the lipase derived from the genus Thermomyces is 1.0 to 5.0% by weight of the high melting point oil or fat, and the content of the lipase derived from the genus Rhizomucor is 1.0 to 5.0% by weight of the high melting point oil or fat.
In one or more embodiments, the lipase is a lipase derived from rhizomucor and a partial glyceride lipase. In these embodiments, the lipase derived from Rhizomucor is present in an amount of 1.0 to 5.0%, for example 2.0 to 5.0%, by weight of the high melting point oil or fat; the content of partial glyceride lipase is 1.0-5.0%, for example 2.0-5.0% of the weight of the high melting point oil.
Detailed Description
The invention aims to improve the hydrolysis rate of high-melting-point grease (such as hydrogenated castor oil, palm oil stearin or lard) and shorten the reaction time in the hydrolysis reaction process. When the method uses high-melting-point grease as a substrate, hydrolysis reaction can be carried out at low temperature by using common lipase, and the final hydrolysis rate can be obviously improved. In the present invention, "high melting point" means a melting point of 40 ℃ or higher, preferably 45 ℃ or higher or 50 ℃ or higher.
In particular, the present invention relates to the hydrolysis of hydrogenated castor oil using at least two lipases from the group consisting of lipases from the genus Thermomyces (Thermomyces sp.), lipases from the genus Rhizomucor sp and partial glyceride lipases.
Herein, "partial glyceride lipase" refers to a lipase which does not have catalytic hydrolysis activity on triglycerides, and preferentially has catalytic hydrolysis activity on diglycerides and/or monoglycerides. Partial glyceride lipases are mainly of two types: monoglyceride-diglyceride lipase and monoglyceride lipase. The present invention preferably uses partial glyceride lipases having catalytic hydrolytic activity for mono-and/or diglycerides. Partial glyceride lipases which are well known in the art may be used, for example, partial glyceride lipases from Aspergillus (Aspergillus), Penicillium (Penicillium) and Malassezia (Malassezia) may be used.
In certain embodiments, partial glyceride lipases suitable for use in the present invention include, but are not limited to, partial glyceride lipases such as those disclosed in CN 201410182887.0, commercially available partial glyceride lipases such as Lipase G50(Lipase G "Amano"50), and lipases GH1(Huang J, Yang Z, Guan F et al, alpha novel mono-and diacylglycerol Lipase hi expressed in Pichia pastoris, and ate application for food emulsifier preparation, Process Biochemistry, 2013, 48(12):1899-, biochemical characterization and molecular basis for substrate selection, Plos One, 2014, 9(7): e102040) and the like.
When used in the method or use of the present invention, the amount of the partial glyceride lipase is usually 0.1 to 6.0% by weight, for example, 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 2.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0%, or 3.0 to 5.0% by weight of the oil.
In certain embodiments, the present invention uses a partial glyceride lipase from Penicillium sp, particularly from Penicillium camemberti (Penicillium camemberti). For example, the partial glyceride Lipase from Penicillium (Penicillium sp.) may be Lipase G50, Lipase G "Amano" 50.
In the present invention, the lipase derived from the genus Thermomyces (Thermomyces sp.) may be a lipase derived from Thermomyces lanuginosus (Thermomyces lanuginosus), for example, the lipase derived from the genus Thermomyces (Thermomyces sp.) is Lipozyme TL IM or a lipase derived from the genus Thermomyces (Thermomyces sp.) having similar enzymatic properties to Lipozyme TL IM.
When used in the method or use of the present invention, the amount of the lipase derived from a thermophilic fungus may be 0.1 to 6.0% by weight of the oil, for example 0.1 to 5.0%, 0.15 to 4.0%, 0.15 to 3.0%, 0.15 to 2.5%, 0.15 to 2.0%, 0.15 to 1.5%, 0.15 to 1.0%, 0.2 to 5.0%, 0.2 to 3.0%, 0.2 to 2.0%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 5.0%, 1.0 to 3.0%, 1.0 to 1.5%, 1.5 to 5.0%, 1.5 to 3.0%, 1.5 to 2.0%, 2.0 to 5.0%, 2.0 to 3.0% or 3.0 to 3.0% by weight of the oil.
In the present invention, the lipase derived from Rhizomucor genus may be a lipase derived from Rhizomucor miehei (Rhizomucor miehei). In certain embodiments, the lipase derived from rhizomucor is Lipozyme RM IM or a rhizomucor-derived lipase having similar enzymatic properties as Lipozyme RM IM.
When used in the method or application of the present invention, the amount of the lipase derived from Rhizomucor is 1.0 to 5.0% by weight, for example, 1.0 to 4.0%, 1.0 to 3.0%, 1.0 to 2.0%, 2.0 to 5.0% by weight, based on the oil or fat.
Any combination of any two of the above listed lipase dosage ranges may be used to practice the present invention. For example, the amount of the partial glyceride lipase may be 0.2-3.0%, such as 0.2-2.0%, by weight of the oil, and the amount of the lipase derived from the genus thermophile used in combination with the partial glyceride lipase may be 0.2-3.0%, such as 0.2-2%, 0.2-1.5%, 0.2-1.0%, 1.0-3.0%, 1.0-2.0%, 1.0-1.5%, or 1.5-3.0%, by weight of the oil; similarly, the amount of the lipase derived from Thermomyces may be 0.15 to 3.0%, for example, 0.2 to 2% by weight of the fat, and the amount of the partial glyceride lipase used in combination with the lipase may be 0.15 to 3.0%, for example, 0.2 to 2%, 0.2 to 1.5%, 0.2 to 1.0%, 1.0 to 3.0%, 1.0 to 2.0%, 1.0 to 1.5%, or 1.5 to 3.0% by weight of the fat.
The hydrolysis is carried out in the presence of an organic solvent. Organic solvents suitable for use in the hydrolysis of the present invention include a first organic solvent and a second organic solvent. The polarity of the first organic solvent is generally between 4 and 6, and the polarity of the second organic solvent is 1 or less. In certain embodiments, the first organic solvent is selected from acetone, chloroform, or mixtures thereof; the second organic solvent is selected from cyclohexane, n-hexane or mixtures thereof.
Generally, the volume ratio of the first organic solvent to the second organic solvent is 40-60: 60-40. In certain embodiments, the volume ratio of the first organic solvent to the second organic solvent is less than or equal to 1, e.g., 40: 60-50: 50 (volume ratio).
In some embodiments, the first organic solvent is acetone, the second organic solvent is n-hexane or cyclohexane, and the volume ratio of acetone to n-hexane or cyclohexane is 40-60: 60-40. In certain embodiments, the volume ratio of acetone to n-hexane or cyclohexane is less than or equal to 1.
In some embodiments, the first organic solvent is chloroform and the second organic solvent is n-hexane, and the volume ratio of chloroform to n-hexane is 40 to 60: 60-40, preferably 40: 60-50: 50.
The polarity of the solvent can be determined based on the dielectric constant of the solvent. The literature reports measurement methods including: katritzky A R, Dan C F, Yang H, et al quantitative Measures of Solvent Polarity [ J ]. ChemInform,2004,104(14): 175-98.
Common solvents with a polarity less than 1 are selected from: isopentane, n-pentane, petroleum ether, n-hexane, cyclohexane, isooctane, trimethylpentane, cyclopentane, heptane, and the like, or combinations of one or more thereof.
Common solvents with a polarity greater than 4 and less than 6 are selected from: butyl acetate, propanol, methyl isobutyl ketone, tetrahydrofuran, ethanol, ethyl acetate, isopropanol, chloroform, methyl ethyl ketone, pyridine, acetone, and the like, or combinations of one or more thereof.
Preferably a mixture of acetone and n-hexane. In some embodiments, the ratio of the acetone to the n-hexane is 40-60: 60-40 (volume ratio). In certain embodiments, the amount of acetone is not greater than the amount of n-hexane, i.e., the volume ratio of acetone to n-hexane is 1 or less. Thus, in these embodiments, the amount of acetone and n-hexane is prepared as 40: 60-50: 50 (volume ratio). In addition, the ratio of the total amount of organic solvent added (in milliliters) to the weight of the oil or fat (in grams) is generally from 1 to 8, for example from 1 to 6.
The hydrolysis is also carried out in the presence of water. In general, an appropriate amount of water may be added to the oil or fat depending on the amount of the enzyme used. Typically, the weight ratio of grease to water is 1: 2 to 6, for example 1: 2-5, 1: 2-5, 1: 3 to 5, etc. For optimal hydrolysis, the mass ratio of oil to water can be determined and adjusted by one skilled in the art according to the amount of enzyme used, the reaction temperature, the amount of solvent used, the reaction time, and other factors.
Similarly, the amount of the organic solvent is adjusted according to the actual reaction conditions.
Upon hydrolysis, water and organic solvent may now be added to the fat, followed by addition of the lipase as described herein. The lipase can be added into the oil and fat respectively or in the form of a mixture. After the addition, the oil and fat, lipase and water are mixed uniformly. For example, the mixture may be stirred uniformly. If the lipase is added to the fat or oil as an aqueous solution, it is preferable to ensure that the mass ratio of the total amount of water in the aqueous solution of fatty acid to the additionally added water to the fat or oil is within the above range.
The hydrolysis reaction may be carried out at a temperature of 35 to 55 ℃ in general, for example, at a temperature of 40 to 50 ℃. Therefore, after the oil and fat, the lipase and the water are uniformly mixed, the temperature of the obtained mixture is gradually increased to be within the range of 35-55 ℃ for reaction. Usually, the temperature is raised with stirring. Stirring can be continuously carried out in the reaction process.
The time of the hydrolysis reaction is not particularly limited, and may be adjusted depending on the amount of the fat or oil to be hydrolyzed, the kind of the lipase to be used, the amount thereof, and the like. Or, the acid value of the sample can be analyzed and the hydrolysis rate can be calculated in the hydrolysis reaction process, and the reaction can be stopped when the expected hydrolysis rate is reached.
When hydrogenated castor oil is hydrolyzed, the 12-hydroxystearic acid obtained can be separated and purified by distillation under reduced pressure.
In certain embodiments, the lipase is a lipase derived from the genus thermophilus and a partial glyceride lipase. In these embodiments, the amount of lipase derived from Thermomyces is 0.2 to 5.0%, such as 1.0 to 3.0%, and the amount of partial glyceride lipase is 0.2 to 5.0%, such as 1.0 to 3.0%, by weight of the oil. In certain preferred embodiments, the ratio of the amount of lipase derived from the genus thermophilic fungi to the amount of partial glyceride lipase is 1 or more.
In certain preferred embodiments, the weight ratio of fat to water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the volume of the organic solvent is 2-8 times of the weight of the grease; the amount of the lipase derived from the thermophilic fungi is 0.2-5.0%, such as 1.0-3.0% of the weight of the grease, the amount of the partial glyceride lipase is 0.2-5.0%, such as 1.0-3.0% of the weight of the grease, and the preferable amount ratio of the lipase derived from the thermophilic fungi to the partial glyceride lipase is more than or equal to 1; preferably, the hydrolysis is carried out at a temperature of 45 to 50 ℃.
In certain embodiments, the lipase is a lipase derived from the genus thermophilus and a lipase derived from the genus rhizomucor. In these embodiments, the amount of lipase derived from Thermomyces is 1.0 to 5.0%, such as 2.0 to 5.0% by weight of the oil; the amount of the lipase derived from Rhizomucor is 1.0-5.0%, such as 2.0-5.0% of the weight of the oil.
In certain preferred embodiments, the weight ratio of fat to water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the volume of the organic solvent is 2-8 times of the weight of the grease; the dosage of the lipase from the thermophilic fungi is 2.0-5.0% of the weight of the grease, and the dosage of the lipase from the rhizomucor is 2.0-5.0% of the weight of the grease.
In certain embodiments, the lipase is a lipase derived from rhizomucor and a partial glyceride lipase. In these embodiments, the amount of lipase derived from Rhizomucor is 1.0 to 5.0%, such as 2.0 to 5.0% by weight of the oil; the amount of partial glyceride lipase is 1.0-5.0%, for example 2.0-5.0% of the weight of the oil.
In certain preferred embodiments, the weight ratio of fat to water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the volume of the organic solvent is 4-8 times of the weight of the grease; the dosage of the lipase from rhizomucor is 2.0-5.0% of the weight of the grease, and the dosage of the lipase from rhizomucor is 2.0-5.0% of the weight of the grease.
The invention also provides application of the partial glyceride lipase and/or the lipase derived from the thermophilic fungus and/or the lipase derived from rhizomucor in hydrolyzing high-melting-point grease, application in preparing 12-hydroxystearic acid by taking hydrogenated castor oil as a substrate, and application in preparing a lipase composition for hydrolyzing high-melting-point grease or preparing 12-hydroxystearic acid by taking hydrogenated castor oil as a substrate.
The present invention also provides a reaction mixture comprising: high-melting-point grease, a first organic solvent, a second organic solvent, water and lipase; wherein the lipase is at least two of a lipase derived from Thermomyces sp, a lipase derived from Rhizomucor sp, and a partial glyceride lipase. In certain embodiments, the amounts and ratios of the high melting point oil, the first organic solvent, the second organic solvent, water and the lipase in the reaction mixture are as described above in the present invention. In certain embodiments, the reaction mixture is a reaction mixture of the present invention for the preparation of 12-hydroxystearic acid. In certain embodiments, the reaction mixture is a reaction mixture obtained during the preparation of 12-hydroxystearic acid according to the present invention, which also contains 12-hydroxystearic acid.
By the process and the reaction mixture of the present invention, a reaction product having an acid value of 100mgKOH/g or more (after removal of the organic solvent and water), preferably 120mgKOH/g or more (after removal of the organic solvent and water) can be obtained. With the process and reaction mixture of the present invention, a hydrolysis rate of at least 50%, such as at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80% can be achieved.
The present invention will be illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. Lipozyme TL IM used in the examples was a product of Novaxin (China) investment Limited, and Lipase G50 was Lipase G "Amano"50, a product of Amano Enzyme preparation Inc. The hydrogenated castor oil used in the examples was obtained from Fengyi Fine chemistry (Hongkong) Ltd and had a melting point of 80-82 ℃; other conventional chemicals were purchased from national pharmaceutical chemicals, ltd, for analytical purity. The percentages in the examples are by weight. Lipase TL is added as an aqueous solution by first dissolving Lipozyme TL IM in water to form an aqueous solution of conventional concentration. The other lipases are all added in powder form.
In the examples, the hydrolysis ratio was calculated by the following formula:
Figure BDA0001190329790000161
wherein, AV0Refers to the acid value of the grease raw material, which is 1.70mgKOH/g for castor oil; AV (Audio video)tMeans sampling acid value at a certain time interval t; SV refers to the saponification value of the grease raw material, which for castor oil is 182.01 mgKOH/g.
COMPARATIVE EXAMPLE 1 (Petroleum ether 60-90)
10G of hydrogenated castor oil is taken into a reactor, 40G of deionized water (400%) and 40ml of petroleum ether (60-90) (400%) are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 4.68mgKOH/g, and the hydrolysis rate was calculated to be 2.53%.
COMPARATIVE EXAMPLE 2 (Isopropanol)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%) and 40ml of isopropanol (400%) are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started and carried out by heating under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 3.22mgKOH/g, and the hydrolysis rate was calculated to be 1.74%.
COMPARATIVE EXAMPLE 3 (ethanol)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%) and 40ml of ethanol (400%) are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 3.76mgKOH/g, and the hydrolysis rate was calculated to be 2.03%.
COMPARATIVE EXAMPLE 4 (n-hexane)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%) and 40ml of normal hexane (400%) are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 69.88mgKOH/g, and the hydrolysis rate was calculated to be 37.81%.
COMPARATIVE EXAMPLE 5 (acetone)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%) and 40ml of acetone (400%) are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started and carried out by heating under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured, the acid value was 25.81mgKOH/g, and the hydrolysis rate was calculated to be 13.37%.
Example 1 (acetone: n-hexane ═ 20:80)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 8ml of acetone and 32ml of normal hexane are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and the acid value was measured after removing the solvent and water, the acid value was 83.55mgKOH/g, and the hydrolysis rate was calculated to be 45.39%.
Example 2 (acetone: n-hexane: 40:60)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured and was 157.14mgKOH/g, and the hydrolysis rate was calculated to be 86.21%.
Example 3 (acetone: n-hexane: 50)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 20ml of acetone and 20ml of n-hexane are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 147.13mgKOH/g, and the calculated hydrolysis rate was 79.61%.
Example 4 (acetone: n-hexane: 60:40)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 24ml of acetone and 16ml of normal hexane are added, then 0.2G of liquid lipase TL IM (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured, the acid value was 95.22mgKOH/g, and the hydrolysis rate was calculated to be 51.52%.
Example 5 (acetone: n-hexane: 40:60)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of normal hexane are added, then 0.15G of liquid lipase TL IM (1.5%) and 0.15G of lipase G50 (1.5%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 137.00mgKOH/g, and the hydrolysis rate was calculated to be 75.04%.
Example 6 (acetone: n-hexane: 40:60, total solvent volume 300%)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 12ml of acetone and 18ml of normal hexane are added, then 0.02G of liquid lipase TL IM (0.2%) and 0.02G of lipase G50 (0.2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured and was 131.41mgKOH/g, and the hydrolysis rate was calculated to be 71.94%.
Example 7 (acetone: n-hexane: 40:60, total solvent volume 500%)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 20ml of acetone and 30ml of normal hexane are added, then 0.02G of liquid lipase TL IM (0.2%) and 0.02G of lipase G50 (0.2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 138.96mgKOH/g, and the hydrolysis rate was calculated to be 76.12%.
Example 8
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.3G of liquid lipase TL (3%) and 0.3G of lipase G50 (3%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 137.84mgKOH/g, and the hydrolysis rate was calculated to be 76.50%.
Example 9
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.1G of liquid lipase TL (1%) and 0.2G of lipase G50 (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 114.67mgKOH/g, and the hydrolysis rate was calculated to be 63.50%.
Example 10
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.2G of liquid lipase TL (2%) and 0.1G of lipase G50 (1%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured and was 135.55mgKOH/g, and the hydrolysis rate was calculated to be 75.25%.
Example 11 (acetone: n-hexane: 40:60, total solvent volume 100%)
Taking 10G of hydrogenated castor oil, adding 40G of deionized water (400%), 4ml of acetone and 6ml of n-hexane into a reactor, then adding 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%), stirring at 400rpm, starting heating for reaction, reacting at 45 ℃, and stopping the reaction after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 122.59mgKOH/g, and the hydrolysis rate was calculated to be 67.96%.
Example 12 (acetone: n-hexane: 40:60, total solvent volume 200%)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 8ml of acetone and 12ml of n-hexane are added, then 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured and was 144.39mgKOH/g, and the hydrolysis rate was calculated to be 80.22%.
Example 13 (acetone: n-hexane: 40:60, total volume of solvent 800%)
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 32ml of acetone and 48ml of n-hexane are added, then 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 141.96mgKOH/g, and the hydrolysis rate was calculated to be 78.85%.
Example 14
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of cyclohexane are added, then 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and the acid value was measured after removing the solvent and water, the acid value was 137.89mgKOH/g, and the hydrolysis rate was calculated to be 76.61%.
Example 15
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of chloroform and 24ml of normal hexane are added, then 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and the acid value was measured after removing the solvent and water, the acid value was 131.60mgKOH/g, and the hydrolysis rate was calculated to be 73.12%.
Example 16
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 40 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured and was 121.78mgKOH/g, and the hydrolysis rate was calculated to be 67.65%.
Example 17
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.2G of liquid lipase TL (2%) and 0.2G of lipase G50 (2%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 50 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and the acid value was measured after removing the solvent and water, the acid value was 143.56mgKOH/g, and the hydrolysis rate was calculated to be 79.76%.
Example 18
10g of hydrogenated castor oil is put into a reactor, 40g of deionized water (400%), 16ml of acetone and 24ml of normal hexane are added, then 0.05g of liquid lipase TL (0.5%) and 0.05g of lipase RM (0.5%) are added, heating is started under the stirring state of 400rpm for reaction, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 55.99mgKOH/g, and the hydrolysis rate was calculated to be 30.49%.
Example 19
10g of hydrogenated castor oil is put into a reactor, 40g of deionized water (400%), 16ml of acetone and 24ml of normal hexane are added, then 0.1g of liquid lipase TL (1%) and 0.1g of lipase RM (1%) are added, the reaction is started and heated under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 108.82mgKOH/g, and the hydrolysis rate was calculated to be 60.21%.
Example 20
Taking 10g of hydrogenated castor oil, adding 20g of deionized water (200%), 16ml of acetone and 24ml of n-hexane into a reactor, then adding 0.2g of liquid lipase TL (2%) and 0.2g of lipase RM (2%), starting heating under the stirring state of 400rpm for reaction, wherein the reaction temperature is 45 ℃, and stopping the reaction after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 103.90mgKOH/g, and the hydrolysis rate was calculated to be 57.44%.
Example 21
10g of hydrogenated castor oil is put into a reactor, 40g of deionized water (400%), 8ml of acetone and 12ml of normal hexane are added, then 0.2g of liquid lipase TL (2%) and 0.2g of lipase RM (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, and after removing the solvent and water, the acid value was measured and was 127.11mgKOH/g, and the hydrolysis rate was calculated to be 70.50%.
Example 22
10g of hydrogenated castor oil is put into a reactor, 40g of deionized water (400%), 32ml of acetone and 48ml of normal hexane are added, then 0.2g of liquid lipase TL (2%) and 0.2g of lipase RM (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 133.71mgKOH/g, and the hydrolysis rate was calculated to be 74.28%.
Example 23
10g of hydrogenated castor oil is put into a reactor, 40g of deionized water (400%), 16ml of acetone and 24ml of normal hexane are added, then 0.5g of liquid lipase TL (5%) and 0.5g of lipase RM (5%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 125.68mgKOH/g, and the hydrolysis rate was calculated to be 69.70%.
Example 24
10G of hydrogenated castor oil is put into a reactor, 20G of deionized water (200%), 16ml of acetone and 24ml of n-hexane are added, then 0.2G of liquid lipase G50 (2%) and 0.2G of lipase RM (2%) are added, the reaction is started and heated under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 113.71mgKOH/g, and the hydrolysis rate was calculated to be 63.17%.
Example 25
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 8ml of acetone and 12ml of n-hexane are added, then 0.2G of liquid lipase G50 (2%) and 0.2G of lipase RM (2%) are added, the reaction is started and heated under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 114.80mgKOH/g, and the hydrolysis rate was calculated to be 63.78%.
Example 26
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 32ml of acetone and 48ml of n-hexane are added, then 0.2G of liquid lipase G50 (2%) and 0.2G of lipase RM (2%) are added, the reaction is started and heated under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 129.90mgKOH/g, and the hydrolysis rate was calculated to be 72.17%.
Example 27
10G of hydrogenated castor oil is put into a reactor, 40G of deionized water (400%), 16ml of acetone and 24ml of n-hexane are added, then 0.5G of liquid lipase G50 (5%) and 0.5G of lipase RM (5%) are added, the reaction is started and heated under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction product was taken out, and after removing the solvent and water, the acid value was measured, the acid value was 140.11mgKOH/g, and the hydrolysis rate was calculated to be 77.84%.
Example 28
10g of hydrogenated castor oil is taken out and put into a reactor, 10g of deionized water (100%), 16ml of acetone and 24ml of normal hexane are added, then 0.2g of lipase TL (2%) and 0.2g of lipase RM (2%) are added, the reaction is started to be heated and carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was measured after removing the solvent and water, the acid value was 62.78mgKOH/g, and the hydrolysis rate was calculated to be 34.88%.
Example 29
Taking 10g of hydrogenated castor oil, adding 60g of deionized water (600%), 16ml of acetone and 24ml of n-hexane into a reactor, then adding 0.2g of lipase TL (2%) and 0.2g of lipase RM (2%), starting heating under the stirring state of 400rpm to carry out reaction, wherein the reaction temperature is 45 ℃, and stopping the reaction after 24 hours of reaction.
The reaction was taken out, and the acid value was measured after removing the solvent and water, the acid value was 115.08mgKOH/g, and the hydrolysis rate was calculated to be 63.93%.
Example 30
10g of palm oil stearin (melting point of 52 ℃) is taken to be put into a reactor, 40g of deionized water (400%), 16ml of acetone and 24ml of normal hexane are added, then 0.2g of lipase TL (2%) and 0.2g of lipase RM (2%) are added, heating is started to carry out reaction under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 151.96mgKOH/g, and the hydrolysis rate was calculated to be 77.92%.
Example 31
10g of lard (the melting point is 40-50 ℃) is put into a reactor, 10g of deionized water (100%), 16ml of acetone and 24ml of normal hexane are added, then 0.2g of lipase TL (2%) and 0.2g of lipase RM (2%) are added, the heating is started and the reaction is carried out under the stirring state of 400rpm, the reaction temperature is 45 ℃, and the reaction is stopped after 24 hours of reaction.
The reaction was taken out, the acid value was detected after removing the solvent and water, the acid value was 148.06mgKOH/g, and the hydrolysis rate was calculated to be 75.93%.

Claims (53)

1. A method for preparing 12-hydroxystearic acid, the method comprising the steps of hydrolyzing hydrogenated castor oil using lipase in the presence of a first organic solvent, a second organic solvent and water;
wherein the lipase is derived from the genus Thermomyces (A)Thermomyces sp.) Lipozyme TL IM of (A), derived from Rhizomucor genus: (A)Rhizomucor sp.) Lipozyme RM IM and a lipase derived from Penicillium (M)Penicillium sp.) At least two of the partial glyceride lipases G50;
wherein the amount of the lipase Lipozyme TL IM derived from the genus thermophilic fungi is 0.2-5.0% of the weight of the hydrogenated castor oil; the usage amount of the lipase Lipozyme RM IM derived from rhizomucor is 1.0-5.0% of the weight of the hydrogenated castor oil; the using amount of the partial glyceride lipase G50 is 0.2-5.0% of the weight of the hydrogenated castor oil;
the polarity of the first organic solvent is 4-6, and the polarity of the second organic solvent is less than or equal to 1;
wherein the first organic solvent is acetone, chloroform or a mixture thereof; the second organic solvent is cyclohexane, n-hexane or a mixture thereof;
wherein the volume ratio of the first organic solvent to the second organic solvent is 40-60: 60-40;
wherein the weight ratio of the hydrogenated castor oil to the water is 1: 2-6;
wherein the ratio of the total addition amount of the first organic solvent and the second organic solvent in milliliters to the mass of the hydrogenated castor oil in grams is 1-8;
wherein the hydrolysis is carried out at a temperature of 35-55 ℃.
2. The method of claim 1, wherein the volume ratio of the first organic solvent to the second organic solvent is less than or equal to 1.
3. The method of claim 1, wherein the weight ratio of hydrogenated castor oil to water is from 1: 2 to 4.
4. The method of claim 1, wherein the weight ratio of hydrogenated castor oil to water is from 1: 4 to 5.
5. The method of claim 1, wherein the partial glyceride lipase G50 is used in an amount of 1.0 to 3.0% by weight of the hydrogenated castor oil.
6. The method of claim 1, wherein the partial glyceride lipase G50 is used in an amount of 1.0 to 5.0% by weight of the hydrogenated castor oil.
7. The method of claim 1, wherein said partial glyceride lipase G50 is used in an amount of 2.0 to 5.0% by weight based on the hydrogenated castor oil.
8. The method according to claim 1, wherein the lipase Lipozyme TL IM derived from the genus Thermomyces is used in an amount of 1.0 to 3.0% by weight of the hydrogenated castor oil.
9. The method according to claim 1, wherein the lipase Lipozyme TL IM derived from the genus Thermomyces is used in an amount of 1.0 to 5.0% by weight of the hydrogenated castor oil.
10. The method according to claim 1, wherein the lipase Lipozyme TL IM derived from the genus Thermomyces is used in an amount of 2.0 to 5.0% by weight of the hydrogenated castor oil.
11. The method according to claim 1, characterized in that the lipase Lipozyme RM IM originating from Rhizomucor sp is used in an amount of 2.0 to 5.0% by weight of the hydrogenated castor oil.
12. The method of any one of claims 1-11,
the lipase is lipase Lipozyme TL IM and partial glyceride lipase G50 from thermophilic fungi; or
The lipase is lipase Lipozyme TL IM derived from thermophilic fungi and lipase Lipozyme RM IM derived from rhizomucor; or
The lipase is lipase Lipozyme RM IM and partial glyceride lipase G50 derived from Rhizomucor.
13. The method of claim 1, wherein the hydrolysis is carried out at a temperature of 40 to 50 ℃.
14. The method of claim 13, wherein the hydrolysis is carried out at a temperature of 45 to 50 ℃.
15. The method according to claim 12, wherein the ratio of the amount of lipase Lipozyme TL IM derived from the genus thermophilus to the amount of partial glyceride lipase G50 is 1 or more.
16. The method of claim 12, wherein the lipase is a lipase Lipozyme TL IM derived from the genus thermophilus and a lipase Lipozyme RM IM derived from the genus rhizomucor; wherein the amount of the lipase Lipozyme TL IM derived from the genus Thermomyces is 1.0-5.0% by weight of the hydrogenated castor oil, and the amount of the lipase Lipozyme RM IM derived from the genus Rhizomucor is 1.0-5.0% by weight of the hydrogenated castor oil.
17. The method according to claim 16, wherein the lipase Lipozyme TL IM derived from the genus thermophilus is used in an amount of 2.0 to 5.0% by weight of the hydrogenated castor oil.
18. The method according to claim 16, wherein the lipase Lipozyme RM IM derived from rhizomucor is used in an amount of 2.0 to 5.0% by weight of the hydrogenated castor oil.
19. The method according to claim 12, wherein the lipase is the lipase Lipozyme RM IM and partial glyceride lipase G50 derived from rhizomucor; wherein the lipase Lipozyme RM IM derived from rhizomucor is 1.0-5.0% of the weight of the hydrogenated castor oil, and the partial glyceride lipase G50 is 1.0-5.0% of the weight of the hydrogenated castor oil.
20. The method according to claim 19, wherein the lipase Lipozyme RM IM derived from rhizomucor is used in an amount of 2.0 to 5.0% by weight of the hydrogenated castor oil.
21. The method of claim 19, wherein the partial glyceride lipase G50 is present in an amount of 2.0 to 5.0% by weight of the hydrogenated castor oil.
22. The method of claim 1, wherein the lipase is the lipase Lipozyme TL IM and partial glyceride lipase G50; wherein the weight ratio of the hydrogenated castor oil grease to the water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the total volume of the acetone and the normal hexane is 2-8 times of the weight of the hydrogenated castor oil; the amount of the lipase Lipozyme TL IM derived from the genus thermophilic fungi is 0.2-5.0% of the weight of the hydrogenated castor oil, and the amount of the partial glyceride lipase G50 is 0.2-5.0% of the weight of the hydrogenated castor oil.
23. The method according to claim 22, wherein the ratio of the amount of Lipozyme TL IM derived from the genus thermophilus to the amount of partial glyceride lipase G50 is 1 or more.
24. The method of claim 22, wherein the hydrolysis is carried out at a temperature of 45 to 50 ℃.
25. The method according to claim 1, wherein the lipase is a lipase Lipozyme TL IM derived from the genus thermophilus and a lipase Lipozyme RM IM derived from the genus rhizomucor; wherein the weight ratio of the hydrogenated castor oil to the water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the total volume of the acetone and the normal hexane is 2-8 times of the weight of the hydrogenated castor oil; the amount of the lipase Lipozyme TL IM derived from the genus Thermomyces is 2.0-5.0% of the weight of the hydrogenated castor oil, and the amount of the lipase Lipozyme RM IM derived from the genus Rhizomucor is 2.0-5.0% of the weight of the hydrogenated castor oil.
26. The method of claim 1, wherein the lipase is the Lipozyme RM IM and partial glyceride lipase G50, derived from rhizomucor; wherein the weight ratio of the hydrogenated castor oil to the water is 1: 4-5; the volume ratio of acetone to n-hexane is 40: 60; the total volume of the acetone and the normal hexane is 4-8 times of the weight of the hydrogenated castor oil; the amount of the lipase Lipozyme RM IM derived from rhizomucor is 2.0-5.0% of the weight of the hydrogenated castor oil, and the amount of the partial glyceride lipase G50 is 2.0-5.0% of the weight of the hydrogenated castor oil.
27. A method for hydrolyzing a high melting point fat, the method comprising the step of hydrolyzing the high melting point fat using a lipase in the presence of a first organic solvent, a second organic solvent and water;
wherein the lipase is derived from the genus Thermomyces (A)Thermomyces sp.) Lipozyme TL IM of (A), derived from Rhizomucor genus: (A)Rhizomucor sp.) Lipozyme RM IM and a lipase derived from Penicillium (M)Penicillium sp.) At least two of the partial glyceride lipases G50;
wherein the usage amount of the lipase Lipozyme TL IM derived from the thermophilic fungus is 0.2-5.0% of the weight of the high melting point grease; the using amount of the lipase Lipozyme RM IM derived from rhizomucor is 1.0-5.0% of the weight of the high-melting-point oil; the using amount of the partial glyceride lipase G50 is 0.2-5.0% of the weight of the high-melting-point grease;
the polarity of the first organic solvent is 4-6, and the polarity of the second organic solvent is less than or equal to 1;
wherein the first organic solvent is acetone, chloroform or a mixture thereof, and the second organic solvent is cyclohexane, n-hexane or a mixture thereof;
wherein the volume ratio of the first organic solvent to the second organic solvent is 40-60: 60-40 parts of;
wherein the weight ratio of the high-melting-point grease to the water is 1: 2-6;
wherein the ratio of the total addition amount of the first organic solvent and the second organic solvent in milliliters to the mass of the high-melting-point grease in grams is 1-8;
wherein, the hydrolysis is carried out at the temperature of 35-55 ℃;
wherein the high-melting-point grease is grease with a melting point of more than 40 ℃.
28. The method of claim 27, wherein the volume ratio of the first organic solvent to the second organic solvent is less than or equal to 1.
29. The method of claim 27, wherein the high melting point fat is hydrogenated castor oil, palm stearin, or lard.
30. The method of claim 27, wherein the weight ratio of the high melting point grease to water is 1: 2 to 4.
31. The method of claim 27, wherein the weight ratio of the high melting point grease to water is 1: 4 to 5.
32. The method according to claim 27, wherein the partial glyceride lipase G50 is used in an amount of 1.0 to 3.0% by weight based on the high melting point fat.
33. The method according to claim 27, wherein the partial glyceride lipase G50 is used in an amount of 1.0 to 5.0% by weight based on the high melting point fat.
34. The method according to claim 27, wherein the partial glyceride lipase G50 is used in an amount of 2.0 to 5.0% by weight based on the high melting point fat.
35. The method of claim 27, wherein the amount of the thermophilic fungus-derived lipase Lipozyme TL IM is 1.0 to 3.0% by weight of the high melting point fat.
36. The method of claim 27, wherein the amount of the thermophilic fungus-derived lipase Lipozyme TL IM is 1.0 to 5.0% by weight of the high melting point fat.
37. The method of claim 27, wherein the thermophilic fungus-derived lipase Lipozyme TL IM is used in an amount of 2.0 to 5.0% by weight of the high melting point fat.
38. The method according to claim 27, wherein the lipase Lipozyme RM IM derived from Rhizomucor is used in an amount of 2.0 to 5.0% by weight based on the high-melting fat or oil.
39. The method of any one of claims 27-38,
the lipase is lipase Lipozyme TL IM and partial glyceride lipase G50 from thermophilic fungi; or
The lipase is lipase Lipozyme TL IM derived from thermophilic fungi and lipase Lipozyme RM IM derived from rhizomucor; or
The lipase is lipase Lipozyme RM IM and partial glyceride lipase G50 derived from Rhizomucor.
40. The method of claim 27, wherein the hydrolysis is carried out at a temperature of 40 to 50 ℃.
41. The method of claim 40, wherein the hydrolysis is carried out at a temperature of 45 to 50 ℃.
42. The method according to claim 39, wherein the ratio of the amount of lipase Lipozyme TL IM to partial glyceride lipase G50 derived from Thermomyces is 1 or more.
43. The method of claim 39, wherein the lipase is Lipozyme TL IM derived from Thermomyces and Lipozyme RM IM derived from Rhizomucor; wherein the amount of the lipase Lipozyme TL IM derived from the thermophilic fungi is 1.0-5.0% of the weight of the high melting point grease; the amount of the lipase Lipozyme RM IM derived from rhizomucor is 1.0-5.0% of the weight of the high-melting-point oil.
44. The method of claim 43, wherein the amount of the thermophilic fungus-derived lipase Lipozyme TL IM is 2.0 to 5.0% by weight of the high melting point fat.
45. The method according to claim 43, wherein the amount of the lipase Lipozyme RM IM derived from Rhizomucor is 2.0 to 5.0% by weight based on the high melting point fat or oil.
46. The method of claim 39, wherein said lipase is the lipase Lipozyme RM IM and partial glyceride lipase G50 derived from Rhizomucor sp; wherein the usage amount of the lipase Lipozyme RM IM derived from rhizopus is 1.0-5.0% of the weight of the high-melting-point oil; the amount of the partial glyceride lipase G50 is 1.0-5.0% of the weight of the high-melting-point oil.
47. The method according to claim 46, wherein the amount of the lipase Lipozyme RM IM derived from Rhizomucor is 2.0 to 5.0% by weight based on the high melting point fat or oil.
48. The method according to claim 46, wherein the partial glyceride lipase G50 is used in an amount of 2.0 to 5.0% by weight based on the high melting point fat.
49. The method of claim 27,
the lipase is lipase Lipozyme TL IM and partial glyceride lipase G50 from thermophilic fungi; wherein the weight ratio of the high-melting-point grease to the water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the total volume of the acetone and the normal hexane is 2-8 times of the weight of the high-melting-point grease; the amount of the lipase Lipozyme TL IM derived from the thermophilic fungi is 0.2-5.0% of the weight of the high-melting-point oil, and the amount of the partial glyceride lipase G50 is 0.2-5.0% of the weight of the high-melting-point oil.
50. The method of claim 48, wherein the ratio of the amount of Lipozyme TL IM derived from Thermomyces to the amount of partial glyceride lipase G50 is 1 or greater.
51. The method of claim 50, wherein the hydrolysis is carried out at a temperature of 45 to 50 ℃.
52. The method of claim 27, wherein the lipase is Lipozyme TL IM derived from the genus thermophilus and Lipozyme RM IM derived from the genus rhizomucor; wherein the weight ratio of the high-melting-point grease to water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the total volume of the acetone and the normal hexane is 2-8 times of the weight of the high-melting-point grease; the amount of the lipase Lipozyme TL IM derived from the genus Thermomyces is 2.0-5.0% of the weight of the high-melting-point oil, and the amount of the lipase Lipozyme TL IM derived from the genus Rhizomucor is 2.0-5.0% of the weight of the high-melting-point oil.
53. The method of claim 27, wherein said lipase is the lipase Lipozyme RM IM and partial glyceride lipase G50 derived from rhizomucor; wherein the weight ratio of the high-melting-point grease to the water is 1: 4-5; the volume ratio of acetone to n-hexane is 40:60, adding a solvent to the mixture; the total volume of the acetone and the normal hexane is 4-8 times of the weight of the high-melting-point grease; the amount of the lipase Lipozyme RM IM derived from rhizomucor is 2.0-5.0% of the weight of the high-melting-point oil, and the amount of the partial glyceride lipase G50 is 2.0-5.0% of the weight of the high-melting-point oil.
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