1. Introduction
Pomegranate (
Punica granatum L.) belongs to the Punicaceae family and is widely grown in many parts of the world, such as Europe, Asia, North Africa, the Mediterranean basin and, in recent times, South Africa [
1,
2]. The increased commercial production of pomegranate from 828 ha in 2016 to 1024 ha in 2019 is highly related to its rich phytochemical compounds in the edible part of the fruit [
3,
4]. Polyphenols, such as flavonoids, condensed tannins and hydrolysable tannins, are major components found in pomegranate arils or juice [
5]. They are the major source of protective compounds that work against the damaging effects of free radicals [
6]. Pomegranate fruit is mostly consumed as fresh juice, flavourings, colourings, concentrates and jellies for recipes [
7]. Epidemiological studies have associated the consumption of pomegranate fruit to a reduced risk of coronary heart disease, diseases that are not transmissible, such as cancer, and diabetes as a result of its high antioxidant capacity [
8,
9]. Pomegranate fruit was noted to be actively used in folk medicine as a result of its high anthocyanin compositions of cyanidin, delphinidin and pelargonidin, which are attributed to the red colour of fruit and aril [
10]. It is essentially useful in the cure of many parasitic diseases such as ulcers, diarrhoea, acidosis, dysentery, and haemorrhage [
11].
Due to its health and nutritional benefits, pomegranate fruit is in demand throughout the year. Thus, the food industry desires a novel process aimed at increasing the shelf-life and improving the pigment stability of pomegranate products. The drying of fruit juice into powder form is a novel way to extend the shelf-life [
12]. As opposed to dried pomegranate arils, pomegranate juice powders have the advantages of easier storage and distribution. Furthermore, the powders can be used as an ingredient to formulate foods. Spray drying is a commonly used method in many food industries for producing food powders and agglomerates [
13,
14]. Along with being an attractive feature of this technological process, the scarce heat damage to the product is essential [
15]. Another challenging factor during spray drying is the clogging of nozzles, especially when drying sugar and acid-rich foods such as honey and natural fruit juices [
16]. The low operating conditions involved in freeze-drying could be an appropriate drying method to produce niche fruit powders from sugar and acid-rich fruit like pomegranate. Freeze-drying is one of the techniques used to produce high-value powder products [
12]. It is a method that results in high-quality dehydrated products due to the low operating temperatures required in the process and the absence of liquid water [
17]. This method reduces thermal damage of nutrients and preserves flavour and colour components of the product [
18].
Studies have reported some factors to be considered during the production of fruit powders: the stickiness of powder particles and safe handling and storage [
19]. Stickiness during drying is mainly due to the high content of sugars such as fructose, glucose, sucrose and acid materials; for example, organic acids such as citric, malic and tartaric acids, which are attributed low molecular weight, contribute more than 90% of solids in fruit juices [
20,
21]. In order to overcome the sticky behaviour of fruit juice powder, high molecular weight carriers or drying aids such as maltodextrin, gum arabic, waxy starch, pectin, vegetable fibres, and starches as encapsulation agents are added [
16,
21,
22,
23]. Studies have shown that carrier agents further preserve some sensitive properties of the food material, such as carotenoids and flavours, and minimise volatile and reactive properties. For instance, mango juice powder obtained through maltodextrin, gum arabic, and waxy starch resulted in characteristic amorphous particles [
24,
25,
26,
27].
Yousefi et al. [
28] reported that gum arabic showed a high colour change and increased glass transition temperature (Tg) of pomegranate powder. Similarly, Seerangurayar et al. [
19] reported that carrier-agent-added date powders had lower hygroscopicity, which offers good storage stability. Fazaeli et al. [
18] reported that additives enhanced the properties of the final product as a result of an increase in Tg and contributed to the high stability of quality attributes of black mulberry juice powder during storage. However, there are limited scientific studies specifically on the processing of pomegranate juice with the use of a freeze-dryer. To further examine the field of application for pomegranate products, this work investigates the freeze-drying of pomegranate juice to evaluate the influence of different carrier agents (maltodextrin, gum arabic and waxy starch) on the physicochemical and technofunctional properties and antioxidant activities of the powders.
2. Materials and Methods
2.1. Raw Material and Sample Preparation
Pomegranate fruit (cv. Wonderful) were harvested at commercial maturity from Blydeverwacht orchard, Wellington, South Africa. The fruit were sorted for uniformity of size, shape, and colour and transported in an air-conditioned vehicle to the Postharvest Technology Laboratory at Stellenbosch University. Fruits were washed, and the juice was extracted using a hand-operated domestic press and frozen at −20 °C for about 24 h.
The fresh juice was thawed and clarified using a centrifuge system (5810 R Eppendorf AG, Hamburg, Germany) at 10,000 rpm for 20 min. The cold, sterile single strength clarified juice with 16.2 °Brix (total soluble solids) was diluted and standardised with distilled water to 12 °Brix and rapidly frozen at −80 °C until experiments were carried out.
In order to obtain a flowable powder from pomegranate juice, a preliminary study was conducted to investigate the amount of carrier that would be added to the pomegranate juice. Each of the carriers (maltodextrin—Sigma Aldrich Co., St. Louis, MO, USA; gum arabic—Sigma Aldrich Co., France; waxy starch—Sigma Aldrich Co., USA) was incorporated in 100 mL pomegranate juice at a range between 10 to 40 g to select a suitable concentration of carrier agent. A 30 g concentration of (maltodextrin, gum arabic or waxy starch)/100 mL pomegranate juice was observed to produce a flowable powder, which was added after standardisation. The mixture was homogenised using a laboratory homogeniser for 5 min [
28].
2.2. Freeze-Drying Procedure
The pomegranate juice was placed in a 90-mL specimen jar and frozen in a static-air freezer at −80 °C. Freeze-drying of frozen samples was carried out in triplicates. A specimen jar containing the samples was carefully taken to a laboratory-scale freeze-dryer (VirTis Co., Gardiner, NY, USA) operating at condenser temperature −85 °C and pressure 18 mTorr and drying continued for 72 h. Dried samples were removed from the freeze-dryer and ground by electrical blender into free-flowing powder (
Figure 1). The pomegranate juice powders (PJPs) were transferred and sealed in plastic bags in a desiccator that contained phosphorus pentoxide to prevent moisture absorption from the surrounding air until further analysis.
2.3. Yield, Water Activity and Physicochemical Attributes of PJP
2.3.1. Powder Yield Determination
The percentage yield of powder was calculated based on the fresh weight [
29]
2.3.2. Determination of Water Activity and Moisture Content
The water activity (aw) of PJP was determined with an electronic dew point water activity meter (CH 8853 Novasina AG, Lachen, Switzerland). The final moisture content of the PJP was measured using a moisture analyser (KERN DBS 60-3 Balingen, Germany) at 120 °C.
2.3.3. Colour Measurement
Colour of PJP was determined by direct reading using a chromometer (Minolta model CR-200, Osaka, Japan) to obtain the colour values:
L* (brightness/darkness),
a* (redness/greenness), and
b* (yellowness/blueness). The measurements were taken at three different times from a colourless petri dish and averaged. The maximum for ‘
L*’ value is 100 (white), and the minimum is zero (black). The colour attributes chroma
C*, hue angle
h° and total colour difference (TCD) were calculated [
1,
30].
L*, a* and b* represent the value after drying at each treatment level and results were expressed as means ± SE of the determinations obtained.
2.3.4. Determination of Total Soluble Solids (TSSs), Titratable Acidity (TA) and pH
Five grams of PJP were extracted in 50 mL distilled water. For 5 min, the mixture was vortexed with the use of a vortex and sonicated for 15 min in an ultrasonic bath (Separation Scientific, Cape Town, South Africa). This was followed by centrifugation at 10,000 rpm for 25 min and recovery of the supernatant for TSS, TA and pH measurements. TSS measurement was determined with the use of a digital hand refractometer (model PT-32; ATAGO, Tokyo, Japan) blanked with distilled water. For TA, 2 mL of supernatant was diluted in 70 mL of distilled water and titrated against 0.2 N of sodium hydroxide (NaOH) to a pH of 8.2 with the use of a Metrohm 862 compact titrosampler (Herisau, Switzerland).
2.4. Technofunctional Characterisation of PJP
2.4.1. Solubility
Solubility (%) was determined using the Eastman and Moore method [
24] and modified slightly. One gram of the sample was uniformly dispersed in H
2O of 50 mL and distilled in a vortex for 30 s. At 3000 rpm for 5 min under 25 °C, the solution was carefully placed in a tube and centrifuged. A 25 mL aliquot of the supernatant was transferred to preweighed Petri dishes and the sample was immediately dried at 105 °C for 5 h. Solubility (%) was determined by subtracting the initial weight from the final weight divided by the initial weight.
2.4.2. Hygroscopicity
Hygroscopicity was calculated according to [
31], with slight modifications. Two grams of the sample were placed inside a hermetic bottle that was controlled with NaCl-saturated solution in a constant relative humidity chamber (MLR-352 H Versatile Environmental Test Chamber, Kyoto, Japan) set at 68.9% RH and 25 °C [
32]. The weight of the sample was calculated to validate the condition for equilibrium between the samples and the environment. The hygroscopicity was expressed as % moisture on wet basis (w.b.).
2.4.3. Bulk Density
In a 100 mL graduated cylinder, PJP (20 g) was weighed and carefully dropped 10 times from a height of 15 cm using a rubber mat. The bulk density was determined by the division of the mass of powder and the volume estimated from the cylinder [
33].
2.4.4. Water- and Oil-Holding Capacity Determination
According to Jalal et al. [
34], the water-holding capacity (WHC) and oil-holding capacity (OHC) of PJP were calculated. A mixture of 25 mL distilled water or sunflower oil and 250 mg of dry sample were slightly vortexed and left at room temperature for 1 h. The solution was placed in a tube and centrifuged at 4000 rpm for 10 min at 25 °C, after which the residue was weighed. The water/oil holding capacity was expressed as g of water/oil held per g of sample. The formula to calculate WHC/OHC is as follows:
2.4.5. Particle Size Distribution
The particle size of the powder was determined with the use of a laser light diffraction instrument (Mastersizer S, model MAM 5005; Malvern Instruments, Malvern, UK). Under magnetic agitation, a small amount of PJP was homogenised in 99% isopropanol, following careful monitoring of the distributed particle size, which was taken in three successive measurements. De Brouckere’s mean diameter was used to express the particle size and the mean diameter over the volume distribution. This is mostly used to characterise a particle [
35].
2.4.6. Microstructure
The microstructure of PJP was examined with the use of a scanning electron microscope (X-Max 51, Oxford Instruments, Concord, MA, USA). SEM images of powder were obtained from uniformly mixed powder samples. Under a high vacuum condition, the samples were coated with a very thin layer of gold. This is often used to provide a reflective surface for the electron beam. The gold coating was carried out in a sputter coater (ACE200 LEICA Mikrosysteme GmbH, Vienna, Austria) under a low vacuum condition while inert argon gas was present. Subsequent viewing of the gold-coated samples was carried out under the microscope.
2.5. Phenolic Contents and Antioxidant Capacity
2.5.1. Determination of Total Phenolic Content (TPC)
TPC was determined by the Folin-Ciocalteu method using a methanolic extract of PJP [
1]. In a test tube, the supernatant (0.05 mL) was mixed with 0.45 mL (50% methanol), followed by adding 0.5 mL Folin-Ciocalteu after 2 min. The mixture was then vortexed and kept in the dark for 10 min before adding 2% Na
2CO
3 and further incubation for 40 min in the dark. The absorbance of each sample was read at 520 nm in a UV-vis spectrophotometer (Thermo Scientific Technologies, Madison, WI, USA) against a blank containing 50% methanol. Absorbance was compared with a standard curve (Gallic acid, 0–10 mg), and results were expressed as mg gallic acid equivalent per gram dry matter (mg GAE/g DM).
2.5.2. Total Anthocyanin Content
Total anthocyanin content (TAC) was quantified differentially by using the pH method [
36]. In triplicates, 1 mL extract was mixed with 9 mL of pH 1.0 and pH 4.5 buffers in separate conditions. In pH 1.0 and 4.5 buffers, absorbance was measured at 520 and 700 nm, expressing the result (cyanidin 3-glucoside) using Equation (6).
where
A = absorbance,
ε = cyd-3-glucoside molar absorbance (26,900), MW = anthocyanin molecular weight (449.2), DF = dilution factor, and
L = cell path length (1 cm). Final results are expressed as equivalent per gram dry matter (mg C
3gE/g DM).
2.5.3. Radical-Scavenging Activity (RSA)
In triplicate, the RSA assay was carried out according to Fawole and Opara [
1]. Briefly, in test tubes, an aqueous methanolic extract of PJP (0.015 mL) was diluted with methanol (0.735 mL) and methanolic DPPH solution (0.75 mL, 0.1 mM) was immediately added. The mixtures were incubated in the dark and at room temperature for 30 min. The absorbance was measured at 517 nm using a UV-vis spectrophotometer (Helios Omega, Thermo Scientific, Waltham, MA, USA) and compared with the standard curve (Trolox equivalent, 0–2.0 mM). The free-radical activity of PJP was expressed as Trolox equivalent (mM) per gram dry matter (mM TE/g DM).
2.5.4. Ferric-Ion Reducing Antioxidant Power (FRAP)
The antioxidant power of PJP was measured using the calorimetric method, according to [
1,
37]. The FRAP working solution was freshly prepared in mixtures of 300 mM acetate buffer (50 mL), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ; 5 mL) and 20 mM ferric chloride (5 mL) at 37 °C. Diluted aqueous methanolic PJP extracts (0.15 mL) were added to 2.85 mL of the FRAP working solution in triplicates, followed by incubating the mixture in the dark for 30 min. Measurement of the absorbance at 593 nm was carried out to monitor the reduction of the Fe
3+-TPTZ complex to a coloured Fe
2+-TPTZ complex at low pH by PJP extracts. Trolox (0–10 mM) was used for the calibration curve, and the results were expressed as Trolox (mM) equivalents per gram dry matter (mM TE/g DM).
2.6. Statistical Analysis
Data were analysed using STATISTICA (Statistica 13.0, StatSoft Inc., Tulsa, OK, USA) and presented as means ± standard error. All analyses were done in triplicates. Data were subjected to analysis of variance (ANOVA), and means were separated according to Fisher’s LSD test at a level of significance of 95%. The graphical presentations were processed by using GraphPad Prism software 4.03 (GraphPad Software, Inc., San Diego, CA, USA). Principal component analysis (PCA) was carried out using XLSTAT software version 2012.04.1 (Addinsoft, Bordeaux, France).
4. Conclusions
The use of three carrier agents (maltodextrin, gum arabic and waxy starch) in the production of freeze-dried PJP was investigated. The results indicated that maltodextrin was more effective in enhancing the yield as well as the physicochemical properties of the PJP, such as colour, TSSs and TA. Similarly, maltodextrin and gum arabic performed better as carriers agents in enhancing the solubility of freeze-dried PJP compared to waxy starch. Maltodextrin was better in the preservation of phenolic content and antioxidant capacity of PJP. Therefore, it could be inferred that maltodextrin resulted in the best carrier agent that retained biochemical activities and maintained the technofunctional properties in the production of freeze-dried pomegranate powder. This study has shown that maltodextrin is the most suitable carrier agent for the formulation or fortification of pomegranate-based food products for baking, candies and ice-cream. This study reports the results of powder produced in a laboratory-scale freeze-dryer. However, a scale-up can be investigated in order to produce, on an industrial scale, powders with similar characteristics. Moreover, further research is required to investigate the storability and optimisation of PJP.