Characterization of Juice Extracted from Ultrasonic-Treated Red Pitaya Flesh

Abstract

Red pitaya is a tropical fruit rich in phytonutrients essential for human health. The fruit is savored either through its processed products or raw consumption. This study aimed to assess the physicochemical properties of red pitaya juice extracted from ultrasonic-pretreated flesh. The red pitaya flesh was cut into cubes and subjected to different durations (20, 40, and 60 min) of ultrasonic treatment. The total soluble solids, pH, and titratable acidity of the juice were unaffected by pretreatment durations. Compared with the non-ultrasonic pretreated sample, the levels of organic acids, phenolics, and anthocyanins in red pitaya juice increased (p < 0.05) following 60 min of ultrasonic pretreatment. The duration of sonication pretreatment influenced the antioxidant activity of red pitaya juice. This pilot study shows that pretreatment of the red pitaya flesh using ultrasound enhances the quality of its juice.

1. Introduction

Pitaya, also known as dragon fruit, strawberry pear, and pitahaya, belongs to the Cactaceae family. It is non-climacteric and is widely cultivated in tropical and subtropical areas of the world. There are two different genera, namely, Hylocereus and Selenicereus. Red pitaya (Hylocereus polyrhizus), white pitaya (Hylocereus undatus), and yellow pitaya (Selenicereus megalanthus) are commercial fruit varieties. Among these, the red pitaya fruit has received particular attention because of its high antioxidant capacity [1]. Red pitaya fruit has an intense red-colored peel, is 13–15 cm in length and 10–15 cm in diameter, and weighs 200–600 g [2,3]. The round-shaped fruit contains numerous small edible seeds embedded throughout the red-violet flesh.

Red pitaya fruit has a short shelf-life after harvest. It is prone to decay, softening, and degradation of nutrients during storage, regardless of whether it is refrigerated. One way to preserve this fruit is to process it into other products, such as juice, powder, jam, jelly, and wine. Red pitaya juice is one of the most valorized products with wide recognition and attractiveness [2]. It is extracted from the flesh of the fruit. Proximate analysis revealed that red pitaya juice contained 85.05–89.98% moisture content, 8.42–12.97% carbohydrate, 0.41–1.45% protein, 0.54–1.19% ash, and 0–2.65% dietary fiber [4]. Fat was not detected [4]. Scientific evidence has shown that the intake of red pitaya juice could ameliorate liver and cardiovascular damage [5] and anemia [6]. Meanwhile, the growing demand for fresh-like products, including fruit juice, has encouraged the food industry to search for food processing methods that preserve and/or enhance the nutritional and organoleptic characteristics of the products.

One of the industrial challenges in red pitaya juice production is to enhance the extraction of functional compounds, such as phytochemicals, from the fruit’s flesh. Pretreatment of the flesh or mash with ultrasound, enzymes, or heat often results in an increase in the extraction of these compounds in the juice [7]. The application of ultrasonic technology in mash pretreatment has received considerable attention lately. Ultrasound waves are organized mechanical vibrations traveling through a solid, liquid, or gas medium. They disrupt cell walls and membranes, facilitating the extraction of intracellular components. Ultrasound duration has a great impact on the quality of fruit extracts [8]; however, limited studies have investigated the effect of ultrasonic pretreatment durations on the quality of fruit juice. A recent study suggested that sonication was a useful approach for preserving bioactive compounds and preventing color degradation of clear red pitaya juice [1]. Ultrasonic treatment of the aforementioned juice was performed after juicing. Little to no published research has investigated the effects of juice obtained from the sonication of red pitaya flesh before juicing. In this sense, the objective of our research was to investigate the physicochemical properties of red pitaya juice extracted from flesh pretreated with ultrasound for different durations (20, 40, and 60 min).

2. Materials and Methods

2.1. Sample Preparation

Red pitaya (Hylocereus polyrhizus) fruits without mechanical damage or visible signs of infection were purchased from a wet market located in Kampar, Perak, Malaysia in February 2022. The fruits were packed into new plastic bags and transported to the laboratory of the Faculty of Science, Universiti Tunku Abdul Rahman. A knife was used to cut the pitaya into halves, and the peels were manually removed. The pitaya flesh was cut into cube sizes of 2 cm³ and separated into four groups of different sonication times (0, 20, 40, and 60 min). Each group had three replicates. An ultrasonic bath (Elma E100H, Baden-Wurttemberg, Germany) was used to sonicate the pitaya cubes at different time intervals (20 min (PT 20), 40 min (PT 40), and 60 min (PT 60)) under an output power of 600 W and a frequency of 37 kHz. The temperature of the sonication process was maintained at 30 °C by water circulation and the thermostat on the ultrasonic bath. The use of mild temperature during the sonication process is an optimal approach to enhance the extraction of phytochemical compounds and, at the same time, prevent the decomposition of these thermolabile compounds [9,10]. Hence, a mild sonication temperature of 30 °C was applied in the present study. The juice extraction of the ultrasonic-treated and non-ultrasonic-treated pitaya cubes was carried out using a commercial juice extractor (Philips, Jakarta, Indonesia). The juice was sieved through gauze, transferred to sterilized air-tight media bottles wrapped with aluminum foil, and kept at 4 °C until further use within 15 days. Juice extracted from pitaya cubes without ultrasonic pretreatment (PT 0) served as the control in the study

2.1.1. Total Soluble Solid (TSS), pH, Titratable Acidity, Color, and Viscosity

The TSS and pH were measured using a portable refractometer (Atago, PAL-3, Tokyo, Japan) and a digital pH meter (Eutech pH 2700, Waltham, MA, USA), respectively. The titratable acidity was measured using the standard procedure of the AOAC 942.15 [11]. The color properties were measured using a colorimeter (Konica Minolta CM-600d, Osaka, Japan). The results are expressed as L* (black/dark (0) to white/light (100)), a* (green (−) to red (+)), and b* (blue (−) to yellow (+)). The chroma, hue angle, browning index, and total color difference (ΔE) were determined using the equations [12] below:

$$\mathrm{Chroma}=\sqrt{{\left( a^{*}\right)}^2+{\left(b^{*}\right)}^2}$$

(1)

$$Hue angle={\tan }^{-1}\left(\frac{b^{*}}{a^{*}}\right)$$

$$\mathrm{Browning} \mathrm{index}=\frac{100 \left(Z-0.31\right)}{0.172} , \mathrm{where} \mathrm{Z}=\frac{\left(a^{*}+1.75L^{*}\right)}{\left(5.645L^{*}+a^{*}-0.3012b^{*}\right)}$$

$$\mathsf{\Delta }\mathrm{E}=\sqrt{ {\left(\mathsf{\Delta }{L}^{*}\right)}^2+{\left(\mathsf{\Delta } a^{*}\right)}^2+{\left( \mathsf{\Delta }{b}^{*}\right)}^2}$$

The ΔE value was classified as a small difference (ΔE < 1.5), distinct (1.5 < ΔE < 3), and very distinct (ΔE > 3) [12]. The viscosity was measured using a viscometer (Brookfield DV2T, Berwyn, IL, USA) equipped with an RV02 spindle (Brookfield DV2T, Berwyn, IL, USA) at a speed of 100 rpm for one minute.

2.1.2. Organic Acids, Total Phenolic Content (TPC), Total Anthocyanin Content (TAC), and 2,2-Diphenyl-1-picrylhydrazyl (DPPH)

Exactly 1 mL of red pitaya juice was added into a falcon tube containing 5 mL of 60% methanol and vortexed for 1 min. The mixture was then centrifuged at 10,000 rpm for 20 min. The supernatant layer (extract) was used for the determination of organic acids, TPC, TAC, and DPPH.

The organic acids were determined according to the method of Scherer et al. [13]. The supernatant layer was initially filtered through a 0.45 μm syringe filter. Exactly 20 μL of the filtered sample was injected into a high-performance liquid chromatography (HPLC) (Shimadzu LC-10AD, Kyoto, Japan) equipped with a UV–Vis detector (Shimadzu SPD-20A, Kyoto, Japan) set at 210 nm. An RP-18 column (150 mm × 4.6 mm with a particle size of 3 μm; Restek Pinnacle, Bellefonte, PA, USA) was used, and the temperature of the column oven was 30 °C. The mobile phase consisted of 0.01 mol/L monopotassium phosphate buffer solution (pH 2.60, adjusted with o-phosphoric acid) with isocratic elution at a flow rate of 0.5 mL/min. Identification and quantification of the organic acids were based on the external standards of the organic acids kit (Merck, Darmstadt, Germany).

The TPC was analyzed according to the method of Choo et al. [14]. Exactly 100 µL of the extract was pipetted into a test tube containing 400 μL of distilled water and 500 μL of 1 N Folin–Ciocalteu reagent. One mL of sodium carbonate solution (7.5%) was added into the test tube after 5 min of incubation at room temperature. After incubating in the dark for 30 min, the absorbance was measured at 765 nm using a spectrophotometer (Thermo Fisher Scientific Genesys 20, Waltham, MA, USA). A gallic acid standard curve was prepared. The results are expressed as mg of gallic acid equivalents (GAE)/100 g juice.

The TAC was evaluated according to the method described by Tan et al. [8]. Exactly 1 mL of the extract was transferred into a falcon tube containing 24 mL of distilled water. The mixture was poured into a cuvette, and the absorbance was measured at 535 nm using a spectrophotometer (Thermo Fisher Scientific Genesys 20, Waltham, MA, USA). The TAC was calculated using the equation below:

TAC = (Absorbance at 535 nm × dilution factor)/98.2

The DPPH was measured using the method of Tan et al. [8]. Exactly 250 μL of the extract was pipetted into a test tube containing 2 mL of methanolic DPPH solution (30 mg/L). The mixture was vortexed and incubated at room temperature for 5 min. The absorbance was measured using a spectrophotometer (Thermo Fisher Scientific Genesys 20, Waltham, MA, USA) set at 517 nm. The percentage of radical scavenging activity (RSA) was calculated using the equation below:

RSA (%) = [(1 − absorbance of sample extract)/absorbance of control] × 100%

2.2. Statistical Analysis

Three repeated experiments were performed on each replicate of a group, and the results were pooled for statistical analysis using IBM SPSS statistical software (Version 20). Data are expressed as mean ± standard deviations. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was conducted to compare the means between groups (PT 0, PT 20, PT 40, and PT 60). The relationship between antioxidant compounds and antioxidant capacity was measured using Pearson’s correlation test, and the corresponding heatmap was visualized using Jamovi software (Version 4.1). A confidence level of 95% was applied in all analyses.

3. Results and Discussion

3.1. Total Soluble Solids, Titratable Acidity, pH, Viscosity, and Color

The sweetness level of fruit juices can be gauged by their total soluble solids (TSS). In

Table 1. Total soluble solids, titratable acidity, pH, viscosity, and color.

Variable	Ultrasonic PreTreatment Time (min)
	0	20	40	60
TSS (% Brix)	4.93 ± 0.06 a	5.03 ± 0.06 a	4.93 ± 0.06 a	5.00 ± 0.00 a
TA (%)	0.08 ± 0.01 a	0.07 ± 0.01 a	0.08 ± 0.01 a	0.08 ± 0.01 a
pH	5.07 ± 0.01 a	5.08 ± 0.02 a	5.05 ± 0.01 a	5.07 ± 0.01 a
Viscosity (mP.a.s)	26.47 ± 0.06 a	26.73 ± 0.12 b	26.57 ± 0.06 ab	26.57 ± 0.06 ab
Color
L*	42.42 ± 0.08 a	41.98 ± 0.09 b	41.77 ± 0.01 c	41.60 ± 0.01 d
a*	11.09 ± 0.34 a	8.87 ± 0.06 b	7.99 ± 0.02 c	7.22 ± 0.21 d
b*	−5.17 ± 0.15 a	−3.91 ± 0.14 b	−3.26 ± 0.03 c	−2.75± 0.08 d
Chroma	12.23 ± 0.38 a	8.63 ± 0.03 b	9.69 ± 0.29 c	7.72 ± 0.23 d
Hue angle	−25.00 ± 0.07 a	−22.19 ± 0.18 b	−23.77 ± 0.17 c	−20.88 ± 0.07 d
Browning index	16.51 ± 0.45 a	12.36 ± 0.03 b	13.53 ± 0.33 c	11.32 ± 0.33 d
∆E	REF	2.59 ± 0.08 a	3.69 ± 0.41 b	4.64 ± 0.02 c

TSS: Total soluble solids and TA: titratable acidity, ΔE: total color differences, REF: reference for total color difference calculation. Means in the same row with discrepant superscripts indicate significant differences at p < 0.05.

, the TSS of red pitaya juice was unaffected by the duration of ultrasonic pretreatment and was in the range of 4.93–5.03° Brix. Greater TSS values were reported in the red pitaya juice obtained by centrifugation (12.4° Brix) [1] and fermentation (28.7° Brix) [15], possibly due to the different sample preparation. The centrifuged red pitaya juice was the clear supernatant layer of the juice [1], whereas our study retained the sediment layers of the juice. Meanwhile, Choo et al. [15] added sugar to the pitaya pulp before subjecting it to fermentation. This might have influenced the final TSS value of the juice. Further study is needed to compare the properties of red pitaya juice extracted using different methods.

Titratable acidity (TA) measures the total acid concentration, and pH measures the hydrogen ion concentration. Both TA and pH are commonly used to estimate the acidity of fruit juice. The TA and pH of red pitaya juice were unaffected by ultrasonic pretreatment. These results were in agreement with those of Cheng et al. [16] on the ultrasonic-treated strawberry purees. The same observations were reported for noni juice [14] and elephant apple juice [17] processed with ultrasonics. The pH value of fermented red pitaya juice (3.13) was lower than that in the present study (5.05–5.08), which might have been due to the increase in lactic acid during the fermentation process.

The red pitaya juice had a relatively high viscosity (26.47 mP.a.s) because of the gelatinous pectin that stayed linked with the flesh. The viscosity of red pitaya juice with 20 min of ultrasonic pretreatment (PT 20) was significantly greater (p < 0.05) than that of the control. No differences (p > 0.05) were observed in the viscosity of PT 40 or PT 60 compared with the control and PT 20 samples. It was found that the pectin content of ultrasonic pretreated tomato waste was increased in the first 30 min but stayed at a constant level after 30 min of treatment [18,19]. Hence, a minor increment in viscosity in PT 20 compared with the control sample might have been due to the breakdown of colloidal molecules, such as pectin, in the cell walls.

The color of fruit juice is associated with its organoleptic quality. L* and a* decreased, while b* increased significantly with prolonged ultrasonic pretreatment (p < 0.05). These results are consistent with previous findings [1]. The decrease in L* and a* might have been associated with the degradation of betacyanin. Meanwhile, the increase in b* might have been associated with the release of dark blue-colored pigment compounds, such as anthocyanins, from the pitaya flesh. The color of the juice became less saturated, as indicated by the chroma, following ultrasonic pretreatment. Compared with the control sample, ultrasonic pretreatment enhanced (p < 0.05) the hue angle of the juice samples. The greater the hue angle value of the juice, the brighter its color [20]. The browning index measures the purity of the brown color, which is an important parameter to gauge the occurrence of enzymatic or non-enzymatic browning reactions during juice processing. In the current work, the browning index of juices isolated from the ultrasound-pretreated flesh was significantly lower (p < 0.05) than that of the control sample. This is because ultrasound can inhibit the enzymes, such as polyphenoloxidase and peroxidase, that are responsible for the browning reaction of fruits [20]. ∆E indicated that the longer the ultrasonic pretreatment of the red pitaya flesh, the greater the color variation of the juice in comparison with the control sample. The same trend was observed in clear red pitaya juice treated with ultrasonication [1]. This might have been due to the ultrasound-induced hydroxylation of the phenolic aromatic ring, which in turn changed the visible spectrum area [21].

3.2. Organic Acids, Phenolics, Anthocyanins, and DPPH Radical Scavenging

Organic acids are one of the main components of fruit juice. Within the experimental conditions, two compounds, namely citric acid (689.19–754.95 mg/100 g) and succinic acid (17.37–40.50 mg/100 g), were identified (

Table 2. Organic acids, phenolics, anthocyanins, and DPPH radical scavenging activity.

Variable	Ultrasonic Pretreatment Time (min)
	0	20	40	60
Organic acid (mg/100 g)
Citric acid	689.19 ± 6.67 a	728.29 ± 35.3 ab	741.88 ± 21.99 ab	754.95 ± 13.58 b
Succinic acid	17.37 ± 1.24 a	19.98 ± 7.31 a	23.85 ± 2.08 a	40.50 ± 0.50 b
TPC (mg GAE/100 g)	37.33 ± 0.83 a	42.67 ± 0.83 b	43.83 ± 0.83 c	46.33 ± 0.83 d
TAC (mg CGE/100 g)	1.37 ± 0.00 a	1.54 ± 0.01 b	1.78 ± 0.03 c	1.98 ± 0.04 d
DPPH (%)	12.36 ± 1.82 a	13.80 ± 1.07 b	15.55 ± 1.22 c	17.54 ± 1.47 d

TPC: total phenolic content, TAC: total anthocyanin content, and DPPH: 2,2-diphenyl-1-picrylhydrazyl. Means in the same row with discrepant superscripts indicate significant differences at p < 0.05.

). Compared with the control, 60 min of ultrasonic pretreatment significantly increased (p < 0.05) the amounts of citric acid and succinic acid by 65.76 mg/100 g and 23.13 mg/100 g, respectively. Similarly, Nguyen and Le [22] reported that the organic acids of juice extracted from the ultrasonic-treated pineapple mash were significantly improved in comparison with those of the juice sample without ultrasonic pretreatment. The increase in the organic acid concentrations could be attributable to the breakdown of cell walls and intracellular structures following acoustic cavitation. During juice extraction, these compounds are released into the aqueous medium, resulting in an increment in their concentrations [14].

Phenolics are secondary metabolites of plants. The TPC of red pitaya juice improved significantly (p < 0.05) with the ultrasonic pretreatment durations (Table 2). Nguyen and Nguyen [23] reported that the TPC of juice obtained from the mulberry mash treated with 60 min of ultrasonication was twofold greater than that of the pressed juice. Our present study indicated that 60 min of ultrasonic pretreatment of the pitaya cubes resulted in a 24% increment in the TPC in the juice. The increase in TPC following ultrasonic pretreatment might be associated with the liberation of bound phenolics following the degradation of cell walls by acoustic cavitation [14]. The quantification of the phenolic profile using high-performance liquid chromatography, nuclear magnetic resonance, or gas chromatography could be conducted to provide insight into the influence of ultrasonic pretreatment durations on the individual phenolic compounds of red pitaya juice.

Anthocyanins are water-soluble flavonoids with antioxidant effects. The TAC of red pitaya juice was enhanced significantly (p < 0.05) with the ultrasonic pretreatment durations (Table 2). Barberry juice extracted from mash pretreated with ultrasound showed a drastic increase in TAC by 105 mg CGE/100 g compared with juice extracted from mash without pretreatment [7]. Anthocyanins are not the main phytochemicals present in the red pitaya [24], hence, the ultrasonic pretreatment of the red pitaya cubes resulted in small increments (0.17–0.61 mg CGE/100 g) in the TAC compared with that in the control. More than 80% of the red pitaya flesh was composed of water [4]; it is possible to expect that the sonication could improve the mass transfer rate, facilitating the release of anthocyanins from the intracellular environment to the in situ water of the flesh [9]. This would result in an increment in the anthocyanin level after juicing.

In the current study, the antioxidant capacity of red pitaya juice extracted from ultrasonic-treated flesh was evaluated using a DPPH radical scavenging assay. DPPH· is a stable free radical and exhibits deep violet color in a methanol solution. The antioxidant compounds in pitaya juice scavenge the free radicals, resulting in a change of color from deep violet to yellow over time. The color change can be evaluated by measuring the absorbance at 517 nm [25]. Our present study indicated that the radical scavenging activity of red pitaya juice was significantly enhanced (p < 0.05) with ultrasonic pretreatment time. A correlation test was conducted to understand the relationship between antioxidant compounds (TPC and TAC) and DPPH of red pitaya juice (Figure 1). Strong positive relationships were observed between TPC and DPPH as well as TAC and DPPH, indicating that phenolics and anthocyanins contribute to the high antioxidant activity in the red pitaya juice. This is consistent with the results found in the literature [26]. Our current study indicates that the duration of ultrasonic pretreatment plays a significant role in extracting the antioxidant compounds from the red pitaya flesh, which could be useful in the food industry to produce an array of high-antioxidant pitaya products, such as juice.

4. Conclusions

Red pitaya juice is available year-round, making it a convenient way to meet daily nutrient requirements. Our present study indicated that L* and a* decreased, while b* increased significantly with prolonged ultrasonic pretreatment. Compared with the control, the levels of organic acids, phenolics, and anthocyanins in red pitaya juice increased following 60 min of ultrasonic pretreatment. Phenolics and anthocyanins corresponded to the high antioxidant capacity of red pitaya juice. Further research comparing the physicochemical characteristics of red pitaya juice obtained from ultrasonic pretreatment with those of juice obtained by other pretreatment methods, such as heat or enzymes, is recommended.