WO1990004033A1 - Production of monoglycerides by enzymatic transesterification - Google Patents

Production of monoglycerides by enzymatic transesterification Download PDF

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Publication number
WO1990004033A1
WO1990004033A1 PCT/US1989/004278 US8904278W WO9004033A1 WO 1990004033 A1 WO1990004033 A1 WO 1990004033A1 US 8904278 W US8904278 W US 8904278W WO 9004033 A1 WO9004033 A1 WO 9004033A1
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Prior art keywords
iipase
alcohol
catalyst
monoglycerides
water
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Application number
PCT/US1989/004278
Other languages
French (fr)
Inventor
Aleksey Zaks
Akiva T. Gross
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Enzytech, Inc.
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Publication date
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Publication of WO1990004033A1 publication Critical patent/WO1990004033A1/en

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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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis

Definitions

  • Monoglycerides represent an important class of surfactants which are widely used as additives in the food industry. Being excellent emulsifiers, monoglycerides help to distribute and stabilize droplets of two immiscible liquids in one another, which improves the texture, homogeneity, consistency and overall quality of these products. Useful properties of monoglycerides, such as a high tendency to form complexes with starch, an ability to modify the crystal structure of foods, and significant aerating and stabilizing effects make them indispensable in the production of baked goods, cake mixtures, salad dressings, frozen deserts and other processed foods. Due to their high surface activity, monoglycerides also have various applications in the pharmaceutical and plastics industries.
  • monoglycerides are produced commercially by glycerolysis of fats.
  • the fatty acid groups are transferred from triglycerides to the available hydroxyl groups of the glycerol to give a mixture of mono-, di- and triglycerides.
  • the monoglycerides must then be isolated by molecular distillation, at high vacuum.
  • the major drawback of the chemical process described above are the low product yield and the high cost of molecular distillation. A large fraction of the yield losses is caused by thermal degradation at the high temperatures used during the reaction and purification.
  • Another method of producing monoglycerides is by enzymatic transformations.
  • Several reaction pathways for obtaining fatty acid glycerides can be used: the esterification of glycerol with fatty acid; the glycerolysis of triglycerides; and partial hydrolysis of triglycerides.
  • Japanese Patent No. 118,094 describes the production of mono ⁇ glycerides by a lipase-catalyzed transesterification reaction between the alkyl ester of a fatty acid, in this case methyl oleate, and glycerol.
  • the invention relates to a process for prepar- ing monoglycerides by the lipase-catalyzed trans ⁇ esterification of triglycerides in an alcohol medium.
  • a selected enzyme is added to a solution or an emulsion of triglycer ⁇ ides in alcohol (e.g., ethanol) containing a certain amount of water.
  • alcohol e.g., ethanol
  • a selected Iipase is added to the reaction medium and a suspension is formed, as Upases are insoluble in most organic solvents.
  • the suspension is agitated until the reaction is com ⁇ plete, after which the enzyme is removed, and the monoglyceride products are separated from the reaction mixture.
  • the yield of isolated ⁇ -mono ⁇ glycerides in the present process is about 90%.
  • the present process affords high yields of monoglycerides having a unique structure, namely monoglycerides acylated in the ⁇ -position.
  • traditional chemical methods result in the production of only ⁇ -acylated monoglycerides.
  • the present process has several other advantages, including low by-product formation, mild reaction conditions, easy product separation, and formation of fatty acid esters as a second major product of the reaction.
  • These fatty acid esters are valuable by-products and can be used either directly in the cosmetics industry or as a starting material in the synthesis of various products. such as fatty alcohols, amines, etc.
  • the oper ⁇ ational stability of the Iipase biocatalyst is quite high, and the biocatalyst can be easily reused.
  • the reaction can be carried out at ambient or slightly elevated temperatures.
  • the present process in contrast to prior ones, requires neither prior hydrogenation of highly unsaturated tri ⁇ glycerides, nor high temperature distillation of the product.
  • lipases catalyze the hydrolysis of fats and oils.
  • the following scheme shows the complete hydrolysis of a triglyceride to glycerol and fatty acids:
  • R , R 2 and represent the hydrocarbon backbone chains of fatty acids.
  • lipases can catalyze transesterification reactions between triglycerides and a variety of alcohols.
  • the process of the invention utilizes selected lipases to catalyze the partial transesterification of triglycerides to form monoglycerides in high yields while minimizing the formation of glycerol and diglycerides.
  • the monoglycerides formed are acylated predominantly in the (3-position.
  • Fatty acid esters are a second major product of the reaction.
  • Triglycerides from any source can be used in the present process. Both saturated and unsaturated triglycerides can be used, for example, soybean oil or corn oil may be used as the triglyceride source.
  • Lipases used in the present process should exhibit high operational stability (e.g., can be reused without loss of bioactivity for at least 72 hours) , be active in a near-anhydrous organic medium (e.g., the amount of water is less than 5%) , and efficienty catalyze the transestification reaction between an alcohol and a triglyceride.
  • efficiencyly catalyze means that a yield of about 90% of monoglyceride is obtained when the Iipase is used.
  • Lipases which have been successfully used for the present process are lipases derived from Pseudomonas fluorescens, and porcine pancrease. In the present process, it was found that at least 90% activity of Pseudomonas fluorescens Iipase or porcine pancreatic Iipase was present after continuous use for at least 72 hours.
  • the present reaction is carried out in an alcohol medium.
  • the alcohol serves also as a reactant.
  • Primary or secondary alkyl alcohols can be used, including, for example, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, pent- anol, pentanediol, isopentanol and hexanol. Mixtures of alcohols (e.g., ethanol/butanol) can also be used.
  • Ethanol is a preferred alcohol. When ethanol is used, monoglycerides and ethyl esters of fatty acids are produced in yields of about 90%.
  • the regiospecificity of the Pseudomonas fluorescens Iipase and porcine pancreatic Iipase in the present process is such that up to about 95% of mono ⁇ glycerides are acylated in the ⁇ -position.
  • the presence of a small amount of water in the alcohol accelerates the reaction and affects the distribution of the products.
  • the preferred amount of water in the present process is from about 1 to about 5% by volume of the alcohol.
  • About 3% water by volume is particularly preferred.
  • the reaction rate in an alcohol medium containing about 3% water, when lower alcohols (i.e., 4 carbon atoms or less) are used is at least three times higher than the reaction rate in the presence of 1% water. Lower yields of monoglycerides are obtained when no water is used.
  • the hydrolysis reaction starts to compete with the transesterifi ⁇ cation reaction, resulting in the formation of undesirable free fatty acids.
  • the triglyceride is combined with an alcohol containing a small amount of water.
  • the reaction is started by the addition of Iipase to the reaction mixture.
  • Lipase can be in the form of a dry powder, or immobilized on a support, such as silica or diatomaceous earth, or on a microcarrier, such as polystyrene or dextran beads.
  • the reaction can be carried out in any appropriate reaction vessel, including a tank reactor, a packed column or a membrane bioreactor. If a tank reactor is used, it is necessary to provide sufficient agitation in order to eliminate diffusional limitations. Agitation can be achieved by shaking or stirring; for example, stirring with a magnetic stirrer or an impeller blade, can be used. Agitation speeds should be sufficient to form and maintain the suspension.
  • the temperature of the reaction mixture may range from about 20 ⁇ C to about 60"C. A preferred temperature range is from about 25 ⁇ C to about 45°C.
  • the reaction should be allowed to proceed for a time sufficient to convert most of the triglyceride to monoglyceride. Reaction times can vary from about 2 to about 20 hours depending on the amount of the catalyst. The course of the reaction can be monitored by chromatography (e.g., gas or thin layer chromatography) . After the reaction is complete, Iipase is removed, either by centrifugation or filtration. The reaction products, ethyl esters of the fatty acids, free fatty acids and mono ⁇ glycerides, are then separated. Separation can be 5 accomplished by crystallization, membrane filtration or chromatography. The yield of -monoglycerides using this process can be up to about 90%.
  • Additives can, optionally, be added to the enzyme preparation.
  • Calcium ions for example, can Q be used to improve the stability and activity of the enzyme.
  • the invention is further illustrated by the following exemplification:
  • porcine pancreatic Iipase from Sigma Chemical Co. (St. Louis, MO) and Pseudomonas fluorescens from Amano International Enzyme Co. (Troy, VA) .
  • the porcine pancreatic Iipase had a specific activity of 110 IU/ g solids and Pseudo ⁇ monas fluorescens Iipase had a specific activity of 30 IU/mg solids.
  • Monoolein, diolein, triolein, soybean and corn oil were also purchased from Sigma. All solvents used in this work were of analytical grade and were obtained from Aldrich Chemical Co. (Milwaukee, WI) .
  • the activity of the Iipase in the hydrolysis reaction was determined potentiometrically (Radio- meter RTS-812 recording pH-stat system) using either tributyrin or corn oil as substrates.
  • 10 mL of an 0.1 g/mL aqueous solution of a substrate was placed in the cuvette of a pH-stat, and the pH was adjusted to 7.0.
  • a Iipase sample was then added, and the acid which was liberated as a result of enzymatic hydrolysis was automatically titrated with 0.5 M NaOH. All products of enzymatic conversions were assayed by gas chromatography (GC) using 12-m fused silica capillary column (S.G.E. Australia) .
  • GC gas chromatography
  • Nitro ⁇ gen was used as a carrier gas (5 mL/min) , and the detector and injector port temperatures were 350°C.
  • the starting temperature of the column was 100 " C, and after the injection it increased to 350"C at 20 ⁇ C/min.
  • the retention times were 7.35 minutes for monoolein; 11.3 minutes for diolein, and 19.8 minutes for triolein.
  • the reaction mixture was silylated with hexamethyldisilazane following the standard procedure described by Sweeley et al. in J. Am. Chem. Soc, 85:2495-2507 (1963).
  • Enzymatically prepared monoglycerides were separated either by flash silica gel chromatography or by crystallization.
  • the solvent was evaporated under reduced pressure, and 5 g of the reaction products were applied on a column (diameter: 2.5 inches; length: 2.0 inches) packed with silica gel and equilibrated with a petroleum ether:ether mixture in a ratio of 9:1.
  • the byproducts were eluted with the above mixture at a flow rate of about 70 ml/min.
  • Mono ⁇ glycerides were eluted in the same manner using anhydrous diethyl ether as elutant.
  • the acidity of silica gel stimulates the migration of the acyl moiety from the ⁇ -position to the more stable -position of monoglyceride. Consequently, chromatographic separation results in the formation of a mixture of monoglycerides acyl- ated in the ⁇ or ⁇ position. If exclusively ⁇ -monoglycerides are required, the products should be separated by crystallization using the following procedure: After the completion of the reaction (e.g., transesterification between triolein and ethanol) , the enzyme was separated by centrifugation and the solvent evaporated under reduced pressure.
  • the reaction e.g., transesterification between triolein and ethanol
  • the resultant oily liquid (5 g) containing mono ⁇ glycerides, free fatty acids and their alkyl esters was dissolved in 30 ml hexane at room temperature. The solution was cooled to -18°C and left at this temperature for 1 hour. White crystals formed, were separated by filtration and washed with hexane (at -18°C) . (3-Monoacylated glycerol of 97% purity was obtained using this method.
  • Triolein (4.5 g) was placed into a round-bottom flask containing 45 mL of 97% (v/v) ethanol (3% water) .
  • One g of Pseudomonas fluorescens Iipase in the form of a dry powder was added to the flask.
  • the formed suspension was agitated on an orbit shaker at 400 rpm at a temperature of 20"C.
  • the course of the reaction was monitored by GC, following the disappearance of the triolein and the appearance of the products (i.e., monoolein and ethyl ester of oleic acid) . After 20 hours, no starting material was observed in the reaction mixture.
  • the major products of the reaction were monoolein and ethyl ester of oleic acid.
  • the reaction was stopped by removing the enzyme, which was done by filtering the reaction mixture through a sintered glass filter.
  • the solvent was then evaporated under vacuum using a rotary evaporator, and the monoglyceride product was purified on a silica gel column. As a result, 1.5 g of monoolein was produced.
  • the purity of the product was at least 95%, as determined by GC and TLC.
  • Example 4 The procedure described in Example 1 was followed, except that 97% (v/v) butyl alcohol (3% water) substituted for 97% ethanol. As a result, 1.5 g of 95% pure monoolein were produced.
  • Example 4 The procedure described in Example 1 was followed, except that 97% (v/v) butyl alcohol (3% water) substituted for 97% ethanol. As a result, 1.5 g of 95% pure monoolein were produced.
  • tripalmitin Five grams of tripalmitin were placed in a flask containing 50 ml of 97% (v/v) butanol (3% water) .
  • One gram of Pseudomonas fluorescens Iipase was added, and the suspension was stirred on an orbit shaker at 400 rpm at 45°C for 30 hr.
  • the product was purified by crystallization from hexane. As a result, 1.2 g of monopalmitate (97% purity) were obtained.

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Abstract

A process for the production of high purity monoglycerides by lipase-catalyzed transesterification, and the products of the reaction, are described. In the method of the present invention, oils or pure triglycerides are combined with alcohol, a small amount of water and a lipase. The reaction proceeds under mild conditions, and produces high yields of β-monoglyceride product.

Description

PRODUCTION OF MONOGLYCERIDES BY ENZYMATIC TRANSESTERIFICATION
Background
Monoglycerides represent an important class of surfactants which are widely used as additives in the food industry. Being excellent emulsifiers, monoglycerides help to distribute and stabilize droplets of two immiscible liquids in one another, which improves the texture, homogeneity, consistency and overall quality of these products. Useful properties of monoglycerides, such as a high tendency to form complexes with starch, an ability to modify the crystal structure of foods, and significant aerating and stabilizing effects make them indispensable in the production of baked goods, cake mixtures, salad dressings, frozen deserts and other processed foods. Due to their high surface activity, monoglycerides also have various applications in the pharmaceutical and plastics industries.
Currently, monoglycerides are produced commercially by glycerolysis of fats. In this process, the fatty acid groups are transferred from triglycerides to the available hydroxyl groups of the glycerol to give a mixture of mono-, di- and triglycerides. The monoglycerides must then be isolated by molecular distillation, at high vacuum. The major drawback of the chemical process described above are the low product yield and the high cost of molecular distillation. A large fraction of the yield losses is caused by thermal degradation at the high temperatures used during the reaction and purification.
Another method of producing monoglycerides is by enzymatic transformations. Several reaction pathways for obtaining fatty acid glycerides can be used: the esterification of glycerol with fatty acid; the glycerolysis of triglycerides; and partial hydrolysis of triglycerides. I.L. Gatfield in Ann. N.Y. Acad. Sci., 434:569-72 (1984). Japanese Patent No. 118,094 describes the production of mono¬ glycerides by a lipase-catalyzed transesterification reaction between the alkyl ester of a fatty acid, in this case methyl oleate, and glycerol. The prep- aration of various types of glyceride esters using Iipase is described by G. Lazar in Fette Seifen, Anstrichm., 87(10) :394-400 (1985). The lipase- catalyzed synthesis of glycerides from free fatty acids and glycerol is described by M.K. Tahoun et al., Microbios. Letts. , 28(111-112) :133-139 (1985); M.M. Hoq et al., Agric. Biol. Che . , 49(2) :335-42 (1985); T. Yamane et al., Ann. N.Y. Acad. Sci., 434:558-568 (1984); M. Pina and J. Graille, Bull. Tech./Gattefosse Rep., £:34-36 (1984); M.M. Hoq et al., J. Am. Oil Chem. Soc. , 61(4) :776-781 (1984); N. Muthukumaran and S.C. Dhar, Leather Sci. , 30(3) :97- 100 (1983); Y. Tsujisaka et al., Biochem. Biophys. Acta, 489(3) :415-522 (1977); and R. Bacaloglu et al., Rev. Roum. Biochem. , 22(3) :177-181 (1985). There are several limitations to the previous processes, including, the need for an excess of glycerol, a low degree of conversion, and complex purification procedures.
Summary of the Invention
The invention relates to a process for prepar- ing monoglycerides by the lipase-catalyzed trans¬ esterification of triglycerides in an alcohol medium. In the present process, a selected enzyme is added to a solution or an emulsion of triglycer¬ ides in alcohol (e.g., ethanol) containing a certain amount of water. A selected Iipase is added to the reaction medium and a suspension is formed, as Upases are insoluble in most organic solvents. The suspension is agitated until the reaction is com¬ plete, after which the enzyme is removed, and the monoglyceride products are separated from the reaction mixture. The yield of isolated β-mono¬ glycerides in the present process is about 90%. The present process affords high yields of monoglycerides having a unique structure, namely monoglycerides acylated in the β -position. On the contrary, traditional chemical methods result in the production of only α-acylated monoglycerides.
The present process has several other advantages, including low by-product formation, mild reaction conditions, easy product separation, and formation of fatty acid esters as a second major product of the reaction. These fatty acid esters are valuable by-products and can be used either directly in the cosmetics industry or as a starting material in the synthesis of various products. such as fatty alcohols, amines, etc. The oper¬ ational stability of the Iipase biocatalyst is quite high, and the biocatalyst can be easily reused. The reaction can be carried out at ambient or slightly elevated temperatures. In addition, the present process, in contrast to prior ones, requires neither prior hydrogenation of highly unsaturated tri¬ glycerides, nor high temperature distillation of the product.
Detailed Description of the Invention
In nature, lipases catalyze the hydrolysis of fats and oils. The following scheme shows the complete hydrolysis of a triglyceride to glycerol and fatty acids:
Figure imgf000006_0001
wherein R , R2 and represent the hydrocarbon backbone chains of fatty acids. In addition to the hydrolysis reaction, lipases can catalyze transesterification reactions between triglycerides and a variety of alcohols.
The process of the invention utilizes selected lipases to catalyze the partial transesterification of triglycerides to form monoglycerides in high yields while minimizing the formation of glycerol and diglycerides. The monoglycerides formed are acylated predominantly in the (3-position. Fatty acid esters are a second major product of the reaction.
Triglycerides from any source can be used in the present process. Both saturated and unsaturated triglycerides can be used, for example, soybean oil or corn oil may be used as the triglyceride source.
Different lipases obtained from a variety of sources, including mammals, yeast, mold and bacteria can be employed as catalysts in the present process. Lipases used in the present process should exhibit high operational stability (e.g., can be reused without loss of bioactivity for at least 72 hours) , be active in a near-anhydrous organic medium (e.g., the amount of water is less than 5%) , and efficienty catalyze the transestification reaction between an alcohol and a triglyceride. The term "efficiently catalyze" means that a yield of about 90% of monoglyceride is obtained when the Iipase is used. Lipases which have been successfully used for the present process are lipases derived from Pseudomonas fluorescens, and porcine pancrease. In the present process, it was found that at least 90% activity of Pseudomonas fluorescens Iipase or porcine pancreatic Iipase was present after continuous use for at least 72 hours.
The present reaction is carried out in an alcohol medium. The alcohol serves also as a reactant. Primary or secondary alkyl alcohols can be used, including, for example, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, pent- anol, pentanediol, isopentanol and hexanol. Mixtures of alcohols (e.g., ethanol/butanol) can also be used. Ethanol is a preferred alcohol. When ethanol is used, monoglycerides and ethyl esters of fatty acids are produced in yields of about 90%. The regiospecificity of the Pseudomonas fluorescens Iipase and porcine pancreatic Iipase in the present process is such that up to about 95% of mono¬ glycerides are acylated in the β-position.
The presence of a small amount of water in the alcohol accelerates the reaction and affects the distribution of the products. The preferred amount of water in the present process is from about 1 to about 5% by volume of the alcohol. About 3% water by volume is particularly preferred. For example, the reaction rate in an alcohol medium containing about 3% water, when lower alcohols (i.e., 4 carbon atoms or less) are used, is at least three times higher than the reaction rate in the presence of 1% water. Lower yields of monoglycerides are obtained when no water is used. At higher water concen¬ trations (e.g., > 5% by volume), the hydrolysis reaction starts to compete with the transesterifi¬ cation reaction, resulting in the formation of undesirable free fatty acids. In the present process, the triglyceride is combined with an alcohol containing a small amount of water. The reaction is started by the addition of Iipase to the reaction mixture. Lipase can be in the form of a dry powder, or immobilized on a support, such as silica or diatomaceous earth, or on a microcarrier, such as polystyrene or dextran beads. The reaction can be carried out in any appropriate reaction vessel, including a tank reactor, a packed column or a membrane bioreactor. If a tank reactor is used, it is necessary to provide sufficient agitation in order to eliminate diffusional limitations. Agitation can be achieved by shaking or stirring; for example, stirring with a magnetic stirrer or an impeller blade, can be used. Agitation speeds should be sufficient to form and maintain the suspension.
The temperature of the reaction mixture may range from about 20βC to about 60"C. A preferred temperature range is from about 25βC to about 45°C. The reaction should be allowed to proceed for a time sufficient to convert most of the triglyceride to monoglyceride. Reaction times can vary from about 2 to about 20 hours depending on the amount of the catalyst. The course of the reaction can be monitored by chromatography (e.g., gas or thin layer chromatography) . After the reaction is complete, Iipase is removed, either by centrifugation or filtration. The reaction products, ethyl esters of the fatty acids, free fatty acids and mono¬ glycerides, are then separated. Separation can be 5 accomplished by crystallization, membrane filtration or chromatography. The yield of -monoglycerides using this process can be up to about 90%.
Additives can, optionally, be added to the enzyme preparation. Calcium ions, for example, can Q be used to improve the stability and activity of the enzyme. The invention is further illustrated by the following exemplification:
EXEMPLIFICATION
Materials Lipases (EC 3.1.1.3) were obtained from the following suppliers: porcine pancreatic Iipase from Sigma Chemical Co. (St. Louis, MO) and Pseudomonas fluorescens from Amano International Enzyme Co. (Troy, VA) . The porcine pancreatic Iipase had a specific activity of 110 IU/ g solids and Pseudo¬ monas fluorescens Iipase had a specific activity of 30 IU/mg solids. Monoolein, diolein, triolein, soybean and corn oil were also purchased from Sigma. All solvents used in this work were of analytical grade and were obtained from Aldrich Chemical Co. (Milwaukee, WI) .
Methods
The activity of the Iipase in the hydrolysis reaction was determined potentiometrically (Radio- meter RTS-812 recording pH-stat system) using either tributyrin or corn oil as substrates. In this process, 10 mL of an 0.1 g/mL aqueous solution of a substrate was placed in the cuvette of a pH-stat, and the pH was adjusted to 7.0. A Iipase sample was then added, and the acid which was liberated as a result of enzymatic hydrolysis was automatically titrated with 0.5 M NaOH. All products of enzymatic conversions were assayed by gas chromatography (GC) using 12-m fused silica capillary column (S.G.E. Australia) . Nitro¬ gen was used as a carrier gas (5 mL/min) , and the detector and injector port temperatures were 350°C. The starting temperature of the column was 100 " C, and after the injection it increased to 350"C at 20βC/min. The retention times were 7.35 minutes for monoolein; 11.3 minutes for diolein, and 19.8 minutes for triolein. For precise quantitative analysis, prior to the injection the reaction mixture was silylated with hexamethyldisilazane following the standard procedure described by Sweeley et al. in J. Am. Chem. Soc, 85:2495-2507 (1963).
In addition to GC, the course of the reactions and the purity of all products were follwed by thin-layer chromatography (TLC) using Whatman K6 silica gel sheets. A mixture of petroleum ether (t>.p. 30-60βC), ether and acetic acid in a ratio of 90:10:1 was used as an eluting buffer. The spots were developed by spraying with 50% H,S04, followed by 10 minutes of heating at 180βC.
Enzymatically prepared monoglycerides were separated either by flash silica gel chromatography or by crystallization. For flash silica gel chroma¬ tography, the solvent was evaporated under reduced pressure, and 5 g of the reaction products were applied on a column (diameter: 2.5 inches; length: 2.0 inches) packed with silica gel and equilibrated with a petroleum ether:ether mixture in a ratio of 9:1. The byproducts were eluted with the above mixture at a flow rate of about 70 ml/min. Mono¬ glycerides were eluted in the same manner using anhydrous diethyl ether as elutant. The acidity of silica gel stimulates the migration of the acyl moiety from the β-position to the more stable -position of monoglyceride. Consequently, chromatographic separation results in the formation of a mixture of monoglycerides acyl- ated in the α or β position. If exclusively β-monoglycerides are required, the products should be separated by crystallization using the following procedure: After the completion of the reaction (e.g., transesterification between triolein and ethanol) , the enzyme was separated by centrifugation and the solvent evaporated under reduced pressure. The resultant oily liquid (5 g) containing mono¬ glycerides, free fatty acids and their alkyl esters was dissolved in 30 ml hexane at room temperature. The solution was cooled to -18°C and left at this temperature for 1 hour. White crystals formed, were separated by filtration and washed with hexane (at -18°C) . (3-Monoacylated glycerol of 97% purity was obtained using this method.
Example 1
Triolein (4.5 g) was placed into a round-bottom flask containing 45 mL of 97% (v/v) ethanol (3% water) . One g of Pseudomonas fluorescens Iipase in the form of a dry powder was added to the flask. The formed suspension was agitated on an orbit shaker at 400 rpm at a temperature of 20"C. The course of the reaction was monitored by GC, following the disappearance of the triolein and the appearance of the products (i.e., monoolein and ethyl ester of oleic acid) . After 20 hours, no starting material was observed in the reaction mixture. As determined by GC and TLC, the major products of the reaction were monoolein and ethyl ester of oleic acid. The reaction was stopped by removing the enzyme, which was done by filtering the reaction mixture through a sintered glass filter. The solvent was then evaporated under vacuum using a rotary evaporator, and the monoglyceride product was purified on a silica gel column. As a result, 1.5 g of monoolein was produced. The purity of the product was at least 95%, as determined by GC and TLC.
Example 2
Various oils can also be used as starting material for the production of monoglycerides. The procedure described in Example 1 was followed, except that 4.5 g of soybean oil was substituted for triolein. As a result, 1.4 g of 95% pure monoglycerides were produced.
Example 3
The procedure described in Example 1 was followed, except that 97% (v/v) butyl alcohol (3% water) substituted for 97% ethanol. As a result, 1.5 g of 95% pure monoolein were produced. Example 4
Five grams of tripalmitin were placed in a flask containing 50 ml of 97% (v/v) butanol (3% water) . One gram of Pseudomonas fluorescens Iipase was added, and the suspension was stirred on an orbit shaker at 400 rpm at 45°C for 30 hr. The product was purified by crystallization from hexane. As a result, 1.2 g of monopalmitate (97% purity) were obtained.
Equivalents
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the spec¬ ific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

-13-CLAIMS
1. A method of preparing monoglycerides comprising combining an alcohol, a triglyceride, water, and a selected Iipase catalyst under conditions sufficient for transesterification to occur between the hydroxyl groups of the alcohol and the fatty acid groups of the triglycerides.
2. A method of Claim 1 wherein the alcohol is a primary or secondary alcohol selected from the group consisting of methanol, ethanol, prop- anol, isopropanol, butanol, isobutanol, pent- anol, pentanediol, isopentanol, hexanol, and mixtures thereof.
3. A method of Claim 1 wherein the selected Iipase catalyst is derived from Pseudomonas fluor¬ escens.
4. A method of Claim 1 wherein the selected Iipase catalyst is porcine pancreatic Iipase.
5. A method of Claim 1 wherein the Iipase catalyst is immobilized on a carrier.
6. A method of Claim 1 wherein the amount of water is from about 1 to about 5 percent, by volume.
7. A method of preparing β-monoglycerides compris¬ ing the steps of: a. combining a primary or secondary alkyl alcohol, triglyceride and water; b. adding a selected Iipase catalyst to the combination formed in (a) ; and c. maintaining the combination obtained in (b) under conditions sufficient for transesterification between the hydroxyl groups of the alcohol and the fatty acid groups of the triglyceride to occur.
8. A method of Claim 7 wherein the alcohol is selected from the group consisting of: meth- anol, ethanol, propanol, isopropanol, butanol, isobutanol, pentanol, pentanediol, isopentanol, hexanol, and mixtures thereof.
9. A method of Claim 7 wherein the selected Iipase catalyst is derived from Pseudomonas fluorescens.
10. A method of Claim 7 wherein the selected Iipase catalyst is porcine pancreatic Iipase.
11- A method of Claim 10 wherein the Iipase is immobilized on a carrier.
12. A method of Claim 11 wherein the carrier com¬ prises diatomaceous earth, silica or poly¬ styrene beads. -15-
13. A method of Claim 7 wherein the amount of water is from about 1 to about 5 percent, by volume.
14. In a method of preparing monoglycerides by lipase-catalyzed transesterification the improvement comprising adding Iipase to a mixture containing a triglyceride, alcohol and water, and maintaining the mixture under conditions appropriate for transesterification to occur between the alcohol and the triglyceride.
PCT/US1989/004278 1988-10-04 1989-09-29 Production of monoglycerides by enzymatic transesterification WO1990004033A1 (en)

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EP0492497A2 (en) * 1990-12-24 1992-07-01 Hoechst Aktiengesellschaft Process for acylating alcohols with immobilized pseudomonaslipase
EP0528813A1 (en) * 1990-04-19 1993-03-03 The Procter & Gamble Company Regio and stereoselective synthesis of triglycerides
FR2772391A1 (en) * 1997-12-15 1999-06-18 Toulousaine De Rech Et De Dev Lipase=catalysed alcoholysis of sunflower oil
FR2772392A1 (en) * 1997-12-15 1999-06-18 Toulousaine De Rech Et De Dev Production of alkyl oleate from vegetable oil
CN1079115C (en) * 1993-09-17 2002-02-13 日清制油株式会社 Transesterification method with powdery lipase
KR20020048464A (en) * 2000-12-16 2002-06-24 정대원 Method for selective preparation of monoglyceride by enzymatic process
WO2006077022A2 (en) * 2005-01-19 2006-07-27 Cognis Ip Management Gmbh Production and use of monoglycerides from triglycerides by alcoholising using thermomyces lanuginosus lipase which is activated by alkaline salts
EP1842908A1 (en) * 2005-01-19 2007-10-10 The Nisshin Oillio Group, Ltd. Process for production of purified lipase
WO2008032007A1 (en) 2006-09-14 2008-03-20 Tmo Renewables Ltd Lipase
EP2085469A1 (en) 2008-01-30 2009-08-05 BAM Bundesanstalt für Materialforschung und -prüfung Lipase formulation
US7799544B2 (en) 2005-01-19 2010-09-21 Cognis Ip Management Gmbh Compositions which can be used as biofuel
WO2013114178A1 (en) 2012-01-30 2013-08-08 Arvind Mallinath Lali Enzymatic process for fat and oil hydrolysis

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US5935828A (en) * 1989-05-01 1999-08-10 Opta Food Ingredients, Inc. Enzymatic production of monoglycerides containing omega-3 unsaturated fatty acids
KR20180134904A (en) * 2016-04-27 2018-12-19 가부시키가이샤 아데카 Flavor improver

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EP0528813A1 (en) * 1990-04-19 1993-03-03 The Procter & Gamble Company Regio and stereoselective synthesis of triglycerides
EP0528813A4 (en) * 1990-04-19 1993-07-21 The Procter & Gamble Company Regio and stereoselective synthesis of triglycerides
EP0492497A2 (en) * 1990-12-24 1992-07-01 Hoechst Aktiengesellschaft Process for acylating alcohols with immobilized pseudomonaslipase
EP0492497A3 (en) * 1990-12-24 1993-10-20 Hoechst Ag Process for acylating alcohols with immobilized pseudomonoslipase
CN1079115C (en) * 1993-09-17 2002-02-13 日清制油株式会社 Transesterification method with powdery lipase
FR2772391A1 (en) * 1997-12-15 1999-06-18 Toulousaine De Rech Et De Dev Lipase=catalysed alcoholysis of sunflower oil
FR2772392A1 (en) * 1997-12-15 1999-06-18 Toulousaine De Rech Et De Dev Production of alkyl oleate from vegetable oil
KR20020048464A (en) * 2000-12-16 2002-06-24 정대원 Method for selective preparation of monoglyceride by enzymatic process
EP1842908A1 (en) * 2005-01-19 2007-10-10 The Nisshin Oillio Group, Ltd. Process for production of purified lipase
WO2006077022A3 (en) * 2005-01-19 2007-01-11 Cognis Deutschland Gmbh Production and use of monoglycerides from triglycerides by alcoholising using thermomyces lanuginosus lipase which is activated by alkaline salts
WO2006077022A2 (en) * 2005-01-19 2006-07-27 Cognis Ip Management Gmbh Production and use of monoglycerides from triglycerides by alcoholising using thermomyces lanuginosus lipase which is activated by alkaline salts
EP1842908A4 (en) * 2005-01-19 2008-10-01 Nisshin Oillio Group Ltd Process for production of purified lipase
EP2186886A1 (en) * 2005-01-19 2010-05-19 The Nisshin Oillio Group, Ltd. Process for production of purified lipase
US7799544B2 (en) 2005-01-19 2010-09-21 Cognis Ip Management Gmbh Compositions which can be used as biofuel
US7935508B2 (en) 2005-01-19 2011-05-03 Cognis Ip Management Gmbh Production and use of monoglycerides
WO2008032007A1 (en) 2006-09-14 2008-03-20 Tmo Renewables Ltd Lipase
EP2085469A1 (en) 2008-01-30 2009-08-05 BAM Bundesanstalt für Materialforschung und -prüfung Lipase formulation
DE102008006716A1 (en) 2008-01-30 2009-08-13 BAM Bundesanstalt für Materialforschung und -prüfung Lipasenformulierung
WO2013114178A1 (en) 2012-01-30 2013-08-08 Arvind Mallinath Lali Enzymatic process for fat and oil hydrolysis
US9512451B2 (en) 2012-01-30 2016-12-06 Institute Of Chemical Technology (Deemed University) Enzymatic process for fat and oil hydrolysis

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