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Research Article Microencapsulation of extra-virgin olive oil by spray-drying: Inuence of wall material and olive quality
Patricia Calvo, Teresa Hernandez, Mercedes Lozano and David Gonzalez-Gomez
Technological Institute of Food and Agriculture (INTAEX), Junta de Extremadura, Ctra. San Vicente S/N, Badajoz, Spain

Encapsulation is a process by which small particles of core products are packaged within a wall material to form microcapsules. One common technique to produce encapsulated products is spray-drying which involves the conversion of liquid oils in the form of an emulsion into dry powders. Emulsication conditions, wall components, and spray-drying parameters have been optimized for the microencapsulation of different extra-virgin olive oils. To achieve this goal, the inuences of emulsion conditions have been evaluated for different wall components such as proteins (sodium caseinate and gelatin), hydrocolloids (Arabic gum), and hydrolyzed starches (starch, lactose, and maltodextrin). In addition, for each of the tested conditions the ratio of wall solid-to-oil and spraydrying parameters were as well optimized. The microencapsulation effectiveness was determined based on process yield and the ratio between free and encapsulated oil (microencapsulation efciency). Highest encapsulation yields were achieved when gelatin, Arabic gum and maltodextrin and sodium caseinate and maltodextrin were used as encapsulation agents and the ratio of wall solid-to-oil was 1:4 and 1:2, respectively. Under these conditions, 53% of oil was encapsulated. The inuence of olive oil quality in the microencapsulation process was evaluated in terms of fatty acids prole alteration after the microencapsulation process.
Keywords: Antioxidant activity / Fatty acids / Microencapsulation / Monovarietal extra-virgin olive oil / Spray-drying

Received: February 22, 2010/ Revised: April 27, 2010/ Accepted: May 28, 2010 DOI: 10.1002/ejlt.201000059

1 Introduction
Microencapsulation is dened as a process in which tiny particles or droplets are surrounded by a coating, or embedded in a homogeneous or heterogeneous matrix, to form small capsules [1, 2] and build a barrier between the

lez-Gomez, Instituto Tecnologico Correspondence: Dr. David Gonza Agroalimentario de Extremadura (INTAEX), Ctra. San Vicente S/N. 06007, Badajoz, Spain E-mail: david.gonzalezgo@juntaextremadura.net Fax: 34-924012674 Abbreviations: AA, antioxidant activity; ABTS, 2,20 -azinobis(3ethylbenzothiazoline)-6-sulfonic acid, diamonium salt; C2M, microencapsulated C2; FAME, fatty acid methyl esters; ME, microencapsulation efciency; MM, microencapsulated Morisca; MUFA, monounsaturated fatty acids; OSI, oxidative stability; PC, principal component; PC1, principal component 1; PC2, principal component 2; PCA, principal component analysis; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; SSPS, soluble soybean polysaccharide; TC, total tocopherol; TPC, total phenolic compound

component in the particle and the environment. The core may be composed of just one or several ingredients and the wall may be single or double-layered. Taking into account food industry characteristics, microencapsulation should be dened as a technique by which liquid droplets, solid particle, or gas compounds are entrapped into thin lms of a food grade microencapsulating agent [2]. Although many techniques have been developed to microencapsulate food ingredients, spray-draying is the most common technology used in food industry due to low cost and available equipment. Compared to freeze-drying, the cost of spray-drying method is 30--50 times lower [3]. Microencapsulation by spray-drying has been successfully used in food industry for several decades [4] and this process is one of the oldest encapsulation methods used since the 1930s [5]. An important step in developing microcapsules is the selection of a wall material that meets the required criteria. In fact, the efciency of the protection or controlled release mainly depends on the composition and structure of the established wall. This wall could act as a barrier and it may protect against oxygen, water, light or could avoid contact with other
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ingredients or control diffusion. The selection of wall materials for microencapsulation by spray-drying has traditionally involved trial-and-error procedures in which the microcapsules are formed. Numerous wall materials have been studied and used for their suitability as encapsulating agents in spray-drying. Walls material for microencapsulation of oil by spray-drying must have emulsifying properties, high water solubility, low viscosity, and drying properties [610]. Typical wall materials include proteins (sodium caseinate, whey proteins, soy proteins, and gelatin) and hydrocolloids (modied starch and Arabic gum). Hydrolyzed starches (glucose, lactose, corn syrup solids, and maltodextrin) are generally added as a secondary wall material to improve drying properties of sprayed droplets [7, 9]. Most edible oils are chemically unstable and susceptible to oxidative deterioration, especially when exposed to oxygen, light, moisture, and temperature. That oxidative degradation result in a loss of nutritional quality and a development undesired avors, affecting shelf stability and sensory properties of the oil [11]. Therefore, oil encapsulation may be useful to retard lipid auto-oxidation and increase the range of applications where otherwise oil could not be used. Apart from that, it has been widely reported, that the production of monovarietal olive oils has increased during the last few years due to their favorable chemical and sensorial characteristics [1219]. Among other factors, the abundance of mono-unsatured and poly-unsatured fatty acids, such as oleic acid, provide a high range of health benecial characteristics to olive oil [16]. Avoiding fatty acids prole alteration, through a microencapsulation process, will prevent loosing such healthy properties of olive oil and increase self life during product manufacturing. In this paper, we have studied for the rst time, the effect of different extra-virgin olive oil (Morisca and Picual monovarietal-type extra-virgin olive oil and three blend-type extra-virgin olive oils) in the microencapsulation process by spray-drying. Fatty acid composition has been evaluated for the different extra-virgin olive oil after and before the microencapsulation process in order to evaluate the olive oil attitude to the spray-drying processing. Furthermore, the main phytocompounds, such as phenolic compounds and tochopherol contents, antioxidant activity (AA) and oxidative stability (OSI) have been studied for each studied oils. Different aspect of microencapsulation process; such as wall material composition, wallcore micropasules ratio, microencapsulation efciency (ME) were as well evaluated.

experimental elds to ensure the monovarietal characteristic of the studied oils. The other three studied olive oils were blend-type extra-virgin olive oils bought in local groceries.

2.2 Microencapsulation wall material

Sodium caseinate and starch were purchased from Sigma Aldrich (Spain). Gum Arabic and lactose were supplied by Panreac (Spain). Gelatin and maltodextrin were procured from Sancan (Spain).

2.3 Microencapsulation process

The rst stage to achieve oil microencapsulation was the formation of a ne and stable emulsion of the core material (oil) in the wall solution. Different ratios oil and wall material were evaluated for each studied wall materials. Finally, the emulsions were prepared at room temperature using a laboratory blender.

2.4 Spray-drying
The emulsions prepared were spray dried with a laboratory scale Buchi spray drier (Mini Spray drier B-290, Buchi Labortechnik, Switzerland) equipped with 0.5 mm diameter nozzle. The pressure of compressed air for the ow of the spray was adjusted to 5 bars. The inlet and outlet air temperatures were maintained at 165 5 and 80 58C, respectively and feed rate was adjusted to 360540 mL/h. Emulsions were prepared at the moment of the spray drying process and the emulsion were kept under magnetic stirring during the whole process. The microcapsules were collected from the collecting chamber and transfer to double layer plastic bags and stored in darkness until analysis.

2.5 Microscopy and scanning electron microscopy

After emulsion, an optical microscope (Leica DML) equipped with a digital camera (Leica DC100) was used to check the microcapsules formation and the morphology were evaluated with a scanning electron microscope.

2.6 Surface oil content and microencapsulation efficiency

Tan et al. [20] modied procedure was employed to measure the amount of unencapsulated oil present at the surface of the powders. Briey, 5 g of microcapsules were precisely weighted in a beaker and 50 mL of hexane was added and shaken during 15 s at ambient temperature to extract supercial oil. The solvent mixture was then ltered throughout a lter paper, and after that, unencapsulated oil was collected after vacuum hexane evaporation. To measure the encapsulated oil, oil was extracted by 4 h Soxhlet of the same powders. After that, hexane was evaporated and internal
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2 Material and methods

2.1 Extra-virgin olive oil
A total of ve different extra-virgin olive oils were used in our studies. Two of them were monovarietal type extra-virgin olive oil (Picual and Morisca) and these oils were obtained in our experimental research facilities from olives harvested in
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oil was scaled. ME was calculated using the following formula: ME Total oil Surface oil 100 Total oil 1

2.10 Chromatographic determination of tochopherols

A Lichrosorb Si60 column, 5 mm, 25 cm 4.60 mm id was used for the chromatographic separation of olive oil tochoperols (Agilent 1200 Liquid Chromatography equipped with a uorescence detector), being the mobile phase ow rate of 1.0 mL/min and 10 mL as the injection volume. The isocratic elution was carried out using HPLC-grade n-hexane/isopropanol/ethanol (98.5:1.0:0.5 by volume) and tocopherols were determined by using a uorescence detector with an emission wavelength of 330 nm and excitation wavelength of 290 nm. The different tocopherols were identied by comparing retention times with those of commercial standards (Merck, Spain). Results are expressed as mg/kg of oil.

Free oil content was then calculated as percentage taking into account the total oil.

2.7 Fatty acid olive oil composition

Fatty acid methyl esters (FAMEs) from the oil samples were obtained by alkaline treatment with 2 N KOH in methanol and gas chromatographic analyses was carried out following the European commission regulation CEE 2568/91 [21]. To determine the alteration of fatty acids prole after the microencapsulation process (emulsion and spray draying), FAME determinations were carried out using non-processed olive oil, Soxhlet extracted of non-processes olive oil and Soxhlet extracted microencapsuled olive oil. All the analysis were performed in an Agilent 7890A Gas Chromatographer equipped with an FID detector. Fatty acids were separated in a 60 m cis/trans FAME column.

2.11 Oxidative stability

An 743 Rancimat (Metrohm, Switzerland) eight-channel OSI instrument was used to evaluate the OSI of the studied olive oils. The OSI index was expressed in h (n 4).

2.12 Statistical analysis

The results are expressed as means SD and were analyzed using a one-way analysis of variance (ANOVA). When ANOVA detected signicant differences between mean values, means were compared using Tukeys test. Correlations between the studied parameters were evaluated with the Pearson test at p<0.05 signicance level. For statistical studies SPSS 15.0 software was used (SSPS, Chicago, IL, USA). Principal components (PCs) and classication analysis were assessed by using the Unscrambler 6.11 software (CAMO Process AS, Oslo, Norway).

2.8 Radical scavenging of olive oils

2,2 -Azinobis(3-ethylbenzothiazoline)-6-sulfonic acid, diamonium salt (ABTS) method was used for radical scavenging determination [22, 23]. Briey, the radical was generated using acidied ethanol. Once the radical was formatted, 1 mL of ABTS were mixed with 20 mL of 0.1 g of oil solved in 1 g of hexane, and the absorbance was measured at 730 nm during 20 min. The results were expressed as a TEAC (antioxidant equivalent to Trolox) value as mmol Trolox/kg oil, determined by interpolation the fall in absorbance (taking into account the adequate dilution and the linear range of calibration line) on a calibration curve using different Trolox concentration. All the measurements were performed in a Shimadzu UV spectrophotometer (Kyoto, Japan), ABTS and Trolox reagents were purchased at Sigma Aldrich and results were expressed as mmol Trolox/kg of oil (n 3).
0

3 Results and discussions

3.1 Wall component selection and emulsion preparation
In order to select the most appropriate wall component a total of four different microcapsule models were evaluated (Table 1) and they were selected according to the microencapsulation yield, internal and external fat and the ME. In general terms, the best results were obtained when a protein and hydrolyzed starch were used simultaneously (models 13). Whereas using a combination of starches without proteins (model 4) the microencapsulation yield dropped considerably. Hydrolyzed starches are considered as good encapsulation agents because they exhibit low viscosities at high solids contents and good solubility, but most of them lack the interfacial properties required for high ME and generally must be associated with other encapsulating material such as proteins or gums [25]. The water content of the microcapsule emulsion has to be adjusted, since emulsion viscosity
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2.9 Total phenolic contents

Total phenolic compounds (TPCs) of the virgin olive oil were determined using a UVVis 1204 Shimadzu spectrophotometer (Kyoto, Japan). Phenolic compounds were extracted from oil samples (2 g of oil solved in 50 mL of hexane) by successive extraction (n 3) with methanol/water (60:40 v/ v) solution. Aqueous fractions were collected and methanol was evaporated under vacuum, and total phenolic contents were determined according to Singleton and Rossi [24]. The phenolic compounds were quantied from a calibration curve using caffeic acid (SigmaAldrich) and data were expressed as mg of caffeic acid per kg oil (n 3).
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Table 1. Microcapsules wall material composition and process characterization in terms on encapsulation yield, internal and external fat and microcapsule efficiency for each tested wall component
Models Composition (%) Oil Water Gelatin Sodium caseinate Maltodextrin Modified starch Lactose Gum Arabic Oil:wall material Encapsulation yield Internal fat External fat Microcapsule efficiency 1 1.81 90.93 2.18 4.72 0.36 1:4 51.20 5.90 42.35 5.28 57.65 5.28 42.35 5.28 2 5.88 82.36 5.88 5.88 1:2 49.49 0.67 52.98 3.24 46.98 3.24 52.98 3.24 3 5.88 82.36 5.88 5.88 1:2 51.21 2.38 38.60 2.37 61.40 2.37 38.52 2.37 4 6.41 64.10 23.08 6.41 1:4.6 33.44 3.27 45.23 2.24 54.77 2.24 33.43 2.24

is a limiting factor for using spray-drying methodology. The solid level is typically maximized, since there is little benet in adding unneeded water and in general, the upper limit is the viscosity at which the emulsion cannot be adequately atomized or the material does not dry in the dryer [26]. The combination of sodium caseinate and lactose (model 2) produced a system with a higher internal fat that the system formed by the combination of sodium caseinate and maltodrextrin (model 3). However, the substitution of the protein component (gelatin instead of sodium caseinate) did not change appreciably the results of the measured parameters (model 1). Similar results were obtained by other authors, for instance Sanchez et al. [27] used a combination of maltodextrin, gum Arabic, and gelatin to encapsulate shark liver oil. In this system, the addition of gelatin increased the strength of the microcapsule wall and it was observed that when the amount of this component increased in the microcapsule formulation, the amount of encapsulated oil was higher. In addition, Minemoto et al. [28] used soluble soybean polysaccharide (SSPS), gum Arabic, and a mixture of both together with maltodextrin. When SSPS was mixed with

maltodextrin the encapsulation efciency increased as the weight fraction of SSPS in the mixture was increased. On the other hand, the weight fraction of gum Arabic scarcely affected the encapsulation efciency.

3.2 Olive oil characterization

In order to evaluate the inuence of the chemical oil composition into the microencapsulation process, the studied olive oils were chemically characterized in terms on total phenolic contents (TPCs), AA, total tocopherols (TCs), OSI, and fatty acids prole. These results are summarized in Tables 2 and 3. Regarding the TPCs, Picual, C1, and C3 were the olive oils having the highest contents. In addition, these three olive oils had higher values of OSI, being C3 the oil with the highest OSI. Concerning the AA values, the highest values were be found in the blend oils, while the major contents of TCs were measured in the monovarietal olive oils. Finally, the ratio of oleic acid to linoleic acid among the different type of olive oil was as well evaluated. Picual and C3 olive oils showed the highest values of this ratio, whereas the lowest vale was

Table 2. Chemical characterization of Morisca and Picual monovarietal-type extra-virgin olive oils and three blend-type extra-virgin olive oils
TPCs Morisca Picual C1 C2 C3 150.59 371.55 397.57 254.24 459.64 16.61a 39.42c 35.14c 28.75b 34.60c AA 1.498 1.214 2.320 3.223 2.798 80a 200a 68b 91c 451c,b TCs 231.4 289.9 227.5 141.4 128.1 1.0c 11.5d 1.4c 5.1b,a 15.1a OSI 25.64 40.03 44.79 25.16 83.89 0.42a 0.03b 0.15c 0.00a 1.99d C18:1/C18:2 5.01 0.01a 16.49 0.12e 9.40 0.01c 8.71 0.02b 12.82 0.02d

TPC, total phenolics compounds (mg caffeic acid/kg oil), AA, antioxidant activity (mmol Trolox/kg oil), TCs, total tocopherols (mg/kg oil), OSI, oxidative stability index (h), C18:1/C18:2, ratio of oleic acid to linoleic acid. Different letters in the same column means signicant differences in the values (Tukeys test, p<0.005).
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Table 3. Fatty acids profile of the studied extra-virgin olive oils before and after the microencapsulation process
Initial Average SD Average Morisca SFA MUFA PUFA SFA MUFA PUFA SFA MUFA PUFA SFA MUFA PUFA SFA MUFA PUFA 18.07a 67.80a 14.13a 15.47a 79.01a 5.52b 12.94a 78.15a 8.91c 14.84a 78.48b 9.11b 14.14a 79.15ns 6.71b 0.04 0.04 0.04 0.04 0.07 0.04 0.02 0.02 0.01 0.88 4.46 0.54 0.04 0.05 0.01 20.29b 70.17b 9.54b Picual 16.59b 79.59b 3.81a C1 13.84c 79.50c 6.66a C2 15.87a 76.62b 7.51 C3 14.54a 79.68ns 5.78b 0.03 0.04 0.02 17.11a 78.35ns 4.53a 6.12 4.10 2.23 0.084 ns 0.000 0.39 0.54 0.91 33.82b 62.80a 3.89a 17.24 16.32 4.49 0.000 0.001 0.000 0.11 0.14 0.04 13.19b 78.36b 8.44b 0.07 0.11 0.15 0.000 0.000 0.000 0.14 0.15 0.02 15.65a 79.62b 4.73ab 1.26 0.73 1.96 0.000 0.000 0.000 1.31 1.31 2.62 23.39c 72.32c 4.29c 1.00 0.75 1.34 0.000 0.000 0.000 Soxhlet SD Microcapsules Average SD Sig.

SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Different letters in the same row means signicant differences in the values according to the listed signicance (Tukeys test). ns: no signicant differences were observed among the values in the same row ( p<0.05).

measured in Morisca monovarietal-type olive oil. Similar results were reported by Cerretani et al. [19]. In addition, as in our results, these authors concluded that higher OSI values were found in those oils having higher concentration of TPCs. Regarding the fatty acid prole, our data are in accordance with those reported by Sanchez Casas et al. [29].

3.3 Olive oil microencapsulation

Model 1 was applied to microencapsulate ve different types of extra-virgin olive oil (Figure 1). This model was selected

because of the higher values of encapsulation yield and microcapsule efciency. The inuence of olive oil chemical composition in the microencapsulation process was evaluated in terms of fatty acids prole alteration after the microencapsulation process. Therefore, fatty acids were determined in the studied olive oils after and before the microencapsulation. In addition, since the Soxhlet procedure was used to totally extract oil from microcapsules, the effect of this procedure was as well evaluated and the results are summarized in Table 3. From this table, it can be observed how Picual extra-virgin olive

Figure 1. Pictures of microcapsules emulsion (A and B) and electron microscopy image (C) of the optimized extra-virgin olive oil microcapsules.
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oil is among the monovarietal olive oil the oil more appropriated to be microencapsulated using spray-drying in terms of not alteration of the fatty acids prole. Similar results are obtained for C3 extra-virgin olive oil among the coupage olive oils studied in this research work. The suitability of these two olive oils to be microencapsulate and processed by spraydrying is not explained by a single factor, but a sum of factors. As it can be observed in Table 2, Picual, C1 and C3 were the oils having the highest values of OSI index (performed by OSI), and the highest values of C18:1/C18:2. Previously [16, 19] it was found a positive Persons correlation between the oleic acid to linoleic acid and the OSI time. In addition, similar correlations were also found among the TPCs and the OSI values [19]. A principal component analysis (PCA) was carried out in order to evaluate the inuence of the spray-drying process into the alteration of the fatty acids prole. PCA is a linear transformation of a set of original data to a set of uncorrelated components in such a way that only a few of the resulting variables account for the majority of the variability observed in the original data. Therefore, a reduction in dimensionality is achieved with minimal loss of information. This reduction is signicant in the evaluation of large datasets, containing many interrelated variables generated by instrumental analytical techniques. Visualization of the results of PCA is usually achieved by plotting pairs of the rst few PCs. In our study, the principal component 1 (PC1) and principal component 2 (PC2) represented the 70% of the system

Figure 3. Loading plot (fatty acids) after PCA of the variables in the plane defined by the two first PCs PC1 and PC2. Each fatty acid has been represented according to their number of carbon atoms and unsaturated grade.

variance. Figures 2 and 3 represent the scores (analyzed samples) and loading (variables) plot for all the studied olive oil and fatty acids. From the scores plot (Figure 2) it can be observed how the samples microencapsulated C2 (C2M extra-virgin olive oil) and microencapsulated Morisca (MM extra-virgin olive oil) formed two different clusters separated from the rest of samples according to the PC1. According to the loadings plot the saturate fatty acids explain the variance of the C2M and MM samples. Same information could be read from Table 3, C2 and M were the oils having more contents of saturated acids after the microencapsulation process, whereas before the microencapsulation the contents of the saturated fatty acids (SFA) were similar in all the studied oils. Apart from that, all the oils but C2 and Morisca are grouped without differencing non-encapsulated and microencapsulated oil and according to the loading plot; the variance of these samples is explained by the unsaturated and polyunsaturated fatty acids.

4 Conclusions
A better microcapsule yield, efciency, and internal and external fat ratio were achieved when a combination of proteins and polysaccharides was used as the wall component. No important differences in terms of these parameters were observed when lactose or maltodextrin were used as the starch component. On the other hand, in order to evaluate the suitability of different extra-virgin olive oils for the microencapsulation procedure by spray-drying, the chemical attributes of the different oils were studied. In the olive oils that had higher amounts of total phenolic contents, OSI index and ratio between C18:1/C18:2, the fatty acid prole was unaltered after the microencapsulation process, regardless of the value of the other studied parameters. Finally, no signicant differences were observed among the monovarietal and coupage-type extra-virgin olive oils.
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Figure 2. Score plot (oil samples) after PCA of the individuals in the plane defined by the two first PCs PC1 and PC2. (M, Morisca type oil; MS, Soxhlet extracted Morisca type oil; MM, microencapsulate Morisca type oil; P Picual type oil; PS, Soxhlet extracted Picual type , oil; PM, microencapsulate Picual type oil; C1, C1 type oil; C1S, Soxhlet extracted C1 type oil; C1M, microencapsulate C1 type oil; C2, C2 type oil; C2S, Soxhlet extracted C2 type oil; C2M, microencapsulate C2 type oil; C3, C3 type oil; C3S, Soxhlet extracted C3 type oil; C3M, microencapsulate C3 type oil).
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The authors are grateful to the Consejeria de Economia, Comercio e Innovacion de la Junta de Extremadura, and the European FEDER Funds for the economical support (Project PDT08B010). David Gonzalez-Gomez thanks to the INIA for his research contract. The authors have declared no conict of interest.

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