Optimization of the reaction conditions in the lipase-catalyzed synthesis of structured triglycerides

Article abstract of DOI:10.1007/s11746-998-0089-5

Structured triglycerides of the ABA-type, containing one type of fatty acid (A) in the sn-1 and sn-3 positions and a second type of fatty acid (B) in the sn-2 position of the glycerol, were synthesized using lipases. The highest yields and purities were achieved in a two-step process, where a triglyceride of the B-type was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn-1,3-regiospecific lipases yielding the corresponding 2-monoglyceride (2-MG). Using this strategy, e.g., 2-monopalmitin (2-MP) was obtained in up to 88% yield at >95% purity by crystallization. Esterification of 2-MP with oleic acid resulted in the formation of 1,3-oleyl-2-palmitoyl-glycerol in up to 72% yield containing 94% palmitic acid in the sn-2 position. The best lipases were from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus. Water activity, solvent, and carrier for lipase immobilization strongly influenced the yield and purity of the products in both steps. Furthermore, 2-MG from fish oil were produced by alcoholysis in up to 84% yield at >95% purity.

Full text of DOI:10.1007/s11746-998-0089-5

Optimization of the Reaction Conditions
in the Lipase-Catalyzed Synthesis
of Structured Triglycerides
U. Schmida, U.T. Bornscheuera, M.M. Soumanoua, G.P. McNeillb, and R.D. Schmida,*
aInstitut für Technische Biochemie, Universität Stuttgart, 70569 Stuttgart, Germany, and bUnilever Research,
Colworth House, Sharnbrook, Bedford MK 44 1LQ, United Kingdom
ABSTRACT: Structured triglycerides of the ABA-type, contain- formulas contain palmitic acid predominantly in sn-1,3 posi-
ing one type of fatty acid (A) in the sn-1 and sn-3 positions and
a second type of fatty acid (B) in the sn-2 position of the glyc-
erol, were synthesized using lipases. The highest yields and pu-
rities were achieved in a two-step process, where a triglyceride
of the B-type was subjected to an alcoholysis reaction in an or-
ganic solvent catalyzed by sn-1,3-regiospecific lipases yielding
the corresponding 2-monoglyceride (2-MG). Using this strategy,
e.g., 2-monopalmitin (2-MP) was obtained in up to 88% yield
at >95% purity by crystallization. Esterification of 2-MP with
oleic acid resulted in the formation of 1,3-oleyl-2-palmitoyl-
glycerol in up to 72% yield containing 94% palmitic acid in the
tions, and their release during digestion may cause the forma-
tion of poorly absorbed calcium soaps. In contrast, fats con-
taining palmitic acid exclusively at the sn-2 position are ab-
sorbed more efficiently (3).
Polyunsaturated fatty acids (PUFA) like eicosapentaenoic
acid (EPA, C20:5) and docosohexaenoic acid (DHA, C22:6
)
have been reported to have several advantages compared to
conventional fatty acids, such as reduction of blood pressure
and plasma triglyceride levels, and control of overactive im-
mune functions (4). DHA is recognized as being important
sn-2 position. The best lipases were from Rhizomucor miehei, for brain and eye development in infants (5). It might be ad-
Rhizopus delemar, and Rhizopus javanicus. Water activity, sol-
vent, and carrier for lipase immobilization strongly influenced
the yield and purity of the products in both steps. Futhermore,
2-MG from fish oil were produced by alcoholysis in up to 84%
yield at >95% purity.
vantageous to provide diets containing only one of these
PUFA, because in some studies negative effects on growth
were reported when DHA was fed to infants in combination
with high EPA levels (5).
The common route reported in literature for the synthesis
of structured triglycerides is based on a simple lipase-cat-
alyzed transesterification between two different triglycerides
or between a triglyceride and fatty acids (6–8). Unfortunately,
yields of ABA-type triglycerides are low and a variety of by-
products is formed. These are difficult to separate from the
desired product. Recently, we developed an efficient alterna-
tive for the synthesis of structured triglycerides based on a
JAOCS 75, 1527–1531 (1998).
KEY WORDS: Alcoholysis, betapol, DHA, EPA, fish oil, im-
mobilization, lipase, 2-monopalmitin, structured triglyceride,
water activity.
The nutritional value of triglycerides and their physicochemi- two-step process (9,10). First, triglycerides of the B-type
cal properties are determined not only by the fatty acid com- were subjected to alcoholysis catalyzed by sn-1,3-regiospe-
position, but also by the positional distribution of the acyl cific lipases yielding the corresponding 2-monoglycerides (2-
groups bonded to the glycerol. Special triglycerides of the MG). The 2-MG were isolated by crystallization and esteri-
ABA-type containing medium-chain fatty acids (e.g., C8) in fied with the A-type fatty acid using the same lipases to form
sn-1,3 positions and a long-chain unsaturated fatty acid (e.g., the desired structured triglycerides. This concept was suc-
C16–C22) in the sn-2 position are an effective energy source cessfully used in the synthesis of MLM-type triglycerides
for patients with malabsorption, e.g., pancreatic insufficiency starting from triolein, trilinolein, peanut oil, or cottonseed oil
(1). Synthesis, biochemistry, and use of structured triglyc- followed by esterification of the corresponding 2-MG with
erides were recently documented (2). An important structured caprylic acid. The alcoholysis was performed in methyl-t-
triglyceride in infant nutrition is 1,3-oleyl-2-palmitoyl-glyc- butyl ether and the esterification in n-hexane. In the present
erol (OPO). Human milk contains palmitic acid predomi- paper, we extended this method to the synthesis of OPO and
nantly in the sn-2 position of triglycerides, but most infant also prepared 2-MG from fish oil containing high concentra-
tions of PUFA by alcoholysis. Special emphasis was given to
the influence of water activity, solvent, and choice of carrier
*To whom correspondence should be addressed at Institut für Technische
material for the immobilization of the lipases on product yield
Biochemie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Ger-
and purity during both reactions.
many. E-mail: rolf.d.schmid@rus.uni-stuttgart.de
Copyright © 1998 by AOCS Press
1527
JAOCS, Vol. 75, no. 11 (1998)

1528
U. SCHMID ET AL.
TABLE 2
MATERIALS AND METHODS
Influence of Lipases on the Yield and Purity of 2-MP Obtained
by Alcoholysis of Tripalmitin with Ethanol in MTBE (after 24 h)
Lipase. Lipases (triacylglycerol hydrolases, E.C. 3.1.1.3)
were from Rhizopus delemar (RDL), Rhizopus javanicus
(RJL), Rhizopus niveus (RNL), and Candida rugosa (all from
Amano Pharmaceutical Co. Ltd., Nagoya, Japan). One com-
mercial lipase from RML (Lipozyme IM), immobilized on
anion exchange resin, was from Novo (Bagsvaerd, Denmark).
All chemicals and solvents used were reagent grade and pur-
chased from common commercial suppliers, with the excep-
tion of EP 100, a polypropylene carrier (particle size 200–400
µm) (Akzo, Obernburg, Germany) and Celite 545 (Fluka,
Buchs, Switzerland). Fish oil was supplied by Unilever (Bed-
ford, United Kingdom).
Yield of 2-MP
(%)
Purity of 2-MP
Lipase from
(%)
Rhizomucor mieheia
Rhizopus delemarb
Rhizopus javanicusb
88
95
50
80c
>95
>90d
aLipozyme immobilized on anion exchange resin.
bImmobilized on Celite.
c20% 1-MP.
dContains 1-MP and dipalmitin. See Table 1 for abbreviations.
erides was performed by preparative HPLC or by column
chromatography using boric acid-treated silica gel (11).
Esterification of 2-monopalmitin. This was performed in
principle as described previously (9). Briefly, the purified 2-
MP obtained from alcoholysis was esterified with oleic acid
in n-hexane using Lipozyme or RDL immobilized on Celite
at 38°C. All reaction components were pre-equilibrated to a
water activity of 0.11. Activated molecular sieve was added
to remove water generated during this step.
GC separation of triglycerides. The triglycerides obtained
from the esterification of 2-MP were separated by GC analy-
sis using the same equipment as described above with the fol-
lowing temperature program: 200°C followed by heating
8°C/min to 360°C, and holding for 20 min.
General methods. Determination of hydrolytic activity of
lipases, immobilization of lipases on Celite or EP 100, esteri-
fication of 2-MG, and purification of structured triglycerides
were performed as described previously (9).
Alcoholysis of triglycerides. Alcoholysis of fish oil and tri-
palmitin was essentially performed as described previously
(9) with slight modifications. Tripalmitin (0.35 mmol) was
dissolved in the solvent given in Table 1 and equilibrated to a
water activity of 0.11 over saturated salt solutions in a closed
vessel. Then the reaction was started by addition of 10%
(based on the weight of triglyceride) of immobilized RML,
RDL, RNL, or RJL, pre-equilibrated at water activity 0.11
over saturated LiCl. Dry ethanol (dried over molecular sieve
3Å, which was activated by heating overnight to 250°C) was
added to a final concentration of 3.5 mmol. The reaction was
stopped after 24 h by removal of the immobilized lipase. The
determination of the composition of the reaction mixture as
well as crystallization of 2-MG was performed as described
previously (9). Purity of 2-MG was determined by gas chro-
matography (GC; Fisons Instruments Mega series, Mainz,
Germany) using a nonpolar column (Optima 5, 25 m × 0.25
mm i.d.; Machery & Nagel, Düren, Germany) after derivati-
zation of samples with N-methyl-N-trimethylsilylheptafluo-
robutyramide. Analysis was carried out using the following
temperature program: 200°C for 2 min, then heating by
8°C/min to 360°C, and holding for 5 min. The purity of 2-MG
given in Tables 1 and 2 was also confirmed by 13C nuclear
magnetic resonance spectroscopy.
Regiospecific analysis of 1,3-oleoyl-2-palmitoyl-glycerol
(OPO). This was performed by digestion of OPO with pan-
creatic lipase (12) followed by GC analysis.
RESULTS
Synthesis of OPO. Alcoholysis of tripalmitin. The first step in
the synthesis of OPO was the alcoholysis of tripalmitin using
sn-1,3-regiospecific lipases. Of three lipases [from Rhizomu-
cor miehei (Lipozyme), RDL, and RJL] investigated, RDL
immobilized on Celite gave the best yield (95% after crystal-
lization) of 2-MP in methyl-t-butyl ether (MTBE) (Table 2).
The alcoholysis reaction was very fast; after 8 h reaction time,
tripalmitin was completely converted to 2-MP and only a
small amount of 1,2 (2,3)-dipalmitin remained. After 24 h,
the reaction mixture contained only 2-MP and palmitic acid
ethyl ester and only a small amount of palmitic acid (Fig. 1).
In contrast, a strong acyl migration took place when
Lipozyme was used, because the monopalmitin fraction was
composed of an 8:1 mixture of 2-MP/1-MP. We related this
to the anion exchange resin serving as carrier for RML, which
is known to promote acyl migration in MTBE (11). This acyl
migration could be suppressed by using acetone as solvent;
however, the yield of 2-MP (35%) was significantly lowered.
In neat ethanol, no alcoholysis reaction with Lipozyme was
observed (Table 1).
In a similar manner, alcoholysis of fish oils I–III was per-
formed, but reaction components were equilibrated to the
water activities given in Table 7. Isolation of 2-monoglyc-
TABLE 1
Influence of Organic Solvents on the Yield and Purity
of 2-Monopalmitin (2-MP) Obtained by Alcoholysis
of Tripalmitin with Lipozyme (after 24 h)
Organic
solvent
Yield of 2-MP
(%)
Purity of 2-MP
(%)
80a
>95
n.d.
Yield and purity of 2-MP were also affected by the carrier
material used for lipase immobilization. As shown in Fig-
ure 2, alcoholysis in MTBE was best performed using
Lipozyme or RDL immobilized on Celite. Here, tripalmitin
MTBE
Acetone
Ethanol
88
35
<1
a20% 1-MP; n.d., not detectable; MTBE, methyl-t-butyl ether.
JAOCS, Vol. 75, no. 11 (1998)

STRUCTURED TRIGLYCERIDES BY LIPASE CATALYSIS
1529
TABLE 3
Synthesis of 1,3-Oleyl-2-palmitoyl-glycerol (OPO) by Esterification
of 2-MP with Oleic Acid in n-Hexane with Immobilized Lipases
(after 5 h)
Positional distribution
Yield of OPO
Lipase from
(%)
1(3)-Position
2-Position
Rhizomucor mieheia
Rhizopus delemarb
70
72
89% OAc
83% OAc
92% PAd
94% PAd
aLipozyme immobilized on anion exchange resin.
bImmobilized on Celite.
cOleic acid.
dPalmitic acid. See Table 1 for abbreviation.
uct revealed that between 92 and 94% palmitic acid was lo-
cated in the sn-2 position of OPO, while 83–89% oleic acid
was bound to the sn-1 and sn-3 positions. Another reason for
the low acyl migration observed during the second step might
be the very short reaction time. After only 2 h the 2-MP was
completely converted into OPO and only a minor amount of
diglyceride was left (Fig. 3).
FIG. 1. Time course of the alcoholysis of tripalmitin with lipase from
Rhizopus delemar immobilized on Celite. PE, palmitic acid ethyl ester;
PA, palmitic acid; 2-MP, 2-monopalmitin; DP, dipalmitin; TP, tri-
palmitin; (mol%), mol of fatty acyl groups.
was completely consumed and mainly 2-MP was formed.
Significantly lower yields of 2-MP and large concentrations
of tripalmitin, dipalmitin, and palmitic acid remained when
using RDL immobilized on polypropylene (EP 100) (Fig. 2).
Esterification of 2-MP with oleic acid. The second step in
the synthesis of OPO is the esterification of 2-MP with oleic
acid. The nutritional value of OPO greatly depends on the
concentration of palmitic acid in the sn-2 position of the
structured triglyceride. Even if highly pure 2-MP is used, acyl
migration during esterification might lead to a reduction in
the amount of palmitic acid in the sn-2 position. Water is
known to promote acyl migration; thus, care must be taken
that the water generated during esterification is quickly re-
moved from the reaction mixture. This was achieved by using
activated molecular sieves, n-hexane as solvent, and pre-equi-
libration of all reaction components to a low water activity of
0.11. Using these precautions, high yields of OPO (up to
72%) were obtained with Lipozyme or RDL immobilized on
Celite (Table 3). Regiospecific analysis of the reaction prod-
Alcoholysis of fish oil. In order to synthesize structured
triglycerides containing PUFA like EPA and DHA, three dif-
ferent fish oils were subjected to an alcoholysis reaction. Ini-
tially, the fatty acid composition of the starting materials was
determined by GC. The three fish oils differed considerably
in their amount of PUFA. Fish oil I contained mainly palmitic
acid and oleic acid, and the total PUFA content was 18.3%.
Fish oil II was composed of 45.5% PUFA with DHA as the
major component. Fish oil III lacked DHA, but contained
37.4% EPA (Table 4). In sharp contrast to the alcoholysis of
tripalmitin, significantly lower yields of 2-MG were achieved
in the alcoholysis of all fish oils, despite identical reaction
conditions. The highest purity of 2-MP (>95%) was obtained
using fish oil I, but only at 6.5% yield. Much higher yields
(28–43%) were achieved with fish oils II and III, but here
1(3)-MG (25 and 50%) were formed too (Table 5). This was
related to the difficult isolation of 2-MG. Crystallization was
unsuccessful due to the low melting point of the PUFA, and
FIG. 2. Influence of carrier material for lipase immobilization on the
yield of 2-MP in the alcoholysis of tripalmitin in methyl-t-butyl ether
(after 24 h). RDL, lipase from Rhizopus delemar; RDL EP 100, RDL im-
mobilized on polypropylene; Lipozyme, RML immobilized on anion
exchange resin. For other abbreviations see Figure 1.
FIG. 3. Time course of the formation of 1,3-oleyl-2-palmitoyl-glycerol
(OPO) from 2-MP with oleic acid in n-hexane with Lipozyme. For ab-
breviations, see Figure 1.
JAOCS, Vol. 75, no. 11 (1998)

1530
U. SCHMID ET AL.
TABLE 4
TABLE 6
Fatty Acid Composition of the Three Fish Oils
Used for the Lipase-Catalyzed Alcoholysis
Influence of Lipases (all immobilized on Celite) on the Yield of 2-MG
Obtained by Alcoholysis of Fish Oil I with Ethanol in Acetone
(after 24 h)a
Fatty
acid
Fish oil I
(mol%)
Fish oil II
(mol%)
Fish oil III
(mol%)
Yield of 2-MG
Lipase from
(%)
C14:0
C16:0
C16:1
C18:0
C18:1
C20:5
C22:6
15.2
27.6
11.0
4.2
17.0
10.5
7.8
9.3
13.4
7.3
2.6
9.8
1.0
n.d.a
26.7
n.d.
4.9
Rhizopus delemar
Rhizopus javanicus
Rhizopus niveus
Candida rugosa
20
19
14
5.5
11.4
34.1
37.4
n.d.
aSee Table 5 for abbreviation.
an.d., not detectable.
acid (C16:1), the major components of the starting material
(Fig. 4).
TABLE 5
Yields of 2-MG in the alcoholysis of fish oil I could be fur-
ther increased to 20% by using acetone as solvent (Table 6).
Acetone has the advantage that it is allowed to be used in food
and lowers acyl migration. Whereas other lipases from Rhi-
zopus sp. were found suitable, lipase from Candida rugosa
only led to the formation of 5.5% 2-MG from fish oil I. The
strongest impact on 2-MG yield was observed by performing
the alcoholysis reaction at different water activities (Table 7).
Best results were achieved at a water activity of 0.94, at
which 37 and 84% 2-MG were produced from fish oils I and
III, respectively. In both cases immobilization of RDL on
polypropylene (EP 100) using acetone as solvent was found
Synthesis of 2-Monoglycerides (2-MG) by Alcoholysis of Three Fish
Oils with Ethanol in MTBE with Lipase from Rhizopus delemar
Immobilized on Celite (after 24 h)
Yield of 2-MG
(%)
Purity of 2-MG
(%)
Fish oil
Fish oil I
Fish oil II
Fish oil III
6.5
28
43
>95
75a
50b
aContains 25% 1-MG.
bContains 50% 1-MG. See Table 1 for abbreviation.
the 2-MG fraction could only be isolated by preparative most suitable.
HPLC or silica gel column chromatography. Acyl migration
observed during silica gel column chromatography could be
suppressed by impregnation of silica gel with boric acid prior
DISCUSSION
to the separation procedure (11).
We have shown in this paper that the two-step process allows
An analysis of the fatty acid composition of the 2-MG the synthesis of 1,3-oleyl-2-palmitoyl-glycerol in high yield
clearly shows the enrichment of PUFA (Fig. 4). 2-MG de- and purity. Furthermore, it was demonstrated that 2-MG con-
rived from fish oil I contains more than 70% PUFA, with taining PUFA can be prepared by alcoholysis of fish oils. In
DHA as the major component. In the case of fish oil II, alco- accordance with our previous papers (9,10)—and in contrast
holysis resulted in a significant enrichment of DHA, because to direct synthesis by a one-step interesterification (6–8)—
the 2-MG was mainly composed of this fatty acid (>70%). acyl migration can be suppressed in the alcoholysis reaction.
2-MG from fish oil III contained mainly EPA and palmitoleic However, in order to achieve best results, choice of solvent,
carrier for lipase immobilization, and water activity must be
carefully controlled and optimized for each new substrate.
For instance, alcoholysis of tripalmitin is best performed with
lipase from Rhizopus delemar immobilized on Celite in
TABLE 7
Influence of Solvent and Water Activity on the Yield of 2-MG
Obtained by Alcoholysis of Fish Oils I and III with Ethanol
(after 24 h)a
Rhizopus delemar
immobilized
on lipase
Yield
of 2-MG
(%)
Water
activity
Substrate
Fish oil I
Solvent
Celite
Celite
EP 100
MTBE
Acetone
Acetone
0.43
0.43
0.94
6.5
20
37
Fish oil III
Celite
EP 100
MTBE
Acetone
0.43
0.94
43
84
FIG. 4. Fatty acid composition of 2-monoglycerides obtained by alco-
holysis of different fish oils in acetone using lipase from Rhizopus dele-
mar immobilized on Celite. See Figure 1 for abbreviation.
aSee Tables 1 and 5 for abbreviations.
JAOCS, Vol. 75, no. 11 (1998)

STRUCTURED TRIGLYCERIDES BY LIPASE CATALYSIS
1531
MTBE (to achieve a high yield of 2-MP) or in acetone (to
REFERENCES
achieve a high purity of 2-MP). In contrast, in the alcoholysis
of fish oil III, acetone was the best solvent and polypropylene
was the best lipase support. Furthermore, the most appropri-
ate water activity must always be determined too; it was 0.94
in the alcoholysis of fish oils I and III, but 0.11 in the alcohol-
ysis of tripalmitin.
1. Jandacek, R.J., J.A. Whiteside, B.N. Holcombe, R.A. Volpen-
hein, and J.D. Taulbee, The Rapid Hydrolysis and Efficient Ab-
sorption of Triacylglycerides with Octanoic Acid in the 1 and 3
Positions and Long-Chain Fatty Acid in the 2-Position, Am. J.
Clin. Nutr. 45:940–945 (1987).
2. Christophe, A.B., Structural Modified Food Fats: Synthesis,
Biochemistry, and Use, AOCS Press, Champaign, 1998.
3. Lien, E.L., F.G. Boyle, R. Yuhas, R.M. Tomarelli, and P. Quin-
lan, The Effect of Triglyceride Positional Distribution on Fatty
Acid Absorption in Rats, J. Ped. Gastroenterol. Nutr.
25:167–174 (1997).
4. McNeill, G.P., R.G. Ackman, and S.R. Moore, Lipase-Cat-
alyzed Enrichment of Long-Chain Polyunsaturated Fatty Acids,
J. Am. Oil Chem. Soc. 73:1403–1407 (1996).
5. Moore, S.R., and G.P. McNeill, Production of Triglycerides En-
riched in Long-Chain n-3 Polyunsaturated Fatty Acids from Fish
Oil, Ibid. 73:1409–1414 (1996).
6. Fomuso, L.B., and C.C. Akoh, Enzymatic Modification of Tri-
olein: Incorporation of Caproic and Butyric Acids to Produce
Reduced-Calorie Structured Lipids, Ibid. 74:269–272 (1997).
7. Koehler, P.E., C.J. Shieh, and C.C. Akoh, Four-Factor Response
Surface Optimization of the Enzymatic Modification of Triolein
to Structured Lipids, Ibid. 72:619–623 (1995).
8. Soumanou, M.M., U.T. Bornscheuer, U. Menge, and R.D.
Schmid, Synthesis of Structured Triglycerides from Peanut Oil
with Immobilized Lipase, Ibid. 74:427–433 (1997).
The isolation of 2-MG is easy to perform by crystalliza-
tion as shown here for 2-MP and in previous papers for 2-
monoolein and 2-monolinolein (9,10). 2-MG derived from
fish oil could not be isolated by this manner, because the melt-
ing point of the monoglycerides is much too low. A separa-
tion by HPLC yields highly pure 2-MG, but this method is
not practical on a large scale. We were able to isolate the
2-MG by column chromatography, but only after boric acid
treatment of silica gel to avoid the formation of 1-MG. The
alcoholysis of the three fish oils further allowed the enrich-
ment of PUFA, because they comprised the major fatty acids
present in the 2-MG fraction. It is expected that the formation
of structured triglycerides from 2-MG containing PUFA can
be performed in a similar manner to that shown in this paper
for OPO and other structured triglycerides as described pre-
viously (9,10).
9. Soumanou, M.M., U.T. Bornscheuer, and R.D. Schmid, Two-
Step Enzymatic Reaction for the Synthesis of Pure Structured
Triglycerides, Ibid. 75:703–710 (1998).
10. Soumanou, M.M., U.T. Bornscheuer, U. Schmid, and R.D.
Schmid, Synthesis of Structured Triglycerides by Lipase Catal-
ysis, Fett/Lipid 100:156–160 (1998).
11. Millqvist-Fureby, A., C. Virto, P. Adlercreutz, and B. Mattias-
son, Acyl Group Migrations in 2-Monoolein, Biocatal. Bio-
transform. 14:89–111 (1996).
The esterification of 2-MP with oleic acid proceeded very
fast, within 2 h, and almost no acyl migration occurred, as
proven by positional analysis of the produced OPO. This con-
tains 94% palmitic acid in sn-2 position and thus is much
more pure compared to the currently marketed product Be-
tapol, which contains only 65% palmitic acid in sn-2 position
(13).
12. Gunstone, F.D., J.L. Harwood, and F.B. Padley, The Lipid
Handbook, Chapman & Hall, London, 1994, p. 231.
ACKNOWLEDGMENTS
13. Zock, P.L., J. Gerritsen, and M.B. Katan, Partial Conservation
of the sn-2 Position of Dietary Triglycerides in Fasting Plasma
Lipids in Humans, Eur. J. Clin. Invest. 26:141–150 (1996).
Financial support by the BMBF (Bonn, Germany, project ZSP
B3.2U) and Unilever (Bedford, United Kingdom) is gratefully ac-
knowledged. Furthermore, we would like to thank Novo Nordisk
Co., Bagsvaerd, Denmark and Amano Pharmaceuticals Inc.,
Nagoya, Japan, for the gifts of lipases.
[Received June 15, 1998; accepted August 20, 1998]
JAOCS, Vol. 75, no. 11 (1998)