Food Bioprocess Technol DOI 10.1007/s11947-013-1172-y
ORIGINAL PAPER
Microencapsulation of Andes Berry (Rubus glaucus Benth.) Aqueous Extract by Spray Drying José Luis Villacrez & José G. Carriazo & Coralia Osorio
Received: 6 March 2013 / Accepted: 8 August 2013 # Springer Science+Business Media New York 2013
Abstract The Andes berry (Rubus glaucus Benth.), a tropical fruit with a high anthocyanin content and pleasant aroma, was selected for spray drying to produce solids that preserve the sensory characteristics (colour, odour and taste) of the fresh fruit. A factorial design (9×2) was implemented to evaluate the influence of the encapsulating agent (maltodextrin DE 20, arabic gum, corn starch, yucca starch, Capsul® TA and Hi-CapTM 100, as well as three mixtures of them) and the nozzle diameter (1.0 and 2.0 mm) on the characteristics of solids, anthocyanin content, moisture and water activity. All solids showed water activity values (a w) between 0.199 and 0.422, and particle size ranging from 3 to 15 μm according to microscopy results. The samples exhibiting the highest anthocyanin content were subjected to a discriminative sensory analysis. The Andes berry microencapsulates obtained with Hi-CapTM 100 and maltodextrin DE 20 as wall materials were chosen because of their sensory properties. The release of anthocyanins as well as volatile aroma compounds by water dissolution was confirmed by both liquid and gas chromatography coupled to mass spectrometry (HPLC-MS and GCMS, respectively). The chroma (C*ab) and a* values of the solutions were higher than those of the powders, which indicated a more intense red colour. Thermal analyses (thermogravimetric analyses and differential scanning calorimetry) showed that these two solids were stable up to 100 °C under a nitrogen atmosphere. Remarkably, the anthocyanin content did not change during 180 days at 18 °C and a relative humidity of less than 60 %. However, the shelf life of these products is highly dependent on the humidity during storage because a relative humidity increase causes significant damage to the microcapsule structures and the loss of the encapsulated materials. J. L. Villacrez : J. G. Carriazo : C. Osorio (*) Departamento de Química, Universidad Nacional de Colombia, AA 14490 Bogotá, Colombia e-mail: [email protected]
Keywords Andes berry . Rubus glaucus Benth . Microencapsulates . Spray drying . Anthocyanins
Introduction The dehydration of fruits is a strategy to extend their shelf life, minimise loss during post-harvest handling (Fernandes et al. 2011) and generate new marketing alternatives for producers, as dehydrated fruits are used either for a stand-alone food product (i.e. a snack) or as an ingredient of another food. A wide range of dehydration procedures are employed for fruits and vegetables; among them, the thermal processing technology of spray drying is considered the most common and cheapest technique to produce microencapsulated food materials on a large scale (Murugesan and Orsat 2012; Rawson et al. 2011; Gharsallaoui et al. 2007). Advantages associated with this technology include readily available equipment, a wide choice of carrier solids, good retention of volatiles and suitable stability of the final product (Reineccius 2004). This technique has been used to encapsulate sensitive food ingredients such as volatiles, lipids, pigments and vitamins. Microencapsulation is defined as the process where tiny particles or droplets are surrounded by a coating, or embedded into a homogeneous or heterogeneous matrix, to give small capsules with improved properties. In these capsules, the active principle is protected from external conditions and can be released later in a controlled way. Additionally, microencapsulation can convert liquids into free-flowing powders, which may ease handling them. Different encapsulating agents are available for food applications, with the following as the most commonly used agents: hydrolysed starches, modified starches, arabic gum and gelatin. Maltodextrins are hydrolysed starches produced by the action of either acids or enzymes. They are commonly used as wall materials due to favourable properties of emulsification, film formation, water solubility,
Food Bioprocess Technol
low viscosity at high concentrations and biodegradability, as well as low cost (Bertolini 2010; Embuscado 2011). Spray drying has recently been used to microencapsulate the anthocyanins from fruits for the production of low-humidity powders with higher pigment stability during storage (Laine et al. 2008; Tonon et al. 2010; Osorio et al. 2010; Fang and Bhandari 2012). The Andes berry (Rubus glaucus Benth.) belongs to the so-called exotic fruits, and due to its sensory properties, it has a wide acceptance among consumers (Sinuco et al. 2013). However, this berry is highly perishable during post-harvest handling, which results in a short shelf life (approximately 10 days). The Andes berry fruits are a promising source of natural colourants (anthocyanins), with cyanidin-3-O (2″-O -β-D-xylopyranosyl-6″-O -α-L -rhamnopyranosylβ-D -glucopyranoside) and cyanidin-3-O-(6″-O -αrhamnopyranosyl)-β-glucopyranoside as the main constituents (Osorio et al. 2012). Various attempts have been made to develop dehydrated food products using this fruit as a potential source of natural colourants. The first endeavour involved the osmotic dehydration of Andes berry fruits with three different agents: sucrose (70 %), sucrose (70 %)–glycerol (65 %) 1:1 and ethanol. This process not only decreased the water activity but also promoted the transfer of anthocyanins and volatile constituents to the osmotic solutions. Tristimulus colourimetry was applied to evaluate the colour-rich osmotic solutions (Osorio et al. 2007). Moreover, the ethanol and sucrose osmotic solutions were selected for transformation into solids using either spray drying or co-crystallisation (with sucrose) (Sinuco et al. 2006; Olaya et al. 2009). To evaluate their thermodynamic properties, Giraldo Gómez et al. (2011) obtained different types of maltodextrinencapsulated powders from the Andes berry fruits using vibrofluidised bed drying, spray drying, vacuum drying and freeze drying. Estupiñán et al. (2011) evaluated the stability of Andes berry anthocyanin freeze-dried powders in isotonic model beverages under different illumination conditions. The powders that contained maltodextrin DE 20 as a carrier agent were the most stable during storage in darkness. Recently, Cerón et al. (2012) obtained anthocyanin-rich extracts from Andes berry fruits using traditional liquid extraction with ethanol as the solvent as well as a pilot-scale enhancedfluidity liquid extraction using CO2 and ethanol. Due to the presence of ethanol, an increase in the solubility of cyanidin-3glucoside was observed when supercritical extraction was employed. The aforementioned pilot-scale process improved the yield of anthocyanin extraction when compared to the traditional process. While part of our research focussed on the development of added-value products from tropical fruits (Osorio et al. 2010; Osorio, et al. 2011), the aim of this work was to obtain microencapsulates from Andes berry fruits using spray drying
with different types of coating materials. Microencapsulate’s physical, chemical, morphological and sensory properties were evaluated, as well as their stability under different humidity conditions.
Materials and Methods Materials Fresh Andes berries were collected from different cultivars of Saboyá, Boyacá, Colombia, and were selected according to their ripeness attributes (colour more than 75 % dark-red to red wine). They were characterised by pH, soluble solid content, acidity content (titratable acidity according to AOAC 2006), colour parameters and anthocyanin content. The microencapsulate moisture content was determined gravimetrically by drying the samples in an oven at 105 °C until a constant weight was reached (AOAC 2006). The water activity (a w) of the microencapsulates was measured in a hygrometer HygroPalm AW1 (Rotronic Instruments, Huntington, NY, USA) at 20 °C using 1 g of each solid. The soluble solid content and pH of the Andes berry fruits were measured in an Abbe Atago 8682 refractometer (Atago, Tokyo, Japan) and a Schott CG820 pH-meter (Gemini BV, Langgewann, Germany), respectively. Preparation of Microencapsulates by Spray Drying Before processing, the mature fruits were dipped into a sodium hypochlorite solution (1 %, v/v) for sanitation and then processed without previous freezing. Then, the fruits were homogenised in a blender for 15 min with a 1:1 ratio of distilled water to fruit (w/w). The resultant extract was filtered through 0.8-mm mesh to eliminate the suspended solids. The spray-drying process was performed in a laboratoryscale LabPlant SD-06 spray drier (LabPlant, Huddersfield, UK) with a 1,110 mm×825 mm×600 mm main spray chamber. Data reported in the literature (Gharsallaoui et al. 2007) have shown that the encapsulating agent, nozzle diameter and air inlet temperature have an influence on the encapsulation efficiency as well as on the particle properties (size and morphology). Thus, a factorial screening design (9×2) was constructed to estimate the effect of nine different carrier agents (maltodextrin DE 20, arabic gum, corn starch, yucca starch, Capsul® TA, Hi-CapTM 100, maltodextrin DE 20/ arabic gum (1:1, w/w), maltodextrin DE 20/corn starch (1:1, w/w) and maltodextrin DE 20/yucca starch (1:1, w/w)) and two nozzles with different internal diameters (1.0 and 2.0 mm). The separate aqueous extracts of the Andes berries were combined in a 1:1 (w/w) ratio with each of the different carrier agents. In each case, 1 L of 20–25° Brix feed-mixture was prepared, kept under magnetic stirring (15 min) at 20 °C until
Food Bioprocess Technol
homogeneity and separately spray-dried immediately with air flow of 100 m3/h and a compressor air pressure of 4 bar. The feed flow was 485 mL/h, and the inlet and outlet air temperatures were 120±2 °C and 70±5 °C, respectively. The spraydried powders were collected in plastic containers, weighed and stored in desiccators containing silica gel at room temperature.
in methanol was 26,900 L cm−1 mol−1, and the molar mass is 449.2 g mol−1) (Giusti and Wrolstad 2001). All measurements were performed in triplicate. Sensory Analyses
The colours of the Andes berry purée and of the microencapsulates (as powders and also dissolved in water, 50 mg/mL) were determined using a Cary 5000 UV–vis–NIR spectrophotometer (Varian, Victoria, Australia). The CIE L*a*b* coordinates (D65, 2°) were also obtained. For the powders, the colour was determined using a Diffuse Reflectance Accessory 2500. Blank measurements were made using BaSO4 (high purity) as a white reference. All measurements were performed in triplicate to report the mean±standard deviation. The colour parameters, chroma (C ab *) and hue (h ab ), were calculated according to the following equations (Meléndez-Martínez et al. 2003): h 2 2 i1=2 C * ab ¼ a* þ b* ð1Þ
The sensory panel was composed of 14 trained judges recruited from the staff of the “Laboratorio de Análisis Sensorial de Alimentos, Universidad de Antioquia (Medellín, Colombia)”, who were trained in several sessions prior to analysis of microencapsulates, in which the odour and taste relevant characteristics of mora fruits were defined and evaluated. The panellists evaluated the five Andes berry microencapsulates exhibiting the highest anthocyanin content. and the samples were randomly served in white cups coded with random numbers. The panellists were instructed to rank the samples for their odour and taste, from five (5) to one (1) according to the sensory similarity of the microencapsulates to the fresh fruit. For each sample, the sum of ranks was calculated. These analyses were performed according to standards (Icontec 2011) and under red light. Additionally, a descriptive sensory analysis of the odour and taste of the microencapsulates was also performed.
. * hab ¼ arctan b * :
Characterisation of the Microencapsulates
Colour Measurements
a
ð2Þ
Particle Morphology and Size Anthocyanin Content Measurement The total anthocyanin contents in the fruit and in the microencapsulates were determined using the spectrophotometric pHdifferential method. Anthocyanin extraction from the fruit followed the procedure published by Fan-Chiang and Wrolstad (2005). Consequently, 20 g of Andes berries was frozen in liquid nitrogen. The anthocyanins were extracted with acetone (20 mL), followed by filtration under vacuum. The residual pigments were re-extracted with 7:3 acetone/water (v/v) until the extraction was complete. The filtrates were combined and partitioned with 1:2 acetone/chloroform (v/v), and the aqueous phase was concentrated and then brought to 25 mL in a volumetric flask with a diluted HCl solution (0.01 %). For the microencapsulates, 2.5 g of each sample was diluted in 25 mL of water. For the measurement of anthocyanin content, the dilutions were prepared in 0.025 M potassium chloride and in 0.4 M sodium acetate, and the pH was adjusted with HCl to pH 1.0 and 4.5, respectively. The absorbance of each dilution was measured at 520 and 700 nm against a distilled water blank using a Jenway 7305 UV/visible spectrophotometer (Jenway, Staffordshire, UK). The total monomeric anthocyanin content was calculated as cyanidin-3-glucoside equivalents (in milligrams) per 100 g of fruit or g of solid (ε value (molar absorptivity) of cyanidin-3-glucoside dissolved in 0.1 % HCl
The morphology of the microcapsules was evaluated by using a JEOL JSM-5910LV Scanning Electron Microscope (JEOL, Boston, MA, USA) operating at 30 kV and by coating the samples by gold sputtering before their examination. The particle size determination was carried out by measuring the diameter of each of the 100 particles localised in a selected area (ca. 80 %) of images acquired with a Nikon ECLIPSE E600 Optical Microscope (Nikon, Tokyo, Japan). Thermal Analysis Thermogravimetric measurements were carried out with a simultaneous Analyser NETZSCH STA 409 CD (Netzsch, Selb, Germany). The apparatus was calibrated with highpurity indium (T m =429.8 K, ΔH m =28.4 J g−1). The experiments were performed under nitrogen flow. The samples (2 mg each) were heated from 20 to 250 °C in aluminium crucibles with a linear heating rate of 20 °C/min, and an empty aluminium crucible was used as the reference material. Volatile Compound Analysis The volatile compounds released from the headspace of Andes berry purée as well as the two selected microencapsulates (those exhibiting the highest score in sensory analyses) were
Food Bioprocess Technol
extracted by HS-SPME (Carasek and Pawliszyn 2006). For this analysis, 2.5 g of each powder was mixed with 5 mL of water and then equilibrated for 45 min in a 20-mL sealed vial at 40 °C. The headspace was collected on a DVB/CAR/PDMS fibre (75 μm thickness; Supelco Inc., Bellefonte, PA, USA) over 1 h and directly injected (5 min desorption time) into an HP 5890 series II gas chromatograph (Wilmington, DE, USA) operating in splitless mode and equipped with an FID. A FFAP fused silica column (J&W Scientific, 30 m×0.32 mm i.d., 0.25 μm film thickness) was used. The column oven temperature was programmed to increase from 50 (after 4 min) to 250 °C at 4 °C/min, and the final temperature was held for 5 min. The injector temperature was maintained at 250 °C, the carrier gas was 1.5 mL of He/min, and the make-up gas was N2 at a 30-mL/min flow rate. The linear retention indices were calculated according to the Kovats method using a mixture of normal paraffin C6-C28 as external references. Compound identification was achieved by comparison with authentic reference standards (Barrios and Morales 2005; Sinuco et al. 2013). Anthocyanin Analysis by HPLC-MS HPLC-MS analysis of the anthocyanin-rich acetone fruit extract (obtained as it was explained above) and the microencapsulate solutions was performed using a Shimadzu LCMS-2010 System (Shimadzu, Tokyo, Japan) equipped with an electrospray ionisation (ESI) probe, which was operated in positive ion mode as reported in the literature (Jaramillo et al. 2011). A LUNA RP-18 5-μm column (150×2.0 mm i.d., Phenomenex®, USA) was used for the analysis of the constituents present in each sample. The solvent system was composed of solvent A, a mixture of water/formic acid/acetonitrile (87:10:3, v/v/v), and solvent B, water/formic acid/acetonitrile (40:10:50, v/v/v). The HPLC program started with a linear gradient from 6 to 20 % B for the first 20 min and then increased from 20 to 40 % B for 20–35 min, 40 to 60 % B at 35–40 min, 60 to 90 % B at 40–45 min and 90 to 6 % B at 45– 40 min. The entire program used a flow rate of 0.8 mL/min. Powder Storage For this study, the two Andes berry microencapsulates that were selected exhibited the highest score in the sensory analyses for their similarity in odour and taste to the fresh fruit. They were stored at controlled temperatures (4 °C and 18± 1 °C), at relative humidities (RH; 75 and 95 %) and in the absence of light. The samples (1.5 g of each powder) were spread into a thin layer in Petri dishes (2 cm diameter). The samples were placed in sealed desiccators containing 200 mL of saturated sodium chloride or potassium nitrate solutions to obtain constant humidity values of 75±2 % and 95±2 %, respectively (Rockland 1960). During equilibration, the humidity was measured using a thermohygrometer (model 445815;
Extech Instruments, Nashua, NH, USA). The independent variables considered were the temperature, humidity and time of storage. The dependent variables were the water activity (a w), moisture and anthocyanin content, which were analysed daily in triplicate for one week, removing samples after each measurement. The control samples were stored at 18 °C and 60±2 % RH during the six-month experiments. The error percentage for the water activity measurements was 0.4 and 1.0 % for moisture. Statistical Analysis Data from the characterisation of the microencapsulates are reported as the mean±standard deviation for determinations performed in triplicate. Using the Statgraphics Plus 5.1 software, analysis of variance (ANOVA) and Tukey’s tests were performed to identify differences among the means. Differences at a probability level of P ≤0.05 were considered significant. To establish significant differences between the samples under sensory analyses, the T Friedman statistical function was calculated according to the equation reported in the literature (Peinado et al. 2012). Next, Tukey’s honestly significant difference was calculated to establish which samples exhibit the significant differences from one another (Meilgaard et al. 1999; Peinado et al. 2012).
Results and Discussion The Andes berry fruit was selected for this study because of its economic significance in Colombia and also due to its high anthocyanin content and pleasant aroma (Sinuco et al. 2013). The physicochemical characteristics of this fruit are shown in Table 1, which serves as a reference point for scaling up the Table 1 Physicochemical characterisation of Andes berry fruit Property
Value
Moisture content (% wet basis)a pH °Brix Acidity (% citric acid)b Anthocyanin contentc Colour parameters
L* a*
86.84±0.63 2.79±0.10 9.0±0.1 6.41±0.02 78.5±0.3 31.23±0.01 56.02±0.01
b* C* ab h ab
11.13±0.05 57.11±0.03 11.34±0.02
a
Gram of water/100 g of sample
b
Milligram of ascorbic acid/100 g of fruit
c
Milligram of cyanidin-3-glucoside equivalents/100 g of fruit FW. All data are the mean of triplicate measurements±standard deviation, P
ORIGINAL PAPER
Microencapsulation of Andes Berry (Rubus glaucus Benth.) Aqueous Extract by Spray Drying José Luis Villacrez & José G. Carriazo & Coralia Osorio
Received: 6 March 2013 / Accepted: 8 August 2013 # Springer Science+Business Media New York 2013
Abstract The Andes berry (Rubus glaucus Benth.), a tropical fruit with a high anthocyanin content and pleasant aroma, was selected for spray drying to produce solids that preserve the sensory characteristics (colour, odour and taste) of the fresh fruit. A factorial design (9×2) was implemented to evaluate the influence of the encapsulating agent (maltodextrin DE 20, arabic gum, corn starch, yucca starch, Capsul® TA and Hi-CapTM 100, as well as three mixtures of them) and the nozzle diameter (1.0 and 2.0 mm) on the characteristics of solids, anthocyanin content, moisture and water activity. All solids showed water activity values (a w) between 0.199 and 0.422, and particle size ranging from 3 to 15 μm according to microscopy results. The samples exhibiting the highest anthocyanin content were subjected to a discriminative sensory analysis. The Andes berry microencapsulates obtained with Hi-CapTM 100 and maltodextrin DE 20 as wall materials were chosen because of their sensory properties. The release of anthocyanins as well as volatile aroma compounds by water dissolution was confirmed by both liquid and gas chromatography coupled to mass spectrometry (HPLC-MS and GCMS, respectively). The chroma (C*ab) and a* values of the solutions were higher than those of the powders, which indicated a more intense red colour. Thermal analyses (thermogravimetric analyses and differential scanning calorimetry) showed that these two solids were stable up to 100 °C under a nitrogen atmosphere. Remarkably, the anthocyanin content did not change during 180 days at 18 °C and a relative humidity of less than 60 %. However, the shelf life of these products is highly dependent on the humidity during storage because a relative humidity increase causes significant damage to the microcapsule structures and the loss of the encapsulated materials. J. L. Villacrez : J. G. Carriazo : C. Osorio (*) Departamento de Química, Universidad Nacional de Colombia, AA 14490 Bogotá, Colombia e-mail: [email protected]
Keywords Andes berry . Rubus glaucus Benth . Microencapsulates . Spray drying . Anthocyanins
Introduction The dehydration of fruits is a strategy to extend their shelf life, minimise loss during post-harvest handling (Fernandes et al. 2011) and generate new marketing alternatives for producers, as dehydrated fruits are used either for a stand-alone food product (i.e. a snack) or as an ingredient of another food. A wide range of dehydration procedures are employed for fruits and vegetables; among them, the thermal processing technology of spray drying is considered the most common and cheapest technique to produce microencapsulated food materials on a large scale (Murugesan and Orsat 2012; Rawson et al. 2011; Gharsallaoui et al. 2007). Advantages associated with this technology include readily available equipment, a wide choice of carrier solids, good retention of volatiles and suitable stability of the final product (Reineccius 2004). This technique has been used to encapsulate sensitive food ingredients such as volatiles, lipids, pigments and vitamins. Microencapsulation is defined as the process where tiny particles or droplets are surrounded by a coating, or embedded into a homogeneous or heterogeneous matrix, to give small capsules with improved properties. In these capsules, the active principle is protected from external conditions and can be released later in a controlled way. Additionally, microencapsulation can convert liquids into free-flowing powders, which may ease handling them. Different encapsulating agents are available for food applications, with the following as the most commonly used agents: hydrolysed starches, modified starches, arabic gum and gelatin. Maltodextrins are hydrolysed starches produced by the action of either acids or enzymes. They are commonly used as wall materials due to favourable properties of emulsification, film formation, water solubility,
Food Bioprocess Technol
low viscosity at high concentrations and biodegradability, as well as low cost (Bertolini 2010; Embuscado 2011). Spray drying has recently been used to microencapsulate the anthocyanins from fruits for the production of low-humidity powders with higher pigment stability during storage (Laine et al. 2008; Tonon et al. 2010; Osorio et al. 2010; Fang and Bhandari 2012). The Andes berry (Rubus glaucus Benth.) belongs to the so-called exotic fruits, and due to its sensory properties, it has a wide acceptance among consumers (Sinuco et al. 2013). However, this berry is highly perishable during post-harvest handling, which results in a short shelf life (approximately 10 days). The Andes berry fruits are a promising source of natural colourants (anthocyanins), with cyanidin-3-O (2″-O -β-D-xylopyranosyl-6″-O -α-L -rhamnopyranosylβ-D -glucopyranoside) and cyanidin-3-O-(6″-O -αrhamnopyranosyl)-β-glucopyranoside as the main constituents (Osorio et al. 2012). Various attempts have been made to develop dehydrated food products using this fruit as a potential source of natural colourants. The first endeavour involved the osmotic dehydration of Andes berry fruits with three different agents: sucrose (70 %), sucrose (70 %)–glycerol (65 %) 1:1 and ethanol. This process not only decreased the water activity but also promoted the transfer of anthocyanins and volatile constituents to the osmotic solutions. Tristimulus colourimetry was applied to evaluate the colour-rich osmotic solutions (Osorio et al. 2007). Moreover, the ethanol and sucrose osmotic solutions were selected for transformation into solids using either spray drying or co-crystallisation (with sucrose) (Sinuco et al. 2006; Olaya et al. 2009). To evaluate their thermodynamic properties, Giraldo Gómez et al. (2011) obtained different types of maltodextrinencapsulated powders from the Andes berry fruits using vibrofluidised bed drying, spray drying, vacuum drying and freeze drying. Estupiñán et al. (2011) evaluated the stability of Andes berry anthocyanin freeze-dried powders in isotonic model beverages under different illumination conditions. The powders that contained maltodextrin DE 20 as a carrier agent were the most stable during storage in darkness. Recently, Cerón et al. (2012) obtained anthocyanin-rich extracts from Andes berry fruits using traditional liquid extraction with ethanol as the solvent as well as a pilot-scale enhancedfluidity liquid extraction using CO2 and ethanol. Due to the presence of ethanol, an increase in the solubility of cyanidin-3glucoside was observed when supercritical extraction was employed. The aforementioned pilot-scale process improved the yield of anthocyanin extraction when compared to the traditional process. While part of our research focussed on the development of added-value products from tropical fruits (Osorio et al. 2010; Osorio, et al. 2011), the aim of this work was to obtain microencapsulates from Andes berry fruits using spray drying
with different types of coating materials. Microencapsulate’s physical, chemical, morphological and sensory properties were evaluated, as well as their stability under different humidity conditions.
Materials and Methods Materials Fresh Andes berries were collected from different cultivars of Saboyá, Boyacá, Colombia, and were selected according to their ripeness attributes (colour more than 75 % dark-red to red wine). They were characterised by pH, soluble solid content, acidity content (titratable acidity according to AOAC 2006), colour parameters and anthocyanin content. The microencapsulate moisture content was determined gravimetrically by drying the samples in an oven at 105 °C until a constant weight was reached (AOAC 2006). The water activity (a w) of the microencapsulates was measured in a hygrometer HygroPalm AW1 (Rotronic Instruments, Huntington, NY, USA) at 20 °C using 1 g of each solid. The soluble solid content and pH of the Andes berry fruits were measured in an Abbe Atago 8682 refractometer (Atago, Tokyo, Japan) and a Schott CG820 pH-meter (Gemini BV, Langgewann, Germany), respectively. Preparation of Microencapsulates by Spray Drying Before processing, the mature fruits were dipped into a sodium hypochlorite solution (1 %, v/v) for sanitation and then processed without previous freezing. Then, the fruits were homogenised in a blender for 15 min with a 1:1 ratio of distilled water to fruit (w/w). The resultant extract was filtered through 0.8-mm mesh to eliminate the suspended solids. The spray-drying process was performed in a laboratoryscale LabPlant SD-06 spray drier (LabPlant, Huddersfield, UK) with a 1,110 mm×825 mm×600 mm main spray chamber. Data reported in the literature (Gharsallaoui et al. 2007) have shown that the encapsulating agent, nozzle diameter and air inlet temperature have an influence on the encapsulation efficiency as well as on the particle properties (size and morphology). Thus, a factorial screening design (9×2) was constructed to estimate the effect of nine different carrier agents (maltodextrin DE 20, arabic gum, corn starch, yucca starch, Capsul® TA, Hi-CapTM 100, maltodextrin DE 20/ arabic gum (1:1, w/w), maltodextrin DE 20/corn starch (1:1, w/w) and maltodextrin DE 20/yucca starch (1:1, w/w)) and two nozzles with different internal diameters (1.0 and 2.0 mm). The separate aqueous extracts of the Andes berries were combined in a 1:1 (w/w) ratio with each of the different carrier agents. In each case, 1 L of 20–25° Brix feed-mixture was prepared, kept under magnetic stirring (15 min) at 20 °C until
Food Bioprocess Technol
homogeneity and separately spray-dried immediately with air flow of 100 m3/h and a compressor air pressure of 4 bar. The feed flow was 485 mL/h, and the inlet and outlet air temperatures were 120±2 °C and 70±5 °C, respectively. The spraydried powders were collected in plastic containers, weighed and stored in desiccators containing silica gel at room temperature.
in methanol was 26,900 L cm−1 mol−1, and the molar mass is 449.2 g mol−1) (Giusti and Wrolstad 2001). All measurements were performed in triplicate. Sensory Analyses
The colours of the Andes berry purée and of the microencapsulates (as powders and also dissolved in water, 50 mg/mL) were determined using a Cary 5000 UV–vis–NIR spectrophotometer (Varian, Victoria, Australia). The CIE L*a*b* coordinates (D65, 2°) were also obtained. For the powders, the colour was determined using a Diffuse Reflectance Accessory 2500. Blank measurements were made using BaSO4 (high purity) as a white reference. All measurements were performed in triplicate to report the mean±standard deviation. The colour parameters, chroma (C ab *) and hue (h ab ), were calculated according to the following equations (Meléndez-Martínez et al. 2003): h 2 2 i1=2 C * ab ¼ a* þ b* ð1Þ
The sensory panel was composed of 14 trained judges recruited from the staff of the “Laboratorio de Análisis Sensorial de Alimentos, Universidad de Antioquia (Medellín, Colombia)”, who were trained in several sessions prior to analysis of microencapsulates, in which the odour and taste relevant characteristics of mora fruits were defined and evaluated. The panellists evaluated the five Andes berry microencapsulates exhibiting the highest anthocyanin content. and the samples were randomly served in white cups coded with random numbers. The panellists were instructed to rank the samples for their odour and taste, from five (5) to one (1) according to the sensory similarity of the microencapsulates to the fresh fruit. For each sample, the sum of ranks was calculated. These analyses were performed according to standards (Icontec 2011) and under red light. Additionally, a descriptive sensory analysis of the odour and taste of the microencapsulates was also performed.
. * hab ¼ arctan b * :
Characterisation of the Microencapsulates
Colour Measurements
a
ð2Þ
Particle Morphology and Size Anthocyanin Content Measurement The total anthocyanin contents in the fruit and in the microencapsulates were determined using the spectrophotometric pHdifferential method. Anthocyanin extraction from the fruit followed the procedure published by Fan-Chiang and Wrolstad (2005). Consequently, 20 g of Andes berries was frozen in liquid nitrogen. The anthocyanins were extracted with acetone (20 mL), followed by filtration under vacuum. The residual pigments were re-extracted with 7:3 acetone/water (v/v) until the extraction was complete. The filtrates were combined and partitioned with 1:2 acetone/chloroform (v/v), and the aqueous phase was concentrated and then brought to 25 mL in a volumetric flask with a diluted HCl solution (0.01 %). For the microencapsulates, 2.5 g of each sample was diluted in 25 mL of water. For the measurement of anthocyanin content, the dilutions were prepared in 0.025 M potassium chloride and in 0.4 M sodium acetate, and the pH was adjusted with HCl to pH 1.0 and 4.5, respectively. The absorbance of each dilution was measured at 520 and 700 nm against a distilled water blank using a Jenway 7305 UV/visible spectrophotometer (Jenway, Staffordshire, UK). The total monomeric anthocyanin content was calculated as cyanidin-3-glucoside equivalents (in milligrams) per 100 g of fruit or g of solid (ε value (molar absorptivity) of cyanidin-3-glucoside dissolved in 0.1 % HCl
The morphology of the microcapsules was evaluated by using a JEOL JSM-5910LV Scanning Electron Microscope (JEOL, Boston, MA, USA) operating at 30 kV and by coating the samples by gold sputtering before their examination. The particle size determination was carried out by measuring the diameter of each of the 100 particles localised in a selected area (ca. 80 %) of images acquired with a Nikon ECLIPSE E600 Optical Microscope (Nikon, Tokyo, Japan). Thermal Analysis Thermogravimetric measurements were carried out with a simultaneous Analyser NETZSCH STA 409 CD (Netzsch, Selb, Germany). The apparatus was calibrated with highpurity indium (T m =429.8 K, ΔH m =28.4 J g−1). The experiments were performed under nitrogen flow. The samples (2 mg each) were heated from 20 to 250 °C in aluminium crucibles with a linear heating rate of 20 °C/min, and an empty aluminium crucible was used as the reference material. Volatile Compound Analysis The volatile compounds released from the headspace of Andes berry purée as well as the two selected microencapsulates (those exhibiting the highest score in sensory analyses) were
Food Bioprocess Technol
extracted by HS-SPME (Carasek and Pawliszyn 2006). For this analysis, 2.5 g of each powder was mixed with 5 mL of water and then equilibrated for 45 min in a 20-mL sealed vial at 40 °C. The headspace was collected on a DVB/CAR/PDMS fibre (75 μm thickness; Supelco Inc., Bellefonte, PA, USA) over 1 h and directly injected (5 min desorption time) into an HP 5890 series II gas chromatograph (Wilmington, DE, USA) operating in splitless mode and equipped with an FID. A FFAP fused silica column (J&W Scientific, 30 m×0.32 mm i.d., 0.25 μm film thickness) was used. The column oven temperature was programmed to increase from 50 (after 4 min) to 250 °C at 4 °C/min, and the final temperature was held for 5 min. The injector temperature was maintained at 250 °C, the carrier gas was 1.5 mL of He/min, and the make-up gas was N2 at a 30-mL/min flow rate. The linear retention indices were calculated according to the Kovats method using a mixture of normal paraffin C6-C28 as external references. Compound identification was achieved by comparison with authentic reference standards (Barrios and Morales 2005; Sinuco et al. 2013). Anthocyanin Analysis by HPLC-MS HPLC-MS analysis of the anthocyanin-rich acetone fruit extract (obtained as it was explained above) and the microencapsulate solutions was performed using a Shimadzu LCMS-2010 System (Shimadzu, Tokyo, Japan) equipped with an electrospray ionisation (ESI) probe, which was operated in positive ion mode as reported in the literature (Jaramillo et al. 2011). A LUNA RP-18 5-μm column (150×2.0 mm i.d., Phenomenex®, USA) was used for the analysis of the constituents present in each sample. The solvent system was composed of solvent A, a mixture of water/formic acid/acetonitrile (87:10:3, v/v/v), and solvent B, water/formic acid/acetonitrile (40:10:50, v/v/v). The HPLC program started with a linear gradient from 6 to 20 % B for the first 20 min and then increased from 20 to 40 % B for 20–35 min, 40 to 60 % B at 35–40 min, 60 to 90 % B at 40–45 min and 90 to 6 % B at 45– 40 min. The entire program used a flow rate of 0.8 mL/min. Powder Storage For this study, the two Andes berry microencapsulates that were selected exhibited the highest score in the sensory analyses for their similarity in odour and taste to the fresh fruit. They were stored at controlled temperatures (4 °C and 18± 1 °C), at relative humidities (RH; 75 and 95 %) and in the absence of light. The samples (1.5 g of each powder) were spread into a thin layer in Petri dishes (2 cm diameter). The samples were placed in sealed desiccators containing 200 mL of saturated sodium chloride or potassium nitrate solutions to obtain constant humidity values of 75±2 % and 95±2 %, respectively (Rockland 1960). During equilibration, the humidity was measured using a thermohygrometer (model 445815;
Extech Instruments, Nashua, NH, USA). The independent variables considered were the temperature, humidity and time of storage. The dependent variables were the water activity (a w), moisture and anthocyanin content, which were analysed daily in triplicate for one week, removing samples after each measurement. The control samples were stored at 18 °C and 60±2 % RH during the six-month experiments. The error percentage for the water activity measurements was 0.4 and 1.0 % for moisture. Statistical Analysis Data from the characterisation of the microencapsulates are reported as the mean±standard deviation for determinations performed in triplicate. Using the Statgraphics Plus 5.1 software, analysis of variance (ANOVA) and Tukey’s tests were performed to identify differences among the means. Differences at a probability level of P ≤0.05 were considered significant. To establish significant differences between the samples under sensory analyses, the T Friedman statistical function was calculated according to the equation reported in the literature (Peinado et al. 2012). Next, Tukey’s honestly significant difference was calculated to establish which samples exhibit the significant differences from one another (Meilgaard et al. 1999; Peinado et al. 2012).
Results and Discussion The Andes berry fruit was selected for this study because of its economic significance in Colombia and also due to its high anthocyanin content and pleasant aroma (Sinuco et al. 2013). The physicochemical characteristics of this fruit are shown in Table 1, which serves as a reference point for scaling up the Table 1 Physicochemical characterisation of Andes berry fruit Property
Value
Moisture content (% wet basis)a pH °Brix Acidity (% citric acid)b Anthocyanin contentc Colour parameters
L* a*
86.84±0.63 2.79±0.10 9.0±0.1 6.41±0.02 78.5±0.3 31.23±0.01 56.02±0.01
b* C* ab h ab
11.13±0.05 57.11±0.03 11.34±0.02
a
Gram of water/100 g of sample
b
Milligram of ascorbic acid/100 g of fruit
c
Milligram of cyanidin-3-glucoside equivalents/100 g of fruit FW. All data are the mean of triplicate measurements±standard deviation, P
