2017 Volume 23 Issue 4 Pages 503-509
The objective of this study was to analyze the surface oil ratio and distribution measurement of spray-dried powders during hexane washing, in order to evaluate the properties of fish oil-encapsulated powders. Two types of spray-dried powders were prepared from two differently sized emulsions, namely: (i) nano and (ii) microsized oil droplets. A matrix composed of maltodextrin and sodium caseinate was used as the wall material to encapsulate the fish oil, and Nile red was used to stain the oil. The extraction of the surface oil releases a threshold amount of oil on the surface of the particle. The study of the fluorescence intensity using confocal laser scanning microscopy (CLSM) indicates good correlation between the oil content and fluorescence intensity. It was difficult to remove the surface oil of the nano-sized oil droplets in the spray-dried powders by hexane washing because the smaller oil droplets had higher stability in the spray-dried powders.
Fish oil is well known as a rich source of n-3 polyunsaturated fatty acids with high amounts of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The intake of fish oils seems to play a major role in reducing cardiovascular diseases, and this has been demonstrated in the epidemiological and observation studies worldwide (Kris-Etherton et al., 2002, Simopoulos, 2004, Wall et al., 2010). The intake of EPA and DHA in the diet during the pregnancy period can positively influence baby development and can reduce the risk of allergies in infants (Minihane, 2016).
Therefore, several studies have been conducted on the production of fish oil. One of the study was regarding the encapsulation of emulsified fish oil by spray-drying. Encapsulating emulsified fish oil by the method of spray drying has been reported in order to form stable fish oil with maltodextrin as a wall material and proteins as emulsifier. The advantages of encapsulating fish oil are better stability against oxidation by retarding autoxidation, protection against enzyme hydrolysis, and masking the fishy smell (Gharsallaoui et al., 2007).
In encapsulating functional oil by spray drying, surface oil content is important in evaluating the stability of functional oil in spray-dried powders. Several studies have been reported about the importance of surface oil in spray-dried powders. An investigation of microencapsulated sunflower oil under the storage condition of 60 ± 1°C for 30 days indicated that low microencapsulation efficiency of powder may contribute to more surface oil content and indicated higher lipid oxidation (Ahn et al., 2008). By encapsulating fish oil by spray drying, it has been revealed that nano oil droplets with a diameter range of 210 – 280 nm contain the lowest surface oil content (Jafari et al., 2008). A report on the oxidation of encapsulated bulk coffee oil stored at 25 and 60°C indicated that peroxide values were constant during storage at 25°C, and the pure coffee oil exhibited higher peroxide values than the encapsulated oil at 60°C (Frascareli et al., 2012). Another finding by Carneiro et al. (2013) indicated that high encapsulation efficiency and a minimum amount of surface oil content in the spray-dried powder are essential to produce a stable powder. Kaushik et al. (2015) reviewed microencapsulation of omega-3 fatty acids. A review of microencapsulation and characterization methods indicated the importance of sensory analysis of initial off-flavors for fish-oil encapsulated powders. Encina et al. (2016) summarized the microencapsulation of fish oil by spray drying and suggested that feed emulsion is a key step for fish oil microencapsulation.
Nowadays, an emulsification system with high-pressure homogenization can significantly form nano-sized diameter oil droplets in spray-dried powder. This encapsulation efficiency is affected by several parameters such as emulsion characteristics, especially oil droplet size (Soottitantawat et al., 2003). Furthermore, conditions during spray drying, generally the inlet temperature, also affect the characteristics of the encapsulated powder (Frascareli et al., 2012). A study by Kikuchi et al. (2013) revealed the importance of oil ratio in encapsulation composition and concluded that a lower oil ratio produces lower surface oil content. Therefore, the analytical procedure of surface oil content in a spray-dried powder is important in evaluating the properties of fish oil encapsulated as a powder.
Powdery surface-oil coverage can be estimated by using electron spectroscopy for chemical analysis (ESCA) (Fäldt et al., 1995, Kim et al., 2002, Nijdam et al., 2006). Another method that has been used is solvent washing with petroleum ether, to extract lipids or surface oil from encapsulated vegetable oil powder (Kim et al., 2002, Turchiuli et al., 2005, Domian et al., 2008). Moreover, a report on surface characterization of four industrial spray-dried powders by Kim et al. (2002) revealed that the surface amount measured using ESCA complies well with that measured by using solvent extraction. Extraction of surface oil from spray-dried powders by dispersing with hexane under various conditions has been reported by our group (Soottitantawat et al., 2005, Paramita et al., 2010, Shiga et al., 2014, Abd Ghani et al., 2016). Furthermore, to measure surface oil content, hexane is recognized to be more effective than petroleum ether if the samples are stored for any period of time (Buck et al., 2007).
The CLSM technique has been used for food structure investigations since 1980s, and several researchers have reported this technique in their food science research (Cardona et al., 2013). Samples are illuminated by the excitation of light in the CLSM system, and hence, it is necessary to use suitable stains for labeling target components (Semwogerere et al., 2005). Generally, fat is stained by Nile red, and a CLSM image is captured by the fluorescence of a stained sample. For example, the layer of surface fat in whole milk powder can be clearly observed using CLSM (McKenna, 1997). Using CLSM, microcapsule characteristics of encapsulated oil and vacuoles inside a powder were also investigated (Lamprecht et al., 2000). The CLSM technique has been adapted by several researchers in visualization and characterization of microcapsules, especially in spray-dried powders (Soottitantawat et al., 2007, Paramita et al., 2010, Yang et al., 2014).
In this study, we investigated and measured the surface oil ratio of encapsulated fish oil powders prepared with oil droplets of two sizes by using the hexane washing method. The surface oil distribution in the microcapsules was also measured by using the CLSM technique for spray-dried powders during hexane washing for a selected period of time, in order to characterize the encapsulated fish oil powders.
Materials Fish oil was obtained from Miyoshi Oil & Fat (Tokyo, Japan), Maltodextrin (MD of dextrose equivalent (DE) = 11) was obtained from Matsutani Chemical Industry (Itami, Japan), and sodium caseinate was received as a gift from Miyoshi Oil & Fat. The fluorescent compound Nile red was purchased from Tokyo Chemical Industry (Tokyo, Japan), and n-hexane (96.0%), methanol (99.5%), N,N-dimethylformamide (DMF) (99.5%), and 2-methyl-1-propanol (99.0%) were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Sample preparation Sodium caseinate (7.5 g) was dissolved in distilled water (250 g) by heating at 50°C and mixing. Maltodextrin (92.5 g) was then added to this solution. Fish oil (150 g) containing Nile red (150 mg) as a fluorescent indicator was added to and mixed with the existing solution. Two types of homogenization methods were used. In order to form micro-sized oil droplets in spray-dried powders, homogenization was carried out using a hand held-type rotor-stator homogenizer (Polytron, PT-6100; Kinematica, Littau, Switzerland) at 8000 rpm for 3 min. Further, nano-sized oil droplets were obtained with second step of homogenization using a high-pressure homogenizer (Starburst Mini; Sugino Machine Limited, Sugino Corp., Toyama, Japan) at 100 MPa for 2 cycles.
Microencapsulation of fish oil powder The emulsions were spray dried using an Ohkawara-L8 spray dryer (Ohkawara Kakouki, Yokohama, Japan) equipped with a centrifugal atomizer. Temperatures of the inlet and outlet air at a flow rate of 110 kg/h were about 160°C and 115°C, respectively. The feed rate of the emulsion was 25 mL/min, and the rotational speed of the atomizer was around 30,000 rpm. The prepared microcapsules were stored at −30°C in an aluminum bag in an atmosphere atmosphere containing nitrogen for whole analysis.
Moisture Moisture content in the powder was measured using an HB43-S halogen moisture analyzer (Mettler Toledo Inc., Columbus, USA).
Oil droplet size measurement in original and reconstituted emulsions and particle size distribution The oil droplet size distribution in the feed emulsion and reconstituted emulsion, which was prepared by dissolving spray-dried powders in distilled water, was analyzed using a Shimadzu SALD-7100 laser diffraction particle size analyzer (Shimadzu Corporation, Kyoto, Japan). About 100 µL of emulsion was mixed with distilled water in order to measure the droplet size. The particle size distribution of the powder diameter was measured using the analyzer after dispersing the microcapsules in 2-methyl-1-propanol.
Surface and total oil contents The surface oil ratio was determined by dispersing 300 mg of powders in 5 mL of hexane in a test tube and mixing them for 0, 5, 10, and 15 min with a vortex mixture (Vortex-Genie 2; Scientific Industries, Bohemia, USA) as hexane-washing procedure. The mixture was then centrifuged with a Compact Tabletop Centrifuge 2010 (Kubota Corporation, Tokyo, Japan) for 5 min at 3000 rpm. The supernatant was used to analyze the content of fish oil as surface oil. The surface oil ratio of the powder is defined as the ratio of the surface oil at 5 min hexane washing in the powder to the total oil content in microcapsules, since the surface oil ratios at 5, 10, and 15 min hexane-washing were almost of the same value. The total oil content was measured by dissolving 20 mg of powders in 1 mL of DMF in a glass tube. The fish oil was then extracted from the DMF solution with 1 mL of hexane by vortex mixing for 1 min and then centrifuging for 5 min at 3000 rpm. The oil content is defined as the ratio of the weight of the oil content in spray-dried powder to the powder weight.
A sample containing fish oil was loaded onto a rod (Chromarod-SIII; Mitsubishi Kagaku Iatron, Tokyo, Japan) and analyzed using a TLC-FID (Iatroscan new MK-5; Bechenheim, Germany) at air and hydrogen flow rates of 20 and 160 mL/min, respectively.
Scanning electronic microscopy (SEM) Surface and cross-sectional images of the microcapsules were taken using a JSM 6060 SEM (JEOL, Tokyo, Japan). The microcapsules were placed onto the SEM sample holder using a double-sided tape and coated with Pt-Pd using an MSP-1S magnetron sputter coater (Vacuum Device Inc., Tokyo, Japan).
Confocal laser scanning microscopy (CLSM) CLSM images of the spray-dried microcapsules were taken after hexane washing for 0, 5, 15, and 30 min. The washed microcapsules were centrifuged and vacuum filtrated using a vacuum pump (Laboport vacuum pump; KNF Neuberger, NJ, USA). The CLSM system (Olympus Fluoview FV300; Olympus Optical, Tokyo, Japan) was equipped with a helium-neon (He-Ne) laser and an argon ion laser (Melles Griot Laser Group, Carlsbad, California). The laser supplies 559 nm excitation wavelengths that allow emission of the result in the fluorescence (characterized by excitation/emission wavelengths of 559/629 nm).
Visualization of image The CLSM images were analyzed using a free java software image processing program, ImageJ 1.45 (Schneider et al., 2012). The relative value of the fluorescence intensity is defined as the ratio of fluorescence intensity at the observed point to the maximum value of fluorescence intensity. The CLSM images were observed at the middle in z-direction of particle of hexane washing and trimmed at 45 µm squares. The observed particle images were set on the center of this square and the fluorescence intensities were measured in the x-direction from the end of square at the center line of the particles.
Physical properties of spray-dried powder The powder diameter, feed emulsion oil droplet diameter, reconstituted oil-droplet diameter, oil content, surface oil ratio, and the moisture content are summarized in Table 1. The particle sizes of both the powders were almost the same (about 29 µm). The average diameters of the feed emulsion after applying the rotor-stator homogenization (RS-emulsion) and high-pressure homogenization (HP-emulsion) were 3.6 µm and 0.2 µm. The average diameters of the reconstituted oil droplets in the powder prepared from the emulsion with the RS-emulsion was 1.6 µm and that of the HP-emulsion was 0.8 µm, respectively. The average diameter of the reconstituted oil droplet has become larger after spray drying because coalescence of oil-droplet in emulsion occurred at atomization stage because of high oil content in feed emulsion (Soottitantawat et al., 2003). Conversely, the average diameter of the reconstituted oil droplet of powder was smaller when the large oil droplet broke and dispersed at the outer surface oil (Soottitantawat et al., 2003). The diameter of the reconstituted oil droplets decreased with increase in homogenization pressure because the pressure results in breakage of the primary emulsion (Garcia et al., 2012). The total oil contents in the microcapsules were about 58 wt% in RS-emulsion and about 60 wt% in HP-emulsion. The surface oil ratio of the spray-dried powders prepared from the RS-emulsion was about 48 wt%, which was higher than that of the powders prepared from the HP-emulsion (11 wt%).
| Analysis | Emulsification method | |
|---|---|---|
| Rotor-stator | High-pressure | |
| Powder diameter (µm) | 29.1 ± 0.11 | 29.2 ± 0.11 |
| Feed emulsion diameter (µm) | 3.6 ± 0.135 | 0.2 ± 0.042 |
| Reconstituted oil-droplet diameter (µm) | 1.6 ± 0.133 | 0.8 ± 0.102 |
| Oil content (wt%)a) | 58.3 ± 0.01 | 61.2 ± 0.05 |
| Surface oil ratio (-)b) | 47.9 ± 0.04 | 11.1 ± 0.007 |
| Moisture content (wt%) | 3.2 ± 0.05 | 3.9 ± 0.06 |
Particle morphology Figure 1 shows the SEM images of the fish oil powders prepared using both the homogenization methods. The particles are spherical as shown in Figs. 1A and 1B, and both particles displayed wrinkled surfaces. However, only those microcapsules prepared from RS-emulsion (Fig. 1A) had a dented surface. In cross-sectional images, the microcapsules prepared from the RS-emulsion had larger entrapped oil droplets in the powder (Figs. 1a and b). These images show that the homogenization procedure in the emulsification process significantly affected the physical properties of the spray-dried microcapsules. All cross-sectional images have indicated vacuoles inside the powders, and Fig. 1 (a) shows the “pockmarks” enclosed within the vacuole.
SEM images of fish oil microcapsules prepared with RS-emulsion (A and a) and HP-emulsion (B and b) oil. (A) and (B) show the surface structure, and (a) and (b) show the cross-sectional structure. Arrow in the image indicates the pockmark.
Surface oil with hexane washing Figure 2 shows the changes in the surface oil ratio of the microcapsules with hexane washing. Vortex mixing of spray-dried powder in hexane was essential to remove the surface oil from powder. However, after 5 min of hexane washing, the surface oil ratio of the microcapsules prepared from the RS-emulsion was about 48 wt%, whereas that of the microcapsules prepared from the HP-emulsion was only 11 wt%. The surface oil ratios of the spray-dried fish oil powders remained constant even after 15 min of hexane washing. These results indicated that the surface oil ratio of the spray-dried powders could be practically measured by washing with hexane for 5 to 15 min. A report by Hardas et al. (2000) also indicated the physical and oxidative stability of encapsulated milkfat, which was estimated using hexane washing to measure surface fat in powders. They noticed that one factor affecting the extractability of fat was the blocking of pores by wall material. In our study, the surface oil ratio of the microcapsules prepared from the RS-emulsion was higher than that of the microcapsules prepared from the HP-emulsion, as shown in Table 1. The fish oil in the microcapsules prepared from the RS-emulsion might leak during vortex mixing and was extracted as the surface oil. Therefore, the fish oil in the microcapsules prepared from the HP-emulsion would be more stable against the solvent extraction than that in the microcapsules prepared from the RS-emulsion.
Changes in the surface oil ratio with hexane-washing time for microcapsules prepared with (○) RS-emulsion and (□) HP-emulsion oil droplets.
Oil distribution measurement in spray-dried powder with hexane washing using CLSM The CLSM technique was used to determine the distribution of fish oil in the spray-dried microcapsules. Gallier et al. (2010) demonstrated the surface structure of bovine milk fat globule membrane using CLSM. McKenna (1997) also investigated the structural components of whole milk powder with CLSM and by using the fluorescence of Nile red. Figure 3 shows the CLSM images of the microcapsules prepared from the RS-emulsion and HP-emulsion after 0, 5, 15, and 30 min of hexane washing. These images were focused at the center of powder with a focus depth of 14 µm. Moreover, the focal point process was affected by the amount of fluorescence staining in the sample (Semwogerere et al., 2005).
CLSM images with focus depth of 14 µm for fish oil microcapsules prepared with (i) RS-emulsion and (ii) HP emulsion oil droplets. Hexane-washing times were 0 min for A and E, 5 min for B and F, 15 min for C and G, and 30 min for D and H.
For 0-min hexane washing, the entire images of the spray-dried microcapsules prepared from the RS-emulsion and HP-emulsion are displayed with fluorescent labeling. However, for microcapsules prepared from the RS-emulsion, the fluorescence of Nile red decreased with an increase in the hexane-washing time. In contrast, for the microcapsules prepared from the HP-emulsion, the hexane-washing process did not affect the surface oil ratio as presented in the CLSM images shown in Fig. 3 (ii). This observation indicated that the time suitable for hexane washing was 5 to 15 min for both the microcapsules prepared from emulsions using different emulsification methods. Hardas et al. (2000) and Bae et al. (2008) also used hexane as a solvent to measure surface oil in spray-dried powders. However, in their procedures, the period of vortex mixing was only 2 min. On the other hand, Serfect et al. (2009) used petroleum ether and mixed slowly for 15 min to determine the extractable oil content. This method has been used by several researchers under the heating conditions of 60°C to 80°C (Sankarikutty et al., 1988, Velasco et al., 2006).
Figure 4 shows the distributions of fluorescence intensity in fish oil microcapsules prepared with RS-emulsion (Figure 4A) and HP-emulsion (Figure 4B) at different hexane-washing times. The particle edge was indicated at the increasing point of relative fluorescence intensity from zero to 100%. For microcapsules prepared from the HP-emulsion, the fluorescence intensity distributions were almost the same with different times of hexane washing. The fluorescence intensity of the microcapsules prepared from the RS-emulsion decreased after 5 and 10 min of washing with hexane and became almost zero after 30 min. The analysis indicated that the fish oil in the microcapsules prepared from the HP-emulsion would be more stable against the solvent extraction than that in the microcapsules prepared from the RS-emulsion because the fluorescence of Nile red remained in the microcapsules prepared from the HP-emulsion even after 30-min washing. On the other hand, the fluorescence of Nile red was almost undetectable in the microcapsules prepared from the RS-emulsion after 30-min washing.
Distributions of fluorescence intensity in fish oil microcapsules prepared with (A) RS-emulsion and (B) HP-emulsion at different hexane-washing times. Washing times; △, ........ : 0 min; ●, - - - : 5 min; ▲, _____ : 15 min and ◯, - - - : 30 min.
Fluorescence intensities of Nile red could not correlate to the oil content in the spray-dried powders as shown in Figure 4A and 4B. Fluorescence intensity depends on photomultiplier (PMT) voltage, gain and offset values of CLSM. High voltage of PMT was used to evaluate shapely, the fluorescence intensities of the powder with RS-emulsion and this PMT voltage is a little excess value to describe the fluorescence intensities of Nile red for HP emulsion. Therefore, the fluorescence intensities for HP emulsion were almost the same for hexane washing times as shown in Fig. 4B.
The surface oil ratio in spray-dried microcapsules was analyzed with 5-min hexane washing. The surface oil ratio of the microcapsules prepared from the RS-emulsion was higher than that of the microcapsules prepared from the HP-emulsion. This result may be due to the presence of pockmarks inside the vacuoles because encapsulated oil was extracted during hexane washing. It was also shown that CLSM is a useful tool for studying the movement of oil during hexane washing. Fluorescence intensity can be used to correlate the oil content and allows the analysis of the distribution of oil inside the microcapsules.
Acknowledgements Asmaliza Abd Ghani received a scholarship from the Malaysian Ministry of Education throughout the study period. This study was also supported by JSPS KAKENHI with Grant Number 15K07455.
