Effects of Enzymatic Hydrolysis on Crude Palm

EFFECTS OF ENZYMATIC HYDROLYSIS ON CRUDE PALMOLEIN BY LIPASE FROM CANDIDA RUGOSA

L.L. YOU1 and B.S. BAHARIN

Department of Food TechnologyFaculty of Science and Food Technology

University Putra MalaysiaSelangor D.E., Malaysia

ABSTRACT

The effects of enzymatic hydrolysis on crude palm olein (CPOlein) bylipase from Candida rugosa were investigated. Reaction variables, namelywater content, reaction temperature and enzyme concentration on hydrolysisof CPOlein were examined. Comparison was also made between CPOlein andhydrolyzed crude palm olein (HCPOlein) for melting point, percentage of freefatty acids (FFA) and viscosity. The optimum conditions for the production ofhydrolyzed oil or FFA-rich oil in enzymatic hydrolysis of CPOlein were reac-tion temperature of 50C, 1% (w/w) lipase from C. rugosa and 50% (w/w) watercontent. FFA in CPOlein increased to 97.9% after the hydrolysis process,which showed an increase of 61 folds. The differences in viscosity between theCPOlein and HCPOlein at different temperatures were statistically significant(P � 0.05). The slip melting point of CPOlein was 17C. After hydrolysis, themelting 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 modification, using enzymes instead of chemicalsconfers several advantages such as the specificity of enzymes and the mildconditions under which they function. Furthermore, enzymes are biodegrad-able and could reduce environmental loading (Lai et al. 1998). Chemicalcatalysts randomize fatty acids in triacyglycerol mixtures and fail to yieldproducts with desired physiochemical characteristics. Among attractions inreplacing the current chemical technology with enzyme biotechnology areenergy 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: [email protected]

Journal of Food Lipids 13 (2006) 73–87. 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 nonenzy-matic reactions that could reduce deterioration in the quality and characteris-tics of the oils.

Hydrolysis of fats occurs through the splitting of the fat molecules andthe addition of water, leading to the formation of free glycerol and fatty acids.The hydrolysis process not only produces free fatty acids (FFA), but alsomono- and diacyglycerols. Hydrolysis in oils and fats may be carried out auto-catalytically, catalyzed by either metals or lipase. Lipase hydrolysis is morepromising as an energy-saving process because the reaction can be carried outat room temperature and by using pressure, without denaturation of biologicsubstances such as highly unsaturated fatty acids, tocopherols and carotenoidsin crude palm olein (CPOlein). Enzymes are usually accepted as food addi-tives, especially when they are part of a natural extract, sometimes of otherfood materials. Moreover, most enzymatic processes rely on simple hydro-lases, often carbohydrases or proteases, to hydrolyze macromolecules withoutany 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 purification of polyunsatu-rated fatty acids (Tanaka et al. 1992; McNeill et al. 1996; Shimada et al.1997a,b; Wanasundara and Shahidi 1998). Nonspecific lipases such as lipasefrom Candida rugosa or Pseudomonas cepacia lack stereospecificity for tria-cyglyerols and can catalyze complete hydrolysis. Linfield et al. (1984) reportsthat C. rugosa lipase is a suitable enzyme used in splitting palm oil. Moreover,complete hydrolysis could be achieved in about 5–6 h. Therefore, nonspecificlipase isolated from C. rugosa (mesophile) is used for the hydrolysis processin this work.

In this study, CPOlein was hydrolyzed first to produce an oil rich in FFAs,which is more polar and hence could theoretically enhance the recovery of theless polar carotenes (You et al. 2002). Effects of water content, reactiontemperature and enzyme concentration on the hydrolysis of CPOlein wereexamined. Comparison was also made between CPOlein and HCPOlein(hydrolyzed crude palm olein) for properties such as melting point, percentageof 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).

74 L.L. YOU and B.S. BAHARIN

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 flask was pluggedwith a silicone rubber stopper to prevent evaporation, and each flask was alsowrapped with aluminum foil to minimize carotene degradation by light.Samples were withdrawn at selected time intervals of 30 min, 1, 2, 3, 4, 5, 6and 8 h of reaction. The samples were centrifuged at 6700 ¥ g for 5 min toremove the lipase. The water present in the samples was removed by dryingover 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 magneticstirrer for homogenous mixing.

Effect of Enzyme Concentration. CPOlein was hydrolyzed with lipasefrom C. rugosa at 50C with 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% (w/w) of enzymeand 50% (w/w) of water. The mixture was agitated using a magnetic stirrerfor homogenous mixing.

Preparation of HCPOlein. One hundred grams of CPOlein was hydro-lyzed with 2 g of lipase (1% w/w) from C. rugosa at 50C for 8 h. One hundredgrams of distilled water, essential for hydrolysis, was added into the mixture ofoil 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. Thewater 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 wasdetermined using the Palm Oil Research Institute of Malaysia test method(Siew et al. 1995). The acidity is the content of FFA conventionally expressedas the percentage of palmitic acid for palm oil and fractions; lauric acid forcoconut oil, palm kernel oil and fractions; and oleic acid for corn oil, soybeanoil and other liquid oils. The acid value is the number of milligrams of Na orKOH necessary to neutralize the FFAs in 1 g of sample.

75HYDROLYSIS EFFECT ON PALM OLEIN

The specific amounts of samples were weighed into Erlenmeyer flasks(Kimble/Kontes, Vineland, NJ). Fifty milliliters of neutralized isopropylalcohol was added into the flasks to dissolve the samples, with the temperatureregulated to about 40C. Phenolphthalein (Sigma) was added to the solutionand was neutralized by dropwise addition of 0.1-N Na or KOH (Sigma) untila faint, but permanent pink persisted. The samples were shaken gently whiletitrating with 0.1-M NaOH or KOH to the first 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 NaOHor KOH solution used (mL); and W is the weight of sample (g).

This equation was used to calculate the percentage of FFA producedduring the hydrolysis process in CPOlein. The values were expressed to threedecimal places for FFA below 0.15% and to two decimal places for FFA above0.15% (Siew et al. 1995).

Determination of Slip Melting Point (SMP). SMP is the temperature atwhich fat in an open-ended capillary column of a specified length rises whenincubating at the specified conditions of the test. A prepared capillary tubecontaining the fat was immersed in a water bath warmed at a specified rateuntil the melting point was reached. This method is applicable to all normalfats and vegetable oils that are solid at ambient temperature, particularly palmoil and palm oil products.

The samples were melted and filtered through the no. 4 qualitative filterpaper (Whatman International Limited, Maidstone, England). Three cleancapillary tubes were dipped into the liquid sample of the oil so that columns offat ca. 10-mm high were obtained in the tubes. The column was chilled in iceuntil the fat solidified. The capillaries were placed in test tubes, held in abeaker of water that had been equilibrated at 10C in a thermostated water bathfor 16 h (or 2 h for quick test). The capillary tubes were removed and wereattached to a thermometer such that the lower ends of the tubes leveled off withthe end of the thermometer bulb. The thermometer and the lower end of thecapillary tube were immersed in the water bath to a depth of ca. 30 mm, andthe starting temperature was 8–10C below the expected slip point of thesample. The water bath was agitated with a magnetic stirrer on a hot plate forhomogeneous heat transfer for temperature to increase at a rate of 1 C/min, butwas reduced to 0.5 C/min nearing the slip point. Heating was continued untilthe fat rose in each tube to the upper end of the capillary tube. The averagevalue of two sets of triplicate results was calculated as the SMP and wasexpressed to one decimal place (Siew et al. 1995).

Viscosities of CPOlein and HCPOlein. The determination of viscosityof the oil samples was carried out at different temperatures of 45, 50, 55 and


60C using a programmable rheometer viscometer (Brookfield model DV III,Brookfield Engineering Laboratories, Middleboro, MA) attached to a Ther-momix model 1419 (B. Braun, Bethlehem, PA) for temperature control.

Statistical Analysis

Results show the mean values of at least two replicates with SD. Analysisof variance using t-test for these variables, temperature, enzyme concentrationand water content in the hydrolysis process was carried out using StatisticalAnalysis Software program (SAS Institute, Cary, NC). Mean values withdifferent superscripts were significantly different (P � 0.05).

RESULTS AND DISCUSSION

Hydrolysis Process

Effect of Temperature. Table 1 shows the effect of temperature on thepercentage of FFA produced during the hydrolysis process by lipase from C.rugosa; the percentage of FFA produced showed a significant decrease with anincrease in temperature. The trend for this reaction can be clearly seen inFig. 1. The difference may be attributable to the optimum temperature underwhich the enzymes function. The optimum temperature for lipase from C.rugosa is 45C. With temperature increment from 50C, the activity of lipasefrom C. rugosa may already be suboptimal because of the denaturation of the

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) 50 55 60 65 70

0.0 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a

0.5 85.3 ± 0.6a 81.6 ± 0.8b 78.6 ± 0.3b 49.9 ± 1.7c 23.7 ± 0.4d

1.0 86.0 ± 1.3a 83.8 ± 0.6a 80.0 ± 1.1b 52.4 ± 1.4c 25.5 ± 0.4d

2.0 89.9 ± 1.3a 87.9 ± 0.8a 85.0 ± 0.4a 60.0 ± 1.3b 29.9 ± 1.7a

3.0 91.7 ± 0.3a 89.0 ± 0.7a 88.1 ± 0.1a 62.3 ± 0.4b 31.6 ± 1.6c

4.0 94.5 ± 0.3a 92.2 ± 1.0a 88.6 ± 0.7b 64.6 ± 0.4c 32.2 ± 0.3d

5.0 95.9 ± 0.6a 92.5 ± 0.7b 89.0 ± 0.8c 64.9 ± 1.0d 32.2 ± 0.6e

6.0 95.9 ± 1.4a 92.7 ± 1.1b 92.1 ± 0.1b 65.5 ± 0.7c 32.4 ± 0.6d

8.0 96.3 ± 0.7a 94.8 ± 0.3a 92.4 ± 0.4b 65.9 ± 1.4c 33.4 ± 0.8d

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


enzyme. Lipase from C. rugosa, however, may be a suitable candidate for thehydrolysis process, as the lower temperature would reduce carotene degrada-tion. 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 ratepasses through a maximum. The temperature at which the rate is a maximumis known as the optimum temperature whose values vary with enzyme con-centration and other factors, such as water content. Changing the temperatureaffects the catalyzed reaction itself and the thermal inactivation of the enzyme.However, inactivation is very slow and has no appreciable effect on the rate ofthe catalyzed reaction. Hence, the overall rate of reaction often increases withthe rise in temperature, as commonly reported in ordinary chemical reactions.Inactivation becomes more important at higher temperatures such that theconcentration of active enzyme falls during the course of reaction. During theenzyme inactivation process, protein denaturation could arise from conforma-tional changes. It can be assumed that bonds (perhaps hydrogen bonds) may be

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FIG. 1. THE EFFECT OF TEMPERATURE ON THE HYDROLYSIS PROCESS BY LIPASEFROM CANDIDA RUGOSA

t, time; T, temperature.


broken, and that the unfolded molecule ceases to function as it should in itscorrect conformation (Laidler and Peterman 1979).

Lipase from C. rugosa showed significant reductions in the percentageof FFA produced with every 5C increase starting from 50C. The explanationis that the temperature coefficient of the rate of inactivation must be greaterthan 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 catalyzedreaction, whereas in the high temperature range, it is higher (Laidler andPeterman 1979). Immobilization may increase the stability of lipases at hightemperatures (Haas et al. 1994). Product inhibition could also bring about adramatic decrease in lipase activity. Dunhaupt et al. (1992) reported thatFFA decreased the hydrolytic activity of P. cepacia lipase in olive oil.Similar results are reported by Lenki et al. (1998) for the hydrolysis ofbutterfat fraction.

Effect of Water Content. Table 2 shows the effect of water content onthe percentage of FFA produced during the hydrolysis process at 50C by lipasefrom C. rugosa. The percentage of FFA produced generally increased signifi-cantly with an increase in water content throughout the course of hydrolysis.Further increase in water content from 50% did not significantly increase theFFA production. The effect of water content on FFA production in the hydroly-sis process by lipase from C. rugosa is shown in Fig. 2.

Water is essential in catalyzing the hydrolysis process. With the resultobtained, the water content that is required in the hydrolysis process is 50%

TABLE 2.EFFECT OF PERCENTAGE OF WATER CONTENT ON THE PERCENTAGE OF FREE FATTY

ACIDS PRODUCED DURING THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDARUGOSA AT 50C

t (h) 20 40 50 60 80

0.0 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a

0.5 82.8 ± 1.1a 83.0 ± 1.6a 85.3 ± 0.7b 81.8 ± 0.8a 2.0 ± 0.1c

1.0 84.6 ± 0.8a 84.9 ± 1.1a 86.0 ± 1.4b 85.4 ± 0.6a,b 2.4 ± 0.3c

2.0 85.4 ± 0.3a 88.0 ± 1.6b 89.9 ± 0.7c 90.1 ± 0.6c 2.8 ± 0.4d

3.0 86.1 ± 0.4a 89.5 ± 0.7b 91.7 ± 0.6c 91.1 ± 0.6c 2.8 ± 0.6d

4.0 86.5 ± 0.7a 92.6 ± 0.8b 94.5 ± 0.7c 91.4 ± 0.6b 2.9 ± 0.1d

5.0 86.6 ± 0.6a 94.8 ± 0.8b 95.9 ± 1.0b 93.1 ± 0.6c 3.0 ± 0.1d

6.0 87.4 ± 0.6a 94.9 ± 0.6b 95.9 ± 0.6b 95.2 ± 0.3b 3.3 ± 0.4c

8.0 88.9 ± 0.1a 95.0 ± 0.1b 96.3 ± 0.4c 95.4 ± 0.1b 3.4 ± 0.1d



(w/w) or at 1:1 oil to water ratio. At lower water content (less than 50%), thedegree of hydrolysis was incomplete. Because lipase catalyzes not onlyhydrolysis but also the reverse esterification reaction simultaneously, a largeamount of water is necessary to shift the equilibrium toward hydrolysis.However, when hydrolysis was conducted in the mixture containing more than50% (w/w) water, the degree of hydrolysis decreased (Shimada et al. 1997a).Results showed that at above 50% (w/w) water, there was a significantdecrease in the percentage of FFA production. Therefore, 50% water was usedfor subsequent hydrolysis processes. Lipases act at the oil/water interface ofheterogeneous reaction systems. In an aqueous medium, hydrolysis is thedominant reaction, but in organic media, esterification and interesterificationreactions are predominant. High water levels reduce lipase-catalyzed esterifi-cation or transesterification, presumably by causing hydrolysis of the acyl-enzyme intermediate (Valivety et al. 1993). Therefore, high water levelsincrease the rate of the hydrolysis process. Wehtje and Adlercreutz (1997)report that the activities of many enzymes such as proteases, glycosidases andlipases versus water activities show an increasing curve. Lipase hydrolytic

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FIG. 2. THE EFFECT OF WATER CONTENT ON THE HYDROLYSIS PROCESS BY LIPASEFROM CANDIDA RUGOSA



activity is sharply water-dependent (Haas et al. 1995). Water is a cosubstratein the hydrolysis reactions, with one water molecule being consumed for eachester bond hydrolyzed. Water is also essential for the retention of lipaseactivity in organic solvents (Haas et al. 1993). Because lipase catalyzes notonly hydrolysis but also esterification, sufficient amounts of water are neces-sary to shift the equilibrium toward hydrolysis (Shimada et al. 1997b). Highwater levels, however, reduce the hydrolytic activity dramatically (Haas et al.1995; Shimada et al. 1997b). This may be attributed to the dilution effect oflipase concentration in the water phase (Shimada et al. 1997b). Haas et al.(1994) reported that water requirements are unique to each lipase and vary inproportion to substrate concentration. Thus, it is reasonable to believe thatincreased concentration of water will decrease the activity in the synthesisprocess, but could enhance the hydrolytic process.

Effect of Enzyme Concentration. Table 3 shows the effect of enzymeconcentration on the percentage of FFA produced during the hydrolysisprocess by lipase from C. rugosa. Results show that the percentage of FFAproduced increased with an increase in enzyme concentration, especially from0.5 to 1%, showing a drastic increase in the percentage of FFA producedbecause higher concentration of enzyme enhanced hydrolytic activity. Thelipase from C. rugosa, at a concentration of 1–2% had no significant differ-ential effect on the production yield, possibly because of the limitationof substrate for the first half hour of the hydrolysis process. However, therewas a significant difference in the percentage of FFA produced for enzyme

TABLE 3.EFFECT OF ENZYME CONCENTRATION ON THE PERCENTAGE OF FREE FATTY ACIDSPRODUCED DURING THE HYDROLYSIS PROCESS BY LIPASE FROM CANDIDA RUGOSA

AT 50C

t (h) 0.5 1.0 1.5 2.0 2.5 3.0

0.0 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a 2.0 ± 0.3a

0.5 75.0 ± 0.8a 85.3 ± 1.6b 87.0 ± 0.7b 88.4 ± 0.6b 94.5 ± 0.8c 95.3 ± 0.6c

1.0 80.0 ± 1.6a 86.0 ± 1.6b 88.0 ± 0.1b 90.0 ± 1.1c 97.8 ± 1.0d 96.4 ± 1.0d

2.0 84.0 ± 2.1a 89.9 ± 1.3b 89.0 ± 0.6b 91.4 ± 0.6b 97.9 ± 1.3c 96.9 ± 0.6c

3.0 85.9 ± 2.3a 91.7 ± 2.0b 92.5 ± 1.0b 92.6 ± 2.0b 98.5 ± 0.6c 97.1 ± 0.5c

4.0 86.9 ± 1.4a 94.5 ± 0.6b 94.2 ± 0.6b 96.0 ± 0.3b 98.7 ± 0.7c 98.5 ± 0.7c

5.0 88.9 ± 0.6a 95.9 ± 1.3b 96.0 ± 0.6b 96.5 ± 1.0b 98.7 ± 0.3c 98.7 ± 0.7c

6.0 86.9 ± 1.6a 95.9 ± 1.6b 96.7 ± 0.3b 96.8 ± 0.4b 98.9 ± 1.0c 99.0 ± 0.4c

8.0 88.9 ± 1.3a 96.3 ± 0.6b 96.9 ± 0.3b 98.7 ± 1.0c 99.0 ± 0.1c 99.2 ± 0.1c



concentrations varying from 2 to 3%. However, 1% enzyme concentration wasused for further studies because of economical factors. There is not much FFAproduction during the latter stages of the hydrolysis process, and the enzymeactivity decreased because of the denaturation occurring during the hydrolysisprocess. For higher enzyme concentrations, equilibrium can be achieved at afaster rate. The effect of enzyme concentration on FFA production in thehydrolysis 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 (McNeillet al. 1996; Shimada et al. 1997a,b). To increase the degree of hydrolysis, itwas necessary to add successively high amounts of lipase (Kosugi et al. 1988;McNeill et al. 1996). The degree of hydrolysis increased with an increase inthe amount of lipase until it reached plateau (Shimada et al. 1997a,b). At lowand moderate lipase levels, the addition of water had little impact uponhydrolysis. At high lipase levels, hydrolysis was retarded by increasing theamount of water (Haas et al. 1993).

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FIG. 3. THE EFFECT OF ENZYME CONCENTRATION ON THE HYDROLYSIS PROCESS BYLIPASE FROM CANDIDA RUGOSA



Characteristics of CPOlein and HCPOlein

FFA Production. CPOlein was hydrolyzed by lipase from C. rugosa at50C, shaken at 700 rpm for 8 h. After hydrolysis, the percentage of FFAproduced was determined. The results are shown in Table 4. FFA in CPOleinincreased to 97.9%, reflecting a 61.0% fold increase after the hydrolysisprocess.

SMP. The SMP of CPOlein was 17C. After hydrolysis, the melting pointfor CPOlein was increased by 160% and then reached 44.4C. The relativelylinear form of FFA in HCPOlein, which can lead to good molecular packing ina semisolid phase and can cause a higher melting point. The double bonds inthe oil molecules are geometrically in the cis configuration, which “kicks” thechain and makes it difficult to form an organized solid structure. Oleic acid(C18:1D9), an unsaturated fatty acid, results in a noticeable lowering of themelting point and hence, of liquid oil at room temperature (Bailey and Bailey2000). As a result, the more double bonds in the fatty acids portion in thetriacylglycerol, the lower is the melting point.

Viscosity. Viscosity is a measure of internal friction in molecules thatcreates resistance to flow. The coefficient of viscosity, h, is defined as the forceper unit area required to maintain a unit difference of velocity between twoparallel layers that are a unit distance apart. When force per unit area is indynes per square centimeter, velocity is in centimeter per second, and distanceis in centimeter, h is expressed in poise (Marvin et al. 1979). Viscosity istemperature dependent; hence, temperature control is essential. The relation-ship between temperature and viscosity is inverse; viscosity decreases as thetemperature increases. Oils are normally liquid at ambient temperature and fatsare normally solid, but oils are more viscous than water, and most food-gradeoils exhibit a Newtonian behavior.

TABLE 4.THE PERCENTAGE OF FREE FATTY ACIDS (FFA) AND SLIP

MELTING POINT (SMP) OF CPOLEIN AND HCPOLEIN

Oil FFA (%) SMP (C)

CPOlein 2.0 ± 0.3a 17.0 ± 0.0a

HCPOlein 96.3 ± 1.0b 44.4 ± 0.7b

Note: Each datum represents the mean values ± SD of analysesfrom two replications. Mean values within each column with thesame superscript are not significantly different (P � 0.05).CPOlein, crude palm olein; HCPOlein, hydrolyzed crude palm olein.


In this study, the viscosity of CPOlein and HCPOlein was determined atdifferent temperatures and stirring rates. As viscosity is also a measure of theflow rate, the understanding of the viscosity of each oil type has assisted themonitoring of the flow rate of the oil in the adsorption column chromatog-raphy. In all cases, the viscosity decreased with the increase in temperatureand also with the increase in stirring rate. The results indicate that CPOleinand HCPOlein reached a plateau after 30 rpm. The hydrolyzed oil was lessviscous with a plateau at around 10 centipoise (cP) compared with the crudeoil 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 compari-son of viscosity between CPOlein and HCPOlein at different temperatures at53 rpm. The differences in viscosity between the oils at different tempera-tures were statistically significant (P � 0.05). This can be attributed to thechanges of triacylglycerols, which are more viscous compared with FFA,which are less viscous.

Oils owe their relatively high viscosities to the intermolecular attractionsof the long chains of their triacylglycerol molecules (Marvin et al. 1979).Different oils have different fatty acid compositions; hence, the increase in theamount of long-chain fatty acids and the degree of saturation increase theviscosities. The viscosity of hydrolyzed oil was less than that of the crude oilbecause of changes in oil form, from triacylglycerol to FFA forms that are lessviscous. The molecular size of triacylglycerol is larger than FFA; therefore,triacylglycerols have stronger intermolecular forces compared with FFA andcontribute to friction while stirring or mixing. Therefore, the higher the frictionduring stirring, the higher viscosity values can be obtained (Lewis 1987). Thisexplains the fact that a decrease in molecular size results in a decrease inviscosity; hence, HCPOlein is less viscous than CPOlein.

TABLE 5.COMPARISON OF VISCOSITY BETWEEN CPOLEIN ANDHCPOLEIN AT DIFFERENT TEMPERATURES AT 53 rpm

Temperature (C) Viscosity (cP)

CPOlein HCPOlein

50 24.2 ± 0.1a 12.7 ± 0.1b

55 21.1 ± 0.3a 10.9 ± 0.3b

60 19.3 ± 0.1a 10.7 ± 0.4b

Note: Each datum represents the mean values ± SD of analysesfrom two replications. Mean values within each column with thesame superscript are not significantly different (P � 0.05).CPOlein, crude palm olein; HCPOlein, hydrolized crude palm olein.


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 theproduction of hydrolyzed oil or FFA-rich oil for CPOlein. HCPOlein, which isless viscous compared with CPOlein, is preferred in this hydrolysis process.

ACKNOWLEDGMENTS

Financial support from Intensification of Research in Priority Area GrantNo. 03-02-04-0141-EA001 of the National Council for Research and ScienceDevelopment in Malaysia is acknowledged. The supply of CPOlein fromGolden Jomalina Food Industries Sdn. Bhd., Teluk Panglima Garang,Selangor, Malaysia is also gratefully acknowledged.

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Effects of Enzymatic Hydrolysis on Crude Palm

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