The Effect of Juicing Methods on the Phytochemical and Antioxidant Characteristics of the Purple Prickly Pear ( Opuntia ﬁcus indica )—Preliminary Findings on Juice and Pomace

: Prickly Pear (PP) is often overlooked due to its’ short shelf-life. Juicing may improve marketability but often affects quality, thereby warranting investigation. Purple PP (whole (WF) and ﬂesh (FF)) was juiced using blenders; stick (SB) and jug (JB); and juicers; commercial (CJ) and cold-pressed (CP). Juices and methanolic (70%) The best methods overall for juice were SB (FRAP), JB (TPC, TBC), CJ (TFC) and CP (DPPH, VitC); and for pomace extracts; SB(FRAP), JB (TPC, VitC), CJ(TFC), and CP (TBC, DPPH).


Introduction
The Prickly Pear (PP) is a relatively short-seasoned fruit of the Opuntia spp. cacti family. The cactus is known for its' uses as; a source of food, traditional medicine and as a hedge or ornamental plant [1][2][3][4]. This cacti family is known globally, because of its' thriving nature, particularly in arid and semi-arid regions, which in Australia, has led to classification as a 'Weed of National Significance' (NWOS), apart from the Opuntia ficus indica species [4][5][6]. Although the species has been studied in other countries, the Australian PP has minimal research regarding the nutritional content [7].

Study Design
The effect the various juicing and blending techniques on PP fruit juice and pomace (waste) was determined, in triplicate, by investigating the phytochemical content, specifically antioxidant characteristics (DPPH, FRAP and VitC) and bioactive content (Total Betalain; Phenolic and Flavonoid content). Physicochemical properties (pH, titratable acidity (TA), Total Soluble Sugars (TSS; Brix • ) were also determined and compared.
Fresh purple PP fruits from the same location and harvest were prepared from fresh and randomly assigned to 'pools', representing a typical harvest and variations in maturity available to consumers. Once prepared, pooled fruits were juiced as; whole fruits (WF), and as peeled fruit flesh (FF), containing seeds. Purple type fruits were juiced using four juicing techniques; jug-blender, stick-blender, commercial-juicer and cold-press juicer, following a 'homogenise and filter' protocol [35]. Double-strained juice and waste were collected and stored separately in 50 mL light protected tubes at −18 • C.

Sample Preparation
Fresh purple PP fruits of the same morning harvest (February 2017) were donated from 'Chiron Health Products' (Victoria, Australia) farm (36 • 25 37.2 S; 143 • 37 55.2 E) and within 24 h of the morning harvest, transported on ice to the University of Canberra Food Science Laboratory. On receival, fresh samples were washed under cold running water and gently rubbed with gloved hands to remove the prickles. Fruits mass of randomly pooled samples (~1 kg) was recorded and juiced either as; WF (randomly pooled); or appropriately peeled (randomly pooled). The prepared samples were stored separately, in darkness at −18 • C prior to juicing due the fruit short harvest season and shelf life [33][34][35][36][37][38].

Titratable Acidity (TA), Total Soluble Solids (TSS) and pH
The juice and pomace of WF and FF samples were thawed at 4 • C prior to analysis. Samples that were exceeding the measurement (due to upper limits of instrument detection) were diluted in DI water (18 MΩcm) prepared on the day, prior to analysis.
Titratable Acidity (TA) of diluted juices (10%; WF and FF), were determined by titration against 0.1M NaOH until pH of 8.1 was achieved, where values were expressed as a percentage [42,43]. NaOH was aliquoted until a pH of 8.1 was achieved, determined by a pH meter 'Butech Instruments pH 700 (Metter Toledo, Port Melbourne, VIC, Australia).
The pH of the various juices was determined using a pH meter (Butech Instruments pH 700; Metter Toledo, Victoria, Australia).
The Total Soluble Solids (TSS) were determined using handheld refractometer (OPTi Digital Hand Held Refractometer, Bellingham and Stanley, UK) following the procedures previously described and expressed as • Brix [42].

Extraction of Phytochemicals from Pomace
The phytochemical content of thawed pomace after juicing, were extracted according to Moussa-Ayoub et al., 2014 [44]. Briefly, 1 g of pomace was suspended in 10 mL of Methanol (70% v/v) and sonicated (FXP4, Ultrasonics, Sydney, NSW, Australia) for 15 min at maximum power [44]. Samples were cooled on ice and stored in darkness at 4 • C until further analysis.  [45] and values were calculated using the formula proposed by Moßhammer et al., 2006 [46]. Briefly, the absorbance's of diluted fruit juices and methanolic pomace extracts (10% v/v) a were measured at 482, 532 and 600 nm (NovaspecPlus, GE Healthcare, Castle Hill, NSW, Australia).
TBC was calculated as a sum of BE and IE values, expressed as mg/mL of total betalain content [45,46].

Antioxidant Characteristics (DPPH and FRAP)
The antioxidant characteristics of thawed juices (WF and FF) and methanolic pomace extracts were measured spectrophotometrically (NovaspecPlus, GE Healthcare, Castle Hill, NSW, Australia) following the Free Radical Capacity (DPPH) and antioxidant capacity by ferric reducing antioxidant power (FRAP) [50,51] methods. All values were expressed in µM TE Trolox (TE) equivalents/g.

Statistical Analysis
All samples were analysed in triplicates, where values were represented as 'mean ± standard deviation' if the variance was small, or 'mean ± standard deviation (median)' if not normally distributed. Values were reported as per 'Rule of Decimals' [55], exceptions to this include limitations of the equipment. Juicing methods were compared based on phytochemical content using a Kruskal-Wallis test, where statistical significance was defined as α < 0.05. The differences between (i) style of juicing (blend vs. juice-type processing) and (ii) fruit preparation (WF vs. FF) were investigated using Mann-Whitney U test. Correlations were determined to assess the strength and direction between two antioxidant or bioactive characteristics within PP fruit juice or pomace extracts using a Kendall-tau B (two-tailed; τ) between investigated compounds. Comparison of the four juicing methods based on phytochemical content were determined using the 'Statistical Processing Package for Social Sciences' (SPSSv23, IBM, Australia).

Titratable Acidity
Titratable Acidity (TA; %) is an indicator of total acid content and reported a better predictor of acid contents impact on flavor, than alternate measures including pH [56]. According to Pyo et al. (2014) the main acids in fruit juices to include citric, malic and ascorbic acids [35]. Titratable acidity (TA; %; Table 1) in Australian grown purple PP fruit ranged between 0.14 ± 0.01% (FF; cold-press)-0.24 ± 0.09% (FF; commercial-juice). The results reported in Table 1 were consistent with those of previous literature [37,57] with the highest content using the in commercial juicer (FF; 0.24 ± 0.09%) followed by cold-pressed (WF; 0.23 ± 0.01%), jug-blend (FF; 0.2 ± 0.05%) and stick-blend (WF; 0.19 ± 0.03%). Fruit components processed did not differ significantly (WF vs. FF; p = 0.778), nor did type of processing methods (blend vs. juice; p = 0.664). Although, Pyo et al. (2014) reports different associations when investigating the predominate acids of fruit juices; to be greater in blended kernel fruit juices for malic and citric acid contents, rather than juices, which contained greater amounts of ascorbic acids [35].

Total Soluble Solids (TSS)
Total Soluble Solids (TSS; • Brix), in addition to firmness, are measures of internal quality attributes in determining fruit maturity [58]. The TSS is reported to increase with ripeness and is used as an indicator of soluble simple sugars or acids, reported to predominantly include sugars; glucose, fructose and sucrose; or acids; citric, ascorbic and mallic acids [59]. TSS of purple PP fruit (Table 1) is reported to range between 10.3 • Brix (WF; Jug-blend) to 13.8 • Brix (WF; stick-blend). No significant differences were observed between the fruit components (WF vs. FF; p = 0.426) or between the different types of processing (blend vs. juice; p = 0.147).

pH
In addition to TA, pH can be used in sensory analysis to detect and establish the potential for bitterness and composition of different food products [56,60]. It is well established that pH varies depending on juice composition, particularly with relations to common fruit acids (malic, citric, lactic and ascorbic) [60]. The pH, (Table 1) ranged in purple PP juice between 5.5 (FF; commercial-juice)-5.97 (FF; cold-pressed). No significant differences were determined between fruit preparation methods (WF vs. FF; p = 0.725) or style of processing (blend vs. juice; p = 0.964).

Total Polyphenol Content (TPC) of Prickly Pear Fruit Juice and Methanolic Pomace Extract
Total Phenolic Content (TPC) includes the cumulative content of compounds including subgroups of flavonols and betalains [61]. Phenols are a large group of bioactive-phytochemicals that exhibit numerous effects including antioxidant and anti-inflammatory effects [35]. A study by Pyo et al. (2014) has demonstrated TPC and TFC can be significantly affected by juicing methods (juice vs. blend; p < 0.05) in Korean kernel fruits [35] however, this was not investigated in the juice waste product commonly referred to as pomace.
The juice of the Australian-grown purple PP fruit contained TPC, ranging from 1516 µg GAE /mL (FF; stick-blend-3031 µg GAE /mL (WF; jug-blend) ( Table 2). The highest TPC were in jug-blended juices, followed by commercially-juiced (FF; 2015 ± 142 µg GAE /mL), cold-press (1524 ± 125 µg GAE /mL) and lastly, stick-blended juices (WF; 2cold-press (2301 ± 89.8 µg GAE /mL).The findings of this study indicate that juicing method has a significant effect on purple PP fruit juices, processed as both; WF (p = 0.022) and FF (p = 0.025). Additionally, style of processing (blending vs. juicing) also had an effect (p = 0.001), although fruit preparation (whole fruit vs. fruit flesh) did not (p = 0.100). The pomace generated during the juicing of purple PP was tested as a methanolic extract for the bioactive composition and antioxidant activity. The TPC of the purple PP pomace ranged between 23.0 ± 17.1 µg GAE /mL (WF; jug-blend) to 44.4 µg GAE /mL (WF; stick-blend). The TPC was affected by juicing method, in juice derived from FF (p = 0.015) but not in juice derived from WF (p = 0.015). Additionally, style of processing (blend vs. juice) was also found to have an effect (p = 0.039) overall on TPC in pomace but fruit preparation did not (p = 0.100-0.700).

The Total Flavonoid Content (TFC) of Prickly Pear Fruit Juice and Methanolic Pomace Extracts
A study by Pyo et al. (2014) reported that the TFC of Korean kernel fruit juices vary (p < 0.05) between juice-type and blender-type processing [35]. Additionally, same study has identified that there were significant differences between methods of preparation in blended juices, in three of the four kernel fruit investigated [35]. The findings of our study (Table 2) indicate that juicing methods affects the TFC of purple PP fruit juices, that were processed as FF (p = 0.038), but not WF (p = 0.091). Pyo et al. (2014) observations in kernel fruit were not consistent with our findings in PP fruit juices, as no significant differences in TFC were drawn between blender-style and juice-style processing (p = 0.590), nor preparation of fruit (WF vs. FF; p = 0.100-0.400). The TFC composition of the purple PP ranged between commercially-juiced 266-428 µg CE /mL (FF; WF). Conclusively, the highest TFC contents of the purple PP juices were reported to be using the commercial juicer (428 ± 68.6 µg CE /mL) followed by; jug-blender (396 ± 45.6 µg CE /mL), stick-blender (396 ± 51.6 µg CE /mL), and cold-pressed (382 ± 54.4 µg CE /mL).

Total Betalain, Betacyanin and Betaxanthin Content of Prickly Pear Fruit Juice and Methanolic Pomace Extracts Total Betalain, Betacyanin and Betaxanthin Content of Prickly Pear Fruit Juice
The betalain composition is associated with yellow (betaxanthins) to red (betacyanin's) colored plant materials and it is widely studied in variety of different plants including; beetroot, pomegranate and amaranth [62]. The variation in composition of betalain subgroups (betaxanthin and betacyanin's) allow for the wide variation in colored plant materials today [62]. The betalains are a bioactive group of compounds [62] with in vitro and in vivo antioxidative [63][64][65][66] and antilipidemic [67][68][69][70] effects. The broader stability of betalains, when compared to other natural dyes, has allowed these compounds to be utilised as food dyes, and also promotes the use of purple PP juice as an alternative during the product development [71].
The findings of this study (Table 2; Figure 1) indicate that the juice derived from purple Australian PP contains the TBC with ranges from 0.269 mg/100 mL (WF; stick-blend) to 0.709 mg/100 mL (FF; jug-blend). The juices derived from different fruit components (WF vs. FF) did not demonstrate significant differences in TBC. Juice processing (p = 0.053-0.086) and style of processing (p = 0.100) did not affect the TBC significantly. The highest contents per method were observed in juices derived from fruit flesh for jug-blended (0.560 mg/100 mL) juices followed by; commercially juiced (0.555 mg/100 mL) stick-blend (0.491 mg/100 mL), and lastly, cold-pressed (0.377 mg/100 mL).  The juice derived from the purple PP juice (Table 2; Figure 1) contained 0.086-0.563 mg BE /100 mL of betacyanin and 0.161-0.319 mg IE /100 mL of betaxanthin. In the presented study, the juicing methods were shown to have a significant effect on betalain constituents, betacyanin, juiced as WF (p = 0.029) and FF (p = 0.036) but not betaxanthin. Style of juicing methods (p = 0.178-551) did not influence either sub-groups content, nor did fruit preparation (p = 0.100-0.200). The highest contents of betacyanin were measured in FF juiced using the jug-blender (0.563 ± 0.038 mg BE /100 mL), followed by the juices obtained by; commercial juicer (0.273 ± 0.040 mg BE /100 mL), cold-press (0.182 ± 0.045 mg BE /100 mL), stick blend (0.172 ± 0.013 mg BE /100 mL). The highest betaxanthin contents were reported in juices derived after the use of the stick-blender (0.319 ± 0.017 mg IE /100 mL) followed by juices obtained after the use of; jug-blender (0.309 ± 0.020 mg IE /100 mL), commercial-juicer (0.219 ± 0.012 mg IE /100 mL) and cold-press juiced (0.182 ± 0.045 mg IE /100 mL).

Total Betalain, Betacyanin and Betaxanthin Content of Prickly Pear Methanolic Pomace Extracts
The TBC was found to be in the range of 0.030-0.089 (FF; WF; cold-press). Like the values obtained for fruit juice, the TBC of PP pomace extracts had significant variations between juicing techniques in fruit juiced as FF (p = 0.034) but not as a WF (p = 0.430). The style of processing was also found to not affect the TBC in pomace extracts (p > 0.05). Method of fruit preparation (p = 0.100-0.700) and style of processing (p = 0.551) were found to not be significantly affected. The highest contents, by juicing method, were reported in cold-pressed juice extracts (WF; 0.898 ± 2.29 mg/100 mL) followed by; commercially-juiced (WF; 0.047 ± 0.064 mg/100 mL), jug-blended (WF; 0.040 ± 0.04 mg/100 mL) and stick-blended (FF; 0.014 ± 0.01 mg/100 mL).

Antioxidant Characteristics (DPPH, FRAP and Vitamin C)
Phytochemical-bioactive compounds exhibit antioxidant activities and have antioxidant effects [61]. Antioxidant characteristics; Free Radical Scavenging Activity (DPPH; µM TE ), Antioxidant Capacity by Ferric Reducing Antioxidant Power (FRAP; µM TE ) and Vitamin C (VitC; mg AA /mL) provide an indication of potential antioxidant activity of the bioactive compounds within the PP fruit, including those investigated ( Table 2). The study by Pyo et al. (2014) reported that in three of the four Korean kernel fruits investigated, juicer-processed juice had a higher antioxidant content than juices processed by blenders [35]. It was also reported fruits juiced as a WF to exhibit a higher antioxidant activity [35] probably due to the higher antioxidant content in skin. Our findings (Table 3) however, indicated that style of processing (blend vs. juice) only affected DPPH (juice p = 0.039; pomace; p = 0.028) and FRAP (pomace; p = 0.024). Similarly, the different juicing methods did have significant effects on DPPH (pomace; p = 0.016-0.024), FRAP (juice p = 0.016-0.024; pomace; p = 0.015-0.023) and VitC (juice p = 0.015-0.016; pomace p = 0.016-0.022) antioxidant characteristics. Additionally, preparation of the fruit (WF vs. FF) predominately did not affect the measured characteristics. Supporting associations between bioactives content and antioxidant characteristics were also demonstrated within PP fruit juice and pomace extracts, when compared by Kendall's tau-B analysis of correlations. The PP juices were found to demonstrated significant inverse relationships between; TFC and TBC (p = 0.001; τ = −0.044); and TBC and VitC (p = 0.001; τ = −0.637). Interestingly, this was not observed in the PP fruit pomace although there were significant correlations in PP pomace extracts between; TPC and DPPH (p = 0.003; τ = 0.440); TPC and VitC (p = 0.011; τ = 0.440); and TFC and FRAP (p = 0.001; τ = 0.519).

Free Radical Capacity (DPPH) of Prickly Pear Fruit Juice and Methanolic Pomace Extracts
The DPPH values in the juice of the Australian grown purple PP did not vary in content between the studied juice methods, nor were they affected by style of processing ( Table 3). Preparation of fruit (WF vs. FF) did not have an effect (p = 0.100) on DPPH characteristics. The findings of our study disagreed with those of Pyo et al. (2014) who found significant variations in DPPH content between types of juicing methods, blend and juice, in three of the four fruits studied [35]. Variations between the two studies may include investigation into different fruits, protocol of processing methods or differences in analysis. Conclusively, Australian grown purple PP fruit juices varied in DPPH characteristics between 667 µM TE (FF; stick-blend)-851 µM TE (FF; cold-pressed). The highest content, with consideration of processing methods were reported in juices obtained by cold-press (FF; 851 ± 437 µM TE ), followed by juices obtained by commercial juicer (FF; 753 ± 15.1 µM TE ), jug-blender (709 ± 105 µM TE ) and lastly, stick-blender (667 ± 126 µM TE ).

Antioxidant Capacity by Ferric Reducing Antioxidant Power (FRAP) of Prickly Pear Fruit Juice and Methanolic Pomace Extracts
The FRAP values in juice ranged between 210 µM TE (WF; jug-blend)-383 µM TE (FF; stick-blend) ( Table 3). The different juicing methods had a significant effect (p < 0.05) on FRAP characteristics regardless of fruit preparation method (WF vs. FF) although, variation in content was not significant when considering fruit preparation (p = 0.100) but not style of processing (p = 0.039). Pyo et al. (2014) however did concluded differences in FRAP characteristics between juice-type and blend-type processing [35]. The highest FRAP levels per method were reported in juices obtained by stick-blender (FF; 365 ± 2.52 µM TE ) followed by; cold-pressed juicer (333 ± 1.53 µM TE ) and lastly, commercial -juicer (WF; 9400 µM TE ), Methanolic pomace extracts (Table 3) were also found to have significant variations (p = 0.015-0.023) in FRAP levels between juicing methods, regardless of fruit component (WF vs. FF). Like the investigated juice, investigation into PP pomace extracts were found to be significantly affected in FRAP characteristics by style of processing (blend vs. juice) (p = 0.024), but again not fruit preparation (p < 0.05). The highest FRAP levels were observed in pomace derived after the juicing performed by stick-blender (FF; 382 µM TE ), followed by jug-blender (FF; 365 µM TE ), cold-press juicer (FF; 333 µM TE ) and lastly, commercial juicer (FF; 297 ± 0.578 µM TE ).

Vitamin C Content of Prickly Pear Fruit Juice and Methanolic Pomace Extracts
The VitC content in fruit and vegetable juicing is often studied, mainly due to its sensitivity to degradation during processing and storage [72]. The study by Pyo et al. (2014) investigated a number of fruits to conclude that a greater VitC content was measured in juices processed by a juice-type processing method, rather than blend-type method [35]. The findings of our study indicate that juicing methods have a significant effect (p = 0.015-0.016) on VitC content of the juice derived from the purple PP fruit (Table 2). Interestingly, there was a non-significant effect observed in VitC in PP fruit juices related to the style of processing method (blend vs. juice; p = 0.630), nor did the fruit preparation methods (WF vs. FF) (All p = 0.100). The VitC content of the PP fruit juice ranged between 1.94 (FF; stick-blend) to 0.167 mg AAE /mL (WF; cold-pressed). Conclusively, the highest content of VitC were in juices derived using a cold-pressed juicer, followed by stick-blender (WF; 4.28 mg AAE /mL), jug-blending (WF; 0.135 ± 0.001 mg AAE /mL) and lastly, commercially-juiced products (WF; 0.121 ± 0.001 mg AAE /mL). The VitC content in the pomace obtained after the various juicing methods (Table 3) shared similar patterns to that of the juice itself. Significant differences in VitC content were observed between juicing methods (p = 0.016-0.022) but again this was not significantly affected by type of processing (blend vs. juice; p = 0.266) or fruit component (WF vs. FF) (All p = 0.100). The VitC content of the juice pomace ranged between 1.94 mg AAE /mL (FF; stick-blend)-4.52 mg AAE /mL (WF; jug-blend). The VitC content of the pomace differed to the juice in that the highest contents were reported in the pomace obtained after the jug-blending juicing, followed by; stick-blender (WF; 4.28 ± 0.159 mg AAE /mL), commercial-juicer (WF; 3.53 ± 0.144 mg AAE /mL) and lastly, cold-press juicing (FF; 2.98 ± 0.633 mg AAE /mL).

Conclusions
The PP fruit production season is relatively short, comprising of only few months when the production of this largely underutilised fruit is quite abundant. Therefore, the utilisation of the fruit for the development of different and adequate 'on-site' fruit products is highly desirable. The fruit of the PP is very suitable for juicing and often various processing methods, such as juicing, affect the quality of the product, including the antioxidant, bioactive and overall nutritional content. The findings of this study indicated that the physiochemical parameters varied between juicing methods, where method of fruit preparation (WF vs. FF) did affect physicochemical parameters.
Phytochemical composition in both juice and pomace were also studied to have significant variations in content between the different techniques. Interestingly, type of method (blend vs. juice) significantly affected only TPC, DPPH and FRAP and was also significantly correlate in PP fruit pomace extracts. Similarly, pomace extracts also hosted correlations between; TPC and DPPH; and TPC and VitC; and between TFC and TBC, in PP juice. The most appropriate juicing method is dependent on prioritised compound for preservation. In addition, the use of juice waste has potential to extend to development of nutraceuticals and/or supplements.