Stability of spray-dried microencapsulated anthocyanins extracted from culinary banana bract

ABSTRACT

Culinary banana bracts are a potential source of anthocyanin, which can be widely used in the food industry because of its health beneficial effects. The aim of the present study was to study the stability of microencapsulated anthocyanin extracted from culinary banana bracts using ultrasound-assisted extraction (UAE). Central composite design (CCD) was used to optimize the process variables for the extraction of anthocyanin and the optimum condition was observed at 15:0.5 solvent to solute ratio, 53.97 ml/100 ml ethanol concentration, and 49.4°C. The optimized experimental anthocyanin content was 56.98 mg/100g and it closely agreed with the predicted value (57.29 mg/100g). High-performance liquid chromatography (HPLC) results revealed that cyanidin-3-O-glucoside and peonidin-3-O-glucoside were two constituents of anthocyanin. Encapsulated pigment powder exhibited satisfactory properties in terms of hygroscopicity, solubility, and good encapsulation efficiency with smooth spherical morphology (2–10µm) as confirmed by Scanning Electron Microscope (SEM). The storage stability of microencapsulated anthocyanin did not change noticeably up to 7 days but thereafter gradually decreased at 30°C and 75% relative humidity. During 21 days of storage, anthocyanin stability decreased by 44 %along with the decline in free radical scavenging activity.

KEYWORDS:

• Culinary banana bracts
• Anthocyanin
• Physicochemical properties
• Optimization
• Encapsulation
• Spray drying

Introduction

Anthocyanins are a flavonoid group of phytochemicals, widely distributed among fruits, berries, and flower and provide attractive colours such as orange, red, and blue. These pigments are water-soluble and this property facilitates their incorporation into numerous aqueous food systems. They have been used for various food preparations such as jelly dessert, milk dessert, soft ice cream, hard ice cream, and yogurt.^[1] In addition to their colourant characteristics, anthocyanins possess biological, pharmacological, anti-inflammatory, antioxidant, and chemoprotective properties.^[2] Anthocyanin is also helpful in many chronic diseases.^[3,4] Anthocyanins have recently received increasing attention as natural colourants in food systems, as a consequence of the common trend towards the consumption of natural products instead of synthetic ones.^[5]

Because of the edible character and abundance of bracts as a residue of banana production coupled with the attractive hue of the pigments, it has great potential as an economical source of natural pigments.^[6] During banana harvesting, 300 kg of coloured bracts per hectare are disposed as residues. Since bracts of banana are widely available and has been traditionally used as food without toxic effect, it could be a potential source of anthocyanins.^[7]

Isolation, identification, and use of anthocyanins are mainly based on its extraction method.^[8] Solvent-extraction procedure is frequently used for the extraction of anthocyanin and the solvent types, solvent concentration, temperature, and time are the important parameters to be optimized.^[9] An efficient method is one that reduces solvent consumption, shortens extraction time, and increases extraction yields. Numerous extraction techniques such as ultrasound-assisted extraction (UAE), supercritical fluid extraction, enzymatic extraction, and soxhlet extraction have been developed for the extraction of some active components from plants.^[10–13] UAE is considered as an inexpensive, simple, and efficient technique among these extraction techniques. Advantages imparted by UAE are improved extraction efficiency, low solvent usage, high level of automation, and reduced extraction time.^[14] Cavitation phenomena and mechanical mixing effect are the main mechanisms that increase extraction efficiency and reduce extraction time.^[15] In the process of UAE, the concept utilized is the production of acoustic cavitation that causes the molecular movement of solvent and sample, which could result in the breakdown of sample micelle or matrix to the intracellular hydrophobic compounds due to the frequency of ultrasonic.^[16] Moreover, marked increase in the temperature enhances the solubility of the analytes in the solvent and eases their diffusion from the sample matrix to the outer region.^[17] Optimization for conventional solvent extraction (CE) and UAE for fruit (blueberries, cherries, and red pear peels) with different anthocyanin compositions revealed that the UAE method dissociated polymeric anthocyanins without further degradation of the monomeric anthocyanins, thus enhancing the total monomeric anthocyanin content in blueberry and cherry acetone extracts.^[16] RSM is widely applied in optimizing the extraction process variables like anthocyanin, phenolic compounds, and polysaccharides.^[18–20]

The extraction methods may not be selective for anthocyanins due to the co-extraction of non-phenolic substances such as sugars, organic acids, and proteins and therefore subsequent purification processes are essential.^[21] Amberlite XAD-7 is a non-ionic, moderately polar, acrylic resin that has been used to purify anthocyanin. Lacking ionic groups, it presumably separates compounds through hydrophobic and polar interaction. Anderson (1988)^[22] employed Amberlite XAD 7 column (18 X 2.6 cm) chromatography as a fractional step in the elucidation of anthocyanin from Daycrycapus daycrydioides. High-performance liquid chromatography (HPLC) for separation is a powerful tool and can identify anthocyanin compositions in the fruit.^[23–25] The major problem associated with the storage of anthocyanins is the high unstability of these compounds and therefore the stabilization of anthocyanins is very crucial. Among the existing stabilization methods of anthocyanins, encapsulation is a cost-effective and reliable method. The utilization of encapsulated anthocyanins instead of free compounds can overcome the drawbacks of their unstability and also improve the bioavailability of anthocyanins. Spray drying is the most commonly used encapsulation technique in the food industry. There are several advantages for this technique such as low operating cost, high quality of capsules in good yield, rapid solubility of the capsules, small size, high stability of capsules, and continuous operation.^[26,27] Moreover, spray-drying microencapsulation technology is also used with the purpose of protecting ingredients that are sensitive to light, oxygen, and free radical degradation and this technique can protect the bioactive compounds with a wall material.^[28–30] According to Zuidam and Shimoni,^[31] maltodextrin is extensively used as wall in spray drying as to satisfy demand and low cost. Maltodextrins turned out to be the best thermal defenders, essential to preserve the integrity of the anthocyanins during their encapsulation.^[32] In the present study, the anthocyanin was extracted by employing ultrasonic-assisted extraction and optimized by RSM from culinary banana bract microencapsulated using spray drying and its stability and related properties were studied.

Material and methods

Material

Ethanol, HCl, Amberlite XAD 7N resins, HPLC-grade standards, i.e., cyanidin-3-O-glucoside and peonidin-3-O-glucoside, formic acid, methanol, and maltodextrin 20DE were purchased from Sigma Aldrich and all the chemicals were of high-purity analytical grade.

Collection and preparation of sample

Culinary banana (Musa ABB) flowers were obtained from Tezpur University Campus, Assam, India. The flower was washed with distilled water, the bract was separated from the male bud, and used for further experiments. A total of 50 g culinary banana bract was used for the experiment.

Optimization of the extraction process of anthocyanin from culinary banana bract

The bracts of culinary banana were ground using mortar and pestle with solvent: solute (5:0.5 to 15:0.5) and ethanol concentration (40–60 ml/100ml) with 0.15% of HCl. The ranges of variables were selected based on preliminary trials and the literature.^[33–35] The ground materials were placed in an ultrasonic bath (Bandelin sonorex, Germany) with a frequency of 20 kHz and a temperature range of 40–60°C for 20 min. The extracted samples were cooled and filtered through Whatmann No 1 filter paper and kept at 4°C in the dark until further analysis.

Experimental design

Mathematical design using response surface methodology based on the parameters solvent: solute, solvent concentration, and sonication temperature was carried out. A central composite design (CCD) with three factors and one response (Table 1) was used for optimization of treatment condition. The best combination of solvent to solute ratio, solvent concentration, and sonication temperature was chosen.

Table 1. Independent variables and their levels employed in a central composite design for the optimization of culinary banana bract extract for anthocyanin content.

Variables	Low	Centre	High
Solvent: solute ratio	5	10	15
Ethanol concentration (ml/100ml)	40	50	60
Sonication temperature (°C)	40	50	60

The response (Y) was partitioned into linear, quadratic, and interactive components and the experimental data were fitted to the second-order regression equation as follows:

where b₀ is the intercept; b₁, b₂, and b₃ are linear coefficients; b₁₁, b₂₂, and b₃₃ are squared coefficients; and b₁₂, b_23, and b₁₃ are interaction coefficients. The experimental design and statistical analysis were conducted using Design-Expert Software (Version 9, Stat-Ease, Inc. Minneapolis, MN USA). The model adequacies were checked in terms of the values of R² and adjusted R². Analysis of variance (ANOVA) was used to determine the significance of the models. Verification of the optimized and predicted values was performed in triplicate to confirm the validity of the model.

Anthocyanin content

The total anthocyanin (TA) content was determined according to the spectrophotometric pH differential method.^[36] Samples were diluted separately with 0.025 M potassium chloride buffer (pH 1) and 0.4M sodium acetate buffer (pH 4.5). Absorbance of the mixture was measured at 520 nm and 700 nm using a UV-Vis spectrophotometer. Absorbance was calculated as given in the following equation:

The TA content was calculated as cyanidin-3-glucoside equivalents as shown in the following equation:

where A is the absorbance, MW is the molecular weight (MW=449.2 gmol⁻¹), DF is the dilution factor, Ɛ is the molar absorptivity (Ɛ = 26900 Lcm⁻¹mol⁻¹), V is the volume of solvent in ml, and l is the path length.

Purification of anthocyanin

Column chromatography was used for further purification of anthocyanin. The concentrated anthocyanin extracts were diluted with distilled water and loaded onto Amberlite XAD 7N macro pore absorptive resins. The Amberlite XAD 7N resins were washed with distilled water and the absorbed anthocyanin was recovered with 80% ethanol. To remove the ethanol and part of the water, the ethanol eluate was concentrated using a rotary vacuum evaporator and the anthocyanin extract was stored at 4°C until use.

Identification of anthocyanins by HPLC

Identification of anthocyanin by HPLC was performed following the method given by Gracia-Tejeda et al.^[37] The purified anthocyanin of banana bract was identified by HPLC (Waters Corporation, USA, UV/Visible Detector-2489). The solvent system used was water, methanol, formic acid (14:5:1), and the flow rate was 1.5 ml/min. The elutes were monitored by visible spectrometry at maximum wavelength 530 nm.

Spray drying of purified anthocyanin extract

Spray drying of the purified anthocyanin extract was performed according to Nayak et al.^[38] The feed mixture was prepared by mixing 27 g maltodextrin with 200 ml purified anthocyanin extract for 15 min. The Total Soluble Solids (TSS) of the mixture was adjusted to 20°Brix before feeding in a spray dryer (Model LU 228 advance, Labultima, Mumbai, India). The spray dryer was operated at 170°C inlet and 80°C outlet temperatures. The feed flow rate was maintained at 6 ml/min.

Properties of encapsulated pigment powders

Hygroscopicity

The hygroscopicity of the encapsulated powder samples was determined according to Cai and Corke^[39] and Tonon et al.^[40] with modifications. Samples from each powder were stored at room temperature in a desiccator containing saturated sodium chloride (NaCl) solutions (75% RH; A_w = 0.75). The samples were weighed after one week, and the hygroscopicity was expressed in grams of absorbed moisture per 100 g of dry solids.

Solubility

Solubility of spray-dried powder was determined according to the method of Singh and Singh.^[41] Powder suspension of 1 g/100 ml was agitated for 30 min in a shaker and then it was centrifuged at 3000 rpm for 10 min. The supernatant obtained was deposited in petriplate and subjected to a temperature of 110°C for 4 h in a drying oven. The solubility was calculated according to the following equation:

Encapsulation efficiency

In order to evaluate the effectiveness of microencapsulation, TA and surface anthocyanin (SA) contents of the microparticles were determined after spray drying. For TA determination, 100 mg of samples were weighed in an amber vial with a screw top and about 1 ml of distilled water was added and was ground to destroy the microparticles. Ethanol (9 ml) was added and the samples were extracted for 5 min and then filtered. For determination of SA, 100 mg of samples was directly extracted with 10 ml ethanol and vortexed for 30 s, followed by centrifugation (SIGMA Laborzentrifugen, 3–18 KS, Osterode, Germany) at 3,000 rpm for 10 min. After phase separation, the clear supernatant was collected and filtered. Anthocyanin content for TA and SA values was determined using the pH-differential method.^[36] Encapsulation efficiencies were calculated according to an equation modified from Barbosa et al.^[42] and shown as follows:

SEM analysis

Particle structures of the spray-dried powder microparticles were evaluated by SEM (JEOL JSM-6390 LV, Japan, PN junction type, semiconducting detector). Powders of microparticles were attached to a double-sided adhesive tape on SEM stubs, coated with 3–5 mA palladium under vacuum, and were examined at 20 kV and magnification of 2000X and 5000X.

Storage stability

Pigment powders were stored in ziplock bags inside an airtight container and storage study was conducted at 30°C and 75% RH using saturated salt (NaCl) solution for a period of 21 days and the effects of storage on anthocyanin contents, antioxidant activity, moisture content, and colour characteristics of the powder were analysed at every 7-day interval.

Anthocyanin content and DPPH radical scavenging activity

TA content was determined as referred in the preceding subhead “Encapsulation efficiency”. DPPH scavenging activity of the extract of powder was estimated by the method of Luo et al.^[43] α-Tocopherol was used as the standard antioxidant compound. Briefly, 50 mg of the extracted powder was dissolved in 5 ml of methanol solution and shaken in an incubator shaker at 150 rpm at 25°C for 30 min. The filtered mixture of methanolic extract (2 ml) was mixed with 2 ml of methanolic solution containing 0.1 mM DPPH. The reaction mixture was mixed vigorously and kept in dark for 30 min and the absorbance was recorded at 517 nm.

Moisture content

The moisture content of the anthocyanin encapsulated powder material was determined according to Association of Official Analytical Chemists (AOAC).^[44] The sample (2 g) was placed in a previously dried (at 105°C for 1 h) empty moisture estimation box. The aluminium box was dried in an oven at 105°C until constant weight was attained. After drying, the aluminium box was removed from the oven and cooled in a desiccator and the weight was taken after reaching room temperature. The losses in weight were taken as the moisture loss of the samples and was calculated using the following relationship:

Colour characteristics

The effect of storage on the colour characteristics of the encapsulated anthocyanin powder was analysed after every 7 days of interval. L*, a*, and b* colour values of the encapsulated anthocyanin powder were measured using a Hunter Color Measurement Spectrophotometer (UltraScan VIS, Hunter Lab). Equipment standardization was carried out by placing a black card inside the transmission compartment, and then placing distilled water (as clear liquid) in a cell over the transmission port and a white standard tile at the reflectance port. After standardization the L*, a*, and b* values were measured. Powders (0.5 g) were dissolved completely in 25 ml of distilled water and subjected to a colorimeter in a 1.0 cm path length optical glass cell and the CIE L*, a*, and b* values were measured in total transmission mode, using illuminant C and 10° observer angle. Chroma (C*) and hue angle (H°) were determined using the following equations:

Statistical analysis

Experiments were carried out in triplicate and the means of data obtained were evaluated using Duncan’s multiple range test to identify significant differences at the 0.05 probability (p < 0.05) using SPSS 16.

Results and discussion

Optimization of the UAE process

A CCD was developed for extraction of TA from the bract of culinary banana using sonication. Table 2a presents the experimental values of TA values of banana bract extracts at various experimental conditions. Multiple regression analysis was performed based on the experimental values, and second-order polynomial model (Eq. 1) represents the recoveries of anthocyanin. A final predictive equation was developed after neglecting the non-significant terms (p>0.05). The quality of the fit of the model was expressed by the R² correlation coefficient (0.934), and its statistical significance was confirmed with an F-test. Results were found significant (p<0.001), attesting to the goodness of fit of the models and lack of fit was insignificant (p>0.05) and it further confirmed model validity. Regression coefficients of the models for anthocyanin obtained by the multiple linear regressions are presented (Eq. 1). Three-dimensional response surface plots (Fig. 1) were constructed to predict the effects of the independent variables and their mutual interaction on the response variables. The values of the coefficients for anthocyanin were used for a final predictive equation as follows:

(1)

Table 2a. Experimental design of three factors and one response central composite design and the total anthocyanin content of ultrasonic-assisted banana bract extract.

Run	X1, Solvent: solute	X2, Solvent Conc (ml/100ml)	X3, Sonication Temp (°C)	Y, Anthocyanin Content (mg/100g)
1	5	40	40	40.27
2	15	40	40	51.61
3	5	60	40	46.11
4	15	60	40	54.12
5	5	40	60	42
6	15	40	60	49.54
7	5	60	60	51.5
8	15	60	60	57.49
9	1.59	50	50	42.66
10	18.40	50	50	55.2
11	10	33.18	50	45.51
12	10	66.81	50	51.42
13	10	50	33.18	53.72
14	10	50	66.81	49.72
15	10	50	50	56.74
16	10	50	50	56.08
17	10	50	50	53.5
18	10	50	50	55.01
19	10	50	50	56
20	10	50	50	53.8

The parameter X₁ shows the varying amount of solvent (5–15 ml) as the solute amount was kept constant (0.5 g) during experiment.

Table 2b. Estimated optimum condition for predicted and experimental values of responses.

Factor (Independent variables)	Optimal condition	Predicted Value, anthocyanin(mg/100g)	Experimental value Anthocyanin (mg/100g)
Solvent: Solute	15:0.5
Ethanol Concentration (ml/100ml)	53.97	57.29	56.98
Sonication Temperature (̊C)	49.39

Figure 1. Effect of a) ethanol concentration and solvent solute ratio, b) sonicated temperature and solvent solute ratio, and c) sonicated temperature and ethanol concentration on the yields of total anthocyanin from culinary banana bract.

Based on the above equation, three-dimensional plots that represent the effects of extraction process variables on the anthocyanin from culinary banana bract are illustrated in Figs. 1a–1c. As shown by Dranca et al.,^[45] ultrasound assistance improved anthocyanin extraction, which is attributed to its cavitation phenomena and maximum extraction efficiency at ultrasonic frequency 37.5 kHz. Ultrasonics has a linear positive effect on the extraction of polyphenols and anthocyanins from A. melanocarpa by-products.^[46] However, a known deleterious effect on the active constituents of medicinal plants through the formation of free radicals and consequently undesirable changes in the drug molecules were found at ultrasound energy (> 20 kHz).^[47] In the present study, the sonication frequency was kept constant at 20 kHz and the effect of other parameters on anthocyanin yield at this ultrasonic frequency was observed. The effects of ethanol concentration and the solvent to solute ratio on the anthocyanin contents in the UAE process are depicted in Fig. 1a. Solvent concentration and solvent solute ratio had a stronger effect in the extraction of anthocyanin. There was a linear increase in the TA content with increase in solvent solute ratio at constant ethanol concentration. A larger solvent volume can dissolve the constituents more effectively and thus result in improvement of the extraction yield.^[48] A similar effect of ethanol concentration on anthocyanin was observed. The effect of extraction temperature and solvent solute ratio is elucidated in Fig. 1b.

The combination of temperature with ultrasonic frequency plays an important role in determining the anthocyanin yield. Increase in sonication temperature at constant solvent solute concentration resulted in an increase in the anthocyanin content up to a certain limit of the tested range. However, further increase of extraction temperature resulted in a decrease of the TA content. Fig. 1c illustrates that there was a linear increase in TA with the increase of sonication temperature at a fixed extraction concentration up to a certain limited range, while a rise in ethanol concentration at a fixed extraction temperature also led to a marked increase in TA. Similar linear and quadratic effects of extraction variables in UAE of anthocyanin from mulberry were also observed by Zou et al.^[36]

Optimum extraction conditions

Based on our findings, the predicted UAE conditions were 15:0.5 solvent to solute ratio, 53.97 ml/100ml ethanol concentration, and 49.39°C temperature for the TA (57.29 mg/100g). The R² value of the model was 0.934, R²-adjusted value was 0.876, F value was 15.91, and the p-value was < 0.0001, which represent that the model had adequately represented the actual relationship among the parameters chosen. Under the above-mentioned conditions, the experimental TA content (56.98 mg/100g) agreed well with the predicted value (Table 2b).

Identification of anthocyanins by HPLC

Anthocyanins pigments extracted from culinary banana bract were separated and identified by HPLC (Fig.2). The results revealed the detected types of anthocyanin pigments from culinary banana bract viz., cyanidin-3-O-glucoside (peak 2) and peonidin-3-O-glucoside (peak 5) and represented 4.86% and 7.17% of the total area, respectively. However, the intense peak 4 may be ascribed to cyanidin-3-rutinoside based on the literature.^[6]

Figure 2. Identification of compounds for anthocyanin pigments extracted from culinary banana bract.

Hygroscopicity

The hygroscopicity of the encapsulated pigment powder was found to 28.89 ± 0.09% (Table 3). Similar results were also observed in spray-dried blackberry juice using maltodextrin 20DE with inlet temperatures of 160°C and 180°C and ranged from 28.73% to 29.51%,^[49] whereas slight variation was observed in barberry anthocyanins with maltodextrin as the wall material.^[50] The hygroscopicity values inversely increased with moisture content such that a lower powder moisture content indicated higher hygroscopicity.^[51]

Table 3. The properties of encapsulated pigment powders.

Hygroscopicity (%)	Solubility (%)	Encapsulation efficiency (%)
28.89 ± 0.09	84.10 ± 0.20	96.15 ± 0.31

(Values represent as mean± SD).

Solubility

The solubility of the encapsulated powder was found to be 84.10±0.20 % (Table 3). Maltodextrin of different dextrose equivalent (DE) is commonly used because of its higher water solubility and it contains more hydrophilic group because of the lower molecular weight.^[52] Solubility of the encapsulated anthocyanin with maltodextrin (10 DE) varied (82.79–87.42%) with inlet air temperature from 140°C to 180°C and the highest powder solubility (87.42%) and dispersibility (86.45%) were observed at 180°C inlet air temperature, which are in line with the present study.^[53]

Encapsulation efficiency

The efficiency of encapsulated anthocyanin is presented in Table 3. Robert et al.^[54] reported that the encapsulation efficiencies of maltodextrin-encapsulated pomegranate juice and maltodextrin-encapsulated pomegranate ethanol extract ranged between 89.4–100 and 96.7–100%, respectively. The encapsulation efficiency of encapsulated powder in this study was found in this range and therefore encapsulated powder with good encapsulation efficiency was obtained. Encapsulation efficiencies are also related to the shelf life of the anthocyanin content in the powder.^[55]

Particle size and microstructure

SEM micrographs of encapsulated anthocyanin powder are shown in Fig. 3. The particles of powder that were spray dried at 170°C air inlet temperature with 20°Brix feed solid levels evinced the particle size ranged from 2 to 10 µm approximately. The spray-dried capsules containing maltodextrins looked like smooth spheres but were not uniform, showing minimum agglomeration and dents on the surface, more porous structure, and were well distributed. Similar surface morphology was also observed in the SEM analysis of microencapsulated barberry’s anthocyanin.^[56] SEM analysis of the spray-dried powders of black carrot anthocyanin pigments containing various wall materials showed the particle size ranging from 3 to 20 µm approximately with a smooth spherical shape.^[32] SEM structures also elucidated that the particle size of the microencapsulated anthocyanin pigment present in Garcinia indica ranged from 5 to 50 µm with smooth spheres and are in line with the present study.^[38]

Figure 3. SEM micrographs of anthocyanin-encapsulated spray-dried powder: a) 2000X and b) 5000X.

Storage stability

Stability of anthocyanin, antioxidant activity, moisture content, and colour changes in spray-dried microencapsulated powders stored at 30°C and 75% RH for a period of 21 days were evaluated after every 7 days of interval.

Anthocyanin content

The anthocyanin content of encapsulated pigment powder decreased gradually and decreased by 44% at the end of 21 days at 30°C and 75% RH (Table 4a). The increase of temperature led to a faster anthocyanin degradation, since these pigments are highly thermo-sensitive. This negative influence of temperature on anthocyanin stability has been observed by many researchers. Pacheco-Palencia et al.^[57] evaluated the anthocyanin stability in the whole, semi-clarified, and clarified açai pulp, and verified a degradation rate 3.5 times higher when the samples were stored at 20°C compared to a storage temperature at 4°C. Ersus and Yurdagel^[32] studied the stability of microcapsules of black carrot anthocyanin and verified a loss of 33% after 64 days of storage at 25°C, whereas at 4°C the loss was 11%. The faster anthocyanin degradation at higher temperature can also be related to the presence of sugars and proteins, which can result in the Maillard reaction, and generally occurs during food processing at high temperatures or during food storage for a long time.

Table 4a. Effect of storage of encapsulated pigment powders on anthocyanin content, DPPH radical scavenging activity (%), and moisture content.

Time (days)	Anthocyanin (mg/100g)	DPPH radical scavenging activity (%)	Moisture content (%)
0	57.64 ± 0.71^d	67.08 ± 0.22^c	5.72 ± 0.30^a
7	52.41 ± .93^c	65.73 ± 0.46^b	7.12 ± 0.10^b
14	36.97 ± 0.79^b	61.76 ± 0.37^a	13.42 ± 0.51^c
21	32.30 ± 0.31^a	61.44 ± 0.01^a	20.26 ± 0.45^d

Mean with different superscript letters in the same column represent a significant difference at p < 0.05, values represent mean ± SD; n = 3.

DPPH radical scavenging activity

The antioxidant activity of spray-dried pigment powder decreased with prolongation in storage period and decreased by 8.4% at the end of 21 days of storage (Table 4a). A slow initial decline of antioxidant activity was also mentioned by Bhattacherjee et al.^[58] in spray-dried anola powder up to 5 months (995.1–921.2 mmol g⁻¹). Decrease of the antioxidant capacity during storage might be ascribed to the decrease of anthocyanin content and was also observed in spray-dried bayberry powders.^[59]

Moisture content

Moisture content of the encapsulated pigment powder increased gradually and reached up to 71.8% at 21 days of storage period (Table 4a). The rate of moisture uptake by encapsulated pigment powder depends on the water activity inside the package headspace as well as package permeability.^[60] Moreover, maltodextrin has a high number of ramifications with hydrophilic groups containing shorter chains, and thus can easily bind to water molecules from the ambient air during powder handling.^[50]

Colour characteristics

The initial colour parameters L*, a*, b*, C*, and H° values for encapsulated anthocyanin powder are shown in Table 4b. There was a gradual decrease of the lightness and red colour, visually observed in spray-dried powder during storage and verified by the similar decrease of a* and L* values. However, the increase in yellowness was observed at 7, 14, and 21 days, respectively (Table 4b). The changes of redness (a*) might be attributed to the degradation of anthocyanins during storage.^[61] The reduction of spray-dried betacyanin pigment content during storage was also observed by a reduction in the values of a*, which indicated a decrease in red colour intensity in the stored samples.^[62]

Table 4b. Effect of storage of encapsulated pigment powder on colour characteristics.

Days	L*	a*	b *	C*	H ̊
0	61.38 ± 3.44^b	21.53 ± 2.0^b	–0.08 ± 0.23^a	21.52 ± 2.0^c	0.19 ± 0.01^a
7	60.16 ± 1.11^b	20.26 ± 0.29^b	0.64 ± 0.05^b	20.3 ± 0.26^b	1.79 ± 0.15^b
14	59.15 ± 0.16^b	19.56 ± 0.39^b	0.69 ± 0.01^b	19.57 ± 0.39^b	2.01 ± 0.04^b
21	55 ± 1.50^a	16.63 ± 0.61^a	1.06 ± 0.03^c	16.46 ± 0.62^a	3.69 ± 0.20^c

Mean with different superscript letters in the same column represent a significant difference at p < 0.05, values represent mean ± SD; n = 3.

Conclusion

The present study revealed that culinary banana bract is an excellent source of anthocyanin. The RSM based on CCD was successfully employed to optimize the ultrasonic-assisted extraction and 56.98 mg/100g anthocyanin was obtained. Anthocyanin extracted from banana bract under the optimum condition was further purified and two major constituents, viz. cyanidin-3-O-glucoside and peonidin-3-O-glucoside, were characterized by HPLC. The pigment powder was encapsulated by spray drying and showed acceptable hygroscopicity, suitable solubility, and good encapsulation efficiency. Particle size of the spray-dried encapsulated powder was found in the range of 2–10 µm with a smooth spherical morphology as evidenced from SEM. In addition, anthocyanin content and free radical scavenging activity decreased and the moisture content increased significantly during storage at 30°C and 75% RH. Colour change as revealed by L*, a*, and b* indicated a decrease in anthocyanin stability during storage and studies on moisture sorption isotherm of the encapsulated anthocyanin powder for determining the proper storage condition will help improve its stability and in the development of value-added foods.

Funding

Financial help received as MAN Fellowship from UGC, New Delhi, GoI, during the study is duly acknowledged.

Additional information

Funding

Financial help received as MAN Fellowship from UGC, New Delhi, GoI, during the study is duly acknowledged.