Effects of Emulsifier Addition on the Crystallization and Melting

Article pubs.acs.org/JAFC

Effects of Emulsifier Addition on the Crystallization and Melting Behavior of Palm Olein and Coconut Oil Jessica Mayumi Maruyama,† Fabiana Andreia Schafer De Martini Soares,† Natalia Roque D’Agostinho,† Maria Inês Almeida Gonçalves,‡ Luiz Antonio Gioielli,† and Roberta Claro da Silva*,† †

Department of Biochemical and Pharmaceutical Technology and ‡Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580, B16, CEP 05508-900, São Paulo, SP, Brazil ABSTRACT: Two commercial emulsifiers (EM1 and EM2), containing predominantly monoacylglycerols (MAGs), were added in proportiond of 1.0 and 3.0% (w/w) to coconut oil and palm olein. EM1 consisted of approximately 90% MAGs, whereas EM2 consisted of approximately 50% MAGs. The crystallization behavior of these systems was evaluated by differential scanning calorimetry (DSC) and microscopy under polarized light. On the basis of DSC results, it was clear that the addition of EM2 accelerated the crystallization of coconut oil and delayed the crystallization of palm olein. In both oils EM2 addition led to the formation of smaller spherulites, and these effects improved the possibilities for using these fats as ingredients. In coconut oil the spherulites were maintained even at higher temperatures (20 °C). The addition of EM1 to coconut oil changed the crystallization pattern. In palm olein, the addition of 3.0% (w/w) of this emulsifier altered the pattern of crystallization of this fat. KEYWORDS: microscopy, DSC, monoacylglycerols, microstructure, thermal behavior, crystalline morphology

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food).6−9 In addition to these applications, MCT oils have found useful applications in various dietetic and health food products, and some manufacturers have also started to incorporate them into products such as margarine and chocolate spreads, which consumers perceive as fattening.9 Coconut oil is composed of >80% saturated fatty acids, and this is much more than the major liquid oils. Nutritionally this may be thought of as a great disadvantage, but such simple comparisons can be misleading. Lauric oils are used in foods only when a sharp melting hard fat is needed. When liquid oils are hydrogenated to a similar consistency, they contain not only more saturates but also trans fatty acids, which some recent studies have shown to be even more objectionable with regard to serum cholesterol profiles than the saturated ones.10,11 Coconut oil is an emerging functional food due to its ability to possess various biological activities such as antiviral and antimicrobial12 and to promote human health and reduce the risk of heart or atherogenic diseases.13 Emulsifiers are additives that exhibit wide applicability in the food industry, such as improving the texture, stability, volume, softness, aeration, and shelf life.14 In high-fat products, these compounds can be used to control or modify the crystallization behavior of the lipid phase.15 The study of the effects of emulsifiers in lipid systems is of great interest for improving industrial bases, particularly in relation to fat for use in chocolate, confectionery, and baking. Previous research has demonstrated that the addition of emulsifiers can modify the crystallization behavior of lipids by either delaying or promoting the crystallization either in bulk lipids or in emulsions.16−24 Sato’s group studied the effect of

INTRODUCTION The industrial processing of lipid-based foods such as chocolate, margarine, spreads, confectionery and bakery fats, and dairy products directly suffers from the influence of the crystallization behavior of the lipids. The crystallization of fats determines important properties of the food: (i) the consistency and plasticity of fat-rich products such as butter, margarine, and chocolate during the stages of production and storage; (ii) the sensory properties such as melting sensation in the mouth; (iii) physical stability related to crystal formation or sedimentation, oil exudation, and coalescence of particles and emulsions; (iv) visual appearance, such as gloss in chocolates and coatings.1 In most foods, the isolated crystallization of triacylglycerols (TAGs) is considered the most important event, although the crystallization of minority lipids as diacylglycerols (DAGs) and monoacylglycerols (MAGs) plays a key role in the quality of various products.2 Palm oil and its fractions are important sources of edible oils for the food industry due to their properties of high thermal and oxidative stability and plasticity.3 Palm olein is the liquid fraction obtained by fractionation of palm oil after crystallization at a controlled temperature.4 Palm olein has been identified as a good ingredient for margarine and shortening formulation, but its application is limited by its propensity to form granular crystals on storage. The formation of these big crystals is attributed to the high content of symmetrical triacylglycerols.5 Coconut oil is a basic source of medium-chain triglyceride (MCT) oils. These oils are based on triacylglycerols of caprylic (8:0) and capric (10:0) fatty acids, and they differ from ordinary oils with respect to their digestion and metabolism.6 Because of their low molecular weight, MCT oils are easily absorbed in the digestive tract and are used immediately as energy sources in the body and thus avoid being stored in the adipose tissue. These properties make them useful ingredients in sports foods and infant foods and in clinical nutrition (enteral/parenteral © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2253

June 17, 2013 February 17, 2014 February 18, 2014 February 18, 2014 dx.doi.org/10.1021/jf405221n | J. Agric. Food Chem. 2014, 62, 2253−2263

Journal of Agricultural and Food Chemistry

Article

Table 1. Fatty Acid Composition of Emulsifiers, Coconut Oil, Palm Olein, and Their Mixtures with the Two Commercial Emulsifiersa fatty acidsb (g/100 g) EM1 EM2 CO CO + 1% EM1 CO + 3% EM1 CO + 1% EM2 CO + 3% EM2 PO PO + 1% EM1 PO + 3% EM1 PO + 1% EM2 PO + 3% EM2

EM1 EM2 CO CO + 1% EM1 CO + 3% EM1 CO + 1% EM2 CO + 3% EM2 PO PO+1%EM1 PO + 3% EM1 PO + 1% EM2 PO + 3% EM2

8:0

10:0

12:0

ND ND 2.4 ± 0.0a 2.3 ± 0.0a 2.3 ± 0.0a 2.3 ± 0.0a 2.3 ± 0.0a ND ND ND ND ND

ND ND 3.3 ± 0.0a 3.3 ± 0.0a 3.2 ± 0.0a 3.3 ± 0.0a 3.2 ± 0.0a ND ND ND ND ND

ND ND 42.6 ± 0.2a 42.2 ± 0.2a 41.4 ± 0.2a 42.2 ± 0.2a 41.4 ± 0.2a 0.5 ± 0.0b 0.5 ± 0.0b 0.5 ± 0.0b 0.5 ± 0.0b 0.5 ± 0.0b

14:0

16:0

ND 22.8 ± 0.2 ND 10.5 ± 0.1 21.0 ± 0.5a 12.2 ± 0.1a 20.8 ± 0.0a 12.3 ± 0.1a 20.4 ± 0.0a 12.5 ± 0.1a 20.8 ± 0.0a 12.1 ± 0.1a 20.4 ± 0.0a 12.1 ± 0.1a 0.7 ± 0.0b 36.9 ± 0.5b 36.7 ± 0.5b 0.7 ± 0.0b 0.7 ± 0.0b 36.4 ± 0.4b 0.7 ± 0.0b 36.6 ± 0.5b 0.7 ± 0.0b 36.1 ± 0.4b fatty acidsc (g/100 g)

18:0

18:1

18:2

2.2 ± 0.1 89.2 ± 0.1 1.7 ± 0.0a 1.7 ± 0.0a 1.7 ± 0.0a 1.6 ± 0.0a 1.4 ± 0.0a 4.7 ± 0.0b 4.7 ± 0.0b 4.6 ± 0.0b 4.5 ± 0.0b 4.2 ± 0.0b

16.2 ± 0.1

58.8 ± 0.1 0.3 ± 0.1 5.1 ± 0.1a 6.6 ± 0.1a 6.7 ± 0.1a 6.4 ± 2.7a 6.9 ± 0.1a 10.6 ± 0.3b 10.0 ± 0.3b 10.0 ± 0.3b 10.5 ± 0.3b 10.3 ± 0.3b

11.6 ± 0.0a 11.7 ± 0.1a 11.7 ± 0.1a 11.5 ± 0.1a 11.3 ± 0.1a 47.6 ± 0.2b 47.3 ± 0.2b 46.6 ± 0.2b 47.1 ± 0.2b 46.2 ± 0.2b

SFA

MCSFAC

LCSFA

UNSFA

MUFA

PUFA

IV

24.9 ± 0.2 99.7 ± 0.1 83.3 ± 0.2a 82.7 ± 0.1a 81.5 ± 1.8a 83.5 ± 1.4a 83.8 ± 0.3a 42.8 ± 0.5b 42.7 ± 0.2b 42.3 ± 0.1b 43.4 ± 0.1b 44.6 ± 0.7b

ND ND 48.4 ± 0.3a 47.9 ± 0.0a 46.9 ± 2.7a 47.9 ± 2.5a 46.9 ± 0.1a 0.5 ± 0.0b 0.5 ± 0.0b 0.5 ± 0.0b 0.5 ± 0.0b 0.5 ± 0.7b

24.9 ± 0.2 99.7 ± 0.1 13.9 ± 0.1a 34.8 ± 0.1a 35.6 ± 0.9a 35.6 ± 1.1a 36.9 ± 0.3a 42.3 ± 0.5b 42.1 ± 0.2b 41.8 ± 0.3b 42.9 ± 0.1b 44.0 ± 0.7b

75.0 ± 0.2 0.3 ± 0.1 16.7 ± 0.1a 17.3 ± 0.1a 18.4 ± 1.8a 14.9 ± 1.4a 16.2 ± 0.3a 57.1 ± 0.5b 57.3 ± 02b 57.7 ± 0.1b 56.6 ± 0.1b 55.4 ± 0.7b

16.2 ± 0.1

58.8 ± 0.1 0.3 ± 0.1 5.1 ± 0.0a 5.6 ± 0.0a 6.7 ± 0.6a 3.4 ± 0.4a 4.9 ± 0.2a 10.6 ± 0.3b 10.0 ± 0.1b 11.0 ± 0.1b 10.5 ± 0.1b 10.3 ± 0.0b

115.8 ± 0.2 0.5 ± 0.1 18.8 ± 0.3a 19.8 ± 0.1a 21.7 ± 2.1a 15.8 ± 1.6a 18.2 ± 0.4a 57.5 ± 0.7b 58.1 ± 0.2b 59.2 ± 0.2b 56.9 ± 0.1b 55.8 ± 1.2b

11.6 ± 0.1a 11.6 ± 0.1a 11.7 ± 1.2a 11.5 ± 1.0a 11.3 ± 0.1a 47.6 ± 0.2b 47.3 ± 0.2b 46.6 ± 0.2b 47.1 ± 0.2b 46.2 ± 0.7b

Values are shown as means ± SD of three replications. Means (n = 3) with different lower case letters in the same column are significantly different (p < 0.05). ND, undetected; EM1, emulsifier 1; EM2, emulsifier 2; CO, coconut oil; PO, palm olein. b8:0, caprylic acid; 10:0, capric acid; 12:0, lauric acid; 14:0, myristic acid; 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3 linolenic acid. cSFA, saturated fatty acids; MCSFA, medium-chain saturated fatty acids; LCSFA, long-chain saturated fatty acids; UNSFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; IV, iodine value (g iodine/100 g). a

diacyglycerols on fat crystallization in oil-in-water emulsions.16,17 Herrera’s group studied the effect of different additives on the crystallization behavior of bulk lipids.18−22 Radujko et al. studied the influence of different emulsifiers on the physical and crystallization characteristics of edible fats.14 Basso et al. studied the effect of monoacylglycerols as modifiers in the crystallization of palm oil.24 The main effects of these additives in the crystallization of fats occur during the stages of nucleation and growth of the crystalline network, changing physical properties such as crystal size, solid fat content, and microstructure. The issue of promoting or inhibiting crystallization, however, is still debatable. In general, studies indicate that emulsifiers with acyl groups similar to those of the crystallized fat accelerate this process.23 For the above reasons, two semisolid fats were selected (palm olein and coconut oil), which have great potential for use as fat ingredients in the food industry. This paper aims to study of changes in the crystallization behavior of palm olein and coconut oil after the addition of commercial emulsifiers containing predominantly MAGs.

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>90.0% MAGs produced from edible vegetable oil; EM2 >52.0% MAGs produced from refined fully hydrogenated vegetable fats). The fatty acid compositions of these fats, emulsifiers, and their blends were determined by gas chromatography and are presented in Table 1. The fats were stored at 0 °C prior to use. All other reagents and solvents were of analytical or chromatographical grade. Sample Preparation. Commercial emulsifiers (1.0 and 3.0% w/w) were dispersed in the melted coconut oil and palm olein and stirred with a magnetic stirrer at 70 ± 0.5 °C until a homogeneous sample was obtained. Fatty Acid Composition. The fatty acid composition was determined after conversion of fatty acids into their corresponding methyl esters (FAMES) using the method described by ISO method 5509.25 Analyses of FAMES were carried out in a Varian gas chromatograph (model 430 GC, Varian Chromatograph Systems, Walnut Creek, CA, USA), equipped with a CP 8412 autoinjector. Galaxie software was used for quantification and identification of peaks. Injections were performed into a 100 m fused silica capillary column (i.d. = 0.25 mm) coated with 0.2 μm of polyethylene glycol (SP-2560, Supelco, Bellefonte, PA, USA) using helium as the carrier gas at an isobaric pressure of 37 psi; linear velocity of 20 cm/s; makeup gas, helium at 29 mL/min at a split ratio of 1:50; and volume injected, 1.0 μL. The injector temperature was set at 250 °C, and the detector temperature was set at 280 °C. The oven temperature was initially held at 140 °C for 5 min, then stepped to 240 °C at a rate of 4 °C/min, and held isothermally for 30 min. Pure oils and theirs blends were analyzed in triplicate, and reported values represent the average of the three runs.

MATERIALS AND METHODS

Materials. Palm olein was obtained from Agropalma S/A (Pará, Brazil) and coconut oil from Copra Alimentos Ltd.a. (Alagoas, Brazil). The emulsifiers were provided by DuPont Nutrition & Health (São Paulo, Brazil) and are constituted mainly by monoacylglycerols (EM1 2254

dx.doi.org/10.1021/jf405221n | J. Agric. Food Chem. 2014, 62, 2253−2263


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Statistical Analysis. Results were expressed as the mean ± SD in triplicate. Differences between the samples (melting and crystallization behavior, diameter of crystals, and fatty acids composition) during the experimental period were statistically analyzed using one-way analysis of variance (ANOVA), followed by post hoc Tukey test, taking on p > 0.05. Statistical analysis was performed using Statistica 11.0 software.30

Medium-chain saturated fatty acids (MCSFAs) are expressed as the sum of the amounts of caprylic, capric, and lauric acids. Long-chain saturated fatty acids (LCSFAs) are expressed as the sum of the amounts of myristic, palmitic, and stearic acids. Saturated fatty acids (SFAs) are expressed as the sum of the amounts of caprylic, capric, lauric, myristic, palmitic, and stearic fatty acids. Unsaturated fatty acids (USFAs) content is expressed as the sum of the amounts of oleic, linoleic, and linolenic acids. Monounsaturated fatty acids (MUFAs) content is expressed as the amount of oleic acid. Polyunsaturated fatty acids (PUFAs) content is expressed as the sum of the amounts of linoleic and linolenic acids. Triacylglycerol Composition. Triacylglycerol composition was analyzed in a Varian gas chromatograph (model 3400CX, Varian Ind. Com. Ltd.a., São Paulo, Brazil). A DB-17HT Agilent (catalog no. 1221811) capillary column (50% phenyl-methylpolysiloxane, 15 m in length × 0.25 mm bore and containing 0.15 μm film). The conditions were as follows: split injection, ratio, 1:100; column temperature, 250 °C; programmed to 350 °C at 5 °C/min; carrier gas, helium, at 1.0 mL/min flow rate; injector temperature, 360 °C; detector temperature, 375 °C; injection volume, 1.0 μL; sample concentration, 100 mg/5 mL of hexane.26 All samples were analyzed in triplicate, and the reported values are the average of three runs. TAG profiles were followed throughout the reactions by plotting the percents of the areas of carbon number peaks groups. Regiospecific Distribution of Fatty Acids. A proton-decoupled 13 C NMR was used to analyze the positional distribution of fatty acids on the triacylglycerol backbone. Lipid samples (250 mg) were dissolved in CDCl3 (0.5 mL) in 5 mm NMR tubes, and NMR spectra were recorded on a Bruker Advance DPX spectrometer operating at 300 MHz. The 13C spectra of the lipid samples were acquired with a spectral width of 2332.090 Hz, pulse of 10.2 μs, and a relaxation delay of 30s. Determination of 13C was performed at a frequency of 75.8 MHz with a multinuclear probe of 5 mm operating at 30 °C, using the method described by Vlahov.27 The results showed the compositions of saturated fatty acids, oleic acid, and linoleic plus linolenic acids in sn-2 and sn-1,3 positions. All samples were analyzed in triplicate, and the reported values are the average of three analyses. Polarized Light Microscopy. Samples were melted at the temperature of 70 °C in an oven and, with a capillary tube a drop of sample was placed on a glass slide preheated at a controlled temperature (70 °C) and covered with a cover glass. Samples were kept in the stove at the analysis temperatures (10, 15, and 20 °C) for 24 h. Crystal morphology was evaluated by means of the polarized light microscope (Olympus, model BX 50) coupled to the digital video camera (Media Cybernetics). Glass slides were placed on a hot plate LTS 32; heating and freezing stages were operated by a TP93 temperature programmer (Linkam Scientific Instruments Ltd., Surrey, UK). The images were captured using Image Pro-Plus software version 7.0 for Windows (Media Cybernetics) using polarized light and amplified up to 100 times. For each glass slide, three visual fields were focused, of which only one was chosen to represent the observed crystals. The evaluation parameter selected for quantitative image analysis was the mean diameter of crystals and was measured by the Image Pro-Plus software, version 7.0 for Windows (Media Cybernetics).28 Differential Scanning Calorimetry (DSC). DSC curves were obtained by DSC (DSC 4000, Perkin-Elmer Corp., Norwalk, CT, USA), under a dynamic atmosphere of He (20 mL/min) and a cooling rate of −10 °C/min and heating at 5 °C/min, at temperatures ranging from 80 to −60 °C for cooling with isothermal time of 10 min at 80 °C with an isothermal of 10 min at −60 °C (AOCS Cj 1-94, 2009), using sealed aluminum capsules containing sample mass between 5 and 10 mg. The temperature and heat of melting were calibrated with indium (initial temperature of 156.6 °C). Curves were processed in the program Pyris, and melting and crystallization curves were analyzed for the onset (Tonset, °C) of melting and crystallization, peak crystallization and melting temperatures (Tpc and Tpm, °C), and crystallization and melting enthalpies (ΔHc and ΔHm, J/g). The samples were analyzed in triplicate.29

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RESULTS AND DISCUSSION Fatty Acid Composition. The fatty acid composition is very important because it is directly related to the properties of each fat, such as melting point and crystallization characteristics. Table 2. Triacylglycerol Composition (Grams per 100 g) of Coconut Oil (CO) and Palm Olein (PO)

a

TAG group

CO

CNa 28 (CaCC) CN 30 (CaCLa) CN 32(CCLa) CN 34 (CLaLa) CN 36 (LaLaLa) CN 38 (LaLaM) CN 40 (LaMM) CN 42 (LaMP) CN 44 (LaPP) CN 46 (CaOO/LaPO/PPM/MMO/MML) CN 48 (MPO/MPL/PPP) CN 50 (PPL/MLO/MLL) CN 52 (PLL/PLO) CN 54 (OOO/LOO/LOL)

2.2 ± 0.1 8.4 ± 0.3 12.8 ± 0.5 16.1 ± 0.1 16.5 ± 0.6 12.9 ± 0.2 10.4 ± 0.5 6.5 ± 0.4 4.2 ± 0.2 3.6 ± 0.6 3.1 ± 0.2 2.6 ± 0.2

PO

2.1 ± 0.2 37.7 ± 0.6 48.2 ± 1.1 11.8 ± 1.5

CN, carbon number.

Figure 1. Regiospecific distribution of fatty acids in sn-1,3 positions (a) and sn-2 position (b) in triacylglycerols of coconut oil, palm olein, and their mixture with the two commercial emulsifiers.

Fatty acid compositions of the individual fats and their mixtures with the two commercial emulsifiers are presented in Table 1. Fatty acid compositions of palm olein and coconut oil are in accordance with the results published in the literature.31,32 Saturated fatty acids are predominant in coconut oil (p < 0.05), 2255



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Figure 2. DSC crystallization (a, c) and melting (b, d) curves of the two commercial emulsifiers.

Figure 3. DSC crystallization (a, c) and melting (b, d) curves of coconut oil and its mixture with the two commercial emulsifiers.

Table 3. Onset Melting Temperature (Tonset), Peak Melting Temperature (Tpm), Melting Enthalpy (ΔHm), Onset Crystallization Temperature (Tonset), Peak Crystallization Temperature (Tpc), and Crystallization Enthalpy (ΔHc) with the Two Commercial Emulsifiersa H1 EM1 EM2

EM1 EM2

H2

H3

Tonset (°C)

Tpm (°C)

ΔHm (J/g)

Tonset (°C)

Tpm (°C)

ΔHm (J/g)

Tonset (°C)

Tpm (°C)

ΔHm (J/g)

−30.5 ± 0.0 6.4 ± 0.5

−27.5 ± 0.0 46.1 ± 0.1

9.3 ± 0.8 13.3 ± 1.1 C1

1.25 ± 0.1 56.6 ± 0.0

10.9 ± 0.1 60.9 ± 0.0

37.0 ± 0.1 122.1 ± 0.2

46.7 ± 0.2

78.8 ± 0.0

44.9 ± 0.6

C2

Tonset (°C)

Tpc (°C)

ΔHc (J/g)

Tonset (°C)

Tpc (°C)

ΔHc (J/g)

25.0 ± 0.1 60.4 ± 0.0

22.7 ± 0.1 57.8 ± 0.1

28.2 ± 0.0 117.3 ± 2.5

−30.6 ± 0.0 16.4 ± 0.1

−30.0 ± 0.0 12.2 ± 0.0

42.7 ± 0.3 12.7 ± 0.3

Values are shown as means ± SD of three replications. Means (n = 3) with different lower case letters in the same column are significantly different (p < 0.05). a

mainly lauric, miristic, and palmitic acids, respectively. Palm olein showed predominance of unsaturated fatty acids (p > 0.05), mainly oleic acid, and at the same time has high amounts of palmitic acid (36.9 ± 0.5%). Unsaturated fatty acids are predominant in EM1, mainly linoleic acid, and EM2 showed

the highest content of saturated fatty acids, mainly stearic acid (Table 1). The addition of EM1 and EM2, in both proportions, showed no significant differences (p < 0.05) in the composition of fatty acids in coconut oil and palm olein. 2256



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Table 4. Onset Melting Temperature (Tonset), Peak Melting Temperature (Tpm), Melting Enthalpy (ΔHm), Onset Crystallization Temperature (Tonset), Peak Crystallization Temperature (Tpc), and Crystallization Enthalpy (ΔHc) of Coconut Oil and Its Mixtures with the Two Commercial Emulsifiersa H1 CO CO + 1% EM1 CO + 3% EM1 CO + 1% EM2 CO + 3% EM2

CO CO + 1.0% EM1 CO + 3.0% EM1 CO + 1.0% EM2 CO + 3.0% EM2

Tonset (°C)

Tpm (°C)

ΔHm (J/g)

10.1 ± 0.1a 10.4 ± 0.3a 12.4 ± 0.1b 9.9 ± 0.1a 11.6 ± 0.6b C1

21.2 ± 0.1a 21.3 ± 0.7a 21.6 ± 0.0a 21.4 ± 0.0a 22.2 ± 0.6a

63.7 ± 0.9a 105.5 ± 0.5b 94.9 ± 0.1b 62.8 ± 0.9a 59.0 ± 0.3a C2

Tonset (°C)

Tpc (°C)

ΔHc (J/g)

Tonset (°C)

Tpc (°C)

ΔHc (J/g)

6.7 ± 0.3a 4.9 ± 0.1b 7.1 ± 0.2a 9.1 ± 0.5c 11.3 ± 0.2d

4.7 ± 0.2a 3.3 ± 0.0b 5.6 ± 0.1a 5.1 ± 0.3a 6.3 ± 0.3a

18.8 ± 1.2a 26.7 ± 0.1 a 10.2 ± 0.1 a 58.7 ± 2.9b 56.5 ± 0.4b

−7.1 ± 0.4a −7.8 ± 0.7a 0.7 ± 0.0b

−11.4 ± 0.1a −12.6 ± 0.0b −3.4 ± 0.0c

7.6 ± 4.8a 25.8 ± 0.0b 38.6 ± 0.6c

a Values are shown as means ± SD of three replications. Means (n = 3) with different lower case letters in the same column are significantly different (p < 0.05).

triacylglycerol groups; TAGs C32 (CCLa, 12.8%), C34 (CLaLa, 16.1%), C36 (LaLaLa, 16.6%), C38 (LaLaM, 12.9%), and C40 (LaMM, 10.5%), where C = capric acid, La = lauric acid, and M = myristic acid, were the major mainly saturated TAGs (84.5%). The triunsaturated triacylglycerols in coconut oil were only 0.2%. The triacylglycerol species identified in the coconut oil samples were similar to those reported by other authors.35 Regiospecific Distribution of Fatty Acids. Regiospecific distribution of fatty acids in triacylglycerols has implications for the nutritional quality and technology of oils and fats. Analysis of the regiospecific distribution of fatty acids in triacylglycerols by NMR is desirable, as it does not require hydrolysis by pancreatic lipase, further separation of partial acylglycerols by thin layer chromatography, and finally analysis of fatty acids by gas chromatography.36 The NMR technique shows that the signal of the spectra corresponding to the sn-2 position is always equivalent to around 33.3 g/100 g (an acyl group of three), whereas the signal corresponding to sn-1,3 positions is always equivalent to around 66.6 g/100 g (two acyl groups of three). Results for fatty acid composition by NMR were similar to those obtained by gas chromatography of most samples of this study to saturated fatty acids, 18:1 cis and trans and 18:2 + 18:3. Figure 1 shows the regiospecific distribution of fatty acids in sn1,3 positions (a) and sn-2 position (b) in triacylglycerols of coconut oil, palm olein, and their mixture with the two commercial emulsifiers. Saturated fatty acids were found mainly in the sn-1,3 positions, and polyunsaturated fatty acids were found mainly in the sn-2 position, a typical feature of vegetable oils.37 Coconut oil was composed by 86.5% saturated fatty acids, 8.9% monounsaturated fatty acids, and 4.6% polyunsaturated fatty acids at the sn-1,3 positions, whereas 80.7% saturated fatty acids, 11.6% monounsaturated fatty acids, and 7.7% polyunsaturated fatty acids were found at the sn-2 position. Palm olein was composed by 67.0% saturated fatty acids, 27.3% monounsaturated fatty acids, and 5.5% polyunsaturated fatty acids at the sn-1,3 positions, whereas 18.5% polyunsaturated fatty acids, 72.3% monounsaturated fatty acids, and 9.1% saturated fatty acids were found at the sn-2 position.

Table 5. Onset Melting Temperature (Tonset), Peak Melting Temperature (Tpm), Melting Enthalpy (ΔHm), Onset Crystallization Temperature (Tonset), Peak Crystallization Temperature (Tpc), and Crystallization Enthalpy (ΔHc) of Palm Olein and Its Mixtures with the Two Commercial Emulsifiersa H1 PO PO + 1% EM1 PO + 3% EM1 PO + 1% EM2 PO + 3% EM2

PO PO + 1.0% EM1 PO + 3.0% EM1 PO + 1.0% EM2 PO + 3.0% EM2

Tonset (°C)

Tpm (°C)

ΔHm (J/g)

−1.8 ± 0.3a 0.7 ± 0.0b 2.7 ± 0.0c 0.8 ± 0.0c 2.9 ± 0.1c

2.2 ± 0.1a 2.4 ± 0.1ab 4.5 ± 0.0c 2.5 ± 0.0ab 4.8 ± 0.1c C1

38.0 ± 0.6a 72.9 ± 0.6b 76.5 ± 1.8b 71.8 ± 1.0b 76.7 ± 4.1b

Tonset (°C)

Tpc (°C)

ΔHc (J/g)

5.1 ± 0.2a 4.2 ± 0.0b 3.7 ± 0.0c 4.1 ± 0.0d 2.9 ± 0.1e

−0.4 ± 0.2a −2.2 ± 0.0b −2.5 ± 0.1c −0.8 ± 0.0d −2.0 ± 0.1e

31.3 ± 0.5a 66.8 ± 4.8b 72.4 ± 0.6c 28.7 ± 0.2d 65.0 ± 0.8e

a Values are shown as means ± SD of three replications. Means (n = 3) with different letters in the same column are significantly different (p < 0.05).

Triacylglycerol Composition. TAG composition is responsible for providing specific physical properties of lipids. In a processed food that contains significant fat content, the product’s behavior may depend on the triacylglycerol composition of that fat.28 Oils and fats are considered complex samples, because they are composed of a large number of triacylglycerols. Thus, the identification of triacylglycerols becomes a difficult process, in which the number of possible structural forms is very large compared to the number of fatty acids.33 TAG compositions of the individual fats are presented in Table 2. In palm olein, four triacylglycerol groups were identified and were similar to those reported by other authors.34 Palm olein has triacylglycerols ranging from C48 to C54, mainly C52 (48.2%) and C50 (37.7%). The high diversity of TAGs in coconut oil ranged from C28 to C54. The triacylglycerol profile of coconut oil showed 14 2257



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Figure 4. DSC crystallization (a, c) and melting (b, d) curves of palm olein and its mixture with the two commercial emulsifiers.

According to Hayes and Khosla,38 palm olein is composed by 85% oleic acid and 10% palmitic acid at the sn-2 position. These authors also claim that saturated fatty acids are found in higher proportion at the sn-2 in highly saturated fats such as coconut oil. The addition of EM1 and EM2, in both proportions (1 and 3%), to coconut oil and palm olein did not change the regiospecific distribution of fatty acids compared to pure oils. Differential Scanning Calorimetry. Figure 2 shows the melting and crystallization curves obtained by DSC of two emulsifiers. Figure 3 shows the changes in crystallization profiles of coconut oil crystallized with and without the addition of EM1 (a) and EM2 (b). Coconut oil showed two distinct but overlapping exothermic peaks, one minor exothermic peak at −11.0 ± 0.2 °C (C2) and a second peak at 4.7 ± 0.2 °C (C1). These slightly different temperatures from those reported by Marina et al.39 and Mansor et al.40 can be attributed to the different nature of preparation of the coconut oil, the growing condition of the coconut, cultivar type, and maturity of the coconut.40 The existence of the two major exothermic peaks in coconut oil is related to crystallization to the saturated and unsaturated TAGs. The addition of EM1 in two different ratios (1.0 and 3.0% w/ w) changed the crystallization behavior of coconut oil (Figure 3). The enthalpy of crystallization of the peak related to the unsaturated TAGs (C2) was significantly increased (p < 0.05) with 1.0 and 3.0% (w/w) EM1; this is due to the composition of the emulsifier being predominantly unsaturated. For this reason, the first peak of coconut oil referring to saturated TAGs was reduced with the addition of 3.0% (w/w) EM1. In many investigations, the first thermal effect during the crystallization process is considered the start of nucleation.41 However, in DSC thermograms, for example, a minor component may affect the initial, minor peak, whereas the later

crystallization peak (representing the bulk of crystallization) may not be affected.41 On the basis of the data plotted in Figure 3 and the values obtained for Tonset (°C) and Tpc (°C), it is possible to observe that the addition of 3% EM1 in coconut oil affected the second stage of the crystallization, the crystal growth, and this effect is probably due to the presence of the emulsifier containing 90% MAGs. Basso et al.24 also observed this effect with the addition of 1% MAGs to palm oil. According to Fredrick,42 MAGs accelerate the polymorphic transition to stable forms. The addition of EM2 also accelerated the process of crystallization but, otherwise, crystallization was affected at the beginning of the first peak (Tonset, °C); it may be stated that the addition of this emulsifier affected the nucleation of crystallization. The onset temperatures (Tonset, °C) were significantly higher (p > 0.05), 9.1 ± 0.5 °C for 1.0% (w/w) of addition and 11.3 ± 0.2 °C for 3.0% (w/w), compared with pure coconut oil that showed an onset temperature (Tonset, °C) of 6.7 ± 0.3 °C. With the addition of EM2 (1.0 and 3.0% w/w) was not observed the formation of the second crystallization peak for the coconut oil. Tables 3, 4, and 5 show the parameters regarding the crystallization curves of coconut oil, palm olein, and their mixture with the commercial emulsifiers. The enthalpy of crystallization was also changed, with higher values observed in total ΔHc of crystallization for all mixtures. The greatest variation in enthalpy of coconut oil occurred with the addition EM2, which shows significant difference from coconut oil (p > 0.05). The enthalpy of crystallization shows a significant difference between the peaks (p > 0.05), indicating that more crystals are present in these samples. The melting curves for coconut oil and mixtures with EM1 (b) and EM2 (d) are shown in Figure 3. Tables 3, 4, and 5 show the 2258



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Figure 5. Images of the crystallization of individual commercial emulsifiers, pure coconut oil, and palm olein. The bar represents 200 μm.

The enthalpy of crystallization (ΔHc) suffered the most relevant effects, with an increase of 35.1 ± 0.8 J/g in the energy released. Similar results were observed with the addition of 3.0% (w/w) EM1, which increased the energy delivered to the crystallization peak to 40.4 ± 0.8 J/g. This indicates that adding EM1 to the palm olein has an effect on energy released in the crystallization. On the other hand, the addition of the emulsifier in a proportion of 1.0% (w/w) of EM2 produced no significant (p < 0.05) effect on the enthalpy of crystallization (ΔHc), showing a decrease of 3.0 ± 0.2 J/g in the energy released when compared to the pure olein. A great increase in the release of heat during the crystallization process may be detrimental, because it may result in elevated temperatures that will cause the dissolution of already-formed crystals.24 The melting curves of mixtures showed melting peaks very close to that of palm olein, but the enthalpies of melting (ΔHm) were significantly higher for EM1 and for EM2 added in a proportion of 3.0% (w/w) and also did not present significant differences for EM2 at 1.0% (w/w). In summary, the emulsifiers modified the DSC melting properties of the coconut oil and palm olein that are ascribed to the influences of the emulsifiers on the crystallization properties. The melting profiles were different, indicating that interactions between TAGs changed due to emulsifier addition.

parameters regarding the melting curves of coconut oil, palm olein, and their mixture with the commercial emulsifiers. The addition of EM1 produced a few changes on the melting temperatures (Tonset °C) for coconut oil, and mixtures demonstrated close values in a range that varies from 9.9 ± 0.1 to 12.4 ± 0.2 °C with no significant difference (p > 0.05). On the other hand, enthalpy of melting was significantly affected (p < 0.05) mainly by adding the EM1, in which there was an increase of total ΔHm in both proportions. The crystallization curves of palm olein with added EM1 (a) and EM2 (b) are shown in Figure 4. The formation of only one peak crystallization in mixtures with the two commercial emulsifiers was observed. The addition of emulsifiers (EM1 and EM2) changed the crystallization profile of palm olein, showing significant difference (p > 0.05) in the crystallization temperature (Tonset, °C). The addition of 1.0 and 3.0% (w/w) EM2 showed crystallization temperature (Tonset, °C) lower than observed in pure palm olein and EM1, indicating that the addition of EM2 delayed the crystallization of this oil. Increasing the crystallization temperature (Tonset, °C) by the addition of EM2, rich in saturated MAGs, in coconut oil and palm olein is in agreement with the results of studies by Man et al.43 and Basso et al.,24 in which the crystallization peaks (Tonset, °C) of the compounds containing more saturated triacylglycerols moved to the region of higher temperatures. 2259



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Figure 6. Images of the crystallization of coconut oil their mixture with EM1 and EM2. The bar represents 200 μm.

possibly be explained by the crystallization of a β′-polymorph.47 The increasing of the temperature did not affect the microstructure of EM1. EM2, which has a high content of saturated fatty acids, showed a dense structure of very small crystals. This is probably due to conditions of cooling that affect the crystallization of higher saturated emulsifier. Even at the highest temperature of crystallization (20 °C) the grain structure maintained crystallization. Coconut oil at 10 °C showed a dense structure of large crystals; the image showed very compact spherulites type, with no empty space between them, and low birefringence, but with good definition. At 15 and 20 °C this oil shows extra-large spherulites, the nucleus had higher birefringence, and the edges showed little needles. In coconut oil as the temperature was increased to 15 °C the microstructure of this oil was sharply affected. Temperatures up to 20 °C increased the voids between spherulites, which consequently decrease the crystallized area. Palm olein (Figure 7) showed a dense structure of very small crystals with instantaneous and homogeneous crystallization, which is observed in a large number of small crystals and with the increase in temperature (20 °C) had reduced the total spherulites. From the observation of microscope images it is possible to affirm that the use of these pure fats (coconut oil and palm olein) has many limitations for applications in those foods that require higher storage temperatures because the temperature of 20 °C formed extra-large spherulites type (coconut oil) or hardly

Polarized Light Microscopy. The study of crystalline microstructure of fat systems has become increasingly important because the functional properties of many foods depend on knowledge of their fine structure. Microstructure is dependent on the composition of a fat, as well as its crystallization behavior, including polymorphism, and the characteristics of the microstructure in turn determine the physical properties of the fat. Success in the measurements requires several stages including obtaining a truly representative image of material, analyzing that image properly, and interpreting the resulting data.44 The anisotropic solid phase lattice in the analysis under polarized light refracts light differently from the isotropic liquid phase. The liquid phase appears in the images in black and solid phase in white or grayscale.45 The “crystals” seen by light microscopy in natural fats are actually clusters of many individual crystals. Fast cooling rates create smaller crystals and slow cooling rates create larger visible crystals, respectively. Thus, the conditions of process determine the size of the clusters, but not the size of the crystals.46 The samples were crystallized at 10, 15, and 20 °C. An image was taken directly after 24 h of formation of the fat crystal network, as can be seen in Figures 5, 6, and 7. Crystal mean diameter values of coconut oil, palm olein, and their mixture with the commercial emulsifiers are shown in Table 6. Figure 5 shows the individual commercial emulsifiers, pure coconut oil, and palm olein. The EM1 sample, which has a composition with high unsaturated fatty acids content, gives a network of small needle-shaped and granular crystals, which can 2260



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Figure 7. Images of the crystallization of palm olein and their mixture with EM1 and EM2. The bar represents 200 μm.

and 15 °C (Table 6). At 20 °C the addition of EM1 in coconut oil produced large spherulites that extrapolated the visual field of the slide; therefore, for statistical purposes a maximum value of the visual field has been estimated as 1250 μm. The addition of EM2 to coconut oil considerably changed the behavior of this oil. The spherulites after the addition of 1.0 and 3.0% (w/w) were significantly smaller in size (Table 6) than the pure coconut oil and that with EM1 added. The addition of 3.0% (w/w) also maintained the large number of spherulites even at higher temperatures (20 °C), which makes this percentage addition a more interesting technological standpoint. Figure 6 shows the palm olein addition with 1.0 and 3.0% (w/ w) of EM1 and EM2. In palm olein, the addition of 1.0% (w/w) EM1 did not alter the microstructure of this fat at any of the temperatures, but the increase in this ratio to 3.0% (w/w) of this emulsifier altered the pattern of crystallization of this fat. With the increased temperature occurred a reduction of crystallized area, and at 20 °C no crystallization occurred even after 24 h. These results for microstructure with addition of EM2 are consistent with the findings of Basso et al.,24 who described that the addition of saturated monoacylglycerols leads to the formation of a greater number of crystallization seeds, possibly more stable to the effect of temperature. During the crystallization process, a crystalline network is formed that allows for greater interaction between triacylglycerol molecules, between which intermolecular attractive forces such as the van der Waal’s forces can act, which may promote greater intermolecular attraction, leading to a decrease in system

Table 6. Mean Crystal Diameter (Micrometers) of Coconut Oil (CO), Palm Olein (PO), and Their Mixture with the Two Commercial Emulsifiers sample CO CO + EM1 (1.0% w/w) CO + EM1 (3.0% w/w) CO + EM2 (1.0% w/w) CO + EM2 (3.0% w/w) PO PO + EM1 (1.0% w/w) PO + EM 1 (3.0% w/w) PO + EM2 (1.0% w/w) PO + EM2 (3.0% w/w)

10 °C

15 °C

20 °C

302.8 ± 133.3a 194.5 ± 86.6b

490.7 ± 181.2a 248.9 ± 111.6a

584.6 ± 35.3a 1250.0 ± 9.9b

125.0 ± 27.0b

75.8 ± 20.7a

1250.0 ± 26.4b

37.3 ± 6.0c

456.7 ± 127.0b

297.0 ± 0.0c

3.2 ± 1.1c

3.7 ± 0.2c

49.4 ± 10.8d

2.7 ± 0.0a 1.5 ± 0.0a

5.4 ± 2.6a 4.6 ± 0.3a

37.2 ± 15.6a 18.2 ± 2.7b

1.1 ± 0.0b

14.6 ± 2.2a

0.0 ± 0.0b

1.3 ± 0.3b

2.3 ± 0.5a

6.8 ± 0.8b

2.5 ± 0.6a

1.8 ± 0.5a

6.6 ± 0.9b

crystallized (palm olein), and these microstructures are not suitable for application in foods. Figure 6 shows the coconut oil addition with 1.0 and 3.0% (w/ w) of EM1 and EM2. The addition of EM1 to coconut oil changed the crystallization pattern. The spherulite size was significantly lower when added 1.0 and 3.0% (w/w) of EM1 were added at temperatures of 10 2261



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solubility, more evident at high temperatures, contributing to the crystallization process. According to Herrera et al.,48 fats for food product applications should have crystal diameters of

Effects of Emulsifier Addition on the Crystallization and Melting

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