Rhizopus arrhizus lipase-catalyzed interesterification of the midfraction of palm oil to a cocoa butter equivalent fat

$Page 1: Rhizopus arrhizus lipase-catalyzed interesterification of the midfraction of palm oil to a cocoa butter equivalent fat$
Rhizopus arrhizus lipase-catalyzed interesterification of the midfraction of palm oil to a cocoa butter equivalent fat

Ljiljana Mojovi~,* Slavica Siler-Marinkovi~,* Gojko Kuki~* and Gordana Vunjak-Novakovi~t

Depar tment o f Biochemical Engineering and Biotechnologies* and Depar tmen t o f Chemical Engineering, t Faculty o f Technology and Metallurgy, University o f Belgrade, Yugoslavia

Celite-immobilized lipase from Rhizopus arrhizus was used to interesterify triacylglycerols of the palm oil midfraction with stearic acid in n-hexane. Under optimum conditions, acyl exchange occurred mainly between the palmitoyl group from the palm oil midfraction and the stearoyl group from the reaction mixture, giving an interesterified product whose fatty acyl composition was similar to that of. cocoa butter. Addition of defatted soya lecithin significantly increased the substrate conversion. This was attributed to the formation of reverse micelles around the Celite-immobilized and hydrated enzyme that protected the enzyme from the nonpolar solvent and enhanced substrate and product diffusion in the enzyme microenvironment. The reverse micelle system exhibited higher productivity and operational stability when compared to the Celite-immobilized lipase.

Keywords: Interesterification reaction; palm oil midfraction; Celite-immobilized lipase; reverse micelle

Introduction Producing fats with highly desirable physicochemical properties, such as cocoa butter equivalents by enzymatic interesterification, has become a popular area of biotechnological research.1 Tanaka et al. 2'3 studied the product ion of cocoa butter-like fats from olive oil and stearic or palmitic acid by enzymatic interesterification in organic solvents using immobilized 1,3 regiospecific lipase from Rhizopus delmar. The palm oil midfraction is a very suitable substrate for cocoa butter-like fat production, because it is inexpensive and has an appropriate triacylglycerol composition. 4

Lipase-catalyzed interesterification is based on the manipulation of the chemical equilibrium of a thermo- dynamically reversible reactionfl and it requires a low water content . 6-8 By using organic solvents, it is relatively easy to achieve an optimum low water content

Address reprint requests to Dr. Mojovi~ at the Department of Bio- chemical Engineering and Biotechnologies, Faculty of Technology and Metallurgy, University of Belgrade, Yugoslavia Received 25 June 1992; revised 20 August 1992

necessary to shift the reaction. However , the proper choice of a solvent that will solubilize the substrate but not affect the enzyme is of great importance. 9-~2

Many attempts to protect the enzyme from the solvent nonpolar environment and to enhance its stability by an appropriate immobilization method have been made. 3'13-16 It has been shown that the degree of dispersion of the lipase into the organic solvent affects the rate of the acyl exchange. 2 Therefore , suitable carriers for interesterification in organic solvents are diatomaceous earth, perlite, and silica gel, 13 since they all have a high surface area and a suitable water retention capac- ity 14 for enzyme activation.

Recently, one relatively new method for enzyme immobilization in reverse micelles has been devel- oped.17-~9 Reverse micelles offer a unique possibility to overcome problems caused by medium heterogeneity. They are spontaneously formed in an organic solvent when certain surfactant molecules and a small amount of water are present. 2° The most common system is based on the use of bis (2-ethylhexyl) sodium sulfosuc- cinate (AOT) in isooctane, and it has already been tested for olive oil hydrolysis. 21,22 The other one, which looks promising for food production, is based on phos-

438 Enzyme Microb. Technol., 1993, vol. 15, May © 1993 Butterworth-Heinemann

pholipids in n-hexane, and it has been studied for triacylglycerol synthesis. 23 However, enzyme stability in reverse micelles is rather poor, and the system might not be convenient for practical application.

We studied the interesterification of the palm oil midfraction with stearic acid in n-hexane by the Celite- immobilized lipase from Rhizopus arrhizus. The phos- pholipid reverse micelles that were formed around Celite-immobilized and water-activated lipase in n-hexane were also studied in order to increase the operational stability of the enzyme.

Materials and methods

Materials

Refined Malaysian palm oil (importer Vital Vrbas, Yu- goslavia) was used. The palm oil midfraction was obtained by a double-stage fractionation of the palm oil in n-hexane, according to the procedure described by Tanaka et al. 24 Sn-l,3 regiospecific lipase from Rhizo- pus arrhizus (400,000 units mg-J) was purchased from Sigma (St. Louis, MO). The enzyme was stored as a suspension in ammonium sulfate at 4°C. Celite (545, Serva, Germany) was used as a carrier for enzyme immobilization. Stearic acid (90% purity) was purchased from Sigma. Sigma olive oil emulsion was used to assess the lipolytic activity of lipase. Defatted soya lecithin was purchased from Uljarice DD (Belgrade, Yugoslavia). Cocoa butter (imported from Africa) was kindly supplied by Soko ~tark (Belgrade, Yugoslavia). Silica gel for column chromatography (70-230 mesh) was purchased from Kemika (Zagreb, Croatia) and used for the separation of triacylglycerols from the reaction mixture. All other chemicals were reagent grade.

Lipase immobilization

Lipase was immobilized on Celite by adsorption. An appropriate amount of inorganic support was mixed thoroughly with lipase solution in 0.2 M phosphate buffer (pH 7.0). This pH value was determined as an optimum for the lipase activity. The mixture of immobilized enzyme was dried in vacuum at 40°C overnight.13 Before use, the immobilized enzyme was hydrated by the addition of a known amount of distilled water.

Interesterification reaction

The interesterification of the palm oil midfraction and stearic acid by immobilized lipase was carried out in Erlenmeyer flasks (100 ml volume) with shaking (130 strokes min-~) at 37°C within 20 h. One gram of the substrate (palm oil midfraction) and 0.7 g of the stearic acid were dissolved in water-saturated n-hexane (4 ml). One-tenth gram of the Celite-immobilized lipase of various activities was added to the reaction mixture. The effects of the addition of defatted lecithin were also studied. In trials with lecithin addition, the reaction mixture was mixed for 1 min at the beginning of the reaction, using a vortex mixer.

Lipase-catalyzed interesterification: L. Mojovi6 et al.

Lipase assay

The lipolytic activity of immobilized lipase was as- sessed using the olive oil emulsion. The amount of free fatty acids released from the olive oil was determined by titration with 0.05 M NaOH. 25 The activities were expressed in IU (international units), where 1 IU is defined as the amount of enzyme required to produce 1 /~mol of free fatty acid per minute.

Analyt ical methods

The course of the lipase-catalyzed interesterification reaction, i.e., the substrate conversion, was followed by determining the stearic acid content incorporated in the triacylglycerols of the palm oil midfraction. The triacylglycerols were isolated from the reaction mixture by silica gel column chromatography according to Quinlin and Weiser. z6 The separated triacylglycerols were hydrolyzed, and the free fatty acids that were formed were esterified with boron trifluoride. 27 The acyl composition was determined by a gas chromato- graph (Varian 1400-FID) equipped with a hydrogen ion- ization detector. A stainless steel column (L = 2 m, ID = 2 mm) was packed with 4% Carbowax 20 M on Chromosorb W AW (80-100 mesh). The temperatures of the injection and column were 200 and 180°C, respectively. The flow rates of nitrogen, hydrogen, and air were 25, 25, and 250 ml min-1, respectively.

Results and discussion

Optimization o f the reaction conditions

In order to optimize the reaction conditions of the enzymatic interesterification, the optimum water content of Celite-immobilized lipase was determined. The effects of the activity of immobilized enzyme on substrate conversion were also studied, n-Hexane was selected as the reaction solvent because of its low denaturing effect on the enzyme. According to Laane et al., 13 n- hexane has a low polarity (the logarithm of the partition coefficient, log P, a measure of polarity, is 3.5) and is appropriate for biocatalytic reactions. Water-saturated n-hexane was used to completely avoid extraction of water from the enzyme microenvironment. The amount of water that is involved in this system by using water-saturated n-hexane is very low, about 0.04%.t3

Effect o f water content

This effect was studied by using vacuum-dried immobilized enzyme which was subsequently hydrated with various amounts of distilled water. As shown in Figure l , the highest percent of stearic acid in interesterified triacylglycerols was attained with the addition of 0.1 ml H20 g- l of immobilized enzyme. MacraC found a similar optimum degree of enzyme hydration (about 10%) for the interesterification of the palm oil midfraction with lipase from Aspergillus niger. Tanaka et al. 2 proposed activating the immobilized enzyme by addition of glycerol instead of water or buffer, and in such

Enzyme Microb. Technol., 1993, vol. 15, May 439

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woter content (ml/g) Figure 1 Effect of addit ion of water (ml H20 g 1 of immobi l ized enzyme) on the interesterif ication reaction. Reaction condit ions: t = 37°C,~ - = 20h, v = 130strokesmin 1, 1 g palm oil midfraction, 07 g stearic acid, 01 g Celi te-immobil ized lipase A = 50 IU, 4 ml n-hexane. (©) Stearic acid (%) in interesterified tr iacylglycerols

a way, a higher yield of interesterified triacylglycerols was achieved. Matsuo et a l } 8 suggested preparing the active interesterification catalysts containing low amounts of water (about 2%) by the slow and controlled drying of mixtures of diatomaceous earth and lipase solution. Chi et al. 29 reported that the effective water content depended on the type of inorganic carrier.

In general, a certain water content is necessary for enzyme activation and shifting the biocatalytic reaction. The water content is specific for each system, e.g., the particular enzyme, support, and solvent, and therefore has to be determined experimentally.

E f f e c t o f i m m o b i l i z e d e n z y m e ac t iv i ty

Interesterification was studied at various immobilized enzyme activities. The highest achieved stearic acid content in triacylglycerols was 35.4%, corresponding to the addition of 120 IU of immobilized enzyme per gram of palm oil midfraction (Figure 2). The relation- ship between the lipolytic enzyme activity and the stearic acid content was not linear. The increase of the immobilized enzyme activity above 120 IU had no effect on substrate conversion.

The course of interesterification at the highest stearic acid content (120 IU of enzyme added per 1 g of palm oil midfraction) is shown in Figure 3.

The highest substrate conversion rate was main- tained during the first 6 h of the reaction. The equilibrium state was established after 12-14 h. A decrease in the triacylglycerol content in the interesterified palm oil midfraction from 94% (at the beginning of the reac-

tion) to 76% at the end of the reaction time is, however, due to the hydrolytic reaction. It was estimated that the yield of interesterified triacylglycerols was 80.8%.

Yokozeki et al. 3 achieved a maximum of about 40% of the incorporated stearic acid in olive oil triacylglycerols. From a practical viewpoint, an amount of 33-36% of stearic acid, which is normally present in natural cocoa butter triacylglycerols, is sufficient for cocoa butter-like fats. Tanaka et al. z used 8000 IU of free lipase from Rhizopus delmar for the interesterification of 10 g of olive oil triacylglycerols. A much greater consumption of lipase in this system may be explained by the fact that the lipase was not previously immobilized on an inorganic support, and the disperser (Celite, sand, silica gel) was added separately. It is possible that lipase in this system was not completely adsorbed at the interface and therefore was not active.

The biochemical pathway of the lipase-catalyzed fat interesterification is not yet fully understood. Conse- quently, a mathematical model that would be based on reaction kinetics and that would give an adequate interpretation of the experimental data is still at the investigational level. Kyotani et al. 6'3° proposed a model for fat interesterification based on the formation of the glyceride enzyme complex. However, natural fats such as palm oil midfraction are complex mixtures of triacylglycerols. Therefore, a proper model should take into account lipase-catalyzed reactions of individual triacylglycerols, as well as di- and monoacylglycer- ols and free fatty acids. In addition, the reverse hydrolytic reaction can compete with interesterification to a

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lipolytic ectivity (IU) Figure2 Effect of added l ipolyt ic activity of immobi l ized enzyme on the interesterification reaction. Reaction condit ions: t = 37°C, "r = 20 h, v = 130 strokes min -1, 1 g palm oil midfraction, 0.7 g stearic acid, 0.1 g Cel i te- immobil ized lipase of various activities, 4 ml n-hexane. (©) Stearic acid (%) in interesterif ied tr iacylglycerols

440 Enzyme Microb. Technol., 1993, v o l 15, May

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significant extent. The positional specificity of lipase, its specificity toward fatty acids, and a decreasing water content in the reaction mixture due to the hydrolysis, should also be taken into account in modeling the interesterification of the palm oil midfraction.

Table 1 compares the compositions of the palm oil midfraction, the interesterified product, and cocoa butter (the reaction conditions are the same as in Figure 3). These data show that the acyl exchange during the interesterification was mainly between the palmitoyl group from the palm oil midfraction and the stearoyl group from the reaction mixture. Previous studies 31 with Rhizopus arrhizus lipase have shown that the enzyme was specific for long-chain fatty acids (more than 12 C-atoms). Saturated fatty acids were thus exchanged faster. For all these reasons, the enzyme was consid-

Table 1 Fatty acyl composi t ion of tr iacylglycerols of the palm oil midfraction, interesterif ied product, and cocoa butter

Palm oil Fatty midfraction Interesterified Cocoa butter acyl group (%) product (%)a (%)

14 : 0 0.9 1.0 0.4 16 :0 51.4 23.4 26.4 18 : 0 3.0 35.4 35.2 18 : 1 37.0 34.0 34.1 18 : 2 7.2 6.5 3.0 20 : 0 0.5 0.7 0.9

a Reaction condit ions are the same as in Figure 3

Lipase-catalyzed interesterification: L. Mojovi~ et al.

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Figure 4 Effect of lecithin on the interesterif ication reaction. (©) Without lecithin; ([3) 2% lecithin; (A) 5% lecithin. Reaction condit ions: t = 37°C, ~- = 20 h, v = 130 strokes min -1, 1 g palm oil midfraction, 0.7 g Cel i te- immobil ized lipase A = 50 IU, 4 ml n-hexane. Stearic acid (%) in interesterif ied tr iacylglycerols

ered as appropriate for the system investigated in this article.

As shown in Table 1, the fatty acyl composition of the interesterified product approached that of the cocoa butter. From the prospective of using the interesterified products as cocoa butter equivalents, a further system- atic comparison with cocoa butter will be necessary. It should include comparison of the amounts of individual triacylglycerol species, as well as physical character- ization.

Effect of lecithin addition

The time course of interesterification with and without lecithin addition was compared. As shown in Figure 4, addition of 5% of the defatted soya lecithin per mass of the immobilized enzyme caused a significant increase in the stearic acid content in the interesterified triacylglycerols, i.e., from 22% (without addition) to 36% (with 5% lecithin). Addition of 2% of the deoiled soya lecithin caused a lower increase in the stearic acid content (Figure 4). A further increase of the amount of lecithin to 10% (data not shown) had no significant effect on the substrate conversion. The effect of lecithin is of great importance, as it results in the same degree of interesterification at substantially lower activities of the enzyme.

The increase of the enzyme-catalyzed s ubstrate conversion with lecithin addition is attributed to the formation of the reverse micellar system around the Celite- immobilized and hydrated enzyme. The reverse mi-


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Figure 5 Stabi l i ty of the enzyme. (A) Cel i te- immobi l ized lipase A = 50 IU; (11) Cel i te- immobi l ized l ipase A = 50 IU wi th addi t ion of 5% lecithin per mass of immobi l i zed enzyme. Reaction condi- t ions as in Table 2. Stearic acid (%) is % of stearic acid present in interester i f ied t r iacy lg lycero ls

celles protect the enzyme from the nonpolar solvent, and enhance the solubility and diffusion of the substrate and product.

The molar ratio of water to surfactant (R) was calculated taking an average molecular weight of the defatted soya lecithin. In recent studies of reverse micellar systems with free lipase, ~7'23 an optimum ratio R = 10 was reported. For the system studied in this paper, R = (H20)/(lecithin) > 50 when 5% of the lecithin was added per mass of immobilized enzyme. The amount of the lecithin that was effective for enhancement of the activity of the immobilized enzyme in our studies was much lower when compared with the classical reverse micellar systems with free enzyme. We suppose that in our system lecithin was not consumed for enzyme dispersion in the organic solvent, since the enzyme was already dispersed by immobilization on Cel- ite particles. Therefore, much smaller amounts of lecithin could provide enzyme protection from the solvent and enhance solubility and diffusion of the substrate and product in the enzyme microenvironment.

As shown in Figure 5, the activity of the Celite- immobilized lipase decreased to about half the initial level after use in five successive reaction batches (corresponding to the operational time of 4.2 days), while the lecithin-protected, Celite-immobilized lipase was more stable. More than 60% of the original activity was preserved after six reaction batches (corresponding to the operational time of 5 days). However, the advantage of the lecithin-protected, Celite-immobilized lipase system was not only in the enhanced stability, but also in a higher operational productivity, as shown in

Table 2. The average productivity of the lecithin-protected, Celite-immobilized lipase was 1.6-fold higher than that of the Celite-immobilized lipase. The yield of the triacylglycerois in both systems was similar, and reached about 80%. This study demonstrated that the lecithin reverse micellar system increased the rate of the interesterification, but in turn did not increase the rate of reversible reaction.

Conclusions

In this article the optimum conditions for retention of lipase activity and for lipase-catalyzed palm oil midfraction interesterification were studied.

Under the optimum conditions for the Celite-immobilized, lipase-catalyzed interesterification that are reported in this article, a product with a fatty acid composition similar to cocoa butter was obtained.

By the addition of defatted soya lecithin, a reverse micellar system was formed around Celite-immobilized enzyme that increased the degree of interesterification. This effect was attributed to the protection of enzyme from the nonpolar solvent environment and to the enhancement of the solubility and diffusion of the substrate and product in the enzyme microenvironment. The calculated molar ratio (R) of water to lecithin was above 50. This value is different from that for the ordi- nary reverse micellar systems using free enzymes, and corresponds to a significantly lower amount of lecithin in the reaction mixture. We suppose that the formation of reverse micelles, and the resulting enhancement of interesterification, could be attributed to the combined effects of enzyme immobilization and lecithin addition.

The advantage of the lecithin-protected, Celite-immobilized enzyme system is the higher operational stability and, in the first instance, the higher productivity when compared with Celite-immobilized lipase. Fur- thermore, the lecithin-protected, Celite-immobilized system can hopefully be applied to the production of fats and oils, especially for the food industry.

Table 2 Incorporat ion of stearic acid into t r iacy lg lycero ls of the palm oil midfract ion by l ipase preparat ions in repeated batches

Celite- Leci th in-protected? immobi l i zed a Cel i te- immobi l ized

l ipase l ipase

Total amount of incor- 740 porated stearic acid (mg per 5 batches c)

Average product iv i ty 0.030 (mg stearic acid/h IU)

1212

0.049

a 0.1 g Cel i te- immobi l ized l ipase A = 50 iU b 0.1 g Cel i te- immobi l ized l ipase A = 50 IU + 5% deoi led soya lecithin per mass of immobi l i zed enzyme c Reaction condi t ions: t = 37°C, ~- = 20 h, v = 130 strokes min 1, 1 g palm oil midfract ion, 0.7 g stearic acid, 0.1 g Cel i te- immobi- lized lipase, 4 ml n-hexane

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Rhizopus arrhizus lipase-catalyzed interesterification of the midfraction of palm oil to a cocoa butter equivalent fat