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Antioxidative activity of microcapsules with beetroot juice using gum Arabic as wall material
food and bioproducts processing 8 8 ( 2 0 1 0 ) 253–258
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Antioxidative activity of microcapsules with beetroot juice using gum Arabic as wall material E. Pitalua, M. Jimenez, E.J. Vernon-Carter, C.I. Beristain ∗ Instituto de Ciencias Básicas, Universidad Veracruzana, Luis Castelazo S/N, Col Industrial Animas, Apto Postal 575, Apdo, CP 91192 Xalapa, Veracruz, Mexico
a b s t r a c t The antioxidative activity of encapsulated natural beet root juice was evaluated during storage at different water activities. In microcapsules of beetroot obtained by spray drying and stored at water activities of 0.110, 0.326 and 0.521 there were no significant differences in betalain concentration, color, antioxidant activity and redox potential during 45 days. However, the samples stored at aw s of 0.748 and 0.898 showed significant differences compared to those stored at 0.110, 0.326 and 0.521, but no difference was shown among the constituents of each group. In the samples stored at 0.748 and 0.898 the antioxidant activity increases, whereas the betalain concentration decreases due to the compounds that are formed while degradation of the betalains occurs. Water adsorption influences the stability of the product during storage. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Betalains; Redox potential; Chain-breaking; Water activity; Color
Stability is an important aspect to consider for the use of pigments as antioxidants and colorants in food. There are many factors which affect the stability and intensity of colorants during processing and storage. The colorant present in beetroot tends to degrade easily in solution. It is reported that its stability is affected during processing and storage (Attoe and von Elbe, 1982); therefore spray drying was used to avoid colorant degradation. It is reported that stabilization of betalains and polyphenols could be improved using microencapsulation by spray drying (Desai and Park, 2005). Microencapsulation through spray drying is an economical method for the preservation of natural colorants by entrapping the ingredients in a coating material (Ersus and Yudagel, 2007; Serris and Biliaderis, 2001). Gum Arabic is one of the most important carrier and coating agents used for the spray drying of flavors and colorants (Beristain et al., 1999), so it is important to evaluate the encapsulation of beetroot juice in this material. There are many reports about how antioxidants can be affected as a consequence of storage conditions; in some cases, storage can induce the formation of compounds with antioxidant properties, which can maintain or even enhance the overall antioxidant potential of foods (Jonsson, 1991). The storage stability of powdered antioxidants for extended peri-
∗
ods is of interest to manufacturers and consumers of food packaged for long-term storage. Beetroot is known to have antioxidant properties since it contains nitrogen compounds called betalains, which are classified as betacyanins that confer the red-violet color to beetroot and betaxhantins, a yellow-orange colorant also present in beetroot in lesser proportion to betacyanins. Fruits and vegetables provide substances which facilitate the formation of free radicals in the human body. Epidemiologic studies have demonstrated that there is a relationship between fruit and vegetable consumption and a decrease in mortality rate due to coronary diseases, cancer and other degenerative illnesses (Ames et al., 1993). Legislative action and consumer concern have resulted in an increased interest in the development of food colorants from natural sources as an alternative to synthetic dyes (Giusti and Wrolstad, 1996). Water activity has long been considered as one of the most influential factors in food safety and stability and, together with temperature, controls the physical and chemical properties of powders. Water adsorption is an important process during the storage of food powders and can lead to changes in their antioxidant properties that can affect the quality and stability of many food products. Nicoli et al. (1999) reported
Corresponding author. Tel.: +52 228 8 42 17 09; fax: +52 228 841 89 32. E-mail address: [email protected] (C.I. Beristain). Received 5 May 2009; Received in revised form 12 November 2009; Accepted 1 January 2010 0960-3085/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2010.01.002
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a series of relationships between chain-breaking activity and the redox potential, and they suggested that the processing of foods reduces their content of antioxidative substances when they are stored at high temperatures: This causes a loss of the antioxidants present in the food, but the formation of new compounds with antioxidant activity, such as the products of Maillard’s reaction, is carried out simultaneously. The degradation kinetics of beetroot pigment encapsulation in polymeric matrices has been studied (Serris and Biliaderis, 2001). In view of the foregoing, the objective of the present work was to develop a powder by spray drying that is derived from beetroot juice and to evaluate its antioxidant stability during storage at different water activities.
1.
Materials and methods
1.1.
Materials
The vegetables were purchased at three of the local markets during the winter and transferred to the University. Gum Arabic (GA) was purchased from Universal Flavors Mexico, SA de CV. Analytical grade solvents: n-hexane, methanol and chloroform were purchased from Sigma (México).
1.2.
Extraction of juice from beet root
The beetroots were washed and peeled, and later the juice was extracted with a commercial juice extractor (Moulinex mod. 140-1-03, Naucalpan, State of México, México). In order to eliminate the excess of fiber in the juice, it was filtered in a vacuum to realize the corresponding analyses. The total solid content was determined according to AOAC (1995). The measurements were made in triplicate. 3 g of beetroot juice were desiccated in a vacuum stove (Shel Lab model 1410). The pH was measured in 10 ml of juice; for this, a Hanna Instruments model HI 8424 potentiometer, calibrated with buffer pH 4 and 7, was used.
1.3.
Preparation of feed and spray drying mixtures
A solution of 30% (w/w) of gum Arabic (GA) was prepared, covered, and left to stand overnight at room temperature. Beet root juice was added at a 1:3 ratio (w/w) with respect to the GA contained in the solution. The mixture was homogenized with a high shear mixer model (Cole Parmer Instrument Co., Chicago, IL, USA) at 5000 rpm during 10 min. Then it was dried in a Mini Spray Dryer model Büchi 190 (Büchi Laboratorium Technik AG, Flawil, Switzerland), with an atomization pressure of 5 bars and inlet and outlet air temperatures of 180 ± 5 ◦ C and 85 ± 5 ◦ C, respectively. Three replicates were performed for the spray drying.
1.4.
Moisture in powder
The moisture content of the powder (microcapsules) was determined gravimetrically by oven-drying at 60 ◦ C until constant weight was attained (AOAC, 1995).
Chicago, IL, USA) and filtered, and the operation was repeated to ensure complete extraction. The filtered blend was centrifuged at 160 × g for 10 min and newly filtered through a nylon filter with pores of 0.20 m for use in posterior antioxidant tests. The Betalain concentration was analyzed according to Stintzing et al. (2005), where the sample was diluted in Mcllvaine buffer (pH 6.5 citrate-phosphate), and the absorbancy was measured at 600 nm. Measurements were performed in triplicate.
1.6.
The microcapsules were stored at 30 ◦ C in desiccators containing a saturated solution of LiCl, MgCl2 , Mg(NO3 )2 , NaCl, BaCl2 , which provide water activities of 0.110, 0.326, 0.521, 0.748 and 0.898, respectively. The samples were put in desiccators immediately after they were spray dried and this was taken as the zero time point. Three portions of the samples were withdrawn every week for analysis.
1.7.
Chain-breaking reaction
The antioxidant activity was evaluated at 515 nm by means of the chain-breaking reaction using the stable free radical 2,2diphenyl-1-picryl hidrazyl (DPPH• ), but upon reduction by an antioxidant or radical species its absorption disappeared. A volume of 3.0 mL of 3 × 10−5 mol/L DPPH methanol solution was used. The reaction was started by the addition of 0.1 mL of filtrated solution. The bleaching of DPPH was monitored at 515 nm with a spectrophotometer (Spectronic Genesis 5, Milton Roy Company, Rochester, NY, USA) at 25 ◦ C for at least 180 min. The following equation was chosen in order to obtain the reaction rate, k: 1 1 − 3 = −3kt A3 A0
(1)
where A0 is the initial density and A is the optical density at increasing time, t. The chain-breaking activity was expressed as: k/mgd.m. (−Abs−3 /min/mgd.m. ), assuming that all the dry matter from the sample possesses antioxidant properties. The data are expressed as the mean of at least three repetitions.
1.8.
Redox potential
The Redox potential was analyzed in the filtered matter obtained from microcapsules. Measurements were made with an Orion Potentiometer and an electrode Ag/AgCl,Cl− sat. (Thermo Electron Corporation, Beverly, MA, USA) according to the methodology proposed by Manzocco et al. (1998). Calibration was performed against a redox standard solution (Orion Application Solution ORP Standard, Beverly, MA, USA) having a redox potential value of 420 mV at 25 ◦ C. Electrodes were placed in a 50 mL flask from which the oxygen was removed by using a system of continuous nitrogen flushing for 10 min. The redox potential was monitored for at least 4 h, until a stable potential was achieved; then the analyses were performed.
1.9. 1.5.
Pigment powder storage for stability
Color
Extractions of bioactive compounds
Five grams of microcapsules were dispersed in a blend of 50 ml methanol and water (1:1) v/v for 5 min. This dispersion was agitated in a High shear mixer (Cole Parmer Instrument Co.,
The L, a, b color values were measured using a spectral photometer (Color Flex CX1115 HunterLab, USA). The instrument was standardized each time with a white ceramic plate (L = 92.8, a = −0.8; b = 0.1). After standardization the L (light-
food and bioproducts processing 8 8 ( 2 0 1 0 ) 253–258
Table 1 – Initial values of analysis performed on natural beetroot juice and powder dried by aspersion. Determination
Natural juice
Powder
Antioxidant activity (−Abs−3 /min/mgd.m. ) Betalain concentration (mg/kg) Redox potential (mV)
5.70 ± 0.60
4.70 ± 0.90
135.75 ± 2.70
119.77 ± 1.31
230 ± 2.50
448.2 ± 4.80
Color
L = 5.62 ± 0.23 a = 2.13 ± 0.11 b = −1.26 ± 0.09
L = −6.56 ± 0.11 a = 4.86 ± 0.12 b = 0.18 ± 0.07
pH Soluble solids (%) Acidity (%)
6.5 ± 0.10 12.5 ± 0.30 0.07 ± 0.009
n.d. n.d. n.d.
n.d. = not determined. These values are the mean of three determinations.
ness), a (red component), b (yellow component) values were measured in the juice and in complete microcapsules. H◦ was calculated by the transformation of a and b in the following equation: H◦ = tan−1
1.10.
b a
(2)
Statistical analysis
All measurements were carried out in at least three replicate experiments, and the results are reported as the means with standard deviation. These were analyzed by the Minitab Oneway analysis of variance (ANOVA). Differences between means at the 5% level were considered as significant.
2.
Results and discussion
2.1.
Properties of juice and microcapsules
After being extracted and filtered, the natural juice of beetroot presented the characteristics shown in Table 1. The hydration of the juice dried by aspersion was carried out on the basis of the amount of soluble solids present in the natural juice (12.5%). The chain-breaking activity of the juice (5.7 −Abs−3 /min/mgd.m. ) was similar to that reported for green tea (5.6 −Abs−3 /min/mgd.m. ). The results obtained showed that there was a retention of 82% in the antioxidant activity by chain-breaking activity in microcapsules (4.7 −Abs−3 /min/mgd.m. ) in comparison with the juice (5.7 −Abs−3 /min/mgd.m. ). In regard to the concentration of betalains (135.75 mg/kg) from juice, a retention of 88.2% was observed with respect to the concentration in microcapsules (119.7 mg/kg) compared with the natural juice of beetroot (135.7 mg/kg). Similar retention (88% ca.) was reported for betacyanin pigment obtained by spray drying (Cai and Corke, 2000). An inlet air temperature of 160–210 ◦ C has been reported as giving optimum flavor retention during drying (Thijssen, 1979). An inlet temperature of 140 ◦ C has been found to decrease retention of bioactive compounds such as betacyanins and indicaxanthins (Saenz et al., 2009), and a temperature above 210 ◦ C has been found to decrease flavor retention for some types of carriers as well (Anker and Reineccius, 1988). On the other hand, the redox potential increased by 94% from juice (230 mV) with respect to the filtered matter obtained from microcapsules (448 mV), thus
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indicating the probable formation of compounds able to promote the transference of electrons. It is also reported that redox potential determinations give indications of the overall reducing properties of all food antioxidants, including those which act slowly as radical species (Nicoli et al., 2004). The initial moisture content ranged from 3.59 to 5.33%, which was higher than that reported for black carrot microcapsules (Ersus and Yudagel, 2007). The moisture content depends on the matrix and the drying conditions, however, it is reported that when carrier material reaches a moisture content of <7%, the diffusion coefficient of water is reduced, and this decreases its movement through the dry matrix (Reineccius, 2004). On the other hand, it is reported that with increased water content, the water molecules acquire solvent properties and promote changes in polymer mobility (Lewicki, 2004). The color values of beet root were measured as L*, a*, b* values and were found to be 5.62, 2.13 and −1.26 for the juice of beetroot and −6.56, 4.86 and 0.18 for the powder, respectively. Parameter “a” was higher in the powder (4.86) than in the juice (2.13); this is important since beetroot pigment is typically used as a colorant in confectionery and meat substitute products (Henry, 1992). For the natural juice of beet root, a value of H◦ = 330 was obtained and for juice dried the value was H◦ = 288, which indicates that the red color of natural beet root juice approaches the pure one of 360◦ , whereas the one dried by aspersion has a less red color. Similar values in hue angle have been found by blending the betaxanthin and betacyanin stock solutions with purplish-blue (H◦ = 333) (Moßhammer et al., 2005) and amaranthus betacyanin pigments (H◦ = 355) (Cai and Corke, 2000). One of the important parameters that determine the stability of betalains and their antioxidant activity in solution is pH; in agreement with Pedrero and Escribano (2001), these attributes were the greatest at pH neutral. In the present work, the pH for the natural juice of beet root was 6.5 and 7.1 for the dehydrated juice. No significant differences were found between the samples used, this being an indication that changes in reduced or oxidized compounds capable of modifying the redox potential had been avoided.
2.2.
Stability of microcapsules during storage
The microcapsules were stored at 30 ◦ C during 44 days under different values of water activity. The concentration of betalains determined in the solution recovered during storage is observed in Fig. 1. After 2 days the initial concentrations of betalain decreased 20, 13 and 16% for samples stored at aw of 0.110, 0.326 and 0.521, respectively. No significant differences in betalain concentrations were found between samples stored at 0.326 and 0.521 water activity. A low degradation was estimated for aw of 0.110. This diminution may be due to the degradation of the superficial pigment in the microcapsule formed during aspersion or to the effect of the juice components (Saenz et al., 2009). However, the samples stored at aw of 0.748 and 0.898 presented a higher betalain degradation of approximately 65 and 75%, respectively, in comparison with the initial concentration of the product. This was still appreciable after 20 days of storage, but after 23 days, it was no longer possible to detect the presence of betalains in the product. It is important to emphasize that the powder stored at aw of 0.748 and 0.898 collapsed after 12 days of storage, although the concentration of betalains was still detectable (data not shown). Several deteriorative changes in food depend on molecular mobility, which is related to the glass transi-
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Fig. 1 – Storage stability of betalain concentrations at different water activities exposed to a constant temperature of 30 ◦ C. tion temperature, among other contributing factors (Serris and Biliaderis, 2001). It is reported that the glass transition temperature (Tg ) of gum Arabic varied from 62 to 42.6 ◦ C at water activities of 0.33 and 0.54, respectively, and that the addition of juice from immature acerola to GA decreased the Tg of the juice powder to 8.05 ◦ C at aw of 0.54 (Righetto and Netto, 2005). A number of studies on microencapsulation properties with different wall materials found that the moisture content influences the pore network of the wall material and affects the oxygen diffusion and retention of the core in spray dried microcapsules (Drush et al., 2007). The degradation of betalains is the result of a hydrolytic reaction possibly due to increased moisture in the product dried by aspersion (Serris and Biliaderis, 2001), and this may favor oxygen diffusion towards the encapsulated material, thus causing oxidation (Cai and Corke, 2000). Levine and Slade (1989) claimed that low mobility in a glassy state makes reaction improbable. However, losses of beetroot pigment encapsulated with polymeric matrices at temperatures below the glass transition temperature (Serris and Biliaderis, 2001). In the present work, the water adsorbed probably damaged the structure of the capsule, thereby causing wall dissolution and oxygen diffusion into the microcapsule core. In dry atmospheres, the water is strongly attracted to the polar sites on the surface of the microcapsules and is, therefore, not available for any type of reaction, which explains why low and intermediate values of degradation were found at water activity below 0.521 (aw < 0.521). On the other hand, when water activity values are high (aw > 0.748), the adsorption phenomenon produces conformational swelling and changes in the structure of the dry powder (Serris and Biliaderis, 2001). It is reported that degradation of anthocyanins occurred even at sub-Tg temperatures for all samples, implying significant reactant mobility in the glassy state (Gradinaru et al., 2003).
2.3.
Antioxidant activity during storage
Fig. 2 shows that the antioxidant activity expressed as chainbreaking activity increased during 44 days of storage. No significant differences were found among the samples stored at aw of 0.110–0.521, which showed an increase from 2 to 6 in
Fig. 2 – Chain-breaking activity values as a function of time during storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation. antioxidant activity, whereas those samples stored at 0.748 and 0.898 reached values of 32 and 38 −Abs−3 /min/mgd.m. , respectively. Under these conditions, it is possible that dissolution and water adsorption help the oxygen to produce intermediary compounds with high antioxidant activity. The degradation of betanin is the result of a hydrolytic reaction of the betanin to d-glucoside cycle-DOPA (CDG) and betalamic acid (BA). The degradation involves a Schiff’s base condensation of the amine of CDG with the aldehyde forming intermediate compounds in the reaction of Maillard (Pasch and von Elbe, 1975; Huang and von Elbe, 1987). The extraction of a protein fraction of the gum Arabic may have favored the Maillard reaction, the products of which function as precursors of the polyphenols that increase antioxidant activity. However, it is reported that the antioxidant activity of beetroot microcapsules is only due to the presence of betanin and not to the degradation products (Pedrero and Escribano, 2001). The increase in antioxidant activity during storage in all the samples may occur because, in those periods of time, new compounds with antioxidant activity able to quench radicals are released or formed, such as the intermediary compounds during Maillard’s reaction (Nicoli et al., 1999); these are formed due to the storage and processing of foods, or to the formation of polyphenols during the processing or the storage. Saenz et al. (2009) found recoveries and formation of polyphenols of over 100% with respect to the initial time as a consequence of the hydrolysis of cactus pear polyphenol conjugates during the drying process (Saenz et al., 2009). In regard to the redox potential, no significant differences were found among the samples stored in atmospheres of aw of 0.110, 0.326 and 0.521. Fig. 3 shows a decrement of 450–230 mV, followed by a 10-day period of stability and an increase to 420 mV after day 14. Thereafter, such behavior in the redox potential may be due to compounds with high antioxidant activity that were formed or liberated during storage because of water adsorption by the microcapsules, or to the interaction of some components in the stored sample with the oxygen or other components of the sample. These compounds represent a reservoir of antioxidants in the powder (Anese et al., 2003). It is possible that at this level of water activity, the adsorbed water is not capable of reducing the compounds to promote
food and bioproducts processing 8 8 ( 2 0 1 0 ) 253–258
Fig. 3 – Redox potential (mV) values as a function of time storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation. electron transfer and to keep the redox potential constant; however, the chain-breaking activity increased as betalain oxidation occurred. On the other hand, samples stored at aw up to 0.748 had a constant redox potential, indicating that the water adsorbed favors the overall reducing properties of all food antioxidants. The redox potential is considered a thermodynamic measurement which provides information about the capability of a redox couple of becoming oxidized or reduced; for this reason it is used to evaluate the reduced compound, able to promote electron transference (Nicoli et al., 2004). Fig. 4 shows the relation between the redox potential and the chain-breaking activity of powder stored at water activities of 0.748 and 0.898; it is observed that the redox potential decreased from 450 to 410 when the chain-breaking increased to 38.33 −Abs−3 /min/mgd.m. . It may be that water adsorbed in storage at high water activities facilitates reaction, causing betalains and other compounds to react with oxygen and
Fig. 4 – Chain-breaking values as a function of Redox potential (mV) during storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation.
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Fig. 5 – Chain-breaking values as a function of remaining betalain concentrations (%) during storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation.
producing an enriched medium which possesses strong radical scavenging and reducing properties. Similar results were described by Nicoli et al. (2004), who explained that the phenomenon can be ascribed to the formation of compounds with high molecular weight. In our experiment, high water activityinduced minor changes in the redox potential, indicating that the compounds with antioxidant activity were formed progressively during storage at a higher aw . Higher antioxidant activities coincided with the time of storage at which the microcapsule collapsed and there was a complete loss of structure, allowing a loss of betalains. The degradation reaction of betalains produces CDG and BA, which have an amine and aldehyde group that may be able to quench radicals. Fig. 5 shows the chain-breaking activity as a function of the remaining betalain concentration stored at 30 ◦ C. It is reported that the constant degradation rate of betalains is attributed not only to moisture but also to oxygen concentration (Serris and Biliaderis, 2001). It was found that longer storage time and higher water activity produced a higher antioxidant capacity to quench radicals. No significant differences were found between samples stored at low water activity (0.110, 0.326 and 0.521) and samples stored at higher water activity (0.748 and 0.898). The color was determined by measuring parameters “L”, “a” and “b”. Parameter “a” was the most sensitive parameter to the retention of betalains during storage. No significant changes were observed in parameter “b” during the first 20 days of storage; after that time, the samples showed a slight increase in this parameter, from 0.11 to 1.17. Degradation in the color of microcapsules was observed during storage. The initial color of the powder varied with betalain retention. The encapsulated color disappeared slowly and the color of the gum Arabic matrix appeared. Similar results were found with encapsulated carotene (Desobry et al., 1999). No significant differences were found in the color of powder stored at a water activity range of 0.110–0.521 during 20 days. Instead, Parameter “a” showed a slight decrease in the samples stored at aw of 0.748. The red pigment decreased with time and was reflected by a decrease in the “a” value.
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3.
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Conclusions
The stability of the betalains contained in the powder product and the antioxidant activity of the microcapsules depends on the water activity at which they are stored. The powder stored at aw < 0.521 presented the greatest stability. Although maximum antioxidant activity was observed in samples stored in atmospheres of aw > 0.748, this is not recommended since the samples suffer collapse and the capsule dissolves in a short period of time. Parameter “a” was the best indicator of changes in the product. The stability of the color was influenced by the water activity. Therefore, the microcapsules described in this study represent an interesting food additive for incorporation into functional foods, both as an antioxidant and as a red colorant.
Acknowledgment The authors greatly appreciate the financial support from the National Council for Science and Technology of Mexico (CONACyT) through project 25773 (CB-2005-01-50820).
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Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Antioxidative activity of microcapsules with beetroot juice using gum Arabic as wall material E. Pitalua, M. Jimenez, E.J. Vernon-Carter, C.I. Beristain ∗ Instituto de Ciencias Básicas, Universidad Veracruzana, Luis Castelazo S/N, Col Industrial Animas, Apto Postal 575, Apdo, CP 91192 Xalapa, Veracruz, Mexico
a b s t r a c t The antioxidative activity of encapsulated natural beet root juice was evaluated during storage at different water activities. In microcapsules of beetroot obtained by spray drying and stored at water activities of 0.110, 0.326 and 0.521 there were no significant differences in betalain concentration, color, antioxidant activity and redox potential during 45 days. However, the samples stored at aw s of 0.748 and 0.898 showed significant differences compared to those stored at 0.110, 0.326 and 0.521, but no difference was shown among the constituents of each group. In the samples stored at 0.748 and 0.898 the antioxidant activity increases, whereas the betalain concentration decreases due to the compounds that are formed while degradation of the betalains occurs. Water adsorption influences the stability of the product during storage. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Betalains; Redox potential; Chain-breaking; Water activity; Color
Stability is an important aspect to consider for the use of pigments as antioxidants and colorants in food. There are many factors which affect the stability and intensity of colorants during processing and storage. The colorant present in beetroot tends to degrade easily in solution. It is reported that its stability is affected during processing and storage (Attoe and von Elbe, 1982); therefore spray drying was used to avoid colorant degradation. It is reported that stabilization of betalains and polyphenols could be improved using microencapsulation by spray drying (Desai and Park, 2005). Microencapsulation through spray drying is an economical method for the preservation of natural colorants by entrapping the ingredients in a coating material (Ersus and Yudagel, 2007; Serris and Biliaderis, 2001). Gum Arabic is one of the most important carrier and coating agents used for the spray drying of flavors and colorants (Beristain et al., 1999), so it is important to evaluate the encapsulation of beetroot juice in this material. There are many reports about how antioxidants can be affected as a consequence of storage conditions; in some cases, storage can induce the formation of compounds with antioxidant properties, which can maintain or even enhance the overall antioxidant potential of foods (Jonsson, 1991). The storage stability of powdered antioxidants for extended peri-
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ods is of interest to manufacturers and consumers of food packaged for long-term storage. Beetroot is known to have antioxidant properties since it contains nitrogen compounds called betalains, which are classified as betacyanins that confer the red-violet color to beetroot and betaxhantins, a yellow-orange colorant also present in beetroot in lesser proportion to betacyanins. Fruits and vegetables provide substances which facilitate the formation of free radicals in the human body. Epidemiologic studies have demonstrated that there is a relationship between fruit and vegetable consumption and a decrease in mortality rate due to coronary diseases, cancer and other degenerative illnesses (Ames et al., 1993). Legislative action and consumer concern have resulted in an increased interest in the development of food colorants from natural sources as an alternative to synthetic dyes (Giusti and Wrolstad, 1996). Water activity has long been considered as one of the most influential factors in food safety and stability and, together with temperature, controls the physical and chemical properties of powders. Water adsorption is an important process during the storage of food powders and can lead to changes in their antioxidant properties that can affect the quality and stability of many food products. Nicoli et al. (1999) reported
Corresponding author. Tel.: +52 228 8 42 17 09; fax: +52 228 841 89 32. E-mail address: [email protected] (C.I. Beristain). Received 5 May 2009; Received in revised form 12 November 2009; Accepted 1 January 2010 0960-3085/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2010.01.002
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a series of relationships between chain-breaking activity and the redox potential, and they suggested that the processing of foods reduces their content of antioxidative substances when they are stored at high temperatures: This causes a loss of the antioxidants present in the food, but the formation of new compounds with antioxidant activity, such as the products of Maillard’s reaction, is carried out simultaneously. The degradation kinetics of beetroot pigment encapsulation in polymeric matrices has been studied (Serris and Biliaderis, 2001). In view of the foregoing, the objective of the present work was to develop a powder by spray drying that is derived from beetroot juice and to evaluate its antioxidant stability during storage at different water activities.
1.
Materials and methods
1.1.
Materials
The vegetables were purchased at three of the local markets during the winter and transferred to the University. Gum Arabic (GA) was purchased from Universal Flavors Mexico, SA de CV. Analytical grade solvents: n-hexane, methanol and chloroform were purchased from Sigma (México).
1.2.
Extraction of juice from beet root
The beetroots were washed and peeled, and later the juice was extracted with a commercial juice extractor (Moulinex mod. 140-1-03, Naucalpan, State of México, México). In order to eliminate the excess of fiber in the juice, it was filtered in a vacuum to realize the corresponding analyses. The total solid content was determined according to AOAC (1995). The measurements were made in triplicate. 3 g of beetroot juice were desiccated in a vacuum stove (Shel Lab model 1410). The pH was measured in 10 ml of juice; for this, a Hanna Instruments model HI 8424 potentiometer, calibrated with buffer pH 4 and 7, was used.
1.3.
Preparation of feed and spray drying mixtures
A solution of 30% (w/w) of gum Arabic (GA) was prepared, covered, and left to stand overnight at room temperature. Beet root juice was added at a 1:3 ratio (w/w) with respect to the GA contained in the solution. The mixture was homogenized with a high shear mixer model (Cole Parmer Instrument Co., Chicago, IL, USA) at 5000 rpm during 10 min. Then it was dried in a Mini Spray Dryer model Büchi 190 (Büchi Laboratorium Technik AG, Flawil, Switzerland), with an atomization pressure of 5 bars and inlet and outlet air temperatures of 180 ± 5 ◦ C and 85 ± 5 ◦ C, respectively. Three replicates were performed for the spray drying.
1.4.
Moisture in powder
The moisture content of the powder (microcapsules) was determined gravimetrically by oven-drying at 60 ◦ C until constant weight was attained (AOAC, 1995).
Chicago, IL, USA) and filtered, and the operation was repeated to ensure complete extraction. The filtered blend was centrifuged at 160 × g for 10 min and newly filtered through a nylon filter with pores of 0.20 m for use in posterior antioxidant tests. The Betalain concentration was analyzed according to Stintzing et al. (2005), where the sample was diluted in Mcllvaine buffer (pH 6.5 citrate-phosphate), and the absorbancy was measured at 600 nm. Measurements were performed in triplicate.
1.6.
The microcapsules were stored at 30 ◦ C in desiccators containing a saturated solution of LiCl, MgCl2 , Mg(NO3 )2 , NaCl, BaCl2 , which provide water activities of 0.110, 0.326, 0.521, 0.748 and 0.898, respectively. The samples were put in desiccators immediately after they were spray dried and this was taken as the zero time point. Three portions of the samples were withdrawn every week for analysis.
1.7.
Chain-breaking reaction
The antioxidant activity was evaluated at 515 nm by means of the chain-breaking reaction using the stable free radical 2,2diphenyl-1-picryl hidrazyl (DPPH• ), but upon reduction by an antioxidant or radical species its absorption disappeared. A volume of 3.0 mL of 3 × 10−5 mol/L DPPH methanol solution was used. The reaction was started by the addition of 0.1 mL of filtrated solution. The bleaching of DPPH was monitored at 515 nm with a spectrophotometer (Spectronic Genesis 5, Milton Roy Company, Rochester, NY, USA) at 25 ◦ C for at least 180 min. The following equation was chosen in order to obtain the reaction rate, k: 1 1 − 3 = −3kt A3 A0
(1)
where A0 is the initial density and A is the optical density at increasing time, t. The chain-breaking activity was expressed as: k/mgd.m. (−Abs−3 /min/mgd.m. ), assuming that all the dry matter from the sample possesses antioxidant properties. The data are expressed as the mean of at least three repetitions.
1.8.
Redox potential
The Redox potential was analyzed in the filtered matter obtained from microcapsules. Measurements were made with an Orion Potentiometer and an electrode Ag/AgCl,Cl− sat. (Thermo Electron Corporation, Beverly, MA, USA) according to the methodology proposed by Manzocco et al. (1998). Calibration was performed against a redox standard solution (Orion Application Solution ORP Standard, Beverly, MA, USA) having a redox potential value of 420 mV at 25 ◦ C. Electrodes were placed in a 50 mL flask from which the oxygen was removed by using a system of continuous nitrogen flushing for 10 min. The redox potential was monitored for at least 4 h, until a stable potential was achieved; then the analyses were performed.
1.9. 1.5.
Pigment powder storage for stability
Color
Extractions of bioactive compounds
Five grams of microcapsules were dispersed in a blend of 50 ml methanol and water (1:1) v/v for 5 min. This dispersion was agitated in a High shear mixer (Cole Parmer Instrument Co.,
The L, a, b color values were measured using a spectral photometer (Color Flex CX1115 HunterLab, USA). The instrument was standardized each time with a white ceramic plate (L = 92.8, a = −0.8; b = 0.1). After standardization the L (light-
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Table 1 – Initial values of analysis performed on natural beetroot juice and powder dried by aspersion. Determination
Natural juice
Powder
Antioxidant activity (−Abs−3 /min/mgd.m. ) Betalain concentration (mg/kg) Redox potential (mV)
5.70 ± 0.60
4.70 ± 0.90
135.75 ± 2.70
119.77 ± 1.31
230 ± 2.50
448.2 ± 4.80
Color
L = 5.62 ± 0.23 a = 2.13 ± 0.11 b = −1.26 ± 0.09
L = −6.56 ± 0.11 a = 4.86 ± 0.12 b = 0.18 ± 0.07
pH Soluble solids (%) Acidity (%)
6.5 ± 0.10 12.5 ± 0.30 0.07 ± 0.009
n.d. n.d. n.d.
n.d. = not determined. These values are the mean of three determinations.
ness), a (red component), b (yellow component) values were measured in the juice and in complete microcapsules. H◦ was calculated by the transformation of a and b in the following equation: H◦ = tan−1
1.10.
b a
(2)
Statistical analysis
All measurements were carried out in at least three replicate experiments, and the results are reported as the means with standard deviation. These were analyzed by the Minitab Oneway analysis of variance (ANOVA). Differences between means at the 5% level were considered as significant.
2.
Results and discussion
2.1.
Properties of juice and microcapsules
After being extracted and filtered, the natural juice of beetroot presented the characteristics shown in Table 1. The hydration of the juice dried by aspersion was carried out on the basis of the amount of soluble solids present in the natural juice (12.5%). The chain-breaking activity of the juice (5.7 −Abs−3 /min/mgd.m. ) was similar to that reported for green tea (5.6 −Abs−3 /min/mgd.m. ). The results obtained showed that there was a retention of 82% in the antioxidant activity by chain-breaking activity in microcapsules (4.7 −Abs−3 /min/mgd.m. ) in comparison with the juice (5.7 −Abs−3 /min/mgd.m. ). In regard to the concentration of betalains (135.75 mg/kg) from juice, a retention of 88.2% was observed with respect to the concentration in microcapsules (119.7 mg/kg) compared with the natural juice of beetroot (135.7 mg/kg). Similar retention (88% ca.) was reported for betacyanin pigment obtained by spray drying (Cai and Corke, 2000). An inlet air temperature of 160–210 ◦ C has been reported as giving optimum flavor retention during drying (Thijssen, 1979). An inlet temperature of 140 ◦ C has been found to decrease retention of bioactive compounds such as betacyanins and indicaxanthins (Saenz et al., 2009), and a temperature above 210 ◦ C has been found to decrease flavor retention for some types of carriers as well (Anker and Reineccius, 1988). On the other hand, the redox potential increased by 94% from juice (230 mV) with respect to the filtered matter obtained from microcapsules (448 mV), thus
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indicating the probable formation of compounds able to promote the transference of electrons. It is also reported that redox potential determinations give indications of the overall reducing properties of all food antioxidants, including those which act slowly as radical species (Nicoli et al., 2004). The initial moisture content ranged from 3.59 to 5.33%, which was higher than that reported for black carrot microcapsules (Ersus and Yudagel, 2007). The moisture content depends on the matrix and the drying conditions, however, it is reported that when carrier material reaches a moisture content of <7%, the diffusion coefficient of water is reduced, and this decreases its movement through the dry matrix (Reineccius, 2004). On the other hand, it is reported that with increased water content, the water molecules acquire solvent properties and promote changes in polymer mobility (Lewicki, 2004). The color values of beet root were measured as L*, a*, b* values and were found to be 5.62, 2.13 and −1.26 for the juice of beetroot and −6.56, 4.86 and 0.18 for the powder, respectively. Parameter “a” was higher in the powder (4.86) than in the juice (2.13); this is important since beetroot pigment is typically used as a colorant in confectionery and meat substitute products (Henry, 1992). For the natural juice of beet root, a value of H◦ = 330 was obtained and for juice dried the value was H◦ = 288, which indicates that the red color of natural beet root juice approaches the pure one of 360◦ , whereas the one dried by aspersion has a less red color. Similar values in hue angle have been found by blending the betaxanthin and betacyanin stock solutions with purplish-blue (H◦ = 333) (Moßhammer et al., 2005) and amaranthus betacyanin pigments (H◦ = 355) (Cai and Corke, 2000). One of the important parameters that determine the stability of betalains and their antioxidant activity in solution is pH; in agreement with Pedrero and Escribano (2001), these attributes were the greatest at pH neutral. In the present work, the pH for the natural juice of beet root was 6.5 and 7.1 for the dehydrated juice. No significant differences were found between the samples used, this being an indication that changes in reduced or oxidized compounds capable of modifying the redox potential had been avoided.
2.2.
Stability of microcapsules during storage
The microcapsules were stored at 30 ◦ C during 44 days under different values of water activity. The concentration of betalains determined in the solution recovered during storage is observed in Fig. 1. After 2 days the initial concentrations of betalain decreased 20, 13 and 16% for samples stored at aw of 0.110, 0.326 and 0.521, respectively. No significant differences in betalain concentrations were found between samples stored at 0.326 and 0.521 water activity. A low degradation was estimated for aw of 0.110. This diminution may be due to the degradation of the superficial pigment in the microcapsule formed during aspersion or to the effect of the juice components (Saenz et al., 2009). However, the samples stored at aw of 0.748 and 0.898 presented a higher betalain degradation of approximately 65 and 75%, respectively, in comparison with the initial concentration of the product. This was still appreciable after 20 days of storage, but after 23 days, it was no longer possible to detect the presence of betalains in the product. It is important to emphasize that the powder stored at aw of 0.748 and 0.898 collapsed after 12 days of storage, although the concentration of betalains was still detectable (data not shown). Several deteriorative changes in food depend on molecular mobility, which is related to the glass transi-
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Fig. 1 – Storage stability of betalain concentrations at different water activities exposed to a constant temperature of 30 ◦ C. tion temperature, among other contributing factors (Serris and Biliaderis, 2001). It is reported that the glass transition temperature (Tg ) of gum Arabic varied from 62 to 42.6 ◦ C at water activities of 0.33 and 0.54, respectively, and that the addition of juice from immature acerola to GA decreased the Tg of the juice powder to 8.05 ◦ C at aw of 0.54 (Righetto and Netto, 2005). A number of studies on microencapsulation properties with different wall materials found that the moisture content influences the pore network of the wall material and affects the oxygen diffusion and retention of the core in spray dried microcapsules (Drush et al., 2007). The degradation of betalains is the result of a hydrolytic reaction possibly due to increased moisture in the product dried by aspersion (Serris and Biliaderis, 2001), and this may favor oxygen diffusion towards the encapsulated material, thus causing oxidation (Cai and Corke, 2000). Levine and Slade (1989) claimed that low mobility in a glassy state makes reaction improbable. However, losses of beetroot pigment encapsulated with polymeric matrices at temperatures below the glass transition temperature (Serris and Biliaderis, 2001). In the present work, the water adsorbed probably damaged the structure of the capsule, thereby causing wall dissolution and oxygen diffusion into the microcapsule core. In dry atmospheres, the water is strongly attracted to the polar sites on the surface of the microcapsules and is, therefore, not available for any type of reaction, which explains why low and intermediate values of degradation were found at water activity below 0.521 (aw < 0.521). On the other hand, when water activity values are high (aw > 0.748), the adsorption phenomenon produces conformational swelling and changes in the structure of the dry powder (Serris and Biliaderis, 2001). It is reported that degradation of anthocyanins occurred even at sub-Tg temperatures for all samples, implying significant reactant mobility in the glassy state (Gradinaru et al., 2003).
2.3.
Antioxidant activity during storage
Fig. 2 shows that the antioxidant activity expressed as chainbreaking activity increased during 44 days of storage. No significant differences were found among the samples stored at aw of 0.110–0.521, which showed an increase from 2 to 6 in
Fig. 2 – Chain-breaking activity values as a function of time during storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation. antioxidant activity, whereas those samples stored at 0.748 and 0.898 reached values of 32 and 38 −Abs−3 /min/mgd.m. , respectively. Under these conditions, it is possible that dissolution and water adsorption help the oxygen to produce intermediary compounds with high antioxidant activity. The degradation of betanin is the result of a hydrolytic reaction of the betanin to d-glucoside cycle-DOPA (CDG) and betalamic acid (BA). The degradation involves a Schiff’s base condensation of the amine of CDG with the aldehyde forming intermediate compounds in the reaction of Maillard (Pasch and von Elbe, 1975; Huang and von Elbe, 1987). The extraction of a protein fraction of the gum Arabic may have favored the Maillard reaction, the products of which function as precursors of the polyphenols that increase antioxidant activity. However, it is reported that the antioxidant activity of beetroot microcapsules is only due to the presence of betanin and not to the degradation products (Pedrero and Escribano, 2001). The increase in antioxidant activity during storage in all the samples may occur because, in those periods of time, new compounds with antioxidant activity able to quench radicals are released or formed, such as the intermediary compounds during Maillard’s reaction (Nicoli et al., 1999); these are formed due to the storage and processing of foods, or to the formation of polyphenols during the processing or the storage. Saenz et al. (2009) found recoveries and formation of polyphenols of over 100% with respect to the initial time as a consequence of the hydrolysis of cactus pear polyphenol conjugates during the drying process (Saenz et al., 2009). In regard to the redox potential, no significant differences were found among the samples stored in atmospheres of aw of 0.110, 0.326 and 0.521. Fig. 3 shows a decrement of 450–230 mV, followed by a 10-day period of stability and an increase to 420 mV after day 14. Thereafter, such behavior in the redox potential may be due to compounds with high antioxidant activity that were formed or liberated during storage because of water adsorption by the microcapsules, or to the interaction of some components in the stored sample with the oxygen or other components of the sample. These compounds represent a reservoir of antioxidants in the powder (Anese et al., 2003). It is possible that at this level of water activity, the adsorbed water is not capable of reducing the compounds to promote
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Fig. 3 – Redox potential (mV) values as a function of time storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation. electron transfer and to keep the redox potential constant; however, the chain-breaking activity increased as betalain oxidation occurred. On the other hand, samples stored at aw up to 0.748 had a constant redox potential, indicating that the water adsorbed favors the overall reducing properties of all food antioxidants. The redox potential is considered a thermodynamic measurement which provides information about the capability of a redox couple of becoming oxidized or reduced; for this reason it is used to evaluate the reduced compound, able to promote electron transference (Nicoli et al., 2004). Fig. 4 shows the relation between the redox potential and the chain-breaking activity of powder stored at water activities of 0.748 and 0.898; it is observed that the redox potential decreased from 450 to 410 when the chain-breaking increased to 38.33 −Abs−3 /min/mgd.m. . It may be that water adsorbed in storage at high water activities facilitates reaction, causing betalains and other compounds to react with oxygen and
Fig. 4 – Chain-breaking values as a function of Redox potential (mV) during storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation.
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Fig. 5 – Chain-breaking values as a function of remaining betalain concentrations (%) during storage at different water activities at 30 ◦ C. The mean values of at least three replicates are reported ± standard deviation.
producing an enriched medium which possesses strong radical scavenging and reducing properties. Similar results were described by Nicoli et al. (2004), who explained that the phenomenon can be ascribed to the formation of compounds with high molecular weight. In our experiment, high water activityinduced minor changes in the redox potential, indicating that the compounds with antioxidant activity were formed progressively during storage at a higher aw . Higher antioxidant activities coincided with the time of storage at which the microcapsule collapsed and there was a complete loss of structure, allowing a loss of betalains. The degradation reaction of betalains produces CDG and BA, which have an amine and aldehyde group that may be able to quench radicals. Fig. 5 shows the chain-breaking activity as a function of the remaining betalain concentration stored at 30 ◦ C. It is reported that the constant degradation rate of betalains is attributed not only to moisture but also to oxygen concentration (Serris and Biliaderis, 2001). It was found that longer storage time and higher water activity produced a higher antioxidant capacity to quench radicals. No significant differences were found between samples stored at low water activity (0.110, 0.326 and 0.521) and samples stored at higher water activity (0.748 and 0.898). The color was determined by measuring parameters “L”, “a” and “b”. Parameter “a” was the most sensitive parameter to the retention of betalains during storage. No significant changes were observed in parameter “b” during the first 20 days of storage; after that time, the samples showed a slight increase in this parameter, from 0.11 to 1.17. Degradation in the color of microcapsules was observed during storage. The initial color of the powder varied with betalain retention. The encapsulated color disappeared slowly and the color of the gum Arabic matrix appeared. Similar results were found with encapsulated carotene (Desobry et al., 1999). No significant differences were found in the color of powder stored at a water activity range of 0.110–0.521 during 20 days. Instead, Parameter “a” showed a slight decrease in the samples stored at aw of 0.748. The red pigment decreased with time and was reflected by a decrease in the “a” value.
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3.
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Conclusions
The stability of the betalains contained in the powder product and the antioxidant activity of the microcapsules depends on the water activity at which they are stored. The powder stored at aw < 0.521 presented the greatest stability. Although maximum antioxidant activity was observed in samples stored in atmospheres of aw > 0.748, this is not recommended since the samples suffer collapse and the capsule dissolves in a short period of time. Parameter “a” was the best indicator of changes in the product. The stability of the color was influenced by the water activity. Therefore, the microcapsules described in this study represent an interesting food additive for incorporation into functional foods, both as an antioxidant and as a red colorant.
Acknowledgment The authors greatly appreciate the financial support from the National Council for Science and Technology of Mexico (CONACyT) through project 25773 (CB-2005-01-50820).
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