Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
CH2 - OOCR3 CH2 OH R3 - 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.
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~0(!143
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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
.
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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.
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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.
-,
., '- ::
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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
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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
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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
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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.
