Appl Biochem Biotechnol (2015) 175:757–769 DOI 10.1007/s12010-014-1312-5

Enzymatic Synthesis of Cocoa Butter Equivalent from Olive Oil and Palmitic-Stearic Fatty Acid Mixture Ibrahim O. Mohamed

Received: 24 April 2014 / Accepted: 15 October 2014 / Published online: 24 October 2014 # Springer Science+Business Media New York 2014

Abstract The main goal of the present research is to restructure olive oil triacylglycerol (TAG) using enzymatic acidolysis reaction to produce structured lipids that is close to cocoa butter in terms of TAG structure and melting characteristics. Lipase-catalyzed acidolysis of refined olive oil with a mixture of palmitic-stearic acids at different substrate ratios was performed in an agitated batch reactor maintained at constant temperature and agitation speed. The reaction attained steady-state conversion in about 5 h with an overall conversion of 92.6 % for the olive oil major triacylglycerol 1-palmitoy-2,3-dioleoyl glycerol (POO). The five major TAGs of the structured lipids produced with substrate mass ratio of 1:3 (olive oil/palmiticstearic fatty acid mixture) were close to that of the cocoa butter with melting temperature between 32.6 and 37.7 °C. The proposed kinetics model used fits the experimental data very well. Keywords Cocoa butter equivalent . Triacylglycerol . Acidolysis . Interesterification . Olive oil . Lipase

Introduction A need for structured lipids is continuously growing due to various reasons, structured lipids can be tailored to meet special nutritional and health requirements such as lipids for infant formula, dietary fat with reduced calories, fats rich in essential fatty acids, fats rich in antioxidant, and so on. Structured lipids can also be tailored to meet food industry needs for lipids with special melting characteristics such as plastic fats for making margarines and other products or as cocoa butter equivalent (CBE) for chocolates. Production of structured lipids can be achieved through hydrogenation, enzymatic catalyzed reaction, or chemical catalyzed reaction. Each one of these methods have merits and drawbacks; however, the recent research trends in structured lipids seem to favor enzymatic lipid modification over chemical methods I. O. Mohamed (*) Department of Food Science, College of Food and Agriculture, United Arab Emirates University, P.O. Box 15777, Al-Ain, United Arab Emirates e-mail: [email protected] Ibrahim O. Mohamed e-mail: [email protected]

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obviously due to its regiospecificity and stereospecificity in addition to the mild reaction conditions which do not promote undesirable changes. Cocoa butter (CB) is one of the major ingredients of dark chocolate, white chocolate, milk chocolate, and other forms of chocolates. The amount of cocoa butter is at least 18 % for a product to be labeled as chocolate and up to 31 % in couverture (European Directive 2000/36/ EC). CB gives chocolates the characteristics of sharp melting at body temperature resulting in cooling sensation in the mouth, the typical mouth feeling while eating a high-quality chocolate [1, 2]. These distinct melting characteristic was attributed to its unique major triacylglycerol (TAG) compositions: 1,3-dipalmitoyl-2-oleyl-glycerol (POP) 20 %, 1(3)-palmitoyl-3(1)stearoyl-2-oleoyl-glycerol (POS) 40 %, and 1,3-distearoyl-2-oleoyl-glycerol (SOS) 27 % with oleic fatty acid at the sn-2 position of the glycerol backbone [3]_ENREF_3. Due to high cost and fluctuations in supply and demand of CB, the global supply of CB falls short of the increased demand due to a limited growing area in West Africa, Central America, and South East Asia as dictated by climatic condition requirements; uncertainty in supply due to crop failure for various reasons may increase the gap between supply and demand resulting in price escalation. In addition, cocoa beans contain low amount of CB [4]. To meet the increased demand for cocoa butter, researches were directed towards restructuring of some vegetable oils to produce cocoa butter equivalent (CBE) to be used as partial replacement of natural cocoa butter in chocolate industry. European Union under Directive 2000/36/EC for chocolate products allows the use of six vegetable fats as CBE from mostly exotic plants up to 5 % of the total weight. However, at this stage, enzymatic modified fats are not allowed but could be potentially considered in future amendment if the process of enzymatic modification is well developed, optimized, and economically feasible. CBE can be obtained by physical processes such as blending of some vegetable oils rich in monounsaturated triacylglycerol (TAGs) at sn2 position and rich in palmitic and stearic fatty acids at sn-1,3 positions, mainly (POP), (POS), and (POP) to attain composition of these TAGs close to that of CB [5, 6] or can be produced by fractionational crystallization [5, 7] or by combination of these methods. Alternatively, CBE can be produced by enzymatic interesterification process by which acyl moieties on the glycerol backbone is altered to be similar to that of CB. Structured lipids produced by enzymatic interesterification is often the preferred method by researchers because its regiospecificity and sterospecificity which will help in producing targeted triacylglycerol (TAG) relatively easy compared to chemical interesterification which is completely random in positioning of the acyl groups in the TAG leading to some difficulties in arriving at the targeted TAG in addition to some other problems. Numerous studies were carried out to produce CBE through enzymatic interesterification using different types of substrates, enzymes, reaction conditions, and reaction systems [8–19]. Always the target for such studies is how to produce fats that resemble CB in terms of melting profile, what yield achieved, and under what reaction system and conditions. Due to variability in substrates used, reaction conditions, and reaction systems, a standard production protocol for CBE that meet all the targeted requirements in terms of cost, quality was not yet achieved. This is due to limited research studies related to production of CBE by enzymatic interesterification in addition to the fact that incorporation of acyl moieties into glycerol backbone depends on many factors related to substrate such as chain length, number of double bonds, the location, and geometry of the double bonds in addition to reactivity and specificity of the lipase used [20]. The main goal of this research was to produce structured TAGs from olive oil with melting characteristics similar to that of cocoa butter using enzymatic acidolysis reaction. Commercial olive oil is available in different grades based on quality with the extra virgin, the most expensive, followed by virgin, refined, and pomace oil, respectively. The composition of these grades with regard to TAGs is basically the same, but the extraction method is different. Çiftçi et al.

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[19] reported that the sn-2 oleic content of TAGs of refined olive pomace oil was quite high (85 %). This makes olive oil with its different grades a good starting substrate for enzymatic synthesis of CBE using sn-1,3 specific lipases to incorporate palmitic and stearic fatty acid in position sn-1 and sn-3 of the glycerol backbone. Another starter component for the synthesis of CBE is the palmitic and stearic fatty acid which is a cheap by-product of palm oil physical refining process. Ab Gapor Md [21] reported that the distillate of crude palm oil physical refining contains 81.7 % free fatty acid which contain both palmitic and stearic fatty acid. The saturated fraction of the palm oil distillate mainly palmitic and stearic fatty acids can be easily fractionated from other fatty acids due to their high melting temperature compared to other fatty acid fractions. Production of CBE from such by-product will be an important value added to palm oil industry.

Materials and Methods Materials Refined and deodorized olive oils were purchased from a local supermarket. Commercial immobilized sn-1,3 specific lipase (lipozyme IM from Mucor miehei) and palmitic-stearic fatty acid mixture (PSFAM) were purchased from Fluka Chemie GmbH (product reference number 09586-Fluka). The fatty acid mixture assay consists of 40 % palmitic, 57 % stearic, 1.5 % myristic, and 1.5 % others (AOAC 996.06). This mixture of fatty acids could be obtained from palm oil physical refinery distillate through fractionation and blending. CB-certified reference material IRMM-801 was purchased from the Institute for Reference Materials and Measurements (Geel, Belgium), n-hexane was purchased from Sigma-Aldrich. Solvents used were of chromatographic grade. Enzymatic Reaction Reaction mixture consisting of olive oil (OO) with palmitic-stearic fatty acid mixture (PSFAM) was carried out at various substrate mass ratios OO/PSFAM (1:1, 1:2, 1:3, 1:4, 1:5) using substrate mass 1.0–3.0 g and enzyme loads of 10 % (based on the weight of the substrates). Solvent n-hexane 1 ml/g substrate was added to the reaction mixture, this low level of solvent is used primarily to aid in the separation of the enzyme from the reaction mixture at the end of the reaction and in solubilizing the fatty acid mixture as its melting point temperature as provided by the supplier ranges between 55 and 57 °C; the incubation temperature used is 60 °C, slightly above the fatty acid mixture melting temperature. The high melting temperature of the fatty acid mixture reduces the degree of freedom in varying the reaction temperature, as a higher temperature to what was selected could have negative effects on enzyme activity and could increase the level of acyl migration. Alternatively, the reaction can be carried at different temperatures, but the level of solvent should be increased which is not a preferred alternative by the authors. The enzymes were equilibrated in saturated sodium chloride salt solutions in closed vessels for about 24 h prior to reaction as reported in a previous study [22] to maintain a desired water activity level. Oh et al. [23] reported that the use of water activity of 0.8 resulted in a minimum acyl migration and maximum yield for acidolysis reaction with immobilized enzyme; this is close to the water activity of 0.75 used in this study. Reactions were carried out in 50-ml Erlenmeyer with airtight silicon stopper, the reaction mixture was incubated in an air bath using an orbital shaker (Model 2628-ICE, LabLine Instrument Inc., Melrose Park, IL) operated at a speed of 160 RPM at an incubation

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temperature of 60 °C. Prior to the reaction, the incubator was set to the required temperature and allowed to reach the targeted set temperature then enzymes were added to the substrate in the flasks which were then placed in the incubator to the desired time. To stop the reaction, the reaction mixture was filtered to remove the enzyme and then the products of the reaction were placed in glass dishes and placed in an oven set at 70 °C to remove the solvent from the sample. Reactions were carried out in duplicates and average values were reported. Triacylglycerol Composition The triacylglycerol composition analysis for the samples and the CB-certified reference material (IRMM-801) was performed in gas chromatography (GC) with FID detector (model 3800; Varian, Palo Alto, CA, USA). A capillary high-polar column Rtx-65TG (65 % diphenyl/ 35 % dimethyl polysiloxane, 30 m in length×0.25 mm internal diameter, and 0.1-μm film; Restek Corporation, Bellefonte, PA, USA) was used. This column, because of its high polarity, separates TAGs based on carbon number and degree of unsaturation or in other words based on carbon number and partition number (PN) defined as PN=CN−2DB, where CN is the carbon number and DB is the total number of double on the TAG [24]. None of the five CB major TAGs have the same CN and PN leading to a clear separation of these TAGs without overlapping. For nonpolar column, TAG separation is usually based on chain length or in other words carbon number, some of the CB major TAGs have the same carbon number which might lead to overlap problem and difficulty in TAG identification using a nonpolar column. The operating conditions of the GC were done according to [22], the mass quantification of the five major TAGs of the samples was determined according to the procedure by [25]. The procedure is based on determining the response factor for each of the five major TAGs from the peak area of the standard major TAGs and the corresponding normalized % mass for each TAG as provided in the certified reference material (IRMM-801). These five major TAGs represent about 85–90 % of TAGs of CB [26], the relative proportion of these TAGs in CB is responsible for their unique melting characteristics. In addition, these five TAGs were used for data banking for both CB and CBEs [26]. The method proposed by [25] which is used in the current research for the identification and quantification of cocoa butter major TAGs was validated in international collaborative trials and finally adopted by the International Organization for Standardization (ISO) as an international ISO standards [27]. Kinetics of Acidolysis Reaction For a well-agitated dispersion batch reactor of constant volume and perfect mixing, the overall mass balance on a triacylglycerol (x) over a differential time (dt) yields rX ¼ ½TAGT o FX ¼

d FX dt

½TAGX ½TAGT o

ð1Þ ð2Þ

The reaction rate for a dispersion batch reactor is given by [28–30]. rX ¼ k X ð F X e − F X Þ

ð3Þ

Where rX is the rate of change of [TAG]X by acidolysis reaction in perfectly mixed batch reactor, kX an effective rate constant, FXe an equilibrium molar fraction of [TAG]X, Fx the mole fraction of [TAG]x at any time, and [TAG]To the initial total mole of the five major TAGs,

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which can be easily determined from the initial composition of these TAGs; this model considers that the reaction rate is proportional to the separation from equilibrium for each of the five major TAGs of the CBE (FXe −FX), this is similar to what was proposed by [29] for the rate of change of fatty acids during acidolysis reaction; however, in this reaction, TAGs is monitored rather than fatty acids. Incorporating Eq. (3) in Eq. (1) and integrating yield   kX t ð4Þ F X ¼ F X e þ ð F X o − F X e Þexp − ½TAGT o From knowledge of mole fraction changes of TAG (x) with time, Eq. 4 can be used to determine the effective rate constant (kX). Melting Profile Structured lipid melting profile characteristics were determined by differential scanning calorimeter (Model DSC Q100, TA Instruments.) according to the following protocol: about 4–10 mg of molten sample was placed in an aluminum pan, another identical empty pan was used as a reference. Samples were heated to 80 °C at a rate of 10 °C/min and held at 80 °C for 15 min to allow for destruction of crystal memory. The samples were then cooled to −40 °C at a rate of 10 °C/min and held at −40 °C for 15 min then the samples were heated back to 80 °C at a rate of 5 °C/min. The melting temperature profiles of the structured lipids were determined from the DSC thermograms from the endothermic peaks.

Results and Discussion TAG Identification In order to establish a reliable procedure for the identification of the major TAGs of the structured lipid, the operating condition of the GC was optimized using the CB-certified reference material (IRMM-801). Figure 1 shows a typical profile of the CB-certified reference material major triacylglycerol; it was quite clear from the chromatogram the excellent separation of the TAG, the shape of the chromatogram matches very well with the one that is shown in the ISO/FDIS 2375–1:2006(E) standard for cocoa butter standard reference material (IRMM-801). Table 1 shows the normalized mass fraction of the five major TAGs of the IRMM-801 reference material as reported in the certificate provided with reference material. The excellent agreement in quantification for the major TAGs from this study with that of the reference material certificate is a validation for the GC procedure used in this study. Table 1 also shows the normalized mass fraction of the major TAGs of the olive oil used in this study; 1-palmitoy-2,3-dioleoyl glycerol (POO) is a major TAG in olive oil (86.9 %) while SOS is not detected. Acidolysis Reaction To study the effects of substrate mass ratio on conversion, several runs of reaction were carried out using different substrate mass ratios for a fixed reaction time of about 12 h. For illustration, Fig. 2 shows typical chromatogram of the structured lipids for substrate mass ratio (1:3). The peaks of the targeted major TAGs were clearly identified by matching the retention time of the

762 35,000 34,000

Appl Biochem Biotechnol (2015) 175:757–769

µV

33,000 32,000 31,000

POS

30,000

28,000 27,000 26,000

POP

29,000

25,000 24,000 23,000 22,000 21,000 20,000

SOS

19,000 18,000 17,000 16,000 15,000 14,000 13,000

11,000 10,000

SOO

POO

12,000

9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000

14.5

15

15.5

16

16.5

17

RT [min]

17.5

18

18.5

19

Fig. 1 TAG chromatogram for cocoa butter certified reference material (IRMM-801)

sample with that of the standard (IRMM-801) injected during the sample batch run. The mass quantification for each of the major TAGs in the samples were determined using an average value for the detector response factor for each TAG determined from a minimum of three injections of CB reference material for each run. Figure 3 shows normalized mass fraction of cocoa butter major TAGs as a function of substrate mass ratios. The TAGs of the structured lipids POP, POS, and SOS increase with the increase of the substrate mass ration while POO and 1-stearoyl-2,3-dioleoyl-glycerol (SOO) decrease with the increase of the substrate mass ratio. However, the changes in the TAG composition for a substrate mass ratio of 1:3 and Table 1 Major TAG composition in cocoa butter certified reference material and pure olive oil TAG

Certificate

This study

Olive oil

POP POS

18.14 44.68

18.24±0.12a 44.73±0.03

7.68±0.07a 3.62±0.03 86.91±0.71

POO

2.26

2.26±0.01

SOS

31.63

31.49±0.13

SOO

3.29

3.29±0.02

g of individual TAG/100 g of total TAGs a

Mean±SD, n=3

0 1.79±0.81

Appl Biochem Biotechnol (2015) 175:757–769

17,500 17,000 16,500 16,000

µV

PPS

18,000

763

15,500 15,000

13,000

PSS

13,500

POP

14,000

12,500

POS

14,500

12,000 11,500 11,000 10,500

8,000

SOO

8,500

SOS

PLP

9,000

SSS

9,500

PLS

POO

10,000

7,500 7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000

14.5

15

15.5

16

16.5

RT [min]

17

17.5

18

18.5

19

Fig. 2 TAG chromatogram for structured lipid obtained from olive oil and fatty acid mixture with mass ratio (1:3)

higher is not significant suggesting that substrate mass ratio of 1:3 could be considered as an optimum for further investigation because it requires a less amount of fatty acids and hence minimizes the cost of recovering the CBE from the product of the reaction. Figure 4 shows the effect of reaction time on the formation or loss of the major TAGs; the normalized mass fraction of the TAGs at time zero represent the value of the major TAGs in pure olive oil prior to reaction. It is clear from the figure that POO, the major TAG in olive oil, decreases at high rate during the first hour of the reaction then the rate of change decreases slowly during the later stages of the reaction reaching a steady value of 6.5 % in about 5 h. The TAG, SOO increases during the first half an hour of the reaction to a maximum value then decreases at a later reaction time until it reaches the steady-state value; such trend seems to indicate that SOO acts as an intermediate for the formation of POS, POP, and SOS. The data in Fig. 4 showed that the composition of the five major TAGs after 5 h from the start of the reaction is not significantly different from that at time 10 h (P

Enzymatic synthesis of cocoa butter equivalent from olive oil and palmitic-stearic fatty acid mixture.

The main goal of the present research is to restructure olive oil triacylglycerol (TAG) using enzymatic acidolysis reaction to produce structured lipi...
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