CA2000143A1 - Production of monoglycerides by enzymatic transesterification - Google Patents

Abstract

PRODUCTION OF MONOGLYCERIDES BY
ENZYMATIC TRANSESTERIFICATION

Abstract of the Disclosure A process for the production of high purity monoglycerides by lipase-catalyzed transesterifica-tion, and the products of the reaction, are des-cribed. 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 .beta.-monoglyceride product.

Description

;200~ 3 PRODUCTION OF MONOGLYCERIDES BY
ENZYMATIC TRANSESTERIFICATION

Background Monoglycerides represent an important class of 05 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 10 and overall quality of these products. Useful properties of monoglycerides, such as a high tendency to form aomplexes with starch, an ability to modify the crystal structure of foods, and significant aerating and stabilizing effects make 15 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 20 indus~ries.
Currently, monoglycsr~de~ are produced commercially by glyaerolysis of fats. In this process, the fatty acid groups are transferred from triglycerides to the available hydroxyl groups of 25 the glycerol to give a mixture of mono-, di- and triglycerides. ~he monoglycerides must then be isolated by molecular distillation, at high vacuum.
The ma;or drawback of the ahemical process described above are the low product yield and the high cost of 30 molecular distillation. A large fraction of the .

)0~3 yield losses is caused by thermal degradation at the high temperatures used during the reaction and purification.
Another method of producing monoglycerides is 05 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.
10 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-15 aration of various types of glyceride esters usinglipase is described by G. Lazar in Fette Seifen, Anstrichm., 87(10):394-400 (1985). The lipase-catalyzed synthesis of glycerid0s from free fatty acids and glycerol is described by M.K. Tahoun et 20 al., Microbios. Letts., 28(111-112~:133~139 (1985);
M.M. Hoq et al., Agric. Biol. Chem., 49(2):335-42 (1985): T. Yamane et al., Ann. N.Y. Acad~ Sci., 434:558-568 (1984)5 M. Pina and J. Graille, Bull.
Tech~/Gattefosse Rep., 76:34-36 ~1984)~ M.M. Hoq et 25 al., J. Am. Oil Chem. Soc., 61(4):776-781 (1984); N.
Muthukumaran and S.C. Dhar, Leather Sci., ~ L:97-lOO (1983); Y. Tsujisaka et al., aiochem. Biophvs.
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 , : ;' - "

~00(~ 3 glycerol, a low degree of conversion, and complex purification procedures.

Summary of the Invention The inventio~ relates to a process for prepar-05 ing monoglycerides by the lipase-catalyzed trans-esterification of triglycerides in an alcohol medium. In the present process, a selected en~yme is added to a solution or an emulsion of triglycer-ides in alcohol (e.g., ethanol) containing a certain 10 amount of water. A selected lipase is added to the reaction medium and a suspension is formed, as lipa~e~ are insoluble in most organic solvents. The suspension is agitated until the reaction is com-plete, after which the enzyme is removed, and the 15 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 monoglycerid~s having a unique structure, namely 20 monoglycerides acylated in the ~-position. On the contrary, traditional chemical methods result in the production of only a-acylated monoglycerides.
The present process has several other advantages, including low by-product formation, mild 25 reaation conditions, easy product separation, and formation of fatty acid esters as a second ma~or 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 30 material in the synthesis of various products, ~(~OQ~3 such as fatty alcohols, amines, etc. The oper-ational stability of the lipase biocatalyst is quite high, and the biocatalyst can be easily reused. The reaction can be carried out at ambient or slightly 05 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.

10 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:

15 CH2 - OOCR1 CH2 OH Rl - COOH
CH - OOCR2 + 3H20-~ CH - OH ~ R2 ~ COOH

wherein Rl, R2 and R3 represent the hydrocarbon backbone chains of fatty acids. In addition to the 20 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 25 O~ triglycerides to form monoglycerides in high yields while minimizing the formation of glycerol and diglycerides. The monoglycerides formed are acylated predominantly in the ~-position. Fatty Z000~43 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 05 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 ean be employed as eatalysts in the present proeess.
10 Lipases used in the present proeess should exhibit high operational stability (e.g., can be reused without loss o~ bioactivity for at least 7~ hours), be active in a near-anhydrous organic medium (e.g., the amount of water is less than 5%), and efficienty lS eatalyze the transestification reaetion between an alcohol and a triglyceride. The term "ef~ieiently eatalyze" means that a yield of about 90% of monoglyeeride is obtained when the lipase is used.
Lipases which have been successfully used for the 20 present proeess are lipases derived from Pseudomonas fluoreueen~, and porcine pancrease. In the present proeess, it was found that at least ~0~ aetivity of Pseudomonas fluorescens lipase or poreine panereatic lipase was present after continuous use for at least 25 72 hours.~
The present reaction is earried out in an aleohol medium. The aleohol serves also as a reaetant. Primary or seeondary alkyl aleohols ean be used, ineluding, for example, methanol, ethanol, 30 propanol, isopropanol, butanol, isobutanol, pent-anol, pentanediol, isopentanol and hexanol.

'.

~0(!143 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%.
05 The regiospecificity of the Pseudomonas fluorescens lipase and porcine pancreatic lipase 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 lO 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, 15 the reac~ion 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 20 when no water is used. At higher water concen-trations (e.g., ~ 5% by ~olume), the hydrolysis reaction ~tarts 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 lipase to the reaction mixture. Lipase can be in the form of a dry powder, or immobilized on a 30 support, such as silica or diatomaceous earth, or on a microcarrier, such as polystyrene or dextran .
.

;~001~3 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 05 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 10 maintain the suspension.
The temperature of the reaction mixture may range from about 20C to about 60C. A preferred temperature range is from about 25C to about 45C.
The reaction should be allowed to proceed for a 15 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 ~f the catalyst. The course of the reaction can be monitored by chromatography (e.g., gas or thin layer 20 chromatography). After the reaction is complete, lipase is removed, either by centrifugation or filtration. The reaction products, ethyl esters of the fatty acids, ~ree Æatty acids and mono-glycerides, are then separated. Separation can be 25 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 30 be used to improve the stability and activity of the enzyme.

2no~)l43 The invention is further illustrated by the following exemplification:

EXEMPLIFICATION

Materials 05 Lipases (BC 3.1.1.3) were obtained from the following suppliers: porcine pancreatic lipase from Sigma Chemical Co. (St. Louis, MO) and Pseudomonas fluorescens from Amano International Enzyme Co.
(Troy, VA). The porcine pancreatic lipase had a 10 specific activity of llO IU/mg solids and P eudo-monas fluorescens lipase 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 15 grade and were obtained from Aldrich Chemical Co.
(Milwaukee, WI).

Methods ~ he activity of the lipase in the hydrolysis reaction was determined potentiometrically (Radio-20 meter RTS-812 recording pH-stat system) using either tributyrin or corn oil as substrates. In this process, lO mL of an O.l g/mL aqueous solution of a suhstrate was placed in the cuvette of a pH-stat, and the pH was adjusted to 7Ø A lipase sample was 25 then added, and the acid which was libarated as a result of enzymatic hydrolysis was automatically titrated with 0.5 M NaOH.

-, ., '- ::

- 2no~l43 All products of enzymatic conversions were assayed by gas chromatography tGC) using 12-m fused silica capillary column (S.G.E. Australia). Nitro-gen was used as a carrier gas (5 mL/min), and the 05 detector and injector port temperatures were 350C.
The starting temperature of the column was 100C, and after the injection it increased to 350C at 20CC/min. The retention times were 7.35 minutes for monoolein; 11.3 minutes for diolein, and 19.8 10 minutes for triolein. For precise quantitative analysis, prior to the injeation the reaction mixture was silylated with hexamethyldisilazane following the standard procedure described by Sweeley et al. in J. Am. Chem._Soc., 85:2495-2507 15 (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 20 (b.p. 30-60C), ether and acetic acid in a ratio of 90:10:1 was used as an eluting buf~er. The spots were developed by spraying with 50% H2S04, followed by 10 minutes of heating at 180~C.
Enzymatically prepared monoglycerides were 25 separated either by flash silica gel chromatography or by crystallization. For flash silica gel ahroma-tography, the solvent was evaporated under reduced pres~ure, and 5 g of the reaction products were applied on a column (diameter: 2.5 inches; length:
30 2.0 inches) packed with silica gel and equilibrated with a petroleum ether:ether mixture in a ratio of `- ~OU(~`~43 9:1. The byproducts were eluted with the above mixt:ure at a flow rate of about 70 ml/min. Mono-glyc:erides were eluted in the same manner using anhydrous diethyl ether as elutant.
05 The acidity of silica gel stimulates the migration of the acyl moiety from the ~-position to the more stable a-position of monoglyceride.
Consequently, chromatographic separation results in the formation of a mixture of monoglycerides acyl-10 ated in the a or ~position. If exclusively ~-monoglyoeride~ 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 15 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.
20 The solution was cooled to -18C and left at this temperature for 1 hour. White crystals formed, were separated by filtration and washed with hexane (at -18C). ~-Monoacylated glycerol of 97% purity was obtained using this method.

25 Exam~le 1 Triolein (4.5 g) was plaaed into a round-bottom flas~ containing 45 mL of 97% (v/v) ethanol (3%
water). One g of Pseudomonas fluorescens lipase in the form of a dry powder was added to the flas~.
30 The formed suspension was agitated on an orbit ;~UUi~3 shaker at 400 rpm at a temperature of 20C. 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 05 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 10 removing the enzyme, which was done by filtering the reaction mixture through a sintered glass filter.
~he 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 15 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 20 material for the production of monoglycerides. The procedure described in Example 1 was followed, except that 4.5 g of soybean oil was substituted ~or triolein. A~ a result, 1.4 g of 95% pure monoglycerides were produced.

25 Example 3 The prooedure desoribed in Example l was ~ollowed, except that 97~ ~v/v) butyl alcohol (3%
water) substituted for 97~ ethanol. As a result, 1.5 g oP 95~ pure monoolein were produced.

t~43 ExamPle 4 Five grams of tripalmitin were placed in a flask containing 50 ml of 97% Sv/v) butanol (3%
water). One gram of Pseudomonas fluorescens lipase 05 was added, and the suspension was stirred on an orbit shaker at 400 rpm at 45C for 30 hr. The prvduct was purified by crystallization from hexane.
As a result, 1.2 g of monopalmitate (97% purity) were obtained.

10 Equivalents Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimantation, numerous equivalents to the spec-ific substances and procedures described herein.
15 Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims (14)

1. A method of preparing monoglycerides comprising combining an alcohol, a triglyceride, water, and a selected lipase 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 lipase catalyst is derived from Pseudomonas fluor-escens.

4. A method of Claim 1 wherein the selected lipase catalyst is porcine pancreatic lipase.

5. A method of Claim 1 wherein the lipase 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 .beta.-monoglycerides compris-ing the steps of:

a. combining a primary or secondary alkyl alcohol, triglyceride and water;
b. adding a selected lipase 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 lipase catalyst is derived from Pseudomonas fluorescens.

10. A method of Claim 7 wherein the selected lipase catalyst is porcine pancreatic lipase.

11. A method of Claim 10 wherein the lipase is immobilized on a carrier.

12. A method of Claim 11 wherein the carrier com-prises diatomaceous earth, silica or poly-styrene beads.

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 lipase 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.