EFFECTS OF ENZYMATIC HYDROLYSIS ON CRUDE PALM OLEIN BY LIPASE FROM CANDIDA RUGOSA

L.L. YOU1 and B.S. BAHARIN Department of Food Technology Faculty of Science and Food Technology University Putra Malaysia Selangor D.E., Malaysia

ABSTRACT The effects of enzymatic hydrolysis on crude palm olein (CPOlein) by lipase from Candida rugosa were investigated. Reaction variables, namely water content, reaction temperature and enzyme concentration on hydrolysis of CPOlein were examined. Comparison was also made between CPOlein and hydrolyzed crude palm olein (HCPOlein) for melting point, percentage of free fatty acids (FFA) and viscosity. The optimum conditions for the production of hydrolyzed oil or FFA-rich oil in enzymatic hydrolysis of CPOlein were reaction temperature of 50C, 1% (w/w) lipase from C. rugosa and 50% (w/w) water content. FFA in CPOlein increased to 97.9% after the hydrolysis process, which showed an increase of 61 folds. The differences in viscosity between the CPOlein and HCPOlein at different temperatures were statistically signicant (P 0.05). The slip melting point of CPOlein was 17C. After hydrolysis, the melting point for CPOlein increased by 160%, reaching 44.4C.

INTRODUCTION Fats and oils are esters of the fatty acids with the trihydric alcohol, glycerol. In fats and oils modication, using enzymes instead of chemicals confers several advantages such as the specicity of enzymes and the mild conditions under which they function. Furthermore, enzymes are biodegradable and could reduce environmental loading (Lai et al. 1998). Chemical catalysts randomize fatty acids in triacyglycerol mixtures and fail to yield products with desired physiochemical characteristics. Among attractions in replacing the current chemical technology with enzyme biotechnology are energy savings and minimization of thermal degradation (Akoh 1997). Enzymes selectively lower the activation energies of biochemical reactions
1

Corresponding author. TEL: 603-89468394; FAX: 603-89483552; EMAIL: badli@fsb.upm.edu.my

Journal of Food Lipids 13 (2006) 7387. All Rights Reserved. 2006, The Author(s) Journal compilation 2006, Blackwell Publishing

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that they catalyze with enhancements in reaction rates relative to nonenzymatic reactions that could reduce deterioration in the quality and characteristics of the oils. Hydrolysis of fats occurs through the splitting of the fat molecules and the addition of water, leading to the formation of free glycerol and fatty acids. The hydrolysis process not only produces free fatty acids (FFA), but also mono- and diacyglycerols. Hydrolysis in oils and fats may be carried out autocatalytically, catalyzed by either metals or lipase. Lipase hydrolysis is more promising as an energy-saving process because the reaction can be carried out at room temperature and by using pressure, without denaturation of biologic substances such as highly unsaturated fatty acids, tocopherols and carotenoids in crude palm olein (CPOlein). Enzymes are usually accepted as food additives, especially when they are part of a natural extract, sometimes of other food materials. Moreover, most enzymatic processes rely on simple hydrolases, often carbohydrases or proteases, to hydrolyze macromolecules without any need for expensive cofactors (Wiseman 1975). Hydrolases such as lipases are widely used in the fats and oils industries. Enzymatic lipid hydrolysis is widely used in the purication of polyunsaturated fatty acids (Tanaka et al. 1992; McNeill et al. 1996; Shimada et al. 1997a,b; Wanasundara and Shahidi 1998). Nonspecic lipases such as lipase from Candida rugosa or Pseudomonas cepacia lack stereospecicity for triacyglyerols and can catalyze complete hydrolysis. Lineld et al. (1984) reports that C. rugosa lipase is a suitable enzyme used in splitting palm oil. Moreover, complete hydrolysis could be achieved in about 56 h. Therefore, nonspecic lipase isolated from C. rugosa (mesophile) is used for the hydrolysis process in this work. In this study, CPOlein was hydrolyzed rst to produce an oil rich in FFAs, which is more polar and hence could theoretically enhance the recovery of the less polar carotenes (You et al. 2002). Effects of water content, reaction temperature and enzyme concentration on the hydrolysis of CPOlein were examined. Comparison was also made between CPOlein and HCPOlein (hydrolyzed crude palm olein) for properties such as melting point, percentage of FFA produced and viscosity.

MATERIALS AND METHODS Palm Oil and Chemicals CPOlein was obtained from Jomalina, Teluk Panglima Karang, Selangor, Malaysia. The oil sample was stored in a cold room at 4C. Lipase from C. rugosa was obtained from Amano Co. (Tokyo, Japan).

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Hydrolysis Process Effect of Temperature. CPOlein was hydrolyzed using lipase from C. rugosa at 50, 55, 60, 65 and 70C with 50% (w/w) water and 1% (w/w) enzyme. The mixture was agitated using a Thermolyne cimarec-top hot stir plate (Sigma, St. Louis, MO) for homogeneous mixing. Each ask was plugged with a silicone rubber stopper to prevent evaporation, and each ask was also wrapped with aluminum foil to minimize carotene degradation by light. Samples were withdrawn at selected time intervals of 30 min, 1, 2, 3, 4, 5, 6 and 8 h of reaction. The samples were centrifuged at 6700 g for 5 min to remove the lipase. The water present in the samples was removed by drying over anhydrous sodium sulfate (Sigma). Effect of Water Content. CPOlein was hydrolyzed with lipase from C. rugosa at 50C with 20, 40, 50, 60 and 80% (w/w) of water content and 1% (w/w) enzyme concentration. The mixture was agitated using a magnetic stirrer for homogenous mixing. Effect of Enzyme Concentration. CPOlein was hydrolyzed with lipase from C. rugosa at 50C with 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% (w/w) of enzyme and 50% (w/w) of water. The mixture was agitated using a magnetic stirrer for homogenous mixing. Preparation of HCPOlein. One hundred grams of CPOlein was hydrolyzed with 2 g of lipase (1% w/w) from C. rugosa at 50C for 8 h. One hundred grams of distilled water, essential for hydrolysis, was added into the mixture of oil and enzyme to obtain a total of 200 g of reaction mixture. After hydrolysis, tha samples were centrifuged at 6700 g for 5 min to remove the lipase. The water present in the samples was dried with anhydrous sodium sulfate (Sigma). The HCPOlein was then stored at 4C in the dark.

Analytical Procedures Determination of Acidity. Acidity (% FFA) of the hydrolyzate was determined using the Palm Oil Research Institute of Malaysia test method (Siew et al. 1995). The acidity is the content of FFA conventionally expressed as the percentage of palmitic acid for palm oil and fractions; lauric acid for coconut oil, palm kernel oil and fractions; and oleic acid for corn oil, soybean oil and other liquid oils. The acid value is the number of milligrams of Na or KOH necessary to neutralize the FFAs in 1 g of sample.

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The specic amounts of samples were weighed into Erlenmeyer asks (Kimble/Kontes, Vineland, NJ). Fifty milliliters of neutralized isopropyl alcohol was added into the asks to dissolve the samples, with the temperature regulated to about 40C. Phenolphthalein (Sigma) was added to the solution and was neutralized by dropwise addition of 0.1-N Na or KOH (Sigma) until a faint, but permanent pink persisted. The samples were shaken gently while titrating with 0.1-M NaOH or KOH to the rst permanent (for 30 s) pink. The results were expressed as % FFA as oleic acid = (28.2 Mx V)/W, where M is the molarity of NaOH or KOH solution; V is the volume of NaOH or KOH solution used (mL); and W is the weight of sample (g). This equation was used to calculate the percentage of FFA produced during the hydrolysis process in CPOlein. The values were expressed to three decimal places for FFA below 0.15% and to two decimal places for FFA above 0.15% (Siew et al. 1995). Determination of Slip Melting Point (SMP). SMP is the temperature at which fat in an open-ended capillary column of a specied length rises when incubating at the specied conditions of the test. A prepared capillary tube containing the fat was immersed in a water bath warmed at a specied rate until the melting point was reached. This method is applicable to all normal fats and vegetable oils that are solid at ambient temperature, particularly palm oil and palm oil products. The samples were melted and ltered through the no. 4 qualitative lter paper (Whatman International Limited, Maidstone, England). Three clean capillary tubes were dipped into the liquid sample of the oil so that columns of fat ca. 10-mm high were obtained in the tubes. The column was chilled in ice until the fat solidied. The capillaries were placed in test tubes, held in a beaker of water that had been equilibrated at 10C in a thermostated water bath for 16 h (or 2 h for quick test). The capillary tubes were removed and were attached to a thermometer such that the lower ends of the tubes leveled off with the end of the thermometer bulb. The thermometer and the lower end of the capillary tube were immersed in the water bath to a depth of ca. 30 mm, and the starting temperature was 810C below the expected slip point of the sample. The water bath was agitated with a magnetic stirrer on a hot plate for homogeneous heat transfer for temperature to increase at a rate of 1 C/min, but was reduced to 0.5 C/min nearing the slip point. Heating was continued until the fat rose in each tube to the upper end of the capillary tube. The average value of two sets of triplicate results was calculated as the SMP and was expressed to one decimal place (Siew et al. 1995). Viscosities of CPOlein and HCPOlein. The determination of viscosity of the oil samples was carried out at different temperatures of 45, 50, 55 and

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TABLE 1. EFFECT OF TEMPERATURE (C) ON THE PERCENTAGE OF FREE FATTY ACIDS PRODUCED DURING THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA t (h) 0.0 0.5 1.0 2.0 3.0 4.0 5.0 6.0 8.0 50 2.0 0.3 85.3 0.6a 86.0 1.3a 89.9 1.3a 91.7 0.3a 94.5 0.3a 95.9 0.6a 95.9 1.4a 96.3 0.7a
a

55 2.0 0.3 81.6 0.8b 83.8 0.6a 87.9 0.8a 89.0 0.7a 92.2 1.0a 92.5 0.7b 92.7 1.1b 94.8 0.3a
a

60 2.0 0.3 78.6 0.3b 80.0 1.1b 85.0 0.4a 88.1 0.1a 88.6 0.7b 89.0 0.8c 92.1 0.1b 92.4 0.4b
a

65 2.0 0.3 49.9 1.7c 52.4 1.4c 60.0 1.3b 62.3 0.4b 64.6 0.4c 64.9 1.0d 65.5 0.7c 65.9 1.4c
a

70 2.0 0.3a 23.7 0.4d 25.5 0.4d 29.9 1.7a 31.6 1.6c 32.2 0.3d 32.2 0.6e 32.4 0.6d 33.4 0.8d

Note: Each datum represents the mean values SD of analyses from two replications. Mean values within each column with the same superscript are not signicantly different (P 0.05). t, time.

60C using a programmable rheometer viscometer (Brookeld model DV III, Brookeld Engineering Laboratories, Middleboro, MA) attached to a Thermomix model 1419 (B. Braun, Bethlehem, PA) for temperature control. Statistical Analysis Results show the mean values of at least two replicates with SD. Analysis of variance using t-test for these variables, temperature, enzyme concentration and water content in the hydrolysis process was carried out using Statistical Analysis Software program (SAS Institute, Cary, NC). Mean values with different superscripts were signicantly different (P 0.05).

RESULTS AND DISCUSSION Hydrolysis Process Effect of Temperature. Table 1 shows the effect of temperature on the percentage of FFA produced during the hydrolysis process by lipase from C. rugosa; the percentage of FFA produced showed a signicant decrease with an increase in temperature. The trend for this reaction can be clearly seen in Fig. 1. The difference may be attributable to the optimum temperature under which the enzymes function. The optimum temperature for lipase from C. rugosa is 45C. With temperature increment from 50C, the activity of lipase from C. rugosa may already be suboptimal because of the denaturation of the

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100

80 Free fatty acid (%)

60

40

20

0 8

7 6 5 4 3 2 1 0
t (h)

50 55 60 65 70
T (C)

FIG. 1. THE EFFECT OF TEMPERATURE ON THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA t, time; T, temperature.

enzyme. Lipase from C. rugosa, however, may be a suitable candidate for the hydrolysis process, as the lower temperature would reduce carotene degradation. Unfortunately, HCPOlein starts to harden at temperatures below 47C. Hence, 50C for lipase from C. rugosa was used in subsequent experiments. Over a range of temperatures, the overall enzyme-catalyzed reaction rate passes through a maximum. The temperature at which the rate is a maximum is known as the optimum temperature whose values vary with enzyme concentration and other factors, such as water content. Changing the temperature affects the catalyzed reaction itself and the thermal inactivation of the enzyme. However, inactivation is very slow and has no appreciable effect on the rate of the catalyzed reaction. Hence, the overall rate of reaction often increases with the rise in temperature, as commonly reported in ordinary chemical reactions. Inactivation becomes more important at higher temperatures such that the concentration of active enzyme falls during the course of reaction. During the enzyme inactivation process, protein denaturation could arise from conformational changes. It can be assumed that bonds (perhaps hydrogen bonds) may be

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TABLE 2. EFFECT OF PERCENTAGE OF WATER CONTENT ON THE PERCENTAGE OF FREE FATTY ACIDS PRODUCED DURING THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA AT 50C t (h) 0.0 0.5 1.0 2.0 3.0 4.0 5.0 6.0 8.0 20 2.0 0.3a 82.8 1.1a 84.6 0.8a 85.4 0.3a 86.1 0.4a 86.5 0.7a 86.6 0.6a 87.4 0.6a 88.9 0.1a 40 2.0 0.3a 83.0 1.6a 84.9 1.1a 88.0 1.6b 89.5 0.7b 92.6 0.8b 94.8 0.8b 94.9 0.6b 95.0 0.1b 50 2.0 0.3a 85.3 0.7b 86.0 1.4b 89.9 0.7c 91.7 0.6c 94.5 0.7c 95.9 1.0b 95.9 0.6b 96.3 0.4c 60 2.0 0.3a 81.8 0.8a 85.4 0.6a,b 90.1 0.6c 91.1 0.6c 91.4 0.6b 93.1 0.6c 95.2 0.3b 95.4 0.1b 80 2.0 0.3a 2.0 0.1c 2.4 0.3c 2.8 0.4d 2.8 0.6d 2.9 0.1d 3.0 0.1d 3.3 0.4c 3.4 0.1d

Note: Each datum represents the mean values SD of analyses from two replications. Mean values within each column with the same superscript are not signicantly different (P 0.05). t, time.

broken, and that the unfolded molecule ceases to function as it should in its correct conformation (Laidler and Peterman 1979). Lipase from C. rugosa showed signicant reductions in the percentage of FFA produced with every 5C increase starting from 50C. The explanation is that the temperature coefcient of the rate of inactivation must be greater than that of the rate of the catalyzed reaction. In the low temperature range, the rate of inactivation is negligible compared with the rate of the catalyzed reaction, whereas in the high temperature range, it is higher (Laidler and Peterman 1979). Immobilization may increase the stability of lipases at high temperatures (Haas et al. 1994). Product inhibition could also bring about a dramatic decrease in lipase activity. Dunhaupt et al. (1992) reported that FFA decreased the hydrolytic activity of P. cepacia lipase in olive oil. Similar results are reported by Lenki et al. (1998) for the hydrolysis of butterfat fraction. Effect of Water Content. Table 2 shows the effect of water content on the percentage of FFA produced during the hydrolysis process at 50C by lipase from C. rugosa. The percentage of FFA produced generally increased signicantly with an increase in water content throughout the course of hydrolysis. Further increase in water content from 50% did not signicantly increase the FFA production. The effect of water content on FFA production in the hydrolysis process by lipase from C. rugosa is shown in Fig. 2. Water is essential in catalyzing the hydrolysis process. With the result obtained, the water content that is required in the hydrolysis process is 50%

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100

80 Free fatty acid (%)

60

40

20

0 8
7 6 5 4 3 2 1 0

t (h)

80

70

60

50

40

30

20

Water content (%)

FIG. 2. THE EFFECT OF WATER CONTENT ON THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA t, time; T, temperature.

(w/w) or at 1:1 oil to water ratio. At lower water content (less than 50%), the degree of hydrolysis was incomplete. Because lipase catalyzes not only hydrolysis but also the reverse esterication reaction simultaneously, a large amount of water is necessary to shift the equilibrium toward hydrolysis. However, when hydrolysis was conducted in the mixture containing more than 50% (w/w) water, the degree of hydrolysis decreased (Shimada et al. 1997a). Results showed that at above 50% (w/w) water, there was a signicant decrease in the percentage of FFA production. Therefore, 50% water was used for subsequent hydrolysis processes. Lipases act at the oil/water interface of heterogeneous reaction systems. In an aqueous medium, hydrolysis is the dominant reaction, but in organic media, esterication and interesterication reactions are predominant. High water levels reduce lipase-catalyzed esterication or transesterication, presumably by causing hydrolysis of the acylenzyme intermediate (Valivety et al. 1993). Therefore, high water levels increase the rate of the hydrolysis process. Wehtje and Adlercreutz (1997) report that the activities of many enzymes such as proteases, glycosidases and lipases versus water activities show an increasing curve. Lipase hydrolytic

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TABLE 3. EFFECT OF ENZYME CONCENTRATION ON THE PERCENTAGE OF FREE FATTY ACIDS PRODUCED DURING THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA AT 50C t (h) 0.0 0.5 1.0 2.0 3.0 4.0 5.0 6.0 8.0 0.5 2.0 0.3a 75.0 0.8a 80.0 1.6a 84.0 2.1a 85.9 2.3a 86.9 1.4a 88.9 0.6a 86.9 1.6a 88.9 1.3a 1.0 2.0 0.3a 85.3 1.6b 86.0 1.6b 89.9 1.3b 91.7 2.0b 94.5 0.6b 95.9 1.3b 95.9 1.6b 96.3 0.6b 1.5 2.0 0.3a 87.0 0.7b 88.0 0.1b 89.0 0.6b 92.5 1.0b 94.2 0.6b 96.0 0.6b 96.7 0.3b 96.9 0.3b 2.0 2.0 0.3a 88.4 0.6b 90.0 1.1c 91.4 0.6b 92.6 2.0b 96.0 0.3b 96.5 1.0b 96.8 0.4b 98.7 1.0c 2.5 2.0 0.3a 94.5 0.8c 97.8 1.0d 97.9 1.3c 98.5 0.6c 98.7 0.7c 98.7 0.3c 98.9 1.0c 99.0 0.1c 3.0 2.0 0.3a 95.3 0.6c 96.4 1.0d 96.9 0.6c 97.1 0.5c 98.5 0.7c 98.7 0.7c 99.0 0.4c 99.2 0.1c

Note: Each datum represents the mean values SD of analyses from two replications. Mean values within each column with the same superscript are not signicantly different (P 0.05). t, time.

activity is sharply water-dependent (Haas et al. 1995). Water is a cosubstrate in the hydrolysis reactions, with one water molecule being consumed for each ester bond hydrolyzed. Water is also essential for the retention of lipase activity in organic solvents (Haas et al. 1993). Because lipase catalyzes not only hydrolysis but also esterication, sufcient amounts of water are necessary to shift the equilibrium toward hydrolysis (Shimada et al. 1997b). High water levels, however, reduce the hydrolytic activity dramatically (Haas et al. 1995; Shimada et al. 1997b). This may be attributed to the dilution effect of lipase concentration in the water phase (Shimada et al. 1997b). Haas et al. (1994) reported that water requirements are unique to each lipase and vary in proportion to substrate concentration. Thus, it is reasonable to believe that increased concentration of water will decrease the activity in the synthesis process, but could enhance the hydrolytic process. Effect of Enzyme Concentration. Table 3 shows the effect of enzyme concentration on the percentage of FFA produced during the hydrolysis process by lipase from C. rugosa. Results show that the percentage of FFA produced increased with an increase in enzyme concentration, especially from 0.5 to 1%, showing a drastic increase in the percentage of FFA produced because higher concentration of enzyme enhanced hydrolytic activity. The lipase from C. rugosa, at a concentration of 12% had no signicant differential effect on the production yield, possibly because of the limitation of substrate for the rst half hour of the hydrolysis process. However, there was a signicant difference in the percentage of FFA produced for enzyme

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100 90 80 Free fatty acid (%) 70 60 50 40 30 20 10 0 8

6 4
1 0.5

2 0
t (h)

1.5 2 2.5 3
Enzyme concentration (%)

FIG. 3. THE EFFECT OF ENZYME CONCENTRATION ON THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA t, time; T, temperature.

concentrations varying from 2 to 3%. However, 1% enzyme concentration was used for further studies because of economical factors. There is not much FFA production during the latter stages of the hydrolysis process, and the enzyme activity decreased because of the denaturation occurring during the hydrolysis process. For higher enzyme concentrations, equilibrium can be achieved at a faster rate. The effect of enzyme concentration on FFA production in the hydrolysis process at 50C is shown in Fig. 3. At low lipase concentration, the degree of hydrolysis is normally low. Prolonged reaction would not further increase the hydrolysis yield (McNeill et al. 1996; Shimada et al. 1997a,b). To increase the degree of hydrolysis, it was necessary to add successively high amounts of lipase (Kosugi et al. 1988; McNeill et al. 1996). The degree of hydrolysis increased with an increase in the amount of lipase until it reached plateau (Shimada et al. 1997a,b). At low and moderate lipase levels, the addition of water had little impact upon hydrolysis. At high lipase levels, hydrolysis was retarded by increasing the amount of water (Haas et al. 1993).

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TABLE 4. THE PERCENTAGE OF FREE FATTY ACIDS (FFA) AND SLIP MELTING POINT (SMP) OF CPOLEIN AND HCPOLEIN Oil CPOlein HCPOlein FFA (%) 2.0 0.3 96.3 1.0b
a

SMP (C) 17.0 0.0a 44.4 0.7b

Note: Each datum represents the mean values SD of analyses from two replications. Mean values within each column with the same superscript are not signicantly different (P 0.05). CPOlein, crude palm olein; HCPOlein, hydrolyzed crude palm olein.

Characteristics of CPOlein and HCPOlein FFA Production. CPOlein was hydrolyzed by lipase from C. rugosa at 50C, shaken at 700 rpm for 8 h. After hydrolysis, the percentage of FFA produced was determined. The results are shown in Table 4. FFA in CPOlein increased to 97.9%, reecting a 61.0% fold increase after the hydrolysis process. SMP. The SMP of CPOlein was 17C. After hydrolysis, the melting point for CPOlein was increased by 160% and then reached 44.4C. The relatively linear form of FFA in HCPOlein, which can lead to good molecular packing in a semisolid phase and can cause a higher melting point. The double bonds in the oil molecules are geometrically in the cis conguration, which kicks the chain and makes it difcult to form an organized solid structure. Oleic acid (C18:1D9), an unsaturated fatty acid, results in a noticeable lowering of the melting point and hence, of liquid oil at room temperature (Bailey and Bailey 2000). As a result, the more double bonds in the fatty acids portion in the triacylglycerol, the lower is the melting point. Viscosity. Viscosity is a measure of internal friction in molecules that creates resistance to ow. The coefcient of viscosity, h, is dened as the force per unit area required to maintain a unit difference of velocity between two parallel layers that are a unit distance apart. When force per unit area is in dynes per square centimeter, velocity is in centimeter per second, and distance is in centimeter, h is expressed in poise (Marvin et al. 1979). Viscosity is temperature dependent; hence, temperature control is essential. The relationship between temperature and viscosity is inverse; viscosity decreases as the temperature increases. Oils are normally liquid at ambient temperature and fats are normally solid, but oils are more viscous than water, and most food-grade oils exhibit a Newtonian behavior.

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TABLE 5. COMPARISON OF VISCOSITY BETWEEN CPOLEIN AND HCPOLEIN AT DIFFERENT TEMPERATURES AT 53 rpm Temperature (C) CPOlein 50 55 60 24.2 0.1a 21.1 0.3a 19.3 0.1a Viscosity (cP) HCPOlein 12.7 0.1b 10.9 0.3b 10.7 0.4b

Note: Each datum represents the mean values SD of analyses from two replications. Mean values within each column with the same superscript are not signicantly different (P 0.05). CPOlein, crude palm olein; HCPOlein, hydrolized crude palm olein.

In this study, the viscosity of CPOlein and HCPOlein was determined at different temperatures and stirring rates. As viscosity is also a measure of the ow rate, the understanding of the viscosity of each oil type has assisted the monitoring of the ow rate of the oil in the adsorption column chromatography. In all cases, the viscosity decreased with the increase in temperature and also with the increase in stirring rate. The results indicate that CPOlein and HCPOlein reached a plateau after 30 rpm. The hydrolyzed oil was less viscous with a plateau at around 10 centipoise (cP) compared with the crude oil which had a viscosity plateau at around 20 cP. At 50C and at 53 rpm, HCPOlein was 48% less viscous than CPOlein. Table 5 shows the comparison of viscosity between CPOlein and HCPOlein at different temperatures at 53 rpm. The differences in viscosity between the oils at different temperatures were statistically signicant (P 0.05). This can be attributed to the changes of triacylglycerols, which are more viscous compared with FFA, which are less viscous. Oils owe their relatively high viscosities to the intermolecular attractions of the long chains of their triacylglycerol molecules (Marvin et al. 1979). Different oils have different fatty acid compositions; hence, the increase in the amount of long-chain fatty acids and the degree of saturation increase the viscosities. The viscosity of hydrolyzed oil was less than that of the crude oil because of changes in oil form, from triacylglycerol to FFA forms that are less viscous. The molecular size of triacylglycerol is larger than FFA; therefore, triacylglycerols have stronger intermolecular forces compared with FFA and contribute to friction while stirring or mixing. Therefore, the higher the friction during stirring, the higher viscosity values can be obtained (Lewis 1987). This explains the fact that a decrease in molecular size results in a decrease in viscosity; hence, HCPOlein is less viscous than CPOlein.

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CONCLUSION In conclusion, reaction temperature at 50C, 1% (w/w) lipase used from C. rugosa and 50% (w/w) of water content were the optimum conditions for the production of hydrolyzed oil or FFA-rich oil for CPOlein. HCPOlein, which is less viscous compared with CPOlein, is preferred in this hydrolysis process.

ACKNOWLEDGMENTS Financial support from Intensication of Research in Priority Area Grant No. 03-02-04-0141-EA001 of the National Council for Research and Science Development in Malaysia is acknowledged. The supply of CPOlein from Golden Jomalina Food Industries Sdn. Bhd., Teluk Panglima Garang, Selangor, Malaysia is also gratefully acknowledged.

REFERENCES AKOH, C.C. 1997. Making new structured fats by chemical reaction and enzymatic modication. Lipid Technol. 7, 6166. BAILEY, P.S., JR. and Bailey, C.A. 2000. Bonding inorganic compounds. In Organic Chemistry: A Brief Survey of Concepts and Applications, pp. 736, Prentice Hall, Inc., Upper Saddle River, NJ. DUNHAUPT, A., LANG, S. and WAGNER, F. 1992. Pseudomonas cepacia lipase: Studies on aggregation, purication and the cleavage of olive oil. Biotechnol. Lett. 14, 953958. HAAS, M.J., CICHOWICZ, D.J., JUN, W. and SCOTT, K. 1995. The enzymatic hydrolysis of triglyceride-phospholipid mixtures in an organic solvent. J. Am. Oil Chem. Soc. 72, 519525. HAAS, M.J., CICHOWICZ, D.J., PHILIPS, J. and MOREAU, R. 1993. The hydrolysis of phosphatidylcholine by an immobilized lipase: Optimization of hydrolysis in organic solvents. J. Am. Oil Chem. Soc. 70, 111117. HAAS, M.J., SCOTT, K., JUN, W. and JANSSEN, G. 1994. Enzymatic phosphatidylcholine hydrolysis in organic solvents: An examination of selected commercially available lipases. J. Am. Oil Chem. Soc. 71, 843849. KOSUGI, Y., SUZUKI, H. and FUNADA, T. 1988. Hydrolysis of beef tallow by lipase from Pseudomonas sp. Biotechnol. Bioengineer. 31, 349356. LAI, O.M., GHAZALI, H.M. and CHONG, C.L. 1998. Effect of enzymatic transesterication on the melting points of palm stearin-sunower oil mixtures. J. Am. Oil Chem. Soc. 75, 881885.

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