Abstract
Effects of agglomeration of β-carotene microencapsulated by spray drying on its stability were analysed. Mixtures of Arabic gum (GA), maltodextrin (MD), modified starch (OSA), and whey protein (WP) were used as carriers. GA + MD and OSA + MD microcapsules were subjected to agglomeration. All the samples were stored for 60 days with access to daylight. Stability of the emulsions had a significant effect on efficiency of microencapsulation but had no effect on β-carotene retention during sample storage. Among the tested samples, the highest retention of colorant characterized the samples containing GA + MD. The agglomeration process reduced the content of β-carotene in the microcapsules almost by half. However, retention of the colorant during storage of the microcapsules was increased most of all and half-life of β-carotene was significantly prolonged. Changes in L* and a* colour parameters during storage were more limited in the case of agglomerated samples.
1 Introduction
Carotenoids are natural pigments present in fruits and vegetables where they play key roles in photosynthesis and photo-protection reactions [1]. β-carotene is one of the major types of carotenoid [2]. β-carotene is known not only for its dyeing properties, but also for its health effects as a vitamin A – precursor and effective antioxidant [3, 4].
Interests in carotenoids have also increased recently because of their contribution to prevention of lifestyle diseases, e. g. heart diseases, cancer, or macular degeneration [5].
Utilization of β-carotene as a nutraceutical ingredient or natural colorant within foods is currently limited by a number of factors such as poor water solubility, high melting point, chemical instability, and bioavailability [6, 7, 8]. Beta carotene is highly sensitive to oxygen, light, and heat, and its oxidation products show a very low or even zero level of pigmentation [4]. A method that can be used to protect β-carotene is microencapsulation [9]. It is a technique by which sensitive ingredients are packed within a coating or wall material. The microencapsulated material is protected from external factors, which results in stabilization of unstable substances [6, 10]. Recently, there have been many publications on the microencapsulation of β-carotene [7, 9, 10, 11, 12, 13, 14]. The spray draying process is one of the most popular, attractive, and widely studied encapsulation technologies owing to its high-production capacity and minimal operation cost [15]. The microencapsulation by spray drying involves the emulsification of an active substance in a coating material solution and then spraying of the resulting dispersion in the hot chamber of the spray dryer. As a result of rapid evaporation of water around the core particles, coatings of the carrier material are formed [3]. An undisputable disadvantage of this method is that part of the core remains on the surface of microcapsules and is exposed to environmental factors [16]. Core retention during storage of microcapsules and effectiveness of microencapsulation depends on many factors, including type of wall material (carrier), properties of dried emulsions, and drying parameters [17].
Agglomeration is defined as aggregation of dispersed materials into units of a large size, called agglomerates. The objective of agglomeration technology is systematic production of agglomerates with closely specified properties. Agglomerates will dissolve or disperse more readily in liquids than the original spray-dried product [18].
During the formation of agglomerates from microcapsules obtained by spray drying, a portion of the core remaining on the surface of the powder particles is enclosed within the new structure and access of oxygen to such hidden active ingredient becomes difficult. It can therefore be assumed that this will increase stability of the microencapsulated active substance. Consequently the aim of this work was to analyse the impact of agglomeration on stability of β-carotene microencapsulated be spray drying, and subjecting to agglomeration in a fluidized bed.
2 Materials and methods
2.1 Materials
Carrier materials: Modified starch E1450 CAPSUL (OSA), maltodextrin (DE 15) (MD), obtained from JAR Jaskulski Aromaty, Poland, and whey protein (WP) from Davisco Food International, USA previously tested as a carrier materials.
Colorant: β-carotene as a 1 % FS oil solution obtained from JAR Jaskulski Aromaty Poland.
Additional materials: Arabic Gel INSTANT BB (GA) from National Starch and Chemical Company, USA
2.2 Methods
2.2.1 Microencapsulation method
Microcapsules were produced by spray drying. The material of the microcapsules’ walls (carrier) was constituted by mixtures of GA and MD, OSA and MD at a ratio of 2:1 and WP and the concentration of these substances was 20 %.
In order to obtain the aqueous phase, the carriers were dispersed at 380 rpm for 30 min in distilled water (40 °C) using a RW 20 DZM laboratory stirrer from Janke & Kunkel (Germany). In order to hydrate completely, the continuous phase was left at room temperature (20 ± 2 °C) for about 24 h. A disperse phase (β-carotene solution) was introduced into the continuous phase (aqueous solution of carriers) and stirred for 10 min with a laboratory stirrer at 380 rpm, Janke & Kunkel (Germany). Emulsions were prepared using two-stage homogenization, with APV-1000 high pressure homogenizer from APV (USA). In the first stage of homogenization, pressure of 55 MPa was applied, at the second stage – 18 MPa.
Drying of the emulsions was carried out in a laboratory spray drier type A/S Niro Atomizer (Denmark) (spraying mechanism – disk). The emulsion was fed to a spray disk using a Alstom type 372,1 peristaltic pump (France) at 4 rpm. Counter-current drying was applied, the inlet air temperature was 190 ± 5 °C and the exhaust air 80 ± 5 ºC. The microcapsules were produced in three replicates.
2.2.2 Agglomeration
The agglomeration was carried out using Strefa 1/Nitro-Aeromic AG agglomerator (Denmark). Three hundred grams of powder was moisturized with 80 g of water. The inlet air temperature was 50 ± 2 ºC, the air velocity through the bed was 50–80 m3/h, the air pressure in the spray nozzle 0.05 MPa. The powder was moisturized for 20 min, the agglomerates were dried for 15 min at 50 ± 2 °C.
2.2.3 Storage study
Samples of the microcapsules and agglomerated powders were stored in glass jars at a temperature of 20 ± 2 ºC, with access to daylight. The test time was 60 days.
2.2.4 Stability of the emulsion
Stability of the emulsion was determined by means of the backscattered light method using LUMISIZER apparatus (Germany). SEPView 6 program was used. Process parameters: 10 s interval, refractive index 1, chamber temperature 25 ºC, centrifugal force 1,200 g, and 255 transmission profiles.
2.2.5 Determination of the total content of β-carotene in microcapsules and on their surface
Determination of β-carotene content in the microencapsulated formulation was performed by the spectrophotometric method. Extraction of the dye from microcapsules and from the surface was carried out by the method proposed by Wagner and Warthesen [19]. This method was slightly modified. The change concerned the type of extraction solvent used. For β-carotene extraction, 50 mg of powder (0.0001 g accuracy) was weighed on an analytical weight into a 50 mL conical flask and 1 cm3 of distilled water was added; after dissolving the microcapsules 1 cm3 of acetone and 1 cm3 of chloroform were also added. The solution was stirred for 5 min using a magnetic stirrer (Polamed MM6, Poland). The organic phase was pipetted and filtered through a soft paper filter, rinsing with hexane. The filtrate was diluted with 1.5 cm3 of chloroform and a three-fold absorbance measurement at 454 nm was carried out against chloroform as a control using a Helios β-Thermo Spectronic spectrophotometer. However, when extracting β-carotene from the surface of microcapsules, the difference was that up to 50 mg of microcapsules the 2.5 cm3 of acetone was added and mixed for 5 min using the same magnetic stirrer. After the microcapsules dropped to the bottom of the flask, the organic phase was pipetted, filtered through a soft paper filter and a three-fold absorbance measurement was performed with reference to acetone. On the basis of the determined content of β-carotene in and on the surface of the microcapsules, the microencapsulation efficiency (% ME) was calculated in accordance with the equation proposed by Hogan et al. [16]:
where:
cβ– total β -carotene content in microcapsules,
pβ– amount of β -carotene present on the surface of microcapsules.
2.2.6 Kinetic parameters
The half-life time was calculated on the basis of the regression equation:
where:
y – colourant concentration (mg/100 g)
t – half-life time (days)
The constant rate for β-carotene decay was determined according to the equation:
where:
K – constant rate for β-carotene decay (mg %/100 g/day),
CA0 and CA – initial and final β-carotene content (%), respectively,
T – time (days). T = 60 days.
2.2.7 Colour of the microencapsulated β-carotene powders
The L*, a*, and b* colour components were determined with the use of CIEL*a*b* on the surface of the powder, using a Minolta CR-200 colorimeter (Minolta, Osaka, Japan; light source D65, observer 2°, a measuring head hole of 8 mm). Each measurement was performed 4 times. The mean value was used as the assay result.
2.2.8 Water content
About 3 g of powder was weighed into glass weighing vessels with an accuracy of 0.0001 g. The samples were dried at 130 ± 2 °C for 1 h. The water content was calculated from the difference between weight of the powder before and after drying.
2.2.9 Water activity
Water activity was measured using a Rotronic HygroLab (Rotronic AG, Bassersdorf, Switzerland) device. The test was performed using a cylindrical probe, four probes were introduced at the same time.
2.2.10 Powder solubility
Three grams of powder was added to 200 mL of distilled water (temperature of 20 ± 1 °C). The mixture was mixed with a rod. The time of complete dissolution of the powder was measured.
2.2.11 Statistical analysis
Statistical analysis of the results was performed using IBM SPSS Statistics (version 23) program, the one-way analysis of variance (ANOVA) for independent samples, post hoc test by HSD Tukey, and the student’s t-test for dependent tests at a significance level of α = 0.01. Data of three independent replicates were investigated.
3 Results and discussion
3.1 Stability of β-carotene emulsion
Retention of the active substance, encapsulated in the microcapsule, depends on many factors, such as: type of substance, type of carrier, and microencapsulation method. In the case of microencapsulation by spray drying the core retention, during the drying and storage of microcapsules, is influenced by properties of the emulsion to be dried [13]. Stability and viscosity of the emulsions are important parameters affecting the microencapsulation process and characteristics of the obtained microcapsules. Retention of the active substance depends also on the carrier material. WPs have better emulsifying properties than polysaccharides (biopolymers – modified starch or Arabic gum). They stabilized the emulsion mainly through electrostatic repulsion, but their disadvantage is the susceptibility of these interactions to the effects of pH, ionic strength and temperature. In contrast, polysaccharides stabilize emulsions through steric repulsion. Their effect is not dependent on pH and ionic strength [20, 21]. In the study, stability of β-carotene emulsions before drying and after the microencapsulation process was determined. The emulsions were obtained by dissolving microcapsules in water. The results of emulsion stability determinations are shown in Figure 1 and Figure 2. The emulsions obtained with WP proved to be the most stable. The curves of the backscattered light percentage coincided, which indicates, according to Chanamai and McClements [20] their high stability. They were more stable than the emulsions containing a mixture of gum Arabic and maltodextrin (GA + MD) and a mixture of modified starch and maltodextrin (OSA + MD). In the case of these emulsions, changes in the backscattered light percentage curves were observed, indicating occurrence of sedimentation in the tested samples (Figure 1), except that in the OSA + MD sample where the changes were larger. Thus, the least stable sample was the emulsion stabilized with OSA and MD. While studying the stability of emulsions reconstituted from microcapsules, the same tendencies were observed (Figure 2).
Intensity of backscattered light of emulsions stabilized with: (a) GA + MD; (b) OSA + MD; (c) WP.
Intensity of backscattered light of emulsions stabilized with: (a) GA + MD; (b) OSA + MD; (c) WP obtained by dissolving microcapsules in water.
3.2 The microencapsulation efficiency
The microencapsulation efficiency ranged from 40 to 67 % (Table 2). These values are within the range found in the literature [13, 22], however microencapsulation efficiency might also be higher up to 87 % [15]. The increasing amount of β-carotene encapsulated in the microcapsules and the simultaneously decreasing amount of dye remaining on the surface of the microcapsules obtained during spray drying is related to the greater homogeneity and stability of the emulsion [10]. Our results allow pointing to a positive correlation between the microencapsulation efficiency and emulsion stability. The WP stabilized emulsions showed the highest stability and the largest ME (microencapsulation efficiency). Thus, it can be concluded that the emulsifying properties of the carrier have a substantial impact on the efficiency of microencapsulation by spray drying. This is in line with the agreement of other authors. Gómez-Mascaraque et al. [14] showed that stability of their emulsions was the main factor affecting the microencapsulation efficiency.
Content of total β-carotene and β-carotene on the surface of microcapsules.
| Sample type | Content of β-carotene [mg%] | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 0 day | 20 days | 40 days | 60 days | ||||||
| total (x ± SD) | surface (x ± SD) | total (x ± SD) | surface (x ± SD) | total (x ± SD) | surface (x ± SD) | total (x ± SD) | surface (x ± SD) | ||
| GA + MD | before* | 200 ± 5 | 100 ± 5 | 174 ± 3 | 89 ± 7 | 146 ± 2 | 72 ± 3 | 103 ± 7 | 66 ± 3 |
| after* | 109 ± 4 | 36 ± 3 | 87 ± 4 | 24 ± 2 | 80 ± 15 | 18 ± 2 | 69 ± 2 | 10 ± 4 | |
| OSA + MD | before | 298 ± 6 | 187 ± 3 | 248 ± 2 | 122 ± 2 | 210 ± 5 | 92 ± 3 | 154 ± 3 | 46 ± 1 |
| after | 140 ± 4 | 91 ± 3 | 128 ± 8 | 84 ± 6 | 117 ± 4 | 74 ± 4 | 90 ± 3 | 64 ± 2 | |
| WP | before | 239 ± 3 | 85 ± 2 | 174 ± 4 | 71 ± 1 | 111 ± 3 | 68 ± 2 | 76 ± 2 | 64 ± 6 |
*before and after the agglomeration process.
Microencapsulation efficiency and decomposition rate, half-life of carotene after spray drying and after agglomeration of the obtained microcapsules.
| Carrier type | Spray-dried microcapsules | Agglomerated microcapsules | |||
|---|---|---|---|---|---|
| Microencapsulation efficiency ME [%] | Degradation rate constant K [mg%/day] | Half-life T1/2 [days] | Degradation rate constant K [mg%/day] | Half-life T1/2 [days] | |
| GA + MD | 50.00 ± 2.00 | 1.62 | 64.07 ± 5.00 | 0.61 | 87.20 ± 2.00 |
| OSA + MD | 40.00 ± 2.00 | 2.41 | 63.68 ± 4.00 | 0.82 | 88.65 ± 3.00 |
| WP | 67.00 ± 3.00 | 2.71 | 41.43 ± 2.00 | – | – |
3.3 Total β-carotene content and its content on the surface of microcapsules
3.3.1 Total β-carotene content and its content on the surface of microcapsules before agglomeration
Immediately after receiving the microcapsules, the tested samples differed in terms of β-carotene content (Table 1). There were statistically significant differences in the total β-carotene concentration [F(2, 5) = 65.12, p ˂ 0.001] as well as β-carotene concentration on the surface [F(2, 5) = 198.93, p ˂ 0.001] between the samples. A sample containing OSA + MD was characterized by the highest content of β-carotene.
A significant effect of the carrier type on the content of β-carotene in microcapsules and on their surface was noticed. This is consistent with the literature data [15, 23].
After 60 days of storage, the content of β-carotene in all the samples was reduced. They were still different in terms of overall colorant content. However, there were no statistically significant differences between the samples in terms of carotene concentration on the surface of microcapsules (Table 1). The decrease in the dye content was dependent on the type of carrier. The degradation of β-carotene in microcapsules and on their surface proceeded in accordance with the first-order reaction. Previous studies have shown that the degradation of α- and β-carotenes follows first-order kinetics in stored, spray-dried carotenoid pigments encapsulation in maltodextrin [19]. Similar conclusions for the degradation of spray-dried β-carotene have been reported by Orset et al. [24]. Analysis of kinetic parameters of dye degradation, found that the sample containing GA + MD was characterized by the smallest rate of decomposition K and the longest half-life of β-carotene decay (Table 2).
It can be concluded that the walls of microcapsules made of GA and MD best protected β-carotene from degradation. The loss of β-carotene during storage resulted from an oxidation reaction by oxygen diffusing through the wall materials [12, 25]. Both the carriers (GA and MD) have a high barrier to oxygen, higher than OSA and WP [26]. Half-life of the β-carotene disintegration in the analysed samples was much shorter than the half-life time (145–431 days) which Wagner and Warthesen [19] obtained in their research. In other studies, the half-life for commercial β-carotene encapsulation in 25DE maltodextrin was 50 days [3]. Robert et al. [23] obtained even shorter half-life encapsulating rosa mosqueta carotenoids in starch and gelatine matrices.
3.3.2 Total β-carotene content and its content on the surface of microcapsules after agglomeration
The agglomeration was applied to samples containing GA + MD and OSA + MD, the least stable sample (WP) was rejected. After the agglomeration, the colorant content was half as high as in microcapsules not subjected to this process (Table 1). The reason for the decrease in β-carotene content in the agglomerated powder was probably the specificity of the agglomeration process. In a fluidized bed, microcapsules with a colorant might crack after water addition. Warechowski et al. [27] suggested that during agglomeration of a powder which particles were filled with oil, an amphiphilic solution is a more appropriate moisturizing substance. However, the reduction of β-carotene concentration on the surface of powder particles after agglomeration was a favourable change (Table 1). β-carotene remaining on the surface of the microcapsules quickly oxidizes [6]. Retention of the colorant during storage was greater for agglomerated than for non-agglomerated samples. The total content of β-carotene decreased during 60 days of storage by 97 and 40 mg in agglomerated samples containing GA + MD and OSA + MD, respectively, and by 144 and 50 mg respectively, in the case of microencapsulated samples. Analysis of the kinetic parameters of β-carotene degradation also showed a beneficial effect of agglomeration on stability of the microencapsulated colorant (Table 2). Half-life was 23 days longer in the case of GA + MD microcapsules and more than 25 days after the agglomeration process in the case of microcapsules from OSA + MD.
3.4 Colour parameters of powders before and after agglomeration
Immediately after formation, the samples differed significantly from each other in terms of colour parameters. The values of the colour parameters depended on the type of carrier used (Table 3).
Colour parameters of microcapsules.
| Sample type | 0 days | 60 days | The absolute difference | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| L* (x ± SD) | a* (x ± SD) | b* (x ± SD) | L* (x ± SD) | a* (x ± SD) | b* (x ± SD) | ΔL* (x ± SD) | Δa* (x ± SD) | Δb* (x ± SD) | ||
| GA + MD | before* | 79.61 ± 0.40 | 16.35 ± 0.48 | 66.27 ± 0.65 | 78.89 ± 1.78 | 13.58 ± 1.56 | 58.30 ± 5.03 | 0.72 | 2.77 | 7.97 |
| after* | 75.68 ± 0.06 | 18.50 ± 0.95 | 66.13 ± 0.79 | 73.28 ± 0.23 | 16.88 ± 1.11 | 63.62 ± 0.68 | 2.40 | 1.62 | 2.51 | |
| OSA + MD | before | 74.97 ± 0.95 | 21.82 ± 1.14 | 61.94 ± 1.54 | 73.31 ± 0.86 | 19.15 ± 1.18 | 58.95 ± 0.59 | 1.66 | 2.67 | 2.99 |
| after | 74.31 ± 0.45 | 20.07 ± 0.47 | 63.09 ± 0.44 | 72.15 ± 0.51 | 17.96 ± 0.58 | 60.76 ± 0.54 | 2.16 | 2.11 | 2.33 | |
| WP | before | 76.67 ± 0.87 | 17.95 ± 0.91 | 63.66 ± 1.86 | 73.08 ± 1.41 | 16.25 ± 1.31 | 59.34 ± 1.94 | 3.59 | 1.70 | 4.32 |
*before and after the agglomeration process
The value of L* parameter, describing brightness, ranged from 79.61 to 74.97, while the chromatic colour parameters take positive values for both a* (indicating the dominant shade red) and b* value (indicating the dominant shade of yellow). However the chromatic parameter b* was not a good indicator of β-carotene retention because it characterizes yellowness to blueness and these colours were not dominant in the case of the aforementioned pigment. The chromatic parameter a* is much more sensitive to changes in β-carotene colour [19]. Changes of L*, a*, and b* parameters were observed during storage. In terms of ΔL *, the test samples differed significantly [F(2, 13) = 9.02, p ˂ 0.01]. In the case of the OSA + MD samples an increase in the brightness parameter was observed. As reported by Orest et al. [24] lightening the colour of the carotenoid formulations is due to isomerization of carotenes. As a result of isomerization, which is the first stage of degradation of these colorants, the carotenes transits from typical trans isomers to cis isomers. Cis isomers have less intense colours than the trans forms have [28]. In the case of powders containing GA + MD, the lowering of L* parameter (ΔL* = 0.72) could have been caused by the darkening of the gum Arabic, which was yellow as confirmed by the high value of parameter b*. On the other hand, the decrease in the value of a* parameter for all the tested samples indicated degradation of β-carotene. Reduction of the red chromatic component during storage of β-carotene encapsulated in maltodextrin matrices was observed by Deobry et al. [3]. Also Elizalde et al. [29] investigating colour changes during storage of β-carotene on surfaces of amorphous matrices of gelatine and trehalose observed similar relationships.
After agglomeration the samples were still varied in terms of colour parameters. Analysis of changes of L* parameter during the storage of powders, showed that they darkened, probably due to changes in the colour of carriers used. However, changes in the red colour component were lower than in the case of non-agglomerated powders. Thus, it has been shown that the agglomeration process increases stability of microencapsulated β-carotene, but does not completely stop the degradation process of the colorant. There was still a colorant on the surface of the microcapsules, which rapid oxidized. Therefore, further research is necessary to optimize the agglomeration process, which will result in a total surface coverage of the microcapsules with the matrix formed in this process.
3.5 Solubility of microcapsules
Moisture contents and water activity of spray-dried microcapsules are presented in Table 4. These parameters differed significantly (p ˂ 0.01). The moisture content of the capsules was slightly lower than in the case of microcapsules obtained by Loksuwan [10] with modified tapioca starch and with water content similar to that found in microcapsules with maltodextrin.
Water content and solubility of microcapsules.
| Sample type | Water content [%] (x ± SD) | Water activity [-] | Solubility [min] (x ± SD) |
|---|---|---|---|
| GA + MD | 2.7 ± 0.1 | 0.127 | before* 3.38 ± 0.1 |
| after* 2.06 ± 0.2 | |||
| OSA + MD | 2.1 ± 0.1 | 0.119 | before 2.89 ± 0.2 |
| after 2.03 ± 0.1 |
*before and after the agglomeration process
Regardless of the type of carrier used (Table 4), the dissolution time of the powder was shorter after agglomeration. It was found that the agglomeration had a beneficial effect on solubility of the microencapsulated β-carotene obtained by spray draying.
4 Conclusions
Stability of the microencapsulated β-carotene emulsion depended on the type of carrier. Emulsions with the addition of WP were more stable than those containing GA + MD and OSA + MD. Emulsion stability had an effect on efficiency of microencapsulation, whereas it did not affect the retention of microencapsulated dye. Retention of β-carotene during storage of the microcapsules depended on the type of carrier used. Among the tested samples, the longest half-life (64 days) and the lowest rate of β-carotene decay rate (1.62 mg%/day) characterized the GA + MD sample.
The process of agglomeration decreased the percentage β-carotene in microencapsulated formulation by nearly a half. It mainly increased dye retention during storage of microcapsules. The agglomeration process significantly prolonged half-life of β-carotene. However, further research is required to reduce the concentration of the dye on the surface of the microcapsules. A promising solution seems to involve introduction of another carrier, in the process of agglomeration, in order to cover the surface of β-carotene with a new matrix.
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