ARTICLE
Auteur(s) :, Mohamed M Soumanou1,*,
Aleodjrodo P Edorh2, Uwe T Bornscheuer3
1Research Laboratory for Applied Chemistry and
Biology (LARECBA), Abomey-Calavi Polytechnic School, Abomey-Calavi
University, 01 BP 2009, Cotonou, Benin
2Faculty of Sciences and Technics, Department of
Biochemistry and Cellular Biology, BP 526, Cotonou, Benin
3Institute of Chemistry and Biochemistry, Department of
Technical Chemistry and Biotechnology, University Greifswald,
Soldmannstr, 16, D-17487 Greifswald, Germany
Article reçu le 14 Septembre 2004, accepté le 17 Janvier
2005
Lipid modification strategies for industry include processes such
as fractionation, hydrogenation and interesterification. While each
of the two first processes has specific uses and advantages,
interesterification reaction offers the greatest potential
application [1]. It includes three approaches: acidolysis,
alcoholysis and transesterification. Chemically, it can be induced
by the use of alkali catalysts in a reaction which lacks
specificity and offers little or no control over the positional
distribution of fatty acids in the final product [1, 2].To overcome
such difficulties, enzymatic approach is used. Applied biocatalysts
are microbial lipases and are based mainly on their specificity.
They are divided in two main groups: random lipases, which cleave
fatty acids at all position on the glycerol molecule (e.g. lipases
from Candida antarctica, Candida rugosa, Corynebacterium acnes and
Staphylococcus aureus) and sn-1, 3-specific lipases, which act
preferentially at the sn-1 and sn-3 positions of the glycerol
molecule (e.g. lipases from Rhizomucor miehei, Rhizopus oryzae,
Aspergillus niger, etc.) [3]. The second group of lipases show
specificity for particular fatty acids. An example is the lipase
from Geotrichum candidum, which has a marked specificity for
long-chain fatty acids that contain cis-9 unsaturation [4].The
microbial lipases described above have been used successfully as
biocatalysts in modification of oils and fats via hydrolysis,
esterification and interesterification [5, 6].The lipase-catalyzed
interesterification process in food industry can be used for the
production of triacylglycerols with specific physical properties
and it also opens possibilities for making so-called structured
triacylglycerols (ST). In the literature, because of its
nutritional importance, ST has received many attentions [7-11].
Reactions were performed for the production of pure ST using one or
two-step enzymatic process. In terms of reaction conditions,
crucial role of support for immobilization and water activity on
the synthesis of ST were also reported [12-15]. Between the two
enzymatic processes used for the production of ST, one step
reaction, namely interesterification of a mixture of triglycerides
received many attentions, because of its application for industrial
purposes. The one step interesterification reaction appears very
easy to carry out, but many reactions involved among various
triglycerides molecules make difficult analytical analysis of
products. However, species fractions of the products under
substrate molar ratio have been theoretically proposed and the
kinetic model of the transesterification of a mixture of
medium-chain fatty acid triglycerides (MCT) and long-chain fatty
acid triglycerides (LCT) was developed [16-18]. Based on this
kinetic model, the rate constant of interesterification reaction
was determined [18]. On the other hand, some minor compounds found
in vegetable oils such as lipid hydroperoxides, phospholipids,
emulsifiers, chlorophyll, carotenoids, lipid polymers, heavy metal
ions and even some antioxidants had effects on activity and
stability of immobilized lipases [19].In the present work, activity
of microbial lipases based on released fatty acid profile during
hydrolysis of peanut oil using free and immobilized biocatalysts
was determined. The effects of molar ratios, temperature, organic
solvent on interesterification of peanut oil with tricaprylin was
studied. For industrial production of ST during one step
interesterification reaction, stability of immobilized lipases was
investigated.
Material and methods
Lipases
Lipases used in this work were from Rhizopus sp. (RSL) (Solvay
Enzymes, Hannover, Germany), Rhizomucor miehei (RML), Humicola
lanuginosa (HLL), Pseudomonas fluorescens (PSL) (Biocatalyst,
England), Candida rugosa (CRL) (lipase OF, Meito, Japan),
Geotrichum candidum (GCL) (lipase GC, Amano, Japan), and
Chromobacterium viscosum (CVL) (Asahi Chemical Industry, Japan).
Two commercial lipases from Rhizomucor miehei (Lipozyme) and
Candida sp. (CSL, SP 382) immobilized on an anion exchange resin,
were from Novo (Bagsvaerd, Denmark). All chemicals and solvents
used for analysis of the reaction products were reagent grade and
purchased from common commercial supplier. Peanut oil used was
purified by column chromatography.
Hydrolysis of peanut oil
Lipolysis in aqueous medium was tested with 5% (w/v) peanut oil
containing 2% (w/v) arabic gum at 37 °C, pH 8. To 20 mL
of the emulsion, 470 μL of CaCl2 solution (22% (w/v))
and 50 U of crude lipase (in 50 mM phosphate buffer at pH 8) or
immobilized form on Celite were mixed, and the fatty acid liberated
was titrated automatically with 0.1 N NaOH at constant pH 8.0.
After 10 min, lipolysis reaction was stopped by addition of
20 mL acetone/ethanol mixture (1:1, v/v) and the products were
extracted three times with 10 mL of ether/heptane solution
(75:25, v/v) from the emulsion. Determination of the composition of
free fatty acids released during lipolysis was analysed by gas
chromatography.
Immobilization of lipase on Celite®
The immobilized lipases tested in this work was obtained by
adsorptive binding. Before immobilization, 1.5 g of
Celite® 545 as support material was soaked with
5 mL of ethanol. One gram of crude microbial lipase was
dissolved in 25 mL phosphate buffer pH 6.0, 20 mM. The
solution obtained was added to the wet support and stirred slowly
at room temperature overnight. The immobilized enzyme preparation
was collected by filtration, washed three times with 10 mL
phosphate buffer, pH 6.0, 20 mM and dried overnight under
vacuum.
Interesterification reaction
Interesterification between peanut oil and tricaprylin was studied
as reaction model. The reaction medium was composed of 1 mmoL
of peanut oil and 0.6 mmoL of tricaprylin in 3 mL
n-hexane. 10% (w/w total TG) of immobilized lipases was used. In
order to study the stability of immobilized lipases, ten successive
transesterification reaction were carried out using the same
immobilized lipase. After each batch reaction, the immobilized
lipase was separated from the reaction medium, washed with chilled
acetone, dried under vacuum and was used for the next reaction
composed of fresh substrates.
Determination of free fatty acids composition
The extracted lipid compound from the lipolysis reaction after
evaporation was dissolved in 1 mL n-heptane in a closed test
tube, and 200 μL methanol containing 20% hydrochloric acid was
added. The mixture obtained was shaken in a water bath at
85 °C. After 15 min, the tube was removed from the bath
and centrifuged. One μl was taken from the supernatant for GLC
analysis on polar column (25 m × 0.53 mm i.d.; Macherey
and Nagel, Düren, Germany). Under these conditions, only free fatty
acids were converted to methyl esters [20]. Analysis was carried
out with temperature programming from 150 to 210 °C at
5 °C/min, 200 °C as injection temperature, and
210 °C as detecting temperature (flame-ionisation detector).
High-performance liquid chromatography (HPLC) -Separation of
ST
The composition of the peanut oil triacylglycerols and those formed
during enzymatic interesterification was characterized by HPLC
using a Nucleosil C18 column (5 μm, 250 ×
4 mm; Sykam, Gilching, Germany) and an evaporative
light-scattering detector (Sedere, Vitry/Seine, France) at column
temperature of 50 °C and a flow rate of 1.5 mL/min.
Elution was performed with a linear gradient elution system of the
two solvent mixtures of A (acetonitrile/isooctane (100%) 90: 10,
vol/vol) and B (acetonitrile /dichloromethane/ethanol (26%) 40: 35:
25, vol/vol/vol) over 45 min.
The main structured triacylglycerols identified by this
analytical method are MLM and LML fractions. MLM includes MML and
LMM, whereas LML includes MLL and LLM. The fatty acid M-type is
indicated as medium-chain fatty acid mainly from tricaprylin and
L-type as long-chain fatty acid from peanut oil.
Results and discussion
The exploitation of lipase specificity for the synthesis of various
products can be tailored in aqueous media as well as in organic
solvent. In this work, first, lipases screening was preferential
monitored in aqueous media by pH-stat. To determine the composition
of fatty acids released during peanut oil hydrolysis catalyzed by
free and immobilized lipases, a GLC-analysis of free fatty acids
was performed as described in material and methods. As can be seen
in figures 1 and 2, the main fatty acids released were oleic acid,
linoleic acid and palmitic acid, which are the major fatty acids
found in peanut oil triglycerides.
Particular long-chain fatty acids detected in peanut oil such as
arachidic acid, behenic acid, which have been implicated in
artherogenesis and bound mainly in sn-3 position of glycerol
molecule [21] were not released. As reported in the literature,
triacylglycerols with long-chain fatty acids, both saturated and
unsaturated were hydrolyzed at only marginally different rates
using lipases from Rhizopus sp. [22, 23] and in addition the low
solubility of such fatty acids in aqueous media may explain their
absence in the released fatty acid fractions.
On the other hand, immobilization of lipases from
Chromobacterium viscosum and Humicola lanuginosa contributes to a
higher release of oleic acid than of the free enzyme (( figure 2 )). Concerning
stearic acid, only free lipase from Chromobacterium viscosum and
Rhizomucor miehei were able to cleave it. Using immobilized
enzymes, stearic acid was found in free fatty acids from lipolysis
catalyzed by RSL, GCL and HLL. The highest stearic acid amount
released (3%) was found with immobilized lipase from Rhizomucor
miehei. In such media, immobilization lipase displayed high
catalytic specificity as described previously in both aqueous and
organic solvents [24].
In organic solvent, immobilized lipases from Rhizomucor miehei,
Candida rugosa and Chromobacterium viscosum were used for
transesterification, due to their activity displayed above.
Reaction standard was a transesterification of peanut oil with
tricaprylin in n-hexane at 40 °C. To determine optimum
synthesis of ST from this reaction, the effect of molar ratio of
substrate on the production of ST was investigated using
immobilized RML. As can be seen in ( figure 3 ), the best molar
ratio found was between 0.7 and 0.8 (mole tricaprylin to mole
peanut oil). From molar ratio 0.9 to 1.5, the amount of MLM
fraction increased, followed by an increase in the remaining
concentration of tricaprylin (data not shown).
Interesterification reaction involves several steps and fatty
acids from natural oil as well as from medium-chain triglycerides
such as tricaprylin must be released before subsequent
esterification to ST can occur. The increase of remaining
concentration of tricaprylin mentioned above may be due to the
different specificity showed among natural oil triglycerides and
tricaprylin by immobilized lipases.
Organic solvents are used in most of reaction catalyzed by
immobilized lipases, because enzyme shows high stability in some
organic solvents. Furthermore, organic solvents improve the
solubility of substrates and thus, increase the initial rate of the
reaction. To investigate the effect of organic solvent on the
synthesis of ST, the three immobilized listed above were used as
catalysts in various organic solvents. The results are indicated in
table 1( Table 1 ). In all organic
solvents tested, the concentration of MLM was lower than that for
LML (table 1). Except for RML, in petroleum ether and for all
immobilized lipases in cyclohexane, the concentration of ST was
high in most of cases. On the other hand, a solvent free system for
the production of ST also led to a high yield and in some cases is
quite similar to that found in organic solvents (table 1). The
yield found here was lower than that obtained from the
transesterification of olive oil with trimyristin (90%) and
tristearin with tricaprin (84.7%) [25, 26]. Because of the high
solubility of medium-chain fatty acid such as caprylic acid in
water, their concentration is reduced at the interface and thereby
reduces their exchange in triglycerides [27]. This may explain the
low yield of ST obtained in the present work.
For industrial purposes, repeated interesterification using the
same immobilized biocatalyst is of great importance. To investigate
stability of commercial immobilized RML and CRL, nine batches
interesterification of peanut oil and tricaprylin in a solvent free
system were studied. For this study, the immobilized lipases were
isolated from reaction products as described in material and
methods. figures 4 and 5 show the experimental results of produced
structured triglycerides of MLM and LML types during reused
immobilized lipases
The determination of relative concentration of each fraction was
based on the amount of the structured triacylglycerols produced at
the first use of biocatalysts.
As can be seen in these figures, after 7 repeated uses, no
significant loss in terms of amount of structured produced
triglycerides was observed with RML, whereas with CRL, decrease in
the amount of synthesized triglycerides appeared after batch 5 and
was more pronounced with triglyceride of the LML-type.
Table 1 Effect of reaction system on the synthesis of
structured triacylglycerols from interesterification of peanut oil
and tricaprylin in molar ratio 0.7 (tricaprylin to peanut oil,
mol/mol) after 24 h.
|
Lipase
|
Reaction system
|
Temperature [°C]
|
ST (wt%)
|
Total ST (wt%)
|
|
|
|
MLM
|
LML
|
|
|
CSL
|
without solvent
|
60
|
28
|
39
|
67
|
|
n-hexane
|
50
|
31
|
37
|
68
|
|
isohexane
|
50
|
33
|
39
|
72
|
|
cyclohexane
|
50
|
31
|
29
|
60
|
|
heptane
|
50
|
27
|
40
|
67
|
|
isooctane
|
50
|
31
|
38
|
69
|
|
petroleum ether
|
50
|
30
|
40
|
70
|
|
CVL
|
without solvent
|
60
|
33
|
42
|
75
|
|
n-hexane
|
50
|
32
|
40
|
72
|
|
isohexane
|
50
|
33
|
43
|
76
|
|
cyclohexane
|
50
|
24
|
35
|
59
|
|
heptane
|
50
|
31
|
42
|
73
|
|
isooctane
|
50
|
31
|
42
|
73
|
|
petroleum ether
|
50
|
33
|
41
|
74
|
|
RML
|
without solvent
|
60
|
31
|
42
|
73
|
|
n-hexane
|
50
|
35
|
44
|
79
|
|
isohexane
|
50
|
34
|
42
|
76
|
|
cyclohexane
|
50
|
30
|
39
|
69
|
|
heptane
|
50
|
31
|
43
|
74
|
|
isooctane
|
50
|
31
|
42
|
73
|
|
petroleum ether
|
50
|
28
|
32
|
60
|
Conclusion
Most of informations known about enzymes have been learned from
studies in aqueous solutions. In this medium, determination of
fatty acids released during lipolysis of peanut oil using microbial
free and immobilized lipases was performed through pH-stat. Among
microbial lipases tested in this work, lipase specificity was
altered through immobilization in emulsion media, namely with
Chromobacterium viscosum, Geotrichum candidum, Humicola lanuginosa,
Rhizomucor miehei and Rhizopus sp. lipases.
Although aqueous media is a solvent of choice, non-aqueous
solvents for enzymatic reactions have been introduced and are of
great biotechnological interest, namely for the synthesis of ST. In
the present work, the best concentration of ST (79%) from the
transesterification of peanut oil with tricaprylin was found in
n-hexane with RML. The concentration of ST obtained in a solvent
free system after 24 h (> 65%) is quite similar to that
found in organic solvents. In such medium, RML maintained its
activity during nine successive batch transesterification of peanut
oil with tricaprylin. For industry point of view, the developed
synthesis transesterification can find application for the
modification of coating or nutrional properties of fats and
oils.
References
1 Marangoni AG, Rousseau D. Engineering triacylglycerols:
the role of interesterification. Trends Food Sci Technol 1995; 6:
329-35.
2 Rousseau D, Marangoni AG. Chemical
interesterification. In: Akoh CC, Min DB, eds. Food
Lipids. New York: Marcel Dekker Inc, 1998: 251-81.
3 Chandler JC. Determining the regioselectivity of
immobilized lipases in triacylglycerol acidolysis reactions. J Am
Oil Chem Soc 2001; 78: 737-42.
4 Jensen RG. Characteristics of the lipase from the mold
Geotrichum candidum: a review. Lipids 1974; 6: 149-57.
5 Macrae AR. Lipase-catalyzed interesterification of oils
and fats. J Am Oil Chem Soc 1983; 60: 243-6.
6 Gandhi NN. Applications of lipase. J Am Oil Chem Soc
1997; 74: 621-34.
7 Soumanou MM, Bornscheuer UT, Schmid RD.
Two-step enzymatic reaction for the synthesis of pure structured
triacylglycerides. J Am Oil Chem Soc 1998; 75: 703-10.
8 Miura S, Ogawa A, Konishi H. A rapid method for
enzymatic and purification of the structured triacylglycerol
1,3-Dilauroyl-2-oleoyl-glycerol. J Am Oil Chem Soc 1999; 76:
927-31.
9 Iwasaki Y, Yamane T. Enzymatic synthesis of
structured lipids. J Mol Catal, B Enzym 2000; 10: 129-40.
10 Bornscheuer UT. In: Lipase-catalyzed synthesis of
structured triacylglycerols. Lipid Technol Newsletter, 2001:
104-7.
11 Bornscheuer UT, Adamczak M, Soumanou MM.
Lipase-catalyzed synthesis of modified lipids. In:
Gunstone FD, Barnes PJ, eds. Lipids as Constituents of
Functional Foods. England: Bridgwater, 2003: 149-82; and
Associates.
12 Goldberg M, Thomas D, Legoy MD. Water activity
as a key parameter of synthesis reactions: the example of lipases
in biphasic (liquid/solid) media. Enzyme Microb Technol 1990; 17:
976-81.
13 Halling PJ. Thermodynamic predictions for biocatalysis
in nonconventional media: theory, tests, and recommendations for
experimental design and analysis. Enzyme Microb Technol 1994; 16:
178-206.
14 Soumanou MM, Bornscheuer UT, Schmid U,
Schmid RD. Crutial role of support and water activity on the
lipase-catalyzed synthesis of structured triglycerides. Biocatal
Biotransf 1999; 16: 443-59.
15 Han JJ, Yamane T. Enhancement of both reaction
yield and rate of synthesis of structured triacylglycerol
containing eicosapentaenoic acid under vacuum with water activity
control. Lipds 1999; 34: 989-95.
16 Basheer S, Snape JB, Mogi K, Nakajima M.
Tranesterification kinetics of triglycerides for a modified lipase
in n-hexane. J Am Oil Chem Soc 1995; 72: 231-7.
17 Xu X. Modification of oils and fats by lipase-catalyzed
interesterification: aspects of process engineering. In:
Bornscheuer UT, ed. Enzymes in Lipid Modification. Weinheim:
Wiley-VCH Verlag GmbH, 2000: 190-215.
18 Mogi K, Nakajima M, Mukataba S.
Transesterification reaction between medium- and long-chain acid
triglycerides using surfactant-modified lipase. Biotechnol Bioeng
2000; 67: 513-9.
19 Xu X, Hoy CE, Adler-Nissen J. Effects of
lipid-borne compounds on the activity and stability of lipase in
microaqueous systems for lipase-catalyzed reaction. In:
Ballesteros A, Plou PJ, Iborra JL, Halling P,
eds. Stability and Stabilization of Biocatalysts. Amsterdam:
Elsevier Science, 1998: 441-6.
20 Badings HT, De Jong C. Glass capillary gas
chromatography of fatty acid methyl esters. A study of conditions
for the quantitative analysis of short- and long-chain fatty acids
in lipids. J Chromatogr 1983; 279: 493-506.
21 Sridhar R, Lakshminarayana G, Kaimal TNB.
Modification of selected edible vegetable oils to high oleic oils
by lipase-catalyzed ester interchange. J Agric Food Chem 1991; 39:
2069-71.
22 Huge-Jensen B, Galluzzo DR, Jensen RG. Studies
on free and immobilized lipases from Mucor miehei. J Am Oil Chem
1988; 65: 905-10.
23 Atomi H, Bornscheuer U, Soumanou MM,
Beer HD, Wohlfahrt G, Schmid RD. Microbial
lipases-from screening to design. In: Barnes PJ, ed.
Oils-Fats-Lipids, 21st World Congress Int Soc Fat Res. England:
Bridgwater, 1995: 49-50; and Associates, Vol. 1.
24 Jensen BF, Eigtved P. Safety aspects of microbial
enzyme technology, exemplified by the safety assessment of an
immobilized lipase preparation LipozymeTM. Food
Biotechnol 1990; 4: 699-725.
25 Jung HJ, Bauer W. Determination of process
parameters and modelling of lipase catalyzed transesterification in
a fixed bed reactor. Chem Eng Technol 1992; 15: 341-8.
26 Akoh CC, Yee LN. Enzymatic synthesis of
position-specific low-calorie structured lipids. J Am Oil Chem Soc
1997; 74: 1409-13.
27 Kimura M, Hasegawa K, Takamura H,
Matoba T. Preparation of triacylglycerols molecular species by
interesterifcation using endocellular lipase in n-hexane. Agric
Biol Chem 1991; 55: 3039-43.
|
Printable version
Version PDF
