http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/02652048.2015.1010458
RESEARCH ARTICLE
Effect of wall material on the antioxidant activity and physicochemical properties of Rubus fruticosus juice microcapsules Dafne I. Dı´az1, Cesar I. Beristain1, Ebner Azuara1, Guadalupe Luna2, and Maribel Jimenez1
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1
Instituto de Ciencias Ba´sicas, Universidad Veracruzana, Dr. Luis Castelazo s/n, Col. Industrial Animas, Xalapa Ver., Me´xico and 2Divisio´n de Estudios de Posgrado e Investigacio´n, Instituto Tecnolo´gico de Orizaba, Orizaba Ver., Me´xico Abstract
Keywords
Blackberry (Rubus fruticosus) juice possesses compounds with antioxidant activity, which can be protected by different biopolymers used in the microencapsulation. Therefore, the effects of cell wall material including maltodextrin (MD), Arabic gum (GA) and whey protein concentrate (WPC) were evaluated on the physicochemical and antioxidant properties of encapsulated blackberries using a spray-drying technique. Anthocyanin concentration, polymeric colour, total polyphenols, radical scavenging activity of the 1,1-diphenyl-2-picrilhydrazil radical, reducing power and the stability at different storage conditions were evaluated. GA and MD conferred a similar protection to the antioxidant compounds when the microcapsules were stored at low water activities (aw50.515) in contrast to at a high moisture content (aw40.902), whereas WPC presented a high protection. Therefore, the selection of the best wall material for blackberry juice encapsulation depends of the conditions of storage of the powder.
Antioxidants, blackberry, microencapsulation, spray drying, stability, wall material
Introduction Nowadays, there are serious diseases such as diabetes mellitus, cardiovascular tissue inflammation and neurodegenerative diseases such as Parkinson’s and Alzheimer’s affecting humans, which are caused by the so-called oxidative stress (Kim and Lee, 2004). The importance of antioxidants is that they are substances that retard or inhibit the oxidation of susceptible substrates to free radical attack; in other words, they prevent the mismatch causing the diseases (Lima, 2001). The concentration of vegetal extracts rich in phytochemicals makes possible the development of natural products with improved stability and with high contents of beneficial compounds to humans due to their antioxidant and nutraceutical properties (Dimitrios, 2006). It is known that important sources of obtaining antioxidants are the fruits of red, blue and purple colours, such as the blackberry (Rubus fruticosus). Blackberries have a significant impact because the fruit possesses compounds that offer a great antioxidant capacity, and there are many studies claim that the dietary intake of berry fruits has a positive and profound impact on human health, performance and diseases, due to presence of flavonoids and phenolic acids (Rios de Souza et al., 2014). The flavonoids include compounds such as flavones, isoflavones, flavonols, catechins and red, blue, violet and purple pigments known as anthocyanins. Anthocyanins are a flavonoid of significant interest, which have a chemical structure adapted to act as an antioxidant. Therefore, anthocyanins can donate hydrogens or electrons to the
History Received 3 April 2014 Revised 29 December 2014 Accepted 06 January 2015 Published online 26 May 2015
free radicals or catch them and displace them in the aromatic structure. However, the degradation of these compounds is very rapid; therefore, diverse methods have been proposed to increase their protection. One of these methods is microencapsulation, which is defined as a process whereby certain bioactive substances are introduced into the matrix or wall system in order to prevent loss (Yan˜ez et al., 2002). The most common method used for microencapsulation is spray drying, because it is inexpensive and easily used by the food industry (Barbosa et al., 2005). The protection provided by this method depends greatly on the wall material used. In spray drying, Arabic gum (GA) has been found to be the most common and efficient substance for the protection of active materials (Lopera et al., 2009). Maltodextrin (MD) and GA have been used in the retention of volatile compounds during the spray drying of watermelon juice (Citrullus lanatus Thunb) (Gonzalez et al., 2011). On the other hand, it was shown that whey protein concentrate (WPC) is a wall material that provides greater protection to the stability of any active substance, such as oils (Jimenez et al., 2006). Therefore, the objective of this study was to evaluate the effect of wall materials including MD, GA and WPC on the physicochemical and antioxidant properties of blackberry juice (R. fruticosus) encapsulated by spray drying and determining their stability during storage at different water activities.
Material and methods Materials
Address for correspondence: Maribel Jimenez, Instituto de Ciencias Ba´sicas, Universidad Veracruzana, Dr. Luis Castelazo s/n, Col. Industrial Animas, Xalapa Ver., Me´xico. E-mail: [email protected]
The raw material was blackberries (R. fruticosus) obtained from a local supermarket produced by ‘‘Field-Blackberries Products’’ from the supplier Roberto Juan Davila Pulito, (Iztacalco, Mexico) and kept frozen at 40 C. The wall materials used: MD with a DE 10 (MD) was purchased from INALMALT (Tlalpan, Mexico),
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WPC was purchased from Vilher (Guadalajara, Mexico) and GA was purchased from Cosmopolitan (Mexico). Juice processing To collect the blackberry juice, the blackberries were thawed at a room temperature of 25 C and then were macerated with a pistil. In order to destroy the cell walls of the pulp and extract the greatest amount of anthocyanins present, ultrasound was applied for 10 min in 10-s intervals in a bath of ice to control of temperature at low 25 C at a constant frequency of 42 kHz using a sonifier (Branson, Digital model 250, Hollister, MO). Later, the juice was collect from the pulp using Whatman no. 1 filter paper (Sigma-Aldrich, St Louis, MO).
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Physicochemical analysis of the blackberry (R. fruticosus) juice
b a pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Croma ¼ a2 þ b2
Colour density ¼ ½ðA420 nm A700 nm Þ þ ðA510 nm A700 nm Þ FD
ð4Þ
The calculated equation was used to calculate the polymeric colour of the bleached sample with bisulphite: Polymer colour ¼ ½ðA420 nm A700 nm Þ þ ðA510 nm A700 nm Þ FD
ð5Þ
where A is the absorbance of the diluted sample to specific wavelengths and FD is the dilution factor used in the determinations. Finally, to calculate the percentage of polymer colour, the following equation was used: % Polymer colour ¼ ðpolymer colour=colour densityÞ 100 ð6Þ
The total titratable acidity percentage, Bx and total solids were determined according to the AOAC (1995). The colour was measured with a colorimeter (ColorFlex V1-72 SNHCX 1115 s/n: Cx1115 Hunter Lab, Reston, VA) using parameters a0 (yellow– red), b0 (blue–green) and L0 (intensity and brilliance) on the scale of the system CIE Lab (International Commission on Illumination, Vienna, Austria) (Tiwari et al., 2008). Equations (1–3) were used to calculate the total colour change (DE), hue angle (H ) and Chroma, respectively. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð1Þ D 2¼ ðL L0 Þ2 þ ða a0 Þ2 þ ðb b0 Þ2 Hue angle ¼ tan1
(treated with distilled water) was obtained from the following equation:
ð2Þ ð3Þ
where: L0, a0 and b0 are parameters of colour values of the initial sample and L, a and b parameters are colours of the treated sample values at different times. Monomeric anthocyanin The content of monomeric anthocyanin was evaluated using the differential pH method (Giusti and Wrolstad, 2001). Once it was determined, the dilution factor was prepared for the samples by adding the corresponding millilitres of 0.025 M KCl buffer at pH 1 or 0.4 M acetate buffer at pH 4.5 and 0.1 mL of the sample. The dilutions were kept at rest for 15 min, and the maxima absorbance at 510 (max) and 700 nm was read in a UV/VIS spectrophotometer (Jenway, Model 6305, Tokyo, Japan). The anthocyanin content was expressed as cyanidin-3-glucoside, using the coefficient of extinction of 26 900 L cm1 mg1 and a molecular weight of 449.2 g/L. Colour and polymer colour density Colour is one of the most important quality criteria for selection of the fruit juices, and the percent of polymeric colour indicates high discoloration and losses of monomeric anthocyanins. To analyse the polymer colour of the bisulphite solution, it was prepared by diluting 1 g of sample in 5 mL of distilled water and diluting in potassium chloride buffer 0.025 M pH 1.0 to obtain the proper dilution. Knowing the dilution factor used, diluted with distilled water sample and transferred to two cells of 2.8 mL of diluted sample, and to the first was added 0.2 mL of the solution of metabisulphite and to the second (sample control) was added 0.2 mL of distilled water. The absorbance of the samples was then read in a UV/VIS spectrophotometer (Jenway, model 6305) at 510 (max), 420 and 700 nm against a blank of distilled water (Giusti and Wrolstad, 2001). The colour density of the control sample
Analysis of the antioxidant activity of blackberry (R. fruticosus) juice Total polyphenols In an amber container, 0.1 mL of sample and 0.5 mL of Folin– Ciocalteu reagent were settled, and after five minutes, 0.4 mL of Na2CO3 was added to 7.5% (w/v) and diluted to 5 mL. The sample was left to stand for 20 min and was read at 740 nm in a UV/VIS spectrophotometer (Jenway, model 6305). For the calculations, a calibration curve was constructed with the following levels: 0.2, 0.6, 1.0, 1.4 and 1.8 mg/mL, using as a reference the Gallic acid from the equation of the straight line calculated from the amount of acid present Gallic/L according to the absorbance of the sample (Dominguez et al., 2005). An R2 ¼ 0.9982 value was obtained. Reducing power To 0.25 mL of sample, 2.5 mL of sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferrocyanide were added, and the mixture was incubated for 20 min in a water bath at 50 C. Then, 2.5 mL of 10% trichloroacetic acid was added. An aliquot of 5 mL was taken from the mixture to which 5 mL of distilled water and 1 mL of ferric chloride were added. This solution was read before an hour at 700 gm in a UV/VIS spectrophotometer (Jenway, model 6305) using sodium phosphate buffer as a blank (Oboh et al., 2009). Percentage of inhibition of the radical 1,1-diphenyl-2picrilhydrazil The scavenging activity of anthocyanins was determined using a solution of 0.1 mM radical 1,1-diphenyl-2-picrilhydrazil (DPPH) in methanol. Amounts of 0.1 mL of the samples were settled in 2.9 mL of DPPH solution and rested for 30 min, and the absorbance (Abs) was read at 517 nm in a UV/VIS spectrophotometer (Jenway, model 6305). The control contained methanol instead of the antioxidant solution, while the blank contained methanol instead. The percentage of inhibition of the DPPH radical was calculated with the following equation (Brand-Williams et al., 1995). % inhibition ¼
Absblank Abssample Abscontrol
ð7Þ
Process of encapsulation of blackberry juice (R. fruticosus) A solution with a ratio of 30% (w/w) (7.5 g solid juice, 22.5 g wall material and 70 g water) was mixed with a homogeniser
DOI: 10.3109/02652048.2015.1010458
Effect of wall material on Rubus fruticosus juice microcapsules
(Wiggen, Hauser model D-500, Menden, Germany) at a speed of 5000 rpm during 10 min. On the other hand, the relative viscosity of the solutions was measured with a Brookfield viscometer Model RTV at 100 rpm using a needle number 2 at 25 C according to the methodology described by Beristain et al. (1999). Then, the sample was fed in a twin fluid from mini spray dryer (model B-290, Bu¨chi, Flawil, Switzerland) with 150 ± 5 C and 90 ± 5 C of inlet and output temperature, respectively. The flow rate of the feed solution was 6.66 104 Ls1, and the atomisation pressure of air gas was 5332.89 Pa, a 4 104 m diameter nozzle was used. Once the powder was collected, it was weighed and the amber bottles were kept in a nitrogen atmosphere.
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Evaluation of the physicochemical properties of the microcapsules The moisture was evaluated by desiccation using the stove method, as described by the AOAC (1995). The colour was measured using the same methodology referred to in the physicochemical analysis of blackberry juice (R. fruticosus). The solubility was conducted by suspending 500 g of microcapsules in 50 mL of water and stirring in the thermostat at 25 C with a magnetic stirring. The time of solubility was considered as the time in seconds in which no particles were observed in suspension. Micrographs For the micrographs, 1.0 g of freshly obtained sample was equilibrated to two different relative humidities in desiccators containing glass salts, P2O5 (aw ¼ 0.0) and BaCl2 (aw ¼ 0.902) and stored at 25 C for 15 days to ensure its balance (Labuza et al., 1985). Subsequently, the surface morphology of the balanced microcapsules was analyzed for which samples were coated with 60% gold and 20% palladium and observed in a scanning electron microscope (JSM-5600LV, JEOL, Akishima, Japan) to 25 kV at different magnifications.
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less the total volume of toluene used (Bhandari et al., 1998). The powder flowability was evaluated using the Hausner ratio according to Turchiuli et al. (2005). The angle of repose was determined in 2 g of the sample of the microcapsules, which was weighed and they were added to a funnel and dropped as free samples forming a cone on the flat surface (Geldart et al., 2006). The angle repose was defined as the base angle formed when a given weight of the powder flowing through a funnel of known dimensions forms a pile and it was calculated as: y ¼ cotan
h r
ð8Þ
from the dimensions of the pile where h ¼ height of the powder pile and r ¼ radius of the pile base. Stability of microcapsules during storage Freshly sprayed microcapsules were stored at 35 C by the method of micro-climates, which consisted of placing a known mass of sample (in triplicate) with its atmosphere in a container sealed with a saturated solution of salt water of known activity (phosphorus pentoxide, aw ¼ 0.0; lithium chloride, aw ¼ 0.108; magnesium chloride, aw ¼ 0.318; magnesium nitrate, aw ¼ 0.515; sodium chloride, aw ¼ 0.743; and barium chloride, aw ¼ 0.902). Each third day, the total colour change and anthocyanin concentration were evaluated. In addition, a fractional volume was determined as the ratio volume at any aw/volume at aw ¼ 0 (Aguilera et al., 1993). Statistical analysis All analysis were done in triplicate (n ¼ 3). Data analysis were performed using the program Statistica 7.0 by StatSoft, Inc. (Tulsa, OK) (2004), the analysis of variance and the Tukey test (p50.05). The experimental data are presented as the mean and standard deviation (SD).
Results and discussion Destruction of microcapsules for their analysis Amounts of 1 g of microcapsules were weighed and mixed with 2.3 mL of water, which were then centrifuged at 4000 rpm for 10 min at a temperature of 5 C. This operation was repeated until the wall material of the microcapsules was completely removed. The samples were placed in amber vials, and the following tests were performed: anthocyanin, polymer colour, polyphenols, reducing power and the percentage of inhibition of the radical DPPH were determined according to the methods described for the analysis of the juice. The results are listed in the Table 3. This extraction was carried out for each test, and each wall material and all tests were made in triplicate. Evaluation of the flow properties of the microcapsules The flow properties were evaluated in the microcapsules obtained immediately after drying and during storage. The bulk density was evaluated in a graduated 10-mL cylinder in which 2 g of sample was placed. The density was determined by the weight of the sample dividing by the volume occupied by the sample. The compact density was determined by the method of ‘‘tappin’’ in which 2–5 g of the sample was placed in a 10-mL graduated cylinder. The test tube was hit on the flat up to a constant volume occupied by the sample (Jimenez et al., 2010). The percentage of compressibility was determined according to what was reported by Kagami et al. (2003). The particle density was measured using a pycnometer and toluene. The particle density is the total mass of the particle dividing by the difference of the pycnometer volume
Physical, chemical and antioxidant properties of blackberry juice Table 1 lists the physicochemical properties of blackberry juice; the pH, acidity and Bx were 3.73, 1.03% and 13.40, respectively. These results are slightly different from those reported by other authors for the variety of fruit Rubus glaucus, which were 2.72, 3.36% and 6.94, respectively (Alzate et al., 2010). This is possibly due to the variations in the conditions of production or the type of extraction of components. The pH was 3.73 indicating that anthocyanins have a red–violet range (Hutchings, 1999), which was confirmed through analysis of the parameters of colour, with values of 10.51, 18.24 and 4.95 for ‘‘L’’, ‘‘a’’ and ‘‘b’’, respectively, a chroma value of 42.88 and a hue angle of 15.18 , placing the sample in the first quadrant of the plane of colour (Francis and Clydesdale, 1975) with a deep red colour. This is interesting for some products such as sweets and beverages whose acceptance is closely associated with colour. Another physicochemical property of interest for the blackberry is the concentration of antioxidants, especially of anthocyanins, whose content was 517.00 mg 1001 g of dry sample, which was slightly less than that reported for Rubus idaeus L. (Anttonen and Karjalainen, 2005). The content of polyphenols was 930.99 mg GAE 1001 g of dry sample, and a 91.61% of inhibition of the DPPH radical was obtained. This designates anthocyanins as a product with high antioxidant activity and a greater resistance to the presence of nucleophilic species according to the percentage of polymer colour found (64.80%).
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Table 1. Physicochemical and antioxidant properties of blackberry juice (R. fruticosus). Physicochemical properties Total solids (%) pH Bx Total acidity (%) Colour L a b H Chroma
Antioxidant properties 12.00 ± 0.00 3.73 ± 0.25 13.40 ± 0.17 1.03 ± 0.04
Anthocyanins (mg/100 g) Polymeric colour (%) Radical scavenging activity DPPH (%) Reducing power Total polyphenols (mg GAE/100 g)
517.00 ± 1.03 64.80 ± 8.98 91.61 ± 2.66 1.67 ± 0.20 930.99 ± 1.87
10.51 ± 0.15 18.24 ± 0.29 4.95 ± 0.41 15.18 ± 0.98 42.88 ± 4.34
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Note: Data are expressed as means ± SD, (n ¼ 3).
Table 2. Physicochemical properties of blackberry juice (R. fruticosus) microencapsules using MD, GA and WPC as wall materials. Property Moisture (%) aw Colour L a b H ( ) Chroma DE Solubility time (s)
MD
GA b
WPC c
0.23 ± 0.23 0.24 ± 0.01a
4.94 ± 0.15 0.34 ± 0.02c
0.98 ± 0.10a 0.31 ± 0.00b
62.70 ± 0.10b 30.53 ± 0.23b 0.66 ± 0.28a 1.23a 30.96b 234.70b 13.9 ± 3.00a
55.38 ± 0.05a 33.61 ± 0.12c 1.95 ± 0.11b 3.32b 37.42c 290.10c 135 ± 10.00b
65.61 ± 0.10c 10.16 ± 0.30a 3.29 ± 0.22c 17.97c 21.02a 123.14a 11.4 ± 5.00a
Note: Data are expressed as means ± SD. Superscript letters within the same row mean a significant difference (p5 0.05, n ¼ 3). Table 3. Antioxidant properties of blackberry juice (R. fruticosus) microcapsules using MD, GA and WPC as wall materials. Antioxidant activity Anthocyanins (mg/100 g) Anthocyanin retention (%) Polymeric colour (%) Polyphenols (mg GAE/100 g) DPPH (%) Reducing power
MD
GA b
87.22 ± 0.63 66.45 ± 3.21b 74.21 ± 16.58b 292.36 ± 28.09b 58.61 ± 1.52b 1.283 ± 0.06a
WPC c
106.86 ± 5.91 71.62 ± 2.25c 64.01 ± 8.86a 403.60 ± 33.38c 60.67 ± 2.08b 1.669 ± 0.25b
34.07 ± 5.55a 23.33 ± 7.00a 92.24 ± 6.80c 246.65 ± 23.62a 49.13 ± 1.52a 1.200 ± 0.08a
Note: Data are expressed as means ± SD. Different superscript letters within the same row mean a significant difference (p5 0.05, n ¼ 3).
The solutions (with 30% of solids (w/w), 7.5 solid juice, 22.5 wall material and 70 g water) were analyzed in the Brookfield viscometer before encapsulation, and the values obtained were 11.25, 15.00 and 30.00 for MD, WPC and GA, respectively, indicating an appropriate range for the encapsulation by spray drying (Beristain et al., 1999). Physicochemical properties of the microcapsules The moisture content is one of the main properties of interest for the stability of food powders (Telis and Martinez-Navarrete, 2009). Table 2 shows that the microcapsules of GA had higher moisture content (4.94%), and MD had the lowest moisture content with 0.23%. Although the parameters for the spray drying were the same, the residual moisture varied for all samples possibly because of the different water-binding capacities of all materials. Similarly, results were obtained for water activity, which varied from 0.240 for MD to 0.340 when GA was used as the wall material. This might be due to the ability of each material
to absorb water during and after drying (Lewicki, 2004; Mathlouthi, 2001). The colour parameters presented for the samples encapsulated with different wall materials were in all cases very variable, this may be due to the degree of protection conferred by each one of the wall materials used. The microcapsules of MD and WPC showed shorter solubility time in comparison to GA, which reached maximum solubility at 135 s, probably due to the nature and microstructure of the wall materials. Antioxidant properties of the microcapsules Table 3 lists the results obtained for the antioxidant activity of the microcapsules. The concentration of anthocyanins after drying was higher when GA was used as the wall material (106.86 ± 5.91 mg/100 g of sample), followed by MD (87.22 ± 0.63 mg/ 100 g of sample) and for WPC (34.07 ± 5.55 mg/100 g of sample). Lower anthocyanins concentration (51.20 ± 0.34 mg/100 g) was reported by microcapsules of R. glaucus using MD as wall
Effect of wall material on Rubus fruticosus juice microcapsules
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DOI: 10.3109/02652048.2015.1010458
material (Olaya et al., 2009). The retention of polyphenols was higher for GA (403.60 ± 33.38 mg GAE/100 g of microcapsules), followed by WPC (246.65 ± 23.62 mg GAE/100 g of microcapsules) and finally MD (292.36 ± 28.09 mg GAE/100 g of microcapsules). Similar to the concentration of anthocyanins, GA (60.67 ± 2.08%) had a higher DPPH radical-scavenging activity percentage and WPC (49.13 ± 1.52%) had the lowest. On the other hand, the percentage of polymeric colour expressed the anthocyanin copolymerised indicating a loss of monomeric anthocyanins and formation of brown colour in red products (Turfan et al., 2011). In this work, WPC had a high percentage of polymeric colour of approximately 92.24 ± 6.80%, indicating a high discoloration, and losses of monomeric anthocyanins, while GA and MD had lower percentages of 64.01 ± 8.86% and 74.21 ± 16.58%, respectively, and lower discolorations. It is reported that losses of monomeric anthocyanins during processing of blackberry juice were due to the formation of anthocyanins polymers resulted in significant losses in the antioxidant activity (Brownmiller et al., 2008). Flow properties of blackberry juice microcapsules The Hausner ratio, angle of repose, bulk and compacted density were used to assess the fluency of the microcapsules developed. These properties depend largely on the moisture of the sample after drying, being higher for the GA (4.99%) than the MD (0.23%) and WPC (0.98%) microcapsules (Table 2). The physical properties of the developed microcapsules are listed in Table 4. The bulk density depended on the type of wall material used; GA microcapsules presented the highest density in bulk (392 kg/m3) and WPC the minor (297 kg/m3). Similarly, the compacted density of GA (601 kg/m3) presented a high value with respect to the other materials. However, these results are slightly higher than those obtained for microcapsules of linoleic acid (250 kg/m3) using the same wall material to the same concentration at room temperature, but different core material (Jimenez et al., 2006). This is important because high values for this parameter indicate a greater convenience for the storage of the powders in small containers (Barbosa-Canovas and Juliano, 2005). The particle density is an important parameter because not only is the volume occupied by the microcapsules but it also includes the volume that occupies each of the pores on its surface. According to the results obtained in this study, WPC and GA microcapsules were shown to possess less particle density, which means that the surface of the microcapsules is integrated and non-porous (Jimenez et al., 2010). The measurement of the angle of repose varied in accordance with the wall material used in the microcapsules; powders that exhibit less than 40 angles of repose usually have regular free-flowing properties, while angles above 50 indicate cohesiveness or flow problems (Bhandari et al., 1998; Turchiuli et al., 2005). According to the repose angle obtained in this study, only the microcapsules with MD had
Table 4. Flow properties of blackberry juice (R. microcapsules using MD, GA and WPC as wall materials. Property Bulk density (kg/m3) Compact density (kg/m3) Particle density (kg/m3) Angle of repose ( ) Compressibility (%) Hausner ratio
fruticosus)
MD
GA
WPC
360 ± 0.00b 492 ± 0.01b 739 ± 0.16b 28.07 ± 0.01a 26.72 ± 0.77a 1.36 ± 0.01a
392 ± 0.03c 601 ± 0.03c 130 ± 0.10a 44.98 ± 1.60b 34.91 ± 2.66b 1.53 ± 0.06b
297 ± 0.01a 441 ± 0.01a 134 ± 0.01a 68.03 ± 2.42c 32.60 ± 1.67b 1.48 ± 0.04b
Note: Data are expressed as means ± SD. Different superscript letters within the same row mean a significant difference (p50.05, n ¼ 3).
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a good free-flowing property, while the microcapsules performed with GA and WPC had flow problems. This variation in the angle may be due to the amount and kind of wall material used in each study. Hausner ratio of the microcapsules developed with the three wall materials showed values ranging from 1.3 to 1.60 indicating awful flowability properties (Turchiuli et al., 2005). These results may be due to the amount, type and wettability properties of biopolymer used in micro-encapsulation. Microstructure of microcapsules The protection offered by the wall material and the flow properties of the microcapsules depend on the external and internal microstructure of the microcapsule and how the wall material is organised in the microcapsules. These factors were studied in detail using scanning electron microscopy. Figure 1 shows the micrographs equilibrated to 0.00 and 0.902 of water activity during 15 days of storage at 25 C. Storing microcapsules at 0.00 of water activity exhibited a spherical morphology with a smooth and free of dented surface, independently of the wall material used. It is reported that particle structure depends of particle friability, feed specifications and drying conditions (Walton, 2000). However, physicochemical properties and particle structure also are affected by moisture content. In this work, developed microcapsules with different wall materials presented a different behaviour when they were stored at high water activities (0.743–0.902). GA microcapsules completely lost their original structure since they presented a complete dissolution of the wall material, which allowed the release of encapsulated material and a collapse and caking of the microcapsules. MD microcapsules were slightly deformed, with an irregular shape and a rough and serrated surface. WPC microcapsules were less affected with greater microcapsule integrity and without a round shape but with irregularity and surface roughness. It is reported that microcapsules in emulsions stabilised only by proteins are very stable to coalescence (Jayasundera et al., 2009). It is possibly due to these have a high molecular weight and a high capacity of interaction with water and stabilising emulsion droplets. Proteins also have the ability to produce a much harder layer, and subsequently more impermeable to moisture, will take more time to solubilise of wall of the microcapsule (Walton, 2000; Jafari et al., 2008). Anthocyanin retention in the capsules The efficiency with which the core material is encapsulated determines the success of the microencapsulation, and an ideal microencapsulation process should not lead to the loss of core material. We assessed the effect that the wall material had on anthocyanin retention (Table 3). GA microcapsules had the highest percentage of retention (71.62 ± 2.25%), suggesting that GA used as wall material protects the antioxidant compounds during drying and that has a higher affinity with the anthocyanins, in comparison with the retention in MD (66.45 ± 3.21%) and WPC (23.33 ± 7.00 %) microcapsules. The retention using GA and MD as wall material was higher than those reported to other antioxidant compounds microencapsulated such as eugenol, phenolic compounds and antioxidants, which are found in a range of 60–65% of effectiveness of microencapsulation (Chatterjee et al., 2012). Stability of microcapsules during storage Stability of anthocyanins during storage Anthocyanins are a good source of natural antioxidants but they are unstable to process and storage and easily susceptible to degradation such as pH, light and temperature, lead to its
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Figure 1. Micrographs of blackberry juice microcapsules using MD, GA and WPC as wall material stored 0.0 and 0.902 water activities.
discolouration. In this work, the degradation of the antioxidant compounds after storage at 35 C and different water activities varied in each wall material (Figure 2). GA showed to be stable at low water activities (0.00–0.515); however, at aw40.515, the rate of anthocyanin degradation increased substantially, the microcapsule became unable to keep their structural integrity, led the gradual dissolution of the wall and possibly caused the degradation of antioxidants compounds. On the other hand, MD microcapsules showed a constant degradation of anthocyanins, being greater at higher water activities. Contrary to GA and MD microcapsules, WPC microcapsules had high anthocyanins degradation to low water activities, but high water activities (0.743– 0.902) provided greater protection. It is possibly due at this moisture level, sufficient water was absorbed by the microcapsules to form a dough-like mass, which acts as a shell core in the microcapsule that protected the anthocyanins microencapsulated (Beristain et al., 2002).
Change of colour during storage The change of colour is one the most important quality parameters for the selection and consumption of food products. Samples of GA and MD microcapsules had a total colour change about 20%, and no significant differences (p 0.05) between equilibrated samples in the range of 0.00–0.515 of water activity. Instead, the same samples stored at water activities40.743 presented about 50% of change of the total colour (Figure 3). However, a significant difference (p 0.05) between both groups was observed. This behaviour was similar to the behaviour of the antioxidant activity during storage, and the microstructure of the microcapsules was observed to water activities40.743 then the microcapsule cakes and collapses. On the other hand, microcapsules of WPC did not show a significant difference (p 0.05) in total colour change in equilibrated samples at low (0.515) and high (0.902) water
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DOI: 10.3109/02652048.2015.1010458
Effect of wall material on Rubus fruticosus juice microcapsules
Figure 2. Anthocyanin retention (%) of blackberry juice microcapsules using MD, GA and WPC as wall material to different water activities after 30 days of storage at 35 C.
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Figure 4. Fractional sample volume of blackberry juice microcapsules using MD, GA and WPC as wall material to different water activities after 30 days of storage at 35 C.
at 35 C, so the microcapsules showed a total collapsed. It is possible due to liquid bridging occurs as a result of water vapour condensation onto the surface of particles and formation of a saturated solution which makes the particle sticky and capable of forming bridges, forming aggregates and causing collapse (Aguilera et al., 1993). Caking and sticking were caused by the movement and diffusion of the moisture within the microcapsule until balancing room moisture was reaching. On the other hand, WPC has a structure with greater ability to interact with water molecules so that showed the lowest change in volume when the samples were stored in a wide range of water activities (0.00–0.902), and this explains why the microcapsule structure was maintained.
Conclusions
Figure 3. Total colour change of blackberry juice microcapsules using MD, GA and WPC as wall material to two water activities (0.515 and 0.902) of storage during 30 days at 35 C.
Gum Arabic was the wall material that provided better retention of anthocyanins during spray drying, but this presented collapse and caking during storage at high water activities. On the other hand, the microcapsules of WPC had lower anthocyanin retention during drying but greater stability during storage at high water activities. The total change of colour is a good indicator of the behaviour and stability of the microcapsules during storage. The selection of wall material for encapsulation of blackberry juice depends on of the application and the ambient conditions of storage.
Declaration of interest activities indicating little effect on colour of the sample and their microstructure.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
Collapse during storage
References
Collapse has been defined as a time-, temperature- and moisturedependent viscous flow that results in a loss of structure (Aguilera et al., 1993). Figure 4 show that volumetric shrinkage presented a different behaviour when different wall materials were used. GA and MD showed a drastic decrease in volume for aw40.515
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RESEARCH ARTICLE
Effect of wall material on the antioxidant activity and physicochemical properties of Rubus fruticosus juice microcapsules Dafne I. Dı´az1, Cesar I. Beristain1, Ebner Azuara1, Guadalupe Luna2, and Maribel Jimenez1
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1
Instituto de Ciencias Ba´sicas, Universidad Veracruzana, Dr. Luis Castelazo s/n, Col. Industrial Animas, Xalapa Ver., Me´xico and 2Divisio´n de Estudios de Posgrado e Investigacio´n, Instituto Tecnolo´gico de Orizaba, Orizaba Ver., Me´xico Abstract
Keywords
Blackberry (Rubus fruticosus) juice possesses compounds with antioxidant activity, which can be protected by different biopolymers used in the microencapsulation. Therefore, the effects of cell wall material including maltodextrin (MD), Arabic gum (GA) and whey protein concentrate (WPC) were evaluated on the physicochemical and antioxidant properties of encapsulated blackberries using a spray-drying technique. Anthocyanin concentration, polymeric colour, total polyphenols, radical scavenging activity of the 1,1-diphenyl-2-picrilhydrazil radical, reducing power and the stability at different storage conditions were evaluated. GA and MD conferred a similar protection to the antioxidant compounds when the microcapsules were stored at low water activities (aw50.515) in contrast to at a high moisture content (aw40.902), whereas WPC presented a high protection. Therefore, the selection of the best wall material for blackberry juice encapsulation depends of the conditions of storage of the powder.
Antioxidants, blackberry, microencapsulation, spray drying, stability, wall material
Introduction Nowadays, there are serious diseases such as diabetes mellitus, cardiovascular tissue inflammation and neurodegenerative diseases such as Parkinson’s and Alzheimer’s affecting humans, which are caused by the so-called oxidative stress (Kim and Lee, 2004). The importance of antioxidants is that they are substances that retard or inhibit the oxidation of susceptible substrates to free radical attack; in other words, they prevent the mismatch causing the diseases (Lima, 2001). The concentration of vegetal extracts rich in phytochemicals makes possible the development of natural products with improved stability and with high contents of beneficial compounds to humans due to their antioxidant and nutraceutical properties (Dimitrios, 2006). It is known that important sources of obtaining antioxidants are the fruits of red, blue and purple colours, such as the blackberry (Rubus fruticosus). Blackberries have a significant impact because the fruit possesses compounds that offer a great antioxidant capacity, and there are many studies claim that the dietary intake of berry fruits has a positive and profound impact on human health, performance and diseases, due to presence of flavonoids and phenolic acids (Rios de Souza et al., 2014). The flavonoids include compounds such as flavones, isoflavones, flavonols, catechins and red, blue, violet and purple pigments known as anthocyanins. Anthocyanins are a flavonoid of significant interest, which have a chemical structure adapted to act as an antioxidant. Therefore, anthocyanins can donate hydrogens or electrons to the
History Received 3 April 2014 Revised 29 December 2014 Accepted 06 January 2015 Published online 26 May 2015
free radicals or catch them and displace them in the aromatic structure. However, the degradation of these compounds is very rapid; therefore, diverse methods have been proposed to increase their protection. One of these methods is microencapsulation, which is defined as a process whereby certain bioactive substances are introduced into the matrix or wall system in order to prevent loss (Yan˜ez et al., 2002). The most common method used for microencapsulation is spray drying, because it is inexpensive and easily used by the food industry (Barbosa et al., 2005). The protection provided by this method depends greatly on the wall material used. In spray drying, Arabic gum (GA) has been found to be the most common and efficient substance for the protection of active materials (Lopera et al., 2009). Maltodextrin (MD) and GA have been used in the retention of volatile compounds during the spray drying of watermelon juice (Citrullus lanatus Thunb) (Gonzalez et al., 2011). On the other hand, it was shown that whey protein concentrate (WPC) is a wall material that provides greater protection to the stability of any active substance, such as oils (Jimenez et al., 2006). Therefore, the objective of this study was to evaluate the effect of wall materials including MD, GA and WPC on the physicochemical and antioxidant properties of blackberry juice (R. fruticosus) encapsulated by spray drying and determining their stability during storage at different water activities.
Material and methods Materials
Address for correspondence: Maribel Jimenez, Instituto de Ciencias Ba´sicas, Universidad Veracruzana, Dr. Luis Castelazo s/n, Col. Industrial Animas, Xalapa Ver., Me´xico. E-mail: [email protected]
The raw material was blackberries (R. fruticosus) obtained from a local supermarket produced by ‘‘Field-Blackberries Products’’ from the supplier Roberto Juan Davila Pulito, (Iztacalco, Mexico) and kept frozen at 40 C. The wall materials used: MD with a DE 10 (MD) was purchased from INALMALT (Tlalpan, Mexico),
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WPC was purchased from Vilher (Guadalajara, Mexico) and GA was purchased from Cosmopolitan (Mexico). Juice processing To collect the blackberry juice, the blackberries were thawed at a room temperature of 25 C and then were macerated with a pistil. In order to destroy the cell walls of the pulp and extract the greatest amount of anthocyanins present, ultrasound was applied for 10 min in 10-s intervals in a bath of ice to control of temperature at low 25 C at a constant frequency of 42 kHz using a sonifier (Branson, Digital model 250, Hollister, MO). Later, the juice was collect from the pulp using Whatman no. 1 filter paper (Sigma-Aldrich, St Louis, MO).
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Physicochemical analysis of the blackberry (R. fruticosus) juice
b a pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Croma ¼ a2 þ b2
Colour density ¼ ½ðA420 nm A700 nm Þ þ ðA510 nm A700 nm Þ FD
ð4Þ
The calculated equation was used to calculate the polymeric colour of the bleached sample with bisulphite: Polymer colour ¼ ½ðA420 nm A700 nm Þ þ ðA510 nm A700 nm Þ FD
ð5Þ
where A is the absorbance of the diluted sample to specific wavelengths and FD is the dilution factor used in the determinations. Finally, to calculate the percentage of polymer colour, the following equation was used: % Polymer colour ¼ ðpolymer colour=colour densityÞ 100 ð6Þ
The total titratable acidity percentage, Bx and total solids were determined according to the AOAC (1995). The colour was measured with a colorimeter (ColorFlex V1-72 SNHCX 1115 s/n: Cx1115 Hunter Lab, Reston, VA) using parameters a0 (yellow– red), b0 (blue–green) and L0 (intensity and brilliance) on the scale of the system CIE Lab (International Commission on Illumination, Vienna, Austria) (Tiwari et al., 2008). Equations (1–3) were used to calculate the total colour change (DE), hue angle (H ) and Chroma, respectively. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð1Þ D 2¼ ðL L0 Þ2 þ ða a0 Þ2 þ ðb b0 Þ2 Hue angle ¼ tan1
(treated with distilled water) was obtained from the following equation:
ð2Þ ð3Þ
where: L0, a0 and b0 are parameters of colour values of the initial sample and L, a and b parameters are colours of the treated sample values at different times. Monomeric anthocyanin The content of monomeric anthocyanin was evaluated using the differential pH method (Giusti and Wrolstad, 2001). Once it was determined, the dilution factor was prepared for the samples by adding the corresponding millilitres of 0.025 M KCl buffer at pH 1 or 0.4 M acetate buffer at pH 4.5 and 0.1 mL of the sample. The dilutions were kept at rest for 15 min, and the maxima absorbance at 510 (max) and 700 nm was read in a UV/VIS spectrophotometer (Jenway, Model 6305, Tokyo, Japan). The anthocyanin content was expressed as cyanidin-3-glucoside, using the coefficient of extinction of 26 900 L cm1 mg1 and a molecular weight of 449.2 g/L. Colour and polymer colour density Colour is one of the most important quality criteria for selection of the fruit juices, and the percent of polymeric colour indicates high discoloration and losses of monomeric anthocyanins. To analyse the polymer colour of the bisulphite solution, it was prepared by diluting 1 g of sample in 5 mL of distilled water and diluting in potassium chloride buffer 0.025 M pH 1.0 to obtain the proper dilution. Knowing the dilution factor used, diluted with distilled water sample and transferred to two cells of 2.8 mL of diluted sample, and to the first was added 0.2 mL of the solution of metabisulphite and to the second (sample control) was added 0.2 mL of distilled water. The absorbance of the samples was then read in a UV/VIS spectrophotometer (Jenway, model 6305) at 510 (max), 420 and 700 nm against a blank of distilled water (Giusti and Wrolstad, 2001). The colour density of the control sample
Analysis of the antioxidant activity of blackberry (R. fruticosus) juice Total polyphenols In an amber container, 0.1 mL of sample and 0.5 mL of Folin– Ciocalteu reagent were settled, and after five minutes, 0.4 mL of Na2CO3 was added to 7.5% (w/v) and diluted to 5 mL. The sample was left to stand for 20 min and was read at 740 nm in a UV/VIS spectrophotometer (Jenway, model 6305). For the calculations, a calibration curve was constructed with the following levels: 0.2, 0.6, 1.0, 1.4 and 1.8 mg/mL, using as a reference the Gallic acid from the equation of the straight line calculated from the amount of acid present Gallic/L according to the absorbance of the sample (Dominguez et al., 2005). An R2 ¼ 0.9982 value was obtained. Reducing power To 0.25 mL of sample, 2.5 mL of sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferrocyanide were added, and the mixture was incubated for 20 min in a water bath at 50 C. Then, 2.5 mL of 10% trichloroacetic acid was added. An aliquot of 5 mL was taken from the mixture to which 5 mL of distilled water and 1 mL of ferric chloride were added. This solution was read before an hour at 700 gm in a UV/VIS spectrophotometer (Jenway, model 6305) using sodium phosphate buffer as a blank (Oboh et al., 2009). Percentage of inhibition of the radical 1,1-diphenyl-2picrilhydrazil The scavenging activity of anthocyanins was determined using a solution of 0.1 mM radical 1,1-diphenyl-2-picrilhydrazil (DPPH) in methanol. Amounts of 0.1 mL of the samples were settled in 2.9 mL of DPPH solution and rested for 30 min, and the absorbance (Abs) was read at 517 nm in a UV/VIS spectrophotometer (Jenway, model 6305). The control contained methanol instead of the antioxidant solution, while the blank contained methanol instead. The percentage of inhibition of the DPPH radical was calculated with the following equation (Brand-Williams et al., 1995). % inhibition ¼
Absblank Abssample Abscontrol
ð7Þ
Process of encapsulation of blackberry juice (R. fruticosus) A solution with a ratio of 30% (w/w) (7.5 g solid juice, 22.5 g wall material and 70 g water) was mixed with a homogeniser
DOI: 10.3109/02652048.2015.1010458
Effect of wall material on Rubus fruticosus juice microcapsules
(Wiggen, Hauser model D-500, Menden, Germany) at a speed of 5000 rpm during 10 min. On the other hand, the relative viscosity of the solutions was measured with a Brookfield viscometer Model RTV at 100 rpm using a needle number 2 at 25 C according to the methodology described by Beristain et al. (1999). Then, the sample was fed in a twin fluid from mini spray dryer (model B-290, Bu¨chi, Flawil, Switzerland) with 150 ± 5 C and 90 ± 5 C of inlet and output temperature, respectively. The flow rate of the feed solution was 6.66 104 Ls1, and the atomisation pressure of air gas was 5332.89 Pa, a 4 104 m diameter nozzle was used. Once the powder was collected, it was weighed and the amber bottles were kept in a nitrogen atmosphere.
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Evaluation of the physicochemical properties of the microcapsules The moisture was evaluated by desiccation using the stove method, as described by the AOAC (1995). The colour was measured using the same methodology referred to in the physicochemical analysis of blackberry juice (R. fruticosus). The solubility was conducted by suspending 500 g of microcapsules in 50 mL of water and stirring in the thermostat at 25 C with a magnetic stirring. The time of solubility was considered as the time in seconds in which no particles were observed in suspension. Micrographs For the micrographs, 1.0 g of freshly obtained sample was equilibrated to two different relative humidities in desiccators containing glass salts, P2O5 (aw ¼ 0.0) and BaCl2 (aw ¼ 0.902) and stored at 25 C for 15 days to ensure its balance (Labuza et al., 1985). Subsequently, the surface morphology of the balanced microcapsules was analyzed for which samples were coated with 60% gold and 20% palladium and observed in a scanning electron microscope (JSM-5600LV, JEOL, Akishima, Japan) to 25 kV at different magnifications.
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less the total volume of toluene used (Bhandari et al., 1998). The powder flowability was evaluated using the Hausner ratio according to Turchiuli et al. (2005). The angle of repose was determined in 2 g of the sample of the microcapsules, which was weighed and they were added to a funnel and dropped as free samples forming a cone on the flat surface (Geldart et al., 2006). The angle repose was defined as the base angle formed when a given weight of the powder flowing through a funnel of known dimensions forms a pile and it was calculated as: y ¼ cotan
h r
ð8Þ
from the dimensions of the pile where h ¼ height of the powder pile and r ¼ radius of the pile base. Stability of microcapsules during storage Freshly sprayed microcapsules were stored at 35 C by the method of micro-climates, which consisted of placing a known mass of sample (in triplicate) with its atmosphere in a container sealed with a saturated solution of salt water of known activity (phosphorus pentoxide, aw ¼ 0.0; lithium chloride, aw ¼ 0.108; magnesium chloride, aw ¼ 0.318; magnesium nitrate, aw ¼ 0.515; sodium chloride, aw ¼ 0.743; and barium chloride, aw ¼ 0.902). Each third day, the total colour change and anthocyanin concentration were evaluated. In addition, a fractional volume was determined as the ratio volume at any aw/volume at aw ¼ 0 (Aguilera et al., 1993). Statistical analysis All analysis were done in triplicate (n ¼ 3). Data analysis were performed using the program Statistica 7.0 by StatSoft, Inc. (Tulsa, OK) (2004), the analysis of variance and the Tukey test (p50.05). The experimental data are presented as the mean and standard deviation (SD).
Results and discussion Destruction of microcapsules for their analysis Amounts of 1 g of microcapsules were weighed and mixed with 2.3 mL of water, which were then centrifuged at 4000 rpm for 10 min at a temperature of 5 C. This operation was repeated until the wall material of the microcapsules was completely removed. The samples were placed in amber vials, and the following tests were performed: anthocyanin, polymer colour, polyphenols, reducing power and the percentage of inhibition of the radical DPPH were determined according to the methods described for the analysis of the juice. The results are listed in the Table 3. This extraction was carried out for each test, and each wall material and all tests were made in triplicate. Evaluation of the flow properties of the microcapsules The flow properties were evaluated in the microcapsules obtained immediately after drying and during storage. The bulk density was evaluated in a graduated 10-mL cylinder in which 2 g of sample was placed. The density was determined by the weight of the sample dividing by the volume occupied by the sample. The compact density was determined by the method of ‘‘tappin’’ in which 2–5 g of the sample was placed in a 10-mL graduated cylinder. The test tube was hit on the flat up to a constant volume occupied by the sample (Jimenez et al., 2010). The percentage of compressibility was determined according to what was reported by Kagami et al. (2003). The particle density was measured using a pycnometer and toluene. The particle density is the total mass of the particle dividing by the difference of the pycnometer volume
Physical, chemical and antioxidant properties of blackberry juice Table 1 lists the physicochemical properties of blackberry juice; the pH, acidity and Bx were 3.73, 1.03% and 13.40, respectively. These results are slightly different from those reported by other authors for the variety of fruit Rubus glaucus, which were 2.72, 3.36% and 6.94, respectively (Alzate et al., 2010). This is possibly due to the variations in the conditions of production or the type of extraction of components. The pH was 3.73 indicating that anthocyanins have a red–violet range (Hutchings, 1999), which was confirmed through analysis of the parameters of colour, with values of 10.51, 18.24 and 4.95 for ‘‘L’’, ‘‘a’’ and ‘‘b’’, respectively, a chroma value of 42.88 and a hue angle of 15.18 , placing the sample in the first quadrant of the plane of colour (Francis and Clydesdale, 1975) with a deep red colour. This is interesting for some products such as sweets and beverages whose acceptance is closely associated with colour. Another physicochemical property of interest for the blackberry is the concentration of antioxidants, especially of anthocyanins, whose content was 517.00 mg 1001 g of dry sample, which was slightly less than that reported for Rubus idaeus L. (Anttonen and Karjalainen, 2005). The content of polyphenols was 930.99 mg GAE 1001 g of dry sample, and a 91.61% of inhibition of the DPPH radical was obtained. This designates anthocyanins as a product with high antioxidant activity and a greater resistance to the presence of nucleophilic species according to the percentage of polymer colour found (64.80%).
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Table 1. Physicochemical and antioxidant properties of blackberry juice (R. fruticosus). Physicochemical properties Total solids (%) pH Bx Total acidity (%) Colour L a b H Chroma
Antioxidant properties 12.00 ± 0.00 3.73 ± 0.25 13.40 ± 0.17 1.03 ± 0.04
Anthocyanins (mg/100 g) Polymeric colour (%) Radical scavenging activity DPPH (%) Reducing power Total polyphenols (mg GAE/100 g)
517.00 ± 1.03 64.80 ± 8.98 91.61 ± 2.66 1.67 ± 0.20 930.99 ± 1.87
10.51 ± 0.15 18.24 ± 0.29 4.95 ± 0.41 15.18 ± 0.98 42.88 ± 4.34
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Note: Data are expressed as means ± SD, (n ¼ 3).
Table 2. Physicochemical properties of blackberry juice (R. fruticosus) microencapsules using MD, GA and WPC as wall materials. Property Moisture (%) aw Colour L a b H ( ) Chroma DE Solubility time (s)
MD
GA b
WPC c
0.23 ± 0.23 0.24 ± 0.01a
4.94 ± 0.15 0.34 ± 0.02c
0.98 ± 0.10a 0.31 ± 0.00b
62.70 ± 0.10b 30.53 ± 0.23b 0.66 ± 0.28a 1.23a 30.96b 234.70b 13.9 ± 3.00a
55.38 ± 0.05a 33.61 ± 0.12c 1.95 ± 0.11b 3.32b 37.42c 290.10c 135 ± 10.00b
65.61 ± 0.10c 10.16 ± 0.30a 3.29 ± 0.22c 17.97c 21.02a 123.14a 11.4 ± 5.00a
Note: Data are expressed as means ± SD. Superscript letters within the same row mean a significant difference (p5 0.05, n ¼ 3). Table 3. Antioxidant properties of blackberry juice (R. fruticosus) microcapsules using MD, GA and WPC as wall materials. Antioxidant activity Anthocyanins (mg/100 g) Anthocyanin retention (%) Polymeric colour (%) Polyphenols (mg GAE/100 g) DPPH (%) Reducing power
MD
GA b
87.22 ± 0.63 66.45 ± 3.21b 74.21 ± 16.58b 292.36 ± 28.09b 58.61 ± 1.52b 1.283 ± 0.06a
WPC c
106.86 ± 5.91 71.62 ± 2.25c 64.01 ± 8.86a 403.60 ± 33.38c 60.67 ± 2.08b 1.669 ± 0.25b
34.07 ± 5.55a 23.33 ± 7.00a 92.24 ± 6.80c 246.65 ± 23.62a 49.13 ± 1.52a 1.200 ± 0.08a
Note: Data are expressed as means ± SD. Different superscript letters within the same row mean a significant difference (p5 0.05, n ¼ 3).
The solutions (with 30% of solids (w/w), 7.5 solid juice, 22.5 wall material and 70 g water) were analyzed in the Brookfield viscometer before encapsulation, and the values obtained were 11.25, 15.00 and 30.00 for MD, WPC and GA, respectively, indicating an appropriate range for the encapsulation by spray drying (Beristain et al., 1999). Physicochemical properties of the microcapsules The moisture content is one of the main properties of interest for the stability of food powders (Telis and Martinez-Navarrete, 2009). Table 2 shows that the microcapsules of GA had higher moisture content (4.94%), and MD had the lowest moisture content with 0.23%. Although the parameters for the spray drying were the same, the residual moisture varied for all samples possibly because of the different water-binding capacities of all materials. Similarly, results were obtained for water activity, which varied from 0.240 for MD to 0.340 when GA was used as the wall material. This might be due to the ability of each material
to absorb water during and after drying (Lewicki, 2004; Mathlouthi, 2001). The colour parameters presented for the samples encapsulated with different wall materials were in all cases very variable, this may be due to the degree of protection conferred by each one of the wall materials used. The microcapsules of MD and WPC showed shorter solubility time in comparison to GA, which reached maximum solubility at 135 s, probably due to the nature and microstructure of the wall materials. Antioxidant properties of the microcapsules Table 3 lists the results obtained for the antioxidant activity of the microcapsules. The concentration of anthocyanins after drying was higher when GA was used as the wall material (106.86 ± 5.91 mg/100 g of sample), followed by MD (87.22 ± 0.63 mg/ 100 g of sample) and for WPC (34.07 ± 5.55 mg/100 g of sample). Lower anthocyanins concentration (51.20 ± 0.34 mg/100 g) was reported by microcapsules of R. glaucus using MD as wall
Effect of wall material on Rubus fruticosus juice microcapsules
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DOI: 10.3109/02652048.2015.1010458
material (Olaya et al., 2009). The retention of polyphenols was higher for GA (403.60 ± 33.38 mg GAE/100 g of microcapsules), followed by WPC (246.65 ± 23.62 mg GAE/100 g of microcapsules) and finally MD (292.36 ± 28.09 mg GAE/100 g of microcapsules). Similar to the concentration of anthocyanins, GA (60.67 ± 2.08%) had a higher DPPH radical-scavenging activity percentage and WPC (49.13 ± 1.52%) had the lowest. On the other hand, the percentage of polymeric colour expressed the anthocyanin copolymerised indicating a loss of monomeric anthocyanins and formation of brown colour in red products (Turfan et al., 2011). In this work, WPC had a high percentage of polymeric colour of approximately 92.24 ± 6.80%, indicating a high discoloration, and losses of monomeric anthocyanins, while GA and MD had lower percentages of 64.01 ± 8.86% and 74.21 ± 16.58%, respectively, and lower discolorations. It is reported that losses of monomeric anthocyanins during processing of blackberry juice were due to the formation of anthocyanins polymers resulted in significant losses in the antioxidant activity (Brownmiller et al., 2008). Flow properties of blackberry juice microcapsules The Hausner ratio, angle of repose, bulk and compacted density were used to assess the fluency of the microcapsules developed. These properties depend largely on the moisture of the sample after drying, being higher for the GA (4.99%) than the MD (0.23%) and WPC (0.98%) microcapsules (Table 2). The physical properties of the developed microcapsules are listed in Table 4. The bulk density depended on the type of wall material used; GA microcapsules presented the highest density in bulk (392 kg/m3) and WPC the minor (297 kg/m3). Similarly, the compacted density of GA (601 kg/m3) presented a high value with respect to the other materials. However, these results are slightly higher than those obtained for microcapsules of linoleic acid (250 kg/m3) using the same wall material to the same concentration at room temperature, but different core material (Jimenez et al., 2006). This is important because high values for this parameter indicate a greater convenience for the storage of the powders in small containers (Barbosa-Canovas and Juliano, 2005). The particle density is an important parameter because not only is the volume occupied by the microcapsules but it also includes the volume that occupies each of the pores on its surface. According to the results obtained in this study, WPC and GA microcapsules were shown to possess less particle density, which means that the surface of the microcapsules is integrated and non-porous (Jimenez et al., 2010). The measurement of the angle of repose varied in accordance with the wall material used in the microcapsules; powders that exhibit less than 40 angles of repose usually have regular free-flowing properties, while angles above 50 indicate cohesiveness or flow problems (Bhandari et al., 1998; Turchiuli et al., 2005). According to the repose angle obtained in this study, only the microcapsules with MD had
Table 4. Flow properties of blackberry juice (R. microcapsules using MD, GA and WPC as wall materials. Property Bulk density (kg/m3) Compact density (kg/m3) Particle density (kg/m3) Angle of repose ( ) Compressibility (%) Hausner ratio
fruticosus)
MD
GA
WPC
360 ± 0.00b 492 ± 0.01b 739 ± 0.16b 28.07 ± 0.01a 26.72 ± 0.77a 1.36 ± 0.01a
392 ± 0.03c 601 ± 0.03c 130 ± 0.10a 44.98 ± 1.60b 34.91 ± 2.66b 1.53 ± 0.06b
297 ± 0.01a 441 ± 0.01a 134 ± 0.01a 68.03 ± 2.42c 32.60 ± 1.67b 1.48 ± 0.04b
Note: Data are expressed as means ± SD. Different superscript letters within the same row mean a significant difference (p50.05, n ¼ 3).
5
a good free-flowing property, while the microcapsules performed with GA and WPC had flow problems. This variation in the angle may be due to the amount and kind of wall material used in each study. Hausner ratio of the microcapsules developed with the three wall materials showed values ranging from 1.3 to 1.60 indicating awful flowability properties (Turchiuli et al., 2005). These results may be due to the amount, type and wettability properties of biopolymer used in micro-encapsulation. Microstructure of microcapsules The protection offered by the wall material and the flow properties of the microcapsules depend on the external and internal microstructure of the microcapsule and how the wall material is organised in the microcapsules. These factors were studied in detail using scanning electron microscopy. Figure 1 shows the micrographs equilibrated to 0.00 and 0.902 of water activity during 15 days of storage at 25 C. Storing microcapsules at 0.00 of water activity exhibited a spherical morphology with a smooth and free of dented surface, independently of the wall material used. It is reported that particle structure depends of particle friability, feed specifications and drying conditions (Walton, 2000). However, physicochemical properties and particle structure also are affected by moisture content. In this work, developed microcapsules with different wall materials presented a different behaviour when they were stored at high water activities (0.743–0.902). GA microcapsules completely lost their original structure since they presented a complete dissolution of the wall material, which allowed the release of encapsulated material and a collapse and caking of the microcapsules. MD microcapsules were slightly deformed, with an irregular shape and a rough and serrated surface. WPC microcapsules were less affected with greater microcapsule integrity and without a round shape but with irregularity and surface roughness. It is reported that microcapsules in emulsions stabilised only by proteins are very stable to coalescence (Jayasundera et al., 2009). It is possibly due to these have a high molecular weight and a high capacity of interaction with water and stabilising emulsion droplets. Proteins also have the ability to produce a much harder layer, and subsequently more impermeable to moisture, will take more time to solubilise of wall of the microcapsule (Walton, 2000; Jafari et al., 2008). Anthocyanin retention in the capsules The efficiency with which the core material is encapsulated determines the success of the microencapsulation, and an ideal microencapsulation process should not lead to the loss of core material. We assessed the effect that the wall material had on anthocyanin retention (Table 3). GA microcapsules had the highest percentage of retention (71.62 ± 2.25%), suggesting that GA used as wall material protects the antioxidant compounds during drying and that has a higher affinity with the anthocyanins, in comparison with the retention in MD (66.45 ± 3.21%) and WPC (23.33 ± 7.00 %) microcapsules. The retention using GA and MD as wall material was higher than those reported to other antioxidant compounds microencapsulated such as eugenol, phenolic compounds and antioxidants, which are found in a range of 60–65% of effectiveness of microencapsulation (Chatterjee et al., 2012). Stability of microcapsules during storage Stability of anthocyanins during storage Anthocyanins are a good source of natural antioxidants but they are unstable to process and storage and easily susceptible to degradation such as pH, light and temperature, lead to its
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Figure 1. Micrographs of blackberry juice microcapsules using MD, GA and WPC as wall material stored 0.0 and 0.902 water activities.
discolouration. In this work, the degradation of the antioxidant compounds after storage at 35 C and different water activities varied in each wall material (Figure 2). GA showed to be stable at low water activities (0.00–0.515); however, at aw40.515, the rate of anthocyanin degradation increased substantially, the microcapsule became unable to keep their structural integrity, led the gradual dissolution of the wall and possibly caused the degradation of antioxidants compounds. On the other hand, MD microcapsules showed a constant degradation of anthocyanins, being greater at higher water activities. Contrary to GA and MD microcapsules, WPC microcapsules had high anthocyanins degradation to low water activities, but high water activities (0.743– 0.902) provided greater protection. It is possibly due at this moisture level, sufficient water was absorbed by the microcapsules to form a dough-like mass, which acts as a shell core in the microcapsule that protected the anthocyanins microencapsulated (Beristain et al., 2002).
Change of colour during storage The change of colour is one the most important quality parameters for the selection and consumption of food products. Samples of GA and MD microcapsules had a total colour change about 20%, and no significant differences (p 0.05) between equilibrated samples in the range of 0.00–0.515 of water activity. Instead, the same samples stored at water activities40.743 presented about 50% of change of the total colour (Figure 3). However, a significant difference (p 0.05) between both groups was observed. This behaviour was similar to the behaviour of the antioxidant activity during storage, and the microstructure of the microcapsules was observed to water activities40.743 then the microcapsule cakes and collapses. On the other hand, microcapsules of WPC did not show a significant difference (p 0.05) in total colour change in equilibrated samples at low (0.515) and high (0.902) water
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DOI: 10.3109/02652048.2015.1010458
Effect of wall material on Rubus fruticosus juice microcapsules
Figure 2. Anthocyanin retention (%) of blackberry juice microcapsules using MD, GA and WPC as wall material to different water activities after 30 days of storage at 35 C.
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Figure 4. Fractional sample volume of blackberry juice microcapsules using MD, GA and WPC as wall material to different water activities after 30 days of storage at 35 C.
at 35 C, so the microcapsules showed a total collapsed. It is possible due to liquid bridging occurs as a result of water vapour condensation onto the surface of particles and formation of a saturated solution which makes the particle sticky and capable of forming bridges, forming aggregates and causing collapse (Aguilera et al., 1993). Caking and sticking were caused by the movement and diffusion of the moisture within the microcapsule until balancing room moisture was reaching. On the other hand, WPC has a structure with greater ability to interact with water molecules so that showed the lowest change in volume when the samples were stored in a wide range of water activities (0.00–0.902), and this explains why the microcapsule structure was maintained.
Conclusions
Figure 3. Total colour change of blackberry juice microcapsules using MD, GA and WPC as wall material to two water activities (0.515 and 0.902) of storage during 30 days at 35 C.
Gum Arabic was the wall material that provided better retention of anthocyanins during spray drying, but this presented collapse and caking during storage at high water activities. On the other hand, the microcapsules of WPC had lower anthocyanin retention during drying but greater stability during storage at high water activities. The total change of colour is a good indicator of the behaviour and stability of the microcapsules during storage. The selection of wall material for encapsulation of blackberry juice depends on of the application and the ambient conditions of storage.
Declaration of interest activities indicating little effect on colour of the sample and their microstructure.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
Collapse during storage
References
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