Characterization of spray-dried Gac aril extract and estimated shelf life of β-carotene and lycopene

Overview of the experimental program

Figure 1 shows a schematic diagram of spray-dried Gac aril powder’s production. Briefly, the Gac aril was separated from the whole Gac and kept frozen. The Gac aril was mixed with highly branched cyclodextrin as a drying aid at 5% for spray drying. The condition of inlet temperatures (140 °C, 160 °C, and 180 °C) of spray drying was varied. The spray-dried Gac aril samples were stored at different temperatures (30 °C, 45 °C, and 55 °C) for 45 days. The spray-dried Gac aril samples were then analyzed for physicochemical properties, carotenoid content, and morphological analysis. The results of β-carotene and lycopene were calculated for the spray-dried Gac aril’s shelf-life period.

Purchase of Gac fruit and Custer Dextrin™

Fresh Gac fruit was purchased from a local market in Bangkok, Thailand. Highly branched cyclodextrin (HBCD, Custer Dextrin™) was obtained from a reputable commercial outlet in Bangkok, Thailand.

Fresh Gac fruit aril preparation

Gac fruit’s surface was cleaned with tap water and dried at room temperature. The preparation technique was followed by Kumkong, Thumthanaruk & Banjongsinsiri (2015). The whole fruit was cut and separated from the red aril containing the seeds. The seeds were removed entirely, and the viscous aril pulp was filtered through the cheesecloth. The viscous aril juice was packed in a polypropylene bag and kept frozen at −20 °C until use. The frozen aril was thawed at room temperature for further experiments.

Production of spray-dried Gac aril powder

The preparation of spray-dried Gac aril was modified the method of Kha, Nguyen & Roach (2010). For each experimental run, the red viscous aril juice (500 g) was blended in distilled water (2.5 L) in a ratio of 1:5. The resulting juice was centrifuged using a high-speed centrifuge (Sorval; Dupont, Wilmington, DE, USA) at 8,000 g at 20 °C for 15 min and filtered twice using a No.1 Watchman filtered paper. Next, the filtered Gac aril juice was then concentrated by a rotary evaporator (R-200; Buchi, Flawil, Switzerland) until the soluble solid was 15° Brix. The concentrated Gac aril juice was mixed with HBCD at 5% (w/v) until the HBCD was completely dissolved. The mixture was dried using a spray dryer (B-290; Buchi, Flawil, Switzerland). Three inlet temperatures (140 °C, 160 °C, and 180 °C) of drying air flow rate, compressor air pressure, and feed rate were set at 473 L/h, 40 m³/h, and 3 mL/min, respectively. After the drying process, the Gac aril powder was collected and packed in a zip-lock bag wrapped with aluminum foil and immediately stored in a desiccator at room temperature. The spray-drying process of Gac aril was carried out in duplicate.

Analysis

Water activity (a_w)

The spray-dried powders’ water activity was measured using a water activity meter (AquaLab; Decagon Devices, Pullman, W, USA). The water activity meter was calibrated with a saturated salt standard before use. Measurements were performed at room temperature.

Water solubility index (WSI)

The water solubility index of the spray-dried Gac powders was analyzed by Anderson et al. (1969). The Gac aril powder (2.5 g) was added to distilled water (30 mL) in a 40 mL centrifuge tube, mixed well, incubated in an incubator at 37 °C for 30 min, and then centrifuged for 20 min at 10,000 rpm in a high-speed centrifuge (Sorval, Dupont, USA). The supernatant was dried in an oven dryer at 103 ± 2 °C. The WSI (%) was calculated as the percentage of dried supernatant with respect to the original 2.5 g Gac aril sample.

Bulk density

Bulk density was determined by Goula, Adamopoulos & Kazakis (2004). Gac aril powder (2 g) was added into an empty 10 mL graduated cylinder and vortexed on a vortex mixer for 1 min. The bulk density value (g/mL) was calculated by the powder’s mass ratio to the volume occupied in the cylinder.

Determination of total carotenoid content

The total carotenoid content of Gac aril powder was analyzed by the modified method of Kaisangsri et al. (2016). The Gac powder sample of 80 mg and 4 mL of dimethyl sulfoxide (DMSO) was added to the glass tube, incubated at 75 °C in a water bath for 50 min, and cooled to room temperature. After that, the 4 mL n-hexane, containing 0.1 g/100 mL butylated hydroxytoluene, was added, vortexed for 10 s, and left for 30 min. A few (2-3) drops of ethanol were added to precipitate any protein in the hexane phase. The top layer was removed into another tube using a glass pipette. Approximately 0.5 g of anhydrous sodium sulfate was added to the tube and let stand for 15 min to absorb any residual water in the n-hexane solution. Total carotenoid concentration in the hexane solution was then measured absorbance at 450 nm wavelength using a UV–VIS spectrophotometer (UV-1601, Shimadzu, Japan). Total carotenoid concentration was calculated with a standard curve using the known concentrations of β-carotene (0–1 mg/L) in hexane. Triplicate samples were analyzed.

Determination of β-carotene and lycopene content by HPLC

β-carotene and lycopene contents were measured using Ishida et al. (2004) with some modification. Gac aril powder (1 g) was extracted with 5 mL of 40% methyl-tert-butyl ether (MTBE), 50% methanol, and 10% ethyl acetate (v/v) for 1 hr. The extracts were passed through a 0.45-mm poly(tetrafluoroethylene) filter. Throughout these procedures, care was taken to keep samples ice-cold and protect them from light exposure. β-carotene and lycopene content were quantified using a reversed-phase HPLC system, consisting of Agilent 1200 series, Diode-Array Detector, auto-injector, and column temperature regulator. Separations were accomplished using an analytical polymeric Verticep™ Bio C30 column (250 × 4.6-mm). The C30 column was then conditioned with elution solvent at a 1 mL/min flow rate for 50 min. Carotenoids were separated using an isocratic mobile phase of 40% methyl-tert-butyl ether (MTBE), 50% methanol, and 10% ethyl acetate (v/v). Injection volumes ranged from 10 µL. The column temperature was maintained at 28 °C. Samples were detected at 450 nm.

Morphological analysis

Gac aril powders’ surface morphology was evaluated using scanning electron microscopy (JSM-7600F, Jeol, Tokyo, Japan). The samples were briefly coated with 5 nm gold under vacuum by a fine auto coater (JSM-7600F, Jeol, Tokyo, Japan), and the powder morphology was carried out at an accelerating voltage of 1.0 kV by the scanning electron microscopy. The digital images were captured with magnifications of 1000X–5000X.

Determination of shelf-life of β-carotene and lycopene of spray-dried Gac aril powders

Gac aril powder (10 mg) produced with an inlet temperature of 160 °C was kept in closed vials. The vial samples were then stored at different temperatures of 30 °C, 45 °C, and 55 °C for 45 days. Triplicate samples were periodically withdrawn during the storage to measure β-carotene and lycopene contents by HPLC, as described previously.

β-carotene and lycopene content of storage at 45 °C and 55 °C was used to calculate storage study by accelerated shelf-life tests (ASLT), using the following equation (ASTM, 1980; Mizrahi, 2004). Temperature coefficient Q₁₀ and Q₁ are the reaction rate for a different temperature of 10 and 1 °C, respectively, as illustrated in Eqs. (1) and (2). The storage time of β-carotene and lycopene with a 50% reduction at 25 °C, 10 °C, and 4 °C were also calculated by Eq. (3). (1)${\displaystyle {\mathrm{Q}}_{10}=\frac{{\theta }_{\mathrm{s}\left(\mathrm{T}\right)}}{{\theta }_{\mathrm{s}\left(\mathrm{T}+10\right)}}}$ (2)${\displaystyle {\mathrm{Q}}_1={\mathrm{Q}}_{10}^{0.1}}$ (3)${\displaystyle {\mathrm{Q}}_1^{\Delta \mathrm{T}}=\frac{{\theta }_{\mathrm{s}\left(\mathrm{T}\right)}}{{\theta }_{\mathrm{s}\left(\mathrm{T}+\Delta \mathrm{T}\right)}}}$ where θ_s(T) is the storage time at the temperature T (days), θ_s(T+10) isthe storage time at the temperature T+10 (days), and ΔT is the difference in °C between the predicted temperature and accelerated temperature.

Statistical analysis

All analytical measurements were carried out in triplicate. The results were calculated and reported in the mean value and standard deviation. The statistical differences were compared to the mean values using Duncan’s multiple range test of SPSS for Windows software version 25.0.

Effect of inlet drying temperature on the physicochemical properties of Gac aril juice powder

The spray-dried Gac aril juice powders produced at different inlet temperatures (140 °C, 160 °C, and 180 °C) showed slightly different physicochemical characteristics, as seen in Table 1. The moisture contents ranged from 9.89–10.86%. When the inlet temperature was changed from 140 °C to 160 °C, powders’ water activity values decreased from 0.61 to 0.52. No significant change in a_w was obtained when the temperature was increased to 180 °C. The Gac aril solution’s pH was in acidic pH (5.53–5.63). For bulk density, the increased inlet temperature significantly reduced the density. The WSI relates to the solubility of dried powder in water. In this study, the aril powder spray dried with an inlet temperature of 160 °C had a different result of WSI compared to those of spray-dried aril powders using inlet temperatures of 140 °C and 180 °C. Spray drying at 140 °C may not be microbiologically stable as the a_w was higher than 0.60 and might cause aggregation during storage.

Total carotenoid compounds (TCC) of β-carotene and lycopene significantly decreased at increased inlet drying temperature (Table 1). Increased inlet temperatures showed significantly affected color parameters (Table 2). When reconstituted in water, the solution of Gac aril yielded no significant differences in the lightness (L*), redness (a*), and hue (Table 3). The yellowness (b*) and chroma values increased when the inlet temperature increased from 160 °C to 180 °C, but not from 140 °C to 160 °C. The decreased L* and increased b* could be affected by high drying temperature.

Morphology of spray-dried Gac aril powders by SEM

The morphology of the dried particles showed the cluster of spherical particles with a mixed size of greater than 10 µm when the increased temperature was used (Fig. 2). The scar remaining on the particle resulted from the detached particle. No broken particles were seen in all spray-dried samples.

Effect of storage temperature and time on the reduction of β-carotene and lycopene in spray-dried Gac aril powder

The concentration of β-carotene and lycopene content of spray-dried Gac aril powders reduced as affected by the increasing storage temperature and time. At 30 °C, β-carotene and lycopene were reduced from 1.403 ± 0.019 and 0.225 ± 0.004 to 0.117 ± 0.014 and 0.033 ± 0.004 mg/g in samples kept for 42 and 24 days, respectively (File 1). Increased storage temperature shortened the shelf life of β-carotene and lycopene. The β-carotene reductions were 90.887 and 89.950% at 33 and 21 days, and the lycopene reductions were 65.778 and 91.111% at 21 and 18 days when kept at 45 °C and 55 °C, respectively (Figs. 3A–3B and Figs. 4A–4B). The reduction of β-carotene and lycopene was approximately 12% and 10% between 18–21 days of storage, respectively (Figs. 3A and 4A). The Q₁₀ for the reduction of β-carotene and lycopene were 1.112 and 1.135 (Table 4).

The estimation of β-carotene and lycopene storage times if kept at 25 °C, 10 °C, and 4 °C is in Table 5. The regression equations from Figs. 3A–3B and Figs. 4A–4B, assuming a 50% loss of β-carotene or lycopene, were used for this calculation. Results showed that if the spray-dried aril powders were kept at 4 °C, the shelf-life of β-carotene and lycopene were 92 and 357 days (Table 5). Therefore, the retention of β-carotene and lycopene showed a pronounced effect when encapsulated with the Custer Dextrin™.