Saffron and beetroot extracts encapsulated in maltodextrin, gum Arabic, modified starch and chitosan: Incorporation in a chewing gum system

Carbohydrate Polymers 127 (2015) 252–263

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Saffron and beetroot extracts encapsulated in maltodextrin, gum Arabic, modified starch and chitosan: Incorporation in a chewing gum system Charikleia Chranioti, Aspasia Nikoloudaki, Constantina Tzia ∗ Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Polytechniou, Zografou, 15780 Athens, Greece

a r t i c l e

i n f o

Article history: Received 8 December 2014 Received in revised form 12 March 2015 Accepted 13 March 2015 Available online 30 March 2015 Keywords: Saffron extract Beetroot extract Encapsulation Freeze drying Color stability Chewing gum

a b s t r a c t Maltodextrin (MD-21DE), gum Arabic (GA), gum Arabic–modified starch (GA–MS), modified starch–chitosan (MS–CH) and modified starch–maltodextrin–chitosan (MS–MD–CH) were used as agents for beetroot and saffron coloring-extracts microencapsulation by freeze drying. The produced powders were evaluated in terms of coloring strength (E) during storage at 40 ◦ C for 10 weeks and a first-order kinetic was applied. Color parameters (L* , a* , b* , C* and E* ) and water sorption behavior was also studied. Moreover, incorporation of the powders in a chewing gum model system was conducted. The type of encapsulating agent significantly (P < 0.05) affected the studied parameters with the order of protection in both extracts being as follows: MD > GA > GA–MS > MS–CH > MS–MD–CH. The water sorption study revealed that MD and GA kept their structural integrity up to water activities of 0.66 and 0.82, respectively. The chewing gum samples produced with coloring extracts encapsulated in GA–MS showed the greatest a* (for beetroot) and b* (for saffron) values indicating a better protection. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Color is one of the most important characteristics of foods, being considered as a quality indicator that determines their acceptance (Azeredo, 2009). Although chemical food additives such as artificial colorants have been widely applied for coloring purposes of food products, their use is still a controversial issue in the food industry due to their toxicological potential on human health (Mizutani, 2009; Reyes, Valim, & Vercesi, 1996). The consumption of artificial colorants has been associated with the development of allergies in children (Inomata, Osuna, Fujita, Ogawa, & Ikezawa, 2006) and the increased risk of cancer (Sasaki et al., 2002). Therefore, legislative actions and consumer concerns have resulted in an increased interest toward replacement of chemical colorants with natural pigments, which are considered not only to be harmless but also to possess functional properties that can exert beneficial effect to human health (Moreira et al., 2012; Rutkowska & Stolyhwo, 2009). Beetroot and saffron are basic natural sources of pigments which are typically used as colorants in quite a wide range of food products. Beetroot pigments consist of two major water soluble

∗ Corresponding author. Tel.: +0030 2107723165; fax: +0030 2107723163. E-mail address: [email protected] (C. Tzia). http://dx.doi.org/10.1016/j.carbpol.2015.03.049 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

fractions, betacyanins that confer the red-violet color and betaxhantins, a yellow–orange colorant also present in beetroot in lesser proportion than betacyanins (Pitalua, Jimenez, Vernon-Carter, & Beristain, 2010). They are typically used at levels of 4–25 mg/kg in a wide range of dairy and confectionery products as well as in meat substitutes. However, their use in foods is limited due to their poor stability when exposed to heat and light (Serris & Biliaderis, 2001). On the other hand, saffron pigments include crocins, a group of water soluble carotenoids, which are glycosyl esters of 8,8diapocarotene-8,8-dioic acid (or crocetin) with biologically activity on human health (Tarantilis, Tsoupras, & Polissiou, 1995). Saffron pigments are typically used at levels of 1–260 ppm in a wide range of culinary, bakery and confectionery preparations as well as in alcoholic and non alcoholic beverages. However, as being highly unsaturated components, they are prone to oxidation and isomerization reactions that lead to losses of coloring strength and nutritive value (Tsimidou & Biliaderis, 1997). Therefore, a protective encapsulation technique is needed for the feasible usage of the afore-mentioned natural pigments in food products. Among the common encapsulation techniques used are spray (Akhavan, Jafari, Ghorbani, & Assadpoor, 2014) and freeze–drying of the oil-in-water prepared emulsion containing the encapsulating agent and the pigment-core (Lim, Tan, Bakar, & Ng, 2011).

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Microencapsulation by freeze drying leads to products with excellent sensory characteristics and preserved biofunctionality since changes associated with high temperatures are minimized, due to the low temperatures involved in the process (Minemoto, Adachi, & Matsuno, 2001). Moreover, the selection of the encapsulation agent is very crucial for an efficient process and it depends on the final application of the encapsulated pigment. In our previous works, various edible polymers have been examined (plain and in mixtures) in fennel oleoresin (Chranioti & Tzia, 2013, 2014a) and saffron volatile flavor substances (Chranioti, Papoutsakis, Nikoloudaki, & Tzia, 2012) encapsulation. Among the studied encapsulating agents, plain gum Arabic (GA), binary blends of gum Arabic–modified starch (GA–MS) and chitosanmodified starch (MS–CH) as well as a ternary one of modified starch–maltodextrin–chitosan (MS–MD–CH) were selected as they displayed relatively high encapsulation efficiencies while plain maltodextrin (MD) was chosen since it is colorless and provides good protection against oxidation (Chranioti & Tzia, 2014b). Although there have been some studies on encapsulation of beetroot (Pitalua et al., 2010) and saffron (Cormier, Dufresne, & Dorion, 1995; Dufresne et al., 1999; Khazaei, Jafari, Ghorbani, & Hemmati Kakhki, 2014) extracts, the use of the afore-mentioned mixtures of agents has not been investigated. Therefore, the aim of this study was to produce natural colorants encapsulated in different mixtures of agents with the use of freeze drying technique and then: (i) to examine the color stability of the obtained encapsulated colorants, (ii) to study their sorption behavior at various relative humidity environments and (iii) to evaluate their incorporation in a chewing gum model system. 2. Materials and methods 2.1. Materials Dried stigmas of saffron were provided directly from the ‘Cooperative of saffron, Krokos Kozanis’ while the beetroot vegetable was purchased from a local market. Modified Starch (MS, Cleargum CO-01, an octenyl-succinylated starch), maltodextrin DE-21 (MD, from Waxy Maize) and gum Arabic (GA) were obtained from Chemicotechnica S.A. Chitosan (CH) high molecular weight (viscosity of 800,000 cps, food-grade, water soluble, odorless and tasteless powder as stated by the manufacturer) was obtained from SigmaAldrich Chemical Co. Sorbitol and mannitol were purchased from Cargill whereas gum base and lecithin were kindly donated from Kraft Foods and Biotrek—Greece, respectively. 2.2. Preparation of aqueous saffron and beetroot extracts Saffron (1 g) was extracted with distilled water (50 mL) under continuous shaking in an ultrasound water bath (Elma, S 30H, Elmasonic) at T = 25 ◦ C for 60 min and at fixed-frequency of 30 kHz, while beetroots were washed, peeled and extracted with water in a commercial juice extractor. Both saffron and beetroot aqueous extracts were filtered and kept in the dark at −30 ◦ C until used. 2.3. Microencapsulation of saffron and beetroot extracts by freeze–drying Plain GA and MD, binary (1:1) blends of GA–MS and MS–CH plus a ternary (1:1:1) one of MS–MD–CH were selected as encapsulating agents. An aliquot of 15 g of GA, MD and MS agent was dispersed individually in distilled water to a final volume of 100 mL, while a 2% chitosan solution (CH) in 1% glacial acetic acid was prepared. A fixed weight ratio (w/w) of 0.33 (extract:agent) was tested (Ahn et al., 2008; Hogan, McNamee, O’Riordan, & O’Sullivan, 2001). The extracts were dissolved into the agents under stirring for 15 min

253

at 600 rpm in a rotor–stator. The resulting solutions were frozen overnight at −30 ◦ C (Shimada, Roos, & Karel, 1991) and lyophilized in a freeze dryer (Christ Alpha 1–4 LD Plus) at P = 0.017 mbar and T = −57 ◦ C for 48 h. The freeze-dried encapsulated extracts were converted into powder with help of a pestle and mortar. 2.4. Kinetic studies of beetroot and saffron pigment degradation Freeze-dried beetroot and saffron powders were stored in brown bottles with a screw cap and kept at 40 ◦ C with 20% RH (relatively humidity) as directly measured with a hygrometer for a period of 10 weeks in order to determine the effect of storage temperature on the retention of betacyanins and carotenoids levels, respectively. The degradation of natural pigments was expressed as coloring strength (E) and followed by periodic absorbance measurements of the reconstituted powder (0.2 g) in aqueous solution with distilled water (10 mL, stirring for 10 min and immediate measurement of the absorbance at certain wavelength). The absorbance was measured with a spectrophotometer (DMS 80, Varian Techtron PTY, LTD, Belrose, Australia) at max = 537 nm, the maximum absorption wavelength of betacyanin (Pasch & von Elbe, 1975) and max = 440 nm, the maximum absorption wavelength of crocin (ISO, 1993). The coloring strength, (E), was calculated as follows (Alonso, Varon, Gomez, Navarro, & Salinas, 1990): 1% Emax =

AV pdC

(1)

where A is the absorbance at the max , V is the quantity of solvent added (mL), p is the weight of the sample (g), d is the pathlength of the cell (cm) and C is a constant, the value of which is 100 cm2 /g. Measurements were carried out in triplicate and reaction rate constants (k) and half-life periods (t1/2 ) were determined by applying a first-order reaction model to the data (Tsimidou & Tsatsaroni, 1993). Powder samples were withdrawn every 2 weeks for a period of 10 weeks for betacyanins and carotenoids degradation kinetic analysis. 2.5. Color change during storage L∗ (lightness), a∗ (redness to green), and b∗ (yellow to blue) color parameters were measured immediately after freeze–drying (zero time) and after 12 weeks of storage at 40 ◦ C and the mean of three replicates was reported. Color measurements were performed directly on the encapsulated powdered products using a colorimeter (Minolta CR 200, Tokyo, Japan). The parameters of chroma (C* ) and total color differences (E* ) were also calculated as follows:



C ∗ = (a∗ )2 + (b∗ ) E ∗ =



L0∗ − L∗



2 1/2

2



+ a∗0 − a∗

(2)

2



+ b∗0 − b∗

2 1/2

(3)

2.6. Water sorption studies Approximately 0.4 g of freeze dried microencapsulated saffron and beetroot samples was placed in small glass desiccators (10 cm diameter) which contained different saturated salt solutions at ambient temperature (25 ◦ C). The prepared saturated solutions of LiCl, MgCl2 , K2 CO3 , Mg(NO3 )2 ·6H2 O, KI and KCl provided water activity (aw ) levels of 0.11, 0.31, 0.42, 0.51, 0.66 and 0.82, respectively (Labuza, 1984). The samples were weighed until equilibrium was attained for a period of 15–20 days. The initial moisture content of the samples was measured on a dry weight basis by drying the sample in a vacuum oven at 100 ◦ C until constant weight was obtained (AOAC, 2000). The water activity (aw ) was measured using Aqualab 3TE water activity meter (Decagon Devices

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Inc. Pullman, WA, USA) according to manufacturer instructions. The sorption isotherms which indicate the variation of water activity and moisture content were plotted and fitted to well-known sorption isotherm models such as Brunauer–Emmett–Teller (BET) and Guggenheim Anderson–de Boer (GAB) (Van den Berg & Bruin, 1981). The BET model is described by the following equation:

monitored. The color measurements were performed in triplicate directly on the chewing gum samples using a colorimeter as described in section 2.5. The L* parameter was taken into account for samples with both samples, while a* and b* parameters were taken into account for beetroot and saffron samples, respectively.

aw aw (k − 1) 1 + = mm k mm k (1 − aw ) m

2.9. Statistical analysis

(4)

where m is the equilibrium moisture content (g H2 O/g dry weight), k is a constant and mm is the BET monolayer value. These two constants can be calculated from the linear regression of the experimental data. The GAB isotherm model is described by the following equation: m CKaw = mm (1 − Kaw ) (1 − Kaw + CKaw )

(5)

where m is the equilibrium moisture content (g H2 O/g dry weight), C and K are constants and mm is the GAB monolayer value. This model can also be presented as a second-order polynomial equation for data-fitting purposes, according to the following equation: aw = ˛(aw )2 + ˇaw +  m



3. Results and discussion 3.1. Storage stability evaluation

(6)

The model that was considered most suitable was based on the high coefficient of determination (R2 ) and the low mean relative percentage deviation modulus (E), defined as follows:



100  mi − mpi E= N mi N

Statistical analysis of measurements results was carried out with the Statistica software (Statsoft 224 Inc., Tulsa, USA). Analysis of variance was performed by ANOVA procedure and significant differences (P < 0.05) between the means were determined by Duncan’s multiple range test. Moreover, multivariate analysis was conducted to correlate the studied parameters by means of principal component analysis (PCA) and multiple linear correlation (Pearson’s correlation).

i=1

where mi is the experimental value, mpi is the predicted value and N is the population of experimental data. It is generally assumed that a good fit is obtained when E < 5%. 2.7. Scanning electron microscopy In order to study the morphological properties of the freezedried encapsulated extracts, a scanning electron microscope (Quanta 200, FEI with LFD Detector) was used. Particles were loaded onto a specimen stub, coated with gold with a sputter coater. Examinations were made at 25–30 kV with ×500 magnification. 2.8. Food application—Incorporation as natural colorants in a chewing gum model system 2.8.1. Preparation of chewing gum model The chewing gum ingredient formulation consisted of gum base (29.48 g/100 g), sorbitol powder (37.24 g/100 g), glycerin (7.06 g/100 g), saturated sorbitol solution (25.82 g/100 g) and lecithin (0.40 g/100 g). For chewing gum preparation the gum base was initially melted (raised to 98–104 ◦ C). The extracts (pure or encapsulated) were diluted in the lecithin solution and then added to the molten gum base under mixing and after 2 min the mixture was allowed to cool. During cooling, the saturated sorbitol solution was added (under mixing for 2 min) followed by addition of about 50% of the sorbitol powder and further mixing for 2 min. At approximately 75 ◦ C the remaining sorbitol powder was added and mixed for 2 min. Finally, the glycerin was added and further mixed for 1 min (Potineni & Peterson, 2008). Twelve different formulations of chewing gum were designed based on two types of extracts (beetroot and saffron) either in pure or encapsulated form. 2.8.2. Color stability test of chewing gum samples The chewing gum model samples were stored at 25 and 40 ◦ C and during storage the changes in color parameters were

The stability of the freeze dried microencapsulated beetroot and saffron extracts was evaluated in terms of coloring strength (E) during 10 weeks storage at 40 ◦ C. A linear relationship was observed from the plots of the ln(E) vs time, implying first-order reaction kinetics for beetroot and saffron’s coloring strength degradation (Fig. 1). The rate constant (k) and half-life period (t1/2 ) of the microencapsulated powders were determined and are presented in Table 1. During 10 weeks of storage at 40 ◦ C, MS–CH and MS–MD–CH samples showed higher reaction rate constant and, consequently, shorter half-life in both extracts (23.61, 26.52 and 30.77, 29.01 weeks for beetroot and saffron, respectively). The half-life was found not to be significantly affected by the type of extract used, presenting mean values of 40.43 and 40.30 weeks in the case of beetroot and saffron, respectively. Overall, MD exhibited the greatest protection against stability accompanied also by high half-life period (t1/2 ) with values of 53.03 and 60.03, for beetroot and saffron, respectively. Previous research have shown that the degradation of natural colorants such as betalains follows first order kinetics during the storage (Saenz, Tapia, Chavez, & Robert, 2009; Serris & Biliaderis, 2001). Similar first-order kinetic responses have also been reported for saffron pigment oxidation under various water activity and temperature conditions (Alonso et al., 1990; Selim, Tsimidou, & Biliaderis, 2000; Tsimidou & Biliaderis, 1997; Tsimidou & Tsatsaroni, 1993). The coloring strength stability was strongly influenced (P < 0.05) by the encapsulating agent combination. In particular, the protection order proved as follows: MD > GA > GA–MS > MS–CH > MS–MD–CH, for both extracts. Similar observations for the protective effect of MD and GA on natural pigments against heat during storage have been reported by other researchers (Nayak & Rastogi, 2010). In particular, Cai and Corke (2000) observed that the use of MD (20 or 25DE) provided better storage stability for Amaranthus betacyanin during storage at 25 ◦ C for 16 weeks. Saenz et al. (2009), also, found that MD protected the betacyanin content of cactus pear when stored at 60 ◦ C for 44 days. In another work, Ferrari, Germer, Alvim, and Maurício de Aguirre (2013) found that the use of MD or GA reinforced the stability of spray-dried anthocyanins stored at 25 and 35 ◦ C and relative humidity of 32.8% for a period of 150 days. Recently, Khazaei et al. (2014) concluded that encapsulation of saffron petal’s extract with freeze drying technique and MD and GA as wall materials can protect anthocyanins content during storage at 35 ◦ C for a period of 10 weeks.

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255

(a)

8,5

GA

8,0

MD

ln (E5371%)

GA-MS 7,5

MS-CH MS-MD-CH

7,0 6,5 6,0 2

0

4

6

8

10

12

Storage time (wee ks)

(b)

8,5

GA

ln (E4401%)

8,0

MD GA-MS

7,5

MS-CH 7,0

MS-MD-CH

6,5 6,0 5,5 2

0

4

6

8

10

12

Storage time (wee ks) Fig. 1. First-order degradation plots of coloring strength (ln E vs time) for freeze dried microencapsulated beetroot (a) and saffron (b) extracts prepared with different agents during storage at 40 ◦ C for 10 weeks.

The stabilizing effect of encapsulation on natural pigments against heat during storage is well studied. In particular, Jafari, He, and Bhandari (2007) reported that encapsulating agents can act as physical obstacles that decrease the effects of oxygen, light, heat and moisture on the encapsulated ingredients. Limitation or absence of oxygen, which is the most deteriorative agent, may as well decrease the negative effect of heat on natural pigments (Jackman, Yada, Tung, & Speers, 1987). Moreover, concerning moisture content, decrease in its value which occurs during encapsulation (Jafari, Assadpoor, He, and Bhandari (2008)) is another protective factor, that acts on reducing molecular mobility

and increasing the viscosity of the encapsulating agent in the glassy state (Tonon, Brabet, & Hubinger, 2010). 3.2. Color stability of freeze dried microencapsulated beetroot and saffron extracts In order to investigate the effect of storage on the color of the obtained encapsulated powders, color measurement was performed. The color parameters (a* , b* , L* , C* and E* ) of the microencapsulated beetroot and saffron extracts immediately after production and after 10 weeks storage at 40 ◦ C are presented in Table 2.

Table 1 Regression analysis of coloring strength in freeze dried microencapsulated beetroot and saffron extracts prepared with different agents during storage at 40 ◦ C for 10 weeks. Sample

Beetroot −1

k (week GA MD GA–MS MS–CH MS–MD–CH

0.082 0.079 0.091 0.163 0.139

Saffron )

t1/2 (weeks)

R

k (week−1 )

t1/2 (weeks)

R2

50.12 53.03 44.66 23.61 26.52

0.975 0.983 0.977 0.876 0.941

0.117 0.069 0.086 0.114 0.119

34.59 60.03 47.12 30.77 29.01

0.992 0.879 0.958 0.987 0.969

2

GA: gum Arabic; MD: maltodextrin; GA–MS: gum Arabic and modified starch; MS–CH: modified starch and chitosan; MS–MD–CH: modified starch, maltodextrin and chitosan, k: rate constant and t1/2 : half-life period.

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Table 2 Color parameters (a* , b* , L* , C* and E* ) of freeze dried microencapsulated beetroot and saffron extracts prepared with different agents after production (0) and after storage at 40 ◦ C for 10 weeks (t). Sample

L*

a*

L0∗

Lt∗

b*

a∗0

a∗t

Beetroot GA MD GA–MS MS–CH MS–MD–CH

64.64 61.03 60.48 65.58 64.46

± ± ± ± ±

1.15a 4.61a 3.56a 3.94a 1.95a

54.80 59.84 57.56 61.33 58.83

± ± ± ± ±

0.34a 1.01d 0.39b 0.24e 0.09c

35.27 37.77 34.13 25.78 27.92

± ± ± ± ±

0.45b 1.12c 1.86b 1.54a 0.87a

25.22 26.55 23.54 20.49 17.43

Saffron GA MD GA–MS MS–CH MS–MD–CH

89.79 89.09 90.98 74.93 83.32

± ± ± ± ±

0.67a 3.33a 0.48a 3.09b 0.55c

61.46 59.88 56.95 58.79 58.85

± ± ± ± ±

0.12d 0.29c 0.80b 0.16a 0.41a

−2.49 −6.60 −6.15 −5.69 −7.45

± ± ± ± ±

0.08e 0.05b 0.13c 0.13d 0.05a

1.32 ± 0.03a 2.47 ± 0.31b 2.77 ± 0.40b −1.11 ± 0.07c 1.24 ± 0.07a

± ± ± ± ±

0.20e 0.53d 0.41c 0.37b 0.13a

C*

E*

b∗0

b∗t

−2.91 ± 0.17b −4.05 ± 0.10a −3.57 ± 0.37ab 4.43 ± 1.00c 3.90 ± 0.36c

14.35 11.54 14.26 20.30 20.45

± ± ± ± ±

0.06a 0.41c 0.49a 0.15b 0.15b

35.39 37.99 34.32 26.16 28.19

± ± ± ± ±

0.44b 1.11c 1.88b 1.68a 0.90a

29.02 28.95 27.52 28.85 26.87

± ± ± ± ±

0.17a 0.65a 0.61b 0.37a 0.19b

22.28 19.76 21.23 17.61 20.46

± ± ± ± ±

0.55b 1.24a 1.51ab 0.24c 0.75ab

31.58 31.85 30.82 26.06 21.81

± ± ± ± ±

0.51a 0.12a 0.57d 0.03c 0.20b

41.78 45.19 42.82 40.12 37.06

± ± ± ± ±

0.40ab 1.87b 0.16ab 3.58ac 0.73c

31.61 31.95 30.95 14.44 21.84

± ± ± ± ±

0.51a 0.15a 0.60a 0.03b 0.20c

30.33 33.18 37.03 21.73 29.74

± ± ± ± ±

0.89a 3.57ab 0.53b 3.90c 0.41a

41.71 44.71 42.38 39.71 36.30

± ± ± ± ±

0.40ab 1.89b 0.15ab 3.63a 0.74c

C0∗

Ct∗

Mean of three replicates ± standard error. a,b,c,d Values with different letters in the same column differ significantly (P < 0.05). GA: gum Arabic; MD: maltodextrin; GA–MS: gum Arabic and modified starch; MS–CH: modified starch and chitosan; MS–MD–CH: modified starch, maltodextrin and chitosan.

Based on the initial values of a* and b* parameters, in beetroot case, MD, GA and GA–MS presented a purple shade of red color in comparison with MS–CH and GA– MS–CH which showed a pink one. The same trend was also observed for saffron samples, in which MD, GA and GA–MS had an intense yellow color while MS–CH and GA–MS–CH showed a pale shade of yellow. The initial variations among the samples were related to the different nature of the agents used (Desobry, Netto, & Labuza, 1997). In the case of beetroot, all powders had a high value of color parameter a* which is attributed to their betacyanin content. Jimenez-Aguilar et al. (2011) have similarly correlated a* value with cyanin content in spray-dried blueberry microencapsulated products using mesquite gum. After 10 weeks of storage, the parameter a* showed a decrease in all samples with MS–MD–CH presenting the highest (P < 0.05) decrease of 37.52%. A slight reduction in lightness, expressed by L* parameter, was also observed in all beetroot samples, but without being significantly affected by the type of the agent used for encapsulation. Concerning saffron samples, b* parameter, which characterizes the extent of yellow color, best represented the observed color changes, since this color was dominant. A decrease in its value with storage time was recorded, with MS–CH and MS–MD–CH samples suffering the greater reduction of 34.37 and 39.91%, respectively, while MD showed the lowest reduction of 28.76%. A more profound decrease in lightness (L* parameter) was observed compared to beetroot samples, with reduction levels varying from 29.36 (MS–MD–CH) to 37.40 (GA–MS), respectively. The chroma (C* ) and total color difference (E* ), which are a combination of the chromatic coordinates, were also analyzed (Table 2). These parameters are extensively used to evaluate the color changes in foods during processing (Goncalves, Pinheiro, Abreu, Branda, & Silva, 2007; Karabulut, Topcu, Duran, Turan, & Bólent, 2007; Skrede et al., 1997). Parameter C* which indicates the dullness/vividness of the product, ranging from 0 to 100, was decreased with storage time in all samples, irrespectively of the agent used. Based on the E* values, corresponding to the color difference between initial and final time for a specific formulation, the samples suffered significant changes during storage. High values of E* parameter means more color changes during treatment or storage (Maskan, 2006). Overall, both in beetroot and saffron samples, the type of the agent significantly (P < 0.05) affected almost all the examined color parameters with MD offering the best protection. The parameter a* and b* best characterized the observed color changes in beetroot and saffron samples, respectively. Similar observations have been reported by other researchers as well. Desobry, Netto, and Labuza

(1999) and Desobry et al. (1997) who colorimetrically evaluated the microencapsulation of carotenoids in maltodextrin systems also found that L* (lightness) and a* (redness) were the most sensitive parameters. Moreover, parameter a* was the most sensitive one when studying the retention of betalains, encapsulated in gum Arabic, during storage (Pitalua et al., 2010). In addition, Sutter, Buera, and Elizalde (2007) and Spada, Norenab, Marczaka, and Tessaro (2012) concluded that a* parameter best represented the color changes in carotenoids encapsulated in a mannitol-phosphate and starch matrix, respectively. 3.3. Water sorption isotherms of freeze dried microencapsulated beetroot and saffron extracts The water sorption isotherms describing the relationship between water activity and equilibrium moisture content (Shrestha, Howes, Adhikari, & Bhandari, 2007) are useful for food products to predict their shelf life stability (Martinelli, Gabas, & Telis-Romero, 2007). The experimental moisture adsorption isotherms of the microencapsulated saffron and beetroot extracts at room temperature (25 ◦ C) are given in Fig. 2. The sorption isotherms of all samples were of sigmoidal shape. This form of isotherm is typical for most food systems including encapsulated natural colorants (Desobry et al., 1997; Nayak & Rastogi, 2010; Pavon-Garcia et al., 2011; Rascón, Beristain, García, & Salgado, 2011; Serris & Biliaderis, 2001). The model that best fit to the experimental data (water activity versus moisture content) for all samples was the GAB model, considering both the lowest value of the average relative deviation (E) and the highest coefficient of determination (R2 ). Similar observations on the fitting of sorption models like BET and GAB have also been reported on freeze-dried beetroot pigments (Serris & Biliaderis, 2001) and spray-dried carotene pigments (Wagner & Warthesen, 1995). Moreover, Nayak and Rastogi (2010) and Pavon-Garcia et al. (2011) have obtained similar results during their sorption studies on spray-dried anthocyanins and natural colorants from Justicia spicigera plant, respectively. The resulting parameters values of the BET and GAB model for both saffron and beetroot microencapsulated extracts are given in Table 3. The parameter of monolayer value (mm ) is of particular interest, as it indicates the amount of water that is strongly adsorbed to specific sites and is considered as the optimum value at which a food is more stable against microbial spoilage (Labuza, 1984). In the present study, the estimated monolayer value (mm ) fell within the range of 3.27–14.40 g H2 O/100 g dry weight and 4.70–11.33 g H2 O/100 g dry weight for freeze dried

C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263

(a)

35 g H2O / 100g dry weight

257

GA MD

30

GA-MS

25

MS-CH

20

MS-MD-CH

15 10 5 0 0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0,90

water activity (a w)

g H2O / 100g dry weight

GA

(b)

35

MD GA-MS

30

MS-CH

25

MS-MD-CH

20 15 10 5 0 0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0,90

water activity (a w) Fig. 2. Water isotherms for freeze dried microencapsulated beetroot (a) and saffron (b) extracts at various water activities.

Table 3 Water isotherm model (BET and GAB) parameters for freeze dried microencapsulated beetroot and saffron extracts prepared with different agents. Sample

BET model

GAB model 2

mm

C

R

E%

mm

K

C

R2

E%

Beetroot GA MD GA–MS MS–CH MS–MD–CH

5.46 3.73 3.27 6.04 4.61

−7.04 −7.44 −5.77 −17.08 −6.03

0.884 0.845 0.908 0.905 0.848

14.97 16.55 14.78 8.95 18.28

10.74 7.34 9.59 14.40 10.12

0.78 0.78 0.62 0.38 0.73

19.83 14.45 21.06 18.95 19.21

0.926 0.933 0.943 0.953 0.951

4.82 4.36 5.65 6.46 4.02

Saffron GA MD GA–MS MS–CH MS–MD–CH

5.43 4.69 6.49 5.71 5.24

−6.34 −19.36 −19.25 −12.50 −7.64

0.894 0.956 0.930 0.913 0.845

15.35 8.66 9.84 33.21 19.03

9.70 6.18 9.58 11.13 11.33

0.81 0.93 0.80 0.56 0.75

40.54 34.62 32.37 26.88 16.60

0.980 0.986 0.940 0.949 0.888

2.77 3.33 6.57 5.20 7.06

mm : Monolayer moisture content; C, K: model constants; aw : water activity; R2 : coefficient of determination; E: mean relative deviation modulus.

microencapsulated beetroot and saffron extracts, respectively, depending on the equation used for estimation and on the type of the encapsulating agent. Regarding the type of the encapsulating agent and based on GAB model, MS–CH and MS–MD–CH presented the highest mm values of 14.40 and 11.33 g H2 O/100 g dry weight, for beetroot and saffron samples, respectively. Serris and Biliaderis (2001) obtained lower values of mm (3.03–6.31 g H2 O/100 g dry weight) for beetroot pigments encapsulated in maltodextrin and

pullulan by freeze drying. This could be attributed to the different nature of the agents used that affects their adsorption behavior. For biopolymers, the process not only involves adsorption but also structural changes (crystalline or amorphous) due to swelling, physical and/or chemical changes occurring during the drying process (Pérez-Alonso, Beristain, Lobato-Calleros, Rodríguez-Huezo, & Vernon-Carter, 2006). The parameter C of the GAB model is related to the heat of adsorption of water on the powders.

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Fig. 3. Physical characteristics of freeze dried microencapsulated beetroot (a) and saffron (b) extracts prepared with different agents at various water activities during the determination of the moisture adsorption isotherms.

Lewicki (1997) stated that C values should fall within the range of 5.67 ≤ C < ∞ for describing properly an isotherm mathematically. In our case, the observed C values fell within the aforementioned limit. Particularly, GA–MS followed by GA presented the highest C values of 21.06 and 19.83 for beetroot samples while GA showed also the highest C value of 40.54 for saffron samples. The parameter K provides a measure of the interaction of the molecules in the multilayers with the adsorbent, and tends to fall between the energy value of the molecules in the monolayer and that of liquid water. When K equal 1 the multilayers have the properties of liquid water (Pérez-Alonso et al., 2006). The values of K for both beetroot and saffron microencapsulated extracts (Table 3) fell within the range of 0.24 < K ≤ 1, which according to Lewicki (1997) describe properly an isotherm mathematically. Among the studied encapsulating agents, MS–CH presented the lowest K values of 0.38 and

0.56 for beetroot and saffron samples, respectively, while GA and MD showed similar values for both extracts. Concerning the GAB parameters (especially K), similar values have been reported by other researchers as well (Pérez-Alonso et al., 2006; Rascón et al., 2011). The physical characteristics of the powders subjected to various humidity levels demonstrated that there was apparent phase change in the produced samples (Fig. 3). In particular, both GA and MD microencapsulated beetroot and saffron extracts at aw = 0.82 were in a liquid form of high viscosity while MD presented a liquid form also at aw = 0.66 (Fig. 3). Similar observations have also been reported in other research works (Rascón et al., 2011; Righetto & Netto, 2005), according to which carotenoids and juice from acerola encapsulated in MD and/or GA at aw ≥ 0.74 became unable to keep their structural integrity leading to collapse and liquefaction.

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259

Fig. 4. Representative outer topography of MD (a), GA (b), GA–MS (c), MS–MD–CH (d) and MS–CH (e) freeze dried microencapsulated saffron extract (magnification ×500).

3.4. Microstructure of freeze dried microencapsulated saffron extracts The outer topographies of freeze-dried encapsulated saffron extracts were assessed by SEM (Fig. 4). SEM examination showed an amorphous glass-like formation, which according to some researchers is considered that protects the entrapped molecules from exposure to heat and oxygen (Goubet, Le Quere, & Voiley, 1998; Kaushik & Roos, 2007; Porzio, 2004; Roos, 1995). Similar images, characteristic of freeze-dried encapsulated in GA and MD pigments are reported by other researchers as well (Khazaei et al., 2014; Sousdaleff et al., 2013). The analysis, also, showed that MD presented less particle differences in comparison to GA, in which the differences in size of its particles may be attributed to its highest bulk density and lowest special volume (Khazaei et al., 2014).

3.5. Multivariate analysis The results obtained based on the principal component analysis (PCA) are presented in Fig. 5. In the case of beetroot samples, the two principal components accounted for 93.45% of the variance in the data (Fig. 5a). The first principal component (71.62% of the total data inertia) was positive for Et , a∗t and t1/2 while the parameters b∗t and k presented negative values. Consequently, this component (PC1) was probably related to the degree of betalains degradation. Concerning saffron samples, the two principal components accounted for 86.06% of the variance in the data (Fig. 5b). The first principal component (PC1) was positive for k while parameters Et , b∗t , Ct∗ and a∗t , t1/2 and E* presented negative values. As in the case of beeroot, it could be interpreted in saffron as the indicative axis of the carotenoids degradation.

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(−0.98), b∗t and Ct∗ (0.99) and between a∗t and E* (−0.98). The coloring strength (Et ) showed the highest correlation coefficient with b∗t (0.97) and Ct∗ (0.95), while displayed low correlation coefficients with the other color parameters; Et and a∗t (0.67), Et and E* (0.63) and Et and Lt∗ (−0.34). In the current study, based on the multivariate analysis, colorimetric analysis proved to be a useful tool for evaluating pigment degradation from the natural colorants sources. Other studies have also shown a strong relationship between the color parameters (such as a* ) and pigments content (carotenoid) (Bengtsson, Namutebi, Alminger, & Svanberg, 2008; Ramakrishnan & Francis, 1973; Spada et al., 2012; Takahata, Noda, & Nagata, 1993).

3.6. Application in chewing gum model—Color stability test

Fig. 5. Principal component analysis of the coloring strength and color parameters of freeze dried microencapsulated beetroot (a) and saffron (b) extracts prepared with different agents.

According to the multiple linear correlation, in the case of beetroot samples, the highest Pearson correlation coefficients were observed between k and t1/2 (−0.99), Et and t1/2 (0.98) and between Et and b∗t (−0.98). The coloring strength (Et ) showed the highest correlation coefficient with a∗t (0.97) and a negative relationship with k (−0.94). In contrast, it displayed low correlation coefficients with the other color parameters; Et and Ct∗ (0.50), Et and E* (0.52) and Et and Lt∗ (−0.49). The halt life half-life period (t1/2 ) showed a positive correlation with a∗t (0.91) and a negative one with b∗t (−0.95). Among the color parameters the stronger correlations were observed between Lt∗ and E* (−0.94) and between a∗t and b∗t (−0.94). Concerning the saffron samples, the highest Pearson correlation coefficients were also observed between k and t1/2

The evolution of color parameters of the chewing gum samples with freeze dried microencapsulated beetroot and saffron extracts during storage at 25 and 40 ◦ C is presented in Figs. 6 and 7. The color parameters of the prepared chewing gum samples with both beetroot and saffron extracts depended significantly (P < 0.05) on the form of the extract (pure or encapsulated), the type of agent and the storage temperature. Storage temperature affected significantly (P < 0.05) the L* parameter; increase in storage temperature resulted in a decrease in L* values in all samples (Figs. 6a and c and 7a and c). Concerning a* color parameter (Fig. 6b and d), it decreased with storage time and temperature, suffering greater decrease when samples were stored at 40 ◦ C compared to 20 ◦ C. In particular, it was observed that a* color parameter almost disappeared after two weeks of storage at 40 ◦ C (0.41, 0.83, −0.37 and −0.45 for pure extract, GA, MS–CH and MS–MD–CH, respectively). The chewing gum samples prepared with GA–MS were found to offer the best protection against color degradation at storage temperature or even at accelerated temperature, followed by MD ones. Concerning b* color parameter (Fig. 7b and d), it decreased with time regardless of the storage temperature. A similar behavior of GA–MS protection was observed in both saffron and beetroot samples. In particular, the chewing gum samples with the encapsulated in GA–MS saffron extracts presented the greatest (P < 0.05) b* values (average values of 36.20 and 38.76 at 25 and 40 ◦ C, respectively). The high b* values observed in chewing gum samples with incorporation of GA–MS saffron extract and stored at 40 ◦ C may be attributed only to the inherent color of the GA matrix and chewing gum base since the color of the extract was already diminished. Overall, color parameters of the prepared chewing gum samples as well as their evolution during storage depended greatly on the type of agent used and storage conditions (time and temperature). Among the agents used, GA–MS provided the best color stability to the examined extracts. The chewing gum samples produced with coloring extracts encapsulated in GA–MS showed the greatest a* (for beetroot) and b* (for saffron) values indicating a better protection. Similar observations have been reported by other researchers as well. Marcolino, Zanin, Durrant, Benassi, and Matioli (2011) also received positive results with regard to color stability when incorporating curcumin, encapsulated in bcyclodextrin, into a yogurt food system. According to Gomes, Petito, Costa, Falcão, and de Lima Araújo (2014) the red bell pepper extract encapsulated in b-cyclodextrin could simulate the color of commercial colorants in yogurt, providing high color stability compared to the crude bell pepper extract. Moreover, pasta prepared with potassium norbixinate and ice cream prepared with curcumin, both colorants encapsulated in MD with freeze drying technique, resulted in greater color stability compared with samples prepared with non-encapsulated colorants (Sousdaleff et al., 2013). The current incorporation study demonstrated the feasilbilty of production

C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263

(a)

(b) 35

85

Pure extract

75

GA

70

GA-MS MS-CH

65

Pure extract

30

MD

α * parameter

L * parameter

80

MS-MD-CH

60

MD

25

GA

20

GA-MS

15

MS-CH MS-MD-CH

10 5

55

0

50 0

5

10

15

-5

5

0

10

15

Storage time (days)

Storage time (days)

(c)

(d) 35

80

Pure extract

30

MD

25

75

GA

70

GA-MS

α * parameter

85

L * parameter

261

MS-CH

65

MS-MD-CH

60

Pure extract MD GA

20

GA-MS

15

MS-CH

10

MS-MD-CH

5

55

0

50 5

0

10

-5

15

0

5

10

15

Storage time (days)

Storage time (days)

Fig. 6. Changes of L∗ (Lightness), and a∗ (redness) color parameters of chewing gum samples prepeared with freeze dried microencapsulated beetroot extracts during storage at 25 and 40 ◦ C. (a) L∗ parameter of beetroot samples stored at 25 ◦ C, (b) a∗ parameter of beetroot samples stored at 25 ◦ C, (c) L∗ parameter of beetroot samples stored at 40 ◦ C and (d) a∗ parameter of beetroot samples stored at 40 ◦ C.

(a)

90 88 84

GA-MS

82

MS-CH

80

MS-MD-CH

78

b * parameter

L * parameter

GA

Pure extract

40

MD

86

(b)

45

Pure extract

MD GA

35

GA-MS

30

MS-CH

25

MS-MD-CH

20

76 74

15 0

5

10

0

15

5

(c)

90

15

(d)

45 Pure extract

85 80

GA

75

GA-MS

70

MS-CH MS-MD-CH

65

Pure extract

40

MD b * parameter

L * parameter

10

Storage time (days)

Storage time (days)

MD GA

35

GA-MS

30

MS-CH

25

MS-MD-CH

20

60

15

55 0

5

10

Storage time (days)

15

0

5

10

15

Storage time (days)

Fig. 7. Changes of L∗ (Lightness) and b∗ (yellowness) color parameters of chewing gum samples prepeared with freeze dried microencapsulated saffron extracts during storage at 25 and 40 ◦ C. (a) L∗ parameter of saffron samples stored at 25 ◦ C, (b) b∗ parameter of saffron samples stored at 25 ◦ C, (c) L∗ parameter of saffron samples stored at 40 ◦ C and (d) b∗ parameter of saffron samples stored at 40 ◦ C.

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of freeze dried microencapsulated beetroot and saffron extracts that can attain their coloring strength and thus could be used as food-grade colorants. However, the incorporation procedure needs to be evaluated for other kinds of foods (such as those of high acidity), so as to verify the extension of their use. 4. Conclusion MD along with GA proved to be effective agents for beetroot and saffron coloring extracts microencapsulation by freeze drying. MD presented the greatest protection against heat during storage accompanied also by high half-life period (t1/2 ) with average values of 53.03 and 60.03 weeks for beetroot and saffron, respectively. The water sorption study showed that MD and GA retained their structural integrity up to water activities of 0.66 and 0.82, respectively. The incorporation study demonstrated higher stability for food model of low moisture such as chewing gum prepared with extracts encapsulated in GA–MS. To conclude, microencapsulation by freeze drying could be suggested not only as a suitable method for color stabilizing of beetroot and saffron extracts, but also as a feasible mean of production of food-grade colorants. Acknowledgment Charikleia Chranioti is grateful to the State Scholarships Foundation of Greece (IKY) for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol. 2015.03.049. References Ahn, J. H., Kim, Y. P., Lee, Y. M., Seo, E. M., Lee, K. M., & Kim, H. S. (2008). Optimization of microencapsulation of seed oil by response surface methodology. Food Chemistry, 107(1), 98–105. Akhavan, S., Jafari, S. M., Ghorbani, M., & Assadpoor, E. (2014). Spray drying microencapsulation of anthocyanins by natural biopolymers: A review. Drying Technology, 32(5), 509–518. Alonso, G. L., Varon, R., Gomez, R., Navarro, F., & Salinas, M. R. (1990). Auto-oxidation in saffron at 40 ◦ C and 75% relative humidity. Journal of Food Science, 55(2), 595–596. AOAC. (2000). Official method of analytical chemists (17th ed.). Arlington, TX: The Association of Official Analytical chemists Inc. Azeredo, H. M. C. (2009). Betalains: Properties, sources, applications and stability—A review. International Journal of Food Science and Technology, 44(12), 2365–2376. Bengtsson, A., Namutebi, A., Alminger, M. L., & Svanberg, U. (2008). Effects of various traditional processing methods on the all-trans-␤-carotene content of orange fleshed sweet potato. Journal of Food Composition and Analysis, 21(2), 134–143. Cai, Y. Z., & Corke, H. (2000). Production and properties of spray-dried Amaranthus betacyanin pigments. Journal of Food Science, 65(6), 1248–1252. Chranioti, C., Papoutsakis, S., Nikoloudaki, A., & Tzia, C. (2012). Study of flavor volatile profile in microencapsulated Greek saffron products. In C. Ho, C. Mussinan, F. Shahidi, & E. T. Contis (Eds.), Nutrition, functional and sensory properties of foods (pp. 111–115). Cambridge, UK: RSC Publishing Inc. Chranioti, C., & Tzia, C. (2013). Binary mixtures of modified starch. Maltodextrin and chitosan as efficient encapsulating agents of fennel oleoresin. Food and Bioprocess Technology, 6(11), 3238–3246. Chranioti, C., & Tzia, C. (2014a). Arabic gum mixtures as encapsulating agents of freeze-dried fennel oleoresin products. Food and Bioprocess Technology, 7(4), 1057–1065. Chranioti, C., & Tzia, C. (2014b). Thermooxidative stability of fennel oleoresin microencapsulated in blended biopolymer agents. Journal of Food Science, 79(6), C1091–C1099. Cormier, F. O., Dufresne, C., & Dorion, S. (1995). Enhanced crocetin glucosylation bymeans of maltosyl-2-cyclodextrin encapsulation. Biotechnology Techniques, 9(8), 553–556. Desobry, S. A., Netto, F. M., & Labuza, T. P. (1997). Comparison of spray-drying. Drumdrying and freeze–drying for b-carotene encapsulation and preservation. Journal of Food Science, 62(6), 1158–1162. Desobry, S. A., Netto, F. M., & Labuza, T. P. (1999). Influence of maltodextrin systems at an equivalent 25 DE on encapsulated ␤-carotene loss during storage. Journal of Food Process and Preservation, 23(1), 39–55.

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