Introduction

Pummelo (Citrus grandis L. Osbeck) of Rutaceae family, is a citrus fruit native to Southeast Asia and the Indo-China regions. It is also known as pomelo, pommelo, shaddock, limau bali and Chinese grapefruit. The fruit is commonly eaten fresh and the taste varies from mildly sweet and bland to sub-acid with a faint touch of bitterness (Morton 1987). Popular variations of the fruits are; PO51 (Sha Thing), PO52 (Tambun) and KK2 (Melo Mas). In 2010, Department of Agriculture Malaysia has introduced a new hybrid known as Ledang variety (PO55), where the fruit is sweeter and has less bitter aftertaste which pummelo are usually known for. In Malaysia, about 1895 ha of pummelo are grown commercially and in 2009, production is estimated to be 8830 metric tonnes (Anim 2012).

However, pummelo is still considered an under-exploited horticultural crop. This non-climatic and non-seasonal fruit is only being harvested in January and September annually and only for the Chinese festive season. As a result of government initiatives production is expected to reach 15000 metric tonnes annually and pummelo farmers have been encouraged to diversify the industry, starting with juice production. With proposed usage of ultraviolet light treatment as an alternative method of pasteurization of pummelo fruit juice, the clarity and viscosity of the juice has to be improved in order to increase the efficiency of the machine. Ultraviolet light treatment is highly dependent on juice’s absorption coefficient, transmissivity of the product, the product profile and the radiation path length (Koutchma and Parisi 2004). Furthermore, with high amount of pectin in the juice, maintaining a stable cloud in fruit juice presents a considerable challenge to fruit processors. In the presence of calcium ions, insoluble calcium pectate is formed in the juice, leading to undesirable precipitation of haze particles. One effective procedure that would assist in extraction, clarification and modification of juice is enzymatic treatment which subsequently leads to higher juice yield (Whitaker 1984).

Fungal enzymes (pectinases) have been known to increase juice yield, assisting in pectin hydrolysis, causing a significant increase in juice yield (Sandri et al. 2011). Pectin substances are structural heteropolysaccharides that occur in the middle lamellae and primary cell walls of plants. Pectinases act on and decrease the intracellular adhesivity and tissue rigidity. The activity of pectinases is also influenced by the physical and chemical parameters that are vital for yield increment. Pectinases are acidic polysaccharides consisting of 3 main classes: polymethylesterase, polygalacturonases and pectate lyase (Prathyusha and Suneetha 2011). Enzymatic treatment of pectinases clarifies the juice cloud while maintaining its stability. The clarified juice is also less viscous, effectively reducing membrane fouling and subsequently flux decline (Rai et al. 2004). Baker and Bruemmer (1970) showed that the cloud in orange juice can be stabilized by use of pectic enzymes containing high level of polygalacturonase activity.

Citrus juice, especially from oranges, grapefruits and pummelos, has long been believed to have a correlation with good health, due to their antioxidant potency and plasma lipid metabolism (Amin et al. 2006). Citrus fruit extracts have also been found to demonstrate anti-cancer, anti-inflammatory, anti-tumour and blood clot inhibition activities (Garg et al. 2001). The health benefits of citrus juice have mainly been attributed to the presence of bioactive compounds, such as phenolic, ascorbic acid and carotenoids. Citrus flavonoids have been said to be responsible for the beneficial effects and such compounds are identified as methoxylated flavones, flavonones and flavonone glucosides (Jayaprakasha and Bhimanagouda 2007).

Naringin, neohesperidin and hesperidin are the major flavonoids formed in Citrus grandis, Citrus paradise, Citrus aurantium and Citrus limon (Kawaii et al. 1999). However, as at the time of writing, identification and/or quantification of flavonoids has been mainly carried out on China’s and Thailand’s pummelo variety (Makynen et al. 2013; Pichaiyongvongdee and Haruenkit 2011; Zhang et al. 2011; Chaiwong and Theppakorn 2010; Tsai et al. 2007;) and very little literature is found on Malaysian variety (Toh et al. 2013; Cheong et al. 2010). Pummelo have been shown to contain coumarins, furocoumarins, flavanones, flavones and flavonols in both free and glycosidic form (Chaiwong and Theppakorn 2010; Zhang et al. 2011). The phenolic constituents of pummelo fruit give out the characteristic flavour and also play a big role in the sensory profile of the juice. It was reported by Alper et al. (2011) that these polyphenols also contributed to haze formation during storage through prior polymerization or condensation leading to the formation of polymeric complexes. Thus with enzymatic clarification, it is expected that the phenolic contents will be affected. The flavonoid profile, too, have been shown to vary with the species and cultivars and the profile can be used to distinguish between the different varieties. However, the effect of enzymatic treatment on pummelo’s fruit juice clarification has not been systematically studied and no literature, at the moment of writing, has specifically focused on the phenolic contents of Malaysian varieties of pummelo fruit juice.

The present study aims to identify and quantify the phenolic contents of two Malaysian cultivars of pummelo fruit juice post enzymatic treatments using a High Performance Liquid Chromatography Diode Array Detection (HPLC-DAD) and Folin Ciocalteu’s method. This study will be useful for the food industry for the production and exploitation of natural flavonoids in pummelo fruit juices to minimize the usage of synthetic antioxidants in order to have more natural, organic foods. In addtion this study will discuss methods of increasing the clarity and cloud stability of the juice.

Materials and methods

Standards and chemicals

Milli-Q water (Millipore, Bedford, MA, USA) was used in all work. Pectinex XXL (EC: 4.2.2.10) was procured from Novozymes (Bagsvaerd, Denmark). Folin-Ciocalteu reagent and sodium carbonate (Na2CO3) used in total phenolic measurements were purchased from Merck (Darmstadt, Germany). Citrus pectin and sodium chloride (NaCl) used in pectin methylesterase activity assay were purchased from Sigma-Aldrich (St Louis, MO, USA). HPLC-grade methanol and formic acid (Merck, Darmstadt, Germany) was used after filtration through a 0.45-µm pore size membrane filter and sonication for 30 minutes. Phenolic acids (gallic, caffeic, chlorogenic, p-coumaric, ferulic and sinapic acids) and flavanones (naringin, naringenin, narirutin and hesperidin) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Preparation of juice sample

Seven days old (from the day of harvest and approximately 3 months maturity) pummelo fruits (Citrus Grandis L. Osbeck) of Ledang variety (PO55) were obtained from Jabatan Pertanian Daerah Segamat, Johor Darul Takzim, Malaysia and Tambun variety (PO52) were obtained from Jabatan Pertanian Daerah Kinta, Ipoh, Perak Darul Ridzuan, Malaysia. Pummelo fruits were kept in a chiller room at 10 °C until they were used for experimental works. Prior to peeling, the pummelos were washed with deionized water to eliminate any microbial contaminations to the fruits. The thick fruit skin (flavedo and albedo) was peeled manually after a one cm-deep horizontal incision was made using a knife to reveal the juicy segments. Pummelo was then peeled into segments and the inner skin of each segment was peeled and discarded. The white membrane surrounding the juicy segments, including seeds, were removed completely. The juice was extracted using a screw type extractor and nylon-filtered to remove the pulps. This process was repeated 3 times to optimize the juice extraction. The juice is then kept in a five-litre HDPE bottle in a deep freezer at− 20 °C until further experimental works.

Enzymatic treatment

Two hundred millilitres of pummelo fruit juice was subjected to different enzymatic treatment according to respective varieties. The optimized range of the variables for enzymatic treatment conditions were based on the preliminary RSM experiments conducted earlier (unpublished source). The independent variables were the incubation time, incubation temperature and concentration of enzyme used. The temperature of the enzymatic treatment was adjusted to the desired level using a constant temperature water bath. The pH of pummelo juice was kept at its natural pH value of 4.0 and was excluded from the RSM experimental design as the pH is considered optimal for exo-pectinase (Kashyap et al. 2001). The enzymatic treatment used was the modified method of Rai et al. (2004). Heat treatment to inactivate enzyme was excluded and replaced with flash-freezing the suspension at− 2 °C for 5 min in an ice bath. This method was done to avoid unwanted changes to the flavour and juice characteristics from the conventional thermal effect to inactivate the pectinase (Sandri et al. 2011; Molinari and Silva 1997). It is understood that the enzyme was not fully inactivated thus, all subsequent experiments were done in a controlled temperature (below 20 °C), which is the lowest temperature for pectinase activation. The treated juices were then centrifuged at 3,000 g for 10 min and the supernatant was collected. After that, the juice was filtered through a Whatman no. 1 filter paper using vacuum suction at 25 mmHg. The filtrate was collected for further analysis. Control samples, where enzymatic preparations were substituted by distilled water, were made for all assays.

Standard chemical analysis

The total titrable acidity was assessed by a digital auto titrator (785 DMP Titrino, Metrohm, Switzerland). Ten millititres of juice was mixed with 40 ml of distilled water. Titratable acidity of the juice was obtained by titration 0.1 mol/L NaOH to the endpoint of pH = 8.5. Titratable acidity content was expressed as citric acid percentage reference by the following equation;

$$ Titrable\; acidity=\frac{EP1\times 0.064\times C30\times 10\mathrm{o}}{C00} $$

EP1 = mL NaOH up to the endpoint (pH = 8.5).

C30 = molarity of NaOH.

COO = sample volume in mL.

The pH value was measured using a digital Jenway 3505 pH meter (Bibby Scientific Limited, Staffordshire, UK). Total soluble solids were measured as Brix using a digital refractometer (AR-2008, Kruss, Germany). Ascorbic acid was determined using a digital autotitrator. The titrator operates using the Bi-voltametric titration method with 2, 6-dichlorophenolindophenol as the titrant (Shui and Leong 2004). Ten millilitres of distilled water, 15 ml of oxalic acid and 1 ml of sodium acetate solution (10 %) were pipette into a beaker. Then, 10 ml of sample juice was added to the mixture and titrated with 0.001 mol/L of 2, 6-dichlorophenolindophenol. Colour of pummelo fruit juice was determined using a Spectrophotometer UltrascanPro (D65 Hunter Lab, VA, USA). The L* value was recorded for the change in lightness of the juice. Juice clarity was measured according to the method of Krop and Pilnik (1974). Ten millilitres of juice was shaken and centrifuge at 3,600 g for 10 min to remove pulp and coarse cloud particles. Percent transmittance was determined at 660 nm using a UV–VIS spectrophotometer (Shimadzu, Kyoto, Japan) with distilled water used as a reference. Total phenols were determined by the Folin-Ciocalteu procedure (Singleton and Rossi 1965) as follows; 1 mm of 10-fold diluted Folin-Ciocalteu reagent was added to a 0.2 ml 1:50 juice sample. After 1 min, 0.8 ml of 7.5 % (w/v) Na2CO3 solution was added and the mixture was shaken. After 2 h, the absorbance was measured at 765 nm using a UV–VIS spectrophotometer (Shimadzu, Kyoto, Japan). The phenolic content was expressed as gallic acid equivalents in mg/L (GAE mg/L).

Liquid chromatographic analysis of phenolic compounds

Samples were filtered through a 0.45-m pore size membrane before injection. An Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) operated by Windows NT based ChemStation software was used. The HPLC equipment was used with a diode array detector (DAD). The system consisted of a binary pump, degasser and auto sampler. The column used was a Thermo Scientific C18 column (Waltham, MA, USA): 250 × 4.6 mm × 5 μm. The injection volume of the fruit juice was 20 μL per sample. The mobile phase consisted of 2 solvents: Solvent A, 0.1 % water in formic acid and Solvent B, 100 % methanol. Phenolic compounds were eluted under the following conditions (Shui and Leong 2004) with modifications: gradient conditions 0 to 20 % solvent B (0 mins), 20 to 30 % solvent B (0 to 20 mins), 30 to 50 % solvent B (20 to 30 mins), 50 to 90 % solvent B (30 to 35 mins), 90 to 20 % solvent B (35 to 40 mins), followed by washing and reconditioning of the column. The separations were performed with a flow rate of 1 mL/min, which was directly injected in the ESI source, without any splitting. The column temperature was maintained at 25 °C. The analysis time was 40 min. The HPLC method was tested on 10 phenolic compounds (coumaric acid, sinapic acid, chlorogenic acid, gallic acid, ferulic acid, caffeic acid, narirutin, hesperidin, naringenin and naringin). The polyphenols standard solutions (10 μg/mL) were prepared in methanol. The ultra-violet-visible-spectra (scanning from 200 to 600 nm) were recorded for all peaks. Triplicate analyses were performed for each sample. The identification of phenolic compounds was obtained by using authentic standards while quantification was performed by external calibration with standards.

Statistical analysis

The data obtained in the study were analyzed using Minitab Release 14 (Minitab Inc., PA, USA). Analysis of variance was performed by ANOVA procedure and significant differences (p <0.05) between means were determined using Tukey’s multiple range test. All analyses were done in triplicate.

Results and discussion

Standard chemical analysis

A study done by Zulueta et al. (2007), stated that the differences between fruits parameters were due to the composition and the storage or processing conditions. From Table 1, it can be concluded that compositions of pummelo fruit highly depended on the choice of cultivar. Ledang juice showed higher concentration of total soluble solids (°brix) and ascorbic acid compared to Tambun variety. Vitamin C derived from ascorbic acid, found abundantly in pummelo fruit juice, was found to have a concentration of 537.47 and 509 mg/L respectively in Ledang and Tambun variety. The findings are consistent with the values of ascorbic acid found in nutrient composition tables of Malaysian Foods which states that the vitamin C content of edible portion of pummelo was 448 mg/L. Vitamin C acts as antioxidant against polyphenol oxidation reaction which subsequently leads to non-enzymatic browning. Ascorbic acid not only restores nutritional value lost during processing, but also contributes to products’ appearances and palatability. Browning takes place when enzymes called polyphenolases which occur naturally in fruit tissue catalyze the oxidation of phenols, to form compounds called quinines. The quinines then polymerize to form melanins, which causes the brown pigment. Ascorbic acid can inhibit browning reaction by reducing the quinines back to the original phenol compounds (Kimball 1999). Thus, it is pragmatic that the amount of ascorbic acid in fruit juice must be higher than the total phenolic acids found in a fruit juice. The empirical Brix/acid ratio, derived by dividing the acid-corrected and temperature-corrected Brix by the percent titratable acidity as citric acid, is one of the most commonly used indicators related to the quality of citrus fruit or the maturity index (Xu et al. 2008). It was found that the Brix/acid ratio for Ledang and Tambun variety is 12.54 and 9.11 respectively. According to Kimball (1999), consumers of citrus juice generally prefer a soluble solids/acid ratio of 15 to 18, depending on the product and individual taste. The rate of ratio increase is linked to the rate of changes in the Brix and acid levels in the fruit. In this study, the level of Brix/acid ratio showed a higher total acidity in both varieties. It is noted that the change in acid levels have greater effect on the Brix/acid ratio. Thus, both varieties will be recommended to blend with other varieties with higher sugar content to make it suitable for juice processing. Hasegawa et al. (1992) also reported that the acidity of citrus plays a great role in terms of bitterness, because under low pH conditions, the A-ring lactone (LARL) can be converted into limonin, a bitter limonoid making juice undesirable. The rate of this reaction is primarily heat dependent, with some effect of the juice pH. It was also reported that pectin and other complex components of citrus juice increase this solubility. These values were recorded for the purpose of having an initial value of juice quality prior to enzymatic treatment.

Table 1 Physicochemical properties of ledang and tambun pummelo fruit juice
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Total phenolic compounds

Total phenolic compounds for Ledang and Tambun varieties including the enzymatically treated are given in Table 2. Phenolic present in fruits and vegetables have received considerable attention because of their potential antioxidant activity. Phenolic compounds undergo a complex redox reaction with the phosphotungstic and phosphor-molybdic acids present in the Folin-Ciocalteu reagent (Prasad et al. 2005). It was found that Ledang variety had a considerably lower total phenolic contents compared to Tambun variety (p <0.05). As this study uses a pink-flesh Tambun variety, it is assumed that the amount of lycopene, carotenoid and anthocyanins are higher, naturally increasing the phenolic compounds in comparison to Ledang, a white-flesh fruit. The anthocyanin content as reported by Kelebek et al. (2009), have higher phenolic activity than in blond juices. It could also have been the difference in cultivation location and practices, maturity age, harvesting conditions and stage.

Table 2 Total phenolics content (GAE mg/L ± Standard Deviation) of ledang and tambun pummelo fruit juice
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Both enzymatically clarified juices, have shown a slight decrease (p >0.05) of total phenolic contents for both varieties. This slight difference might be due to a number of reasons; (1) Temperature of enzymatic clarification treatment (Table 1): temperature that was used was not high enough to warrant an optimum enzymatic reactivity which was reported (Sun et al. 2013) in the range of 50 °C. The author also noted that the pH of the juice during the clarification treatment played an important role, where the optimum pH was found to be 5.8. pH and both varieties of pummelo fruit juice was kept at 4.0 to maintain the acidity level of the juice. pH of the juice can cause different folding and unfolding structures and ionization in the active site of prototropic groups (2) It was also concluded that individual phenolic compounds of whole fruits might vary in their browning rate as studied by Rocha and Morais (2002). The low content of total phenolic compounds indicate that they had been subjected to oxidation (browning) to a greater extent since the oxidation products were precipitated during the process since enyzyme degradation of pectins lead to an improvement of juice clarity and to spontaneous flotation and clarification effect. (3) It could also be due to storage conditions after the enzyme treatment where the samples were kept in a deep freezer at a temperature of − 20 °C. As reported by Patthamakanokporn et al. (2008) storage temperature could induce the reaction of the endogenous polyphenol oxidase and total phenolic contents of guava fruits were found to gradually decrease throughout the 3 months storage.

Liquid chromatographic analysis of phenolic compounds

Four common phenolic compounds reported in plants are; cinnamic acid and its derivatives (e.g. chlorogenic acid), flavans (e.g. catechins), anthocyanins where compounds are usually red, blue and purple, soluble in water and highly detected (e.g. cyanidin) and flavanones (e.g. naringin). A total of six phenolic compounds in Ledang and Tambun pummelo fruit juice (Table 3), including hydroxycinnamic acids and flavanones compounds, were identified by comparing retention times and HPLC-DAD chromatograph peaks with those of the standards (Fig. 1). The total amount of phenolic compounds found through liquid chromatographic analysis was 196.7 and 394.3 mg/L in non-enzymatically treated Ledang and Tambun variety respectively, with naringin being the main contributor of the total phenolic compounds. A high difference (p <0.05) of total phenolic contents for the control juice could be affected by the species, growing season, ripening, and environmental factors such as light, temperature as well as, processing treatment. Pink-fleshed Tambun could also be the reason for the high amount of phenolic content found in comparison to Ledang.

Table 3 Phenolics content (mg/L ± Standard Deviation) of ledang and tambun pummelo fruit juice pre and post-enzymatic treatment
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Fig. 1
figure 1

HPLC-DAD chromatogram at 280 nm of LE (LEDANG-ENZYMATIC TREATMENT: 0.021 % ENZYME CONCENTRATION, 31 °C, 99 min) and TE (TAMBUN-ENZYMATIC TREATMENT: 0.065 % ENZYME CONCENTRATION, 33 °C, 83 min)

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Through liquid chromatographic analysis, it was identified that three hydroxycinnamic acids and three flavanones were found in both varieties at 280 nm and 320 nm respectively. Two hydroxybenzoic acids that were tested for identification; gallic and ferulic acid were not detected in both varieties. Gallic acid, the major contributor to hydroxybenzoic acid, is a naturally abundant plant phenolic compound which has been attracting considerable interests for its antioxidant properties (Strlic et al. 2002).

Hidroxycinnamic acids

Three hydroxycinnamic acids identified in the analysis were chlorogenic acid, caffeic acid and coumaric acid. Chlorogenic and coumaric has long been known as the antioxidant that reduced the risk of stomach cancer by reducing the formation of carcinogenic nitrosamines (Nicoli et al. 1997). Sinapic, gallic and ferulic acid were not found in either 280 nm or 320 nm chromatogram, as the amount of acids were too nominal in comparison to other acids in the fruit juice for the chromatograph to detect it at both wavelengths. Chlorogenic acid was the most dominant hydroxycinnamic acid in both varieties, as it accounted for the largest proportion of the total hydroxycinnamic acid contents (Table 3). In both Ledang and Tambun, chlorogenic acid was found with 15.8 − 28.5 mg/L, followed by coumaric acid (10–14.4 mg/L) and caffeic acid (8–11 mg/L). It was found that the enzymatic treatment gave a slight effect to both varieties, although with no significant difference (p >0.05) to the fruit juice, except for coumaric acid that decreased with a significant difference (p <0.05), where in Ledang the decrement was as much as 0.31 % as compared to Tambun with decrement of 0.08 %. It was found that hydroxycinnamic acids were able to transform into volatile phenols during enzymatic treatment (Jackson 2000). Coumaric and ferulic acids are particularly significant, where pectinase can release cinnamic acids derivatives from their ester linkages and metabolized into volatile phenols, which explained the decrease of coumaric acids from the effect of pectinase. It was reported by Matilda and Jorma (2002) that gallic acid which is unstable under alkaline conditions, chlorogenic and other caffeoylquinic acids are hydrolyzed rapidly to caffeic acid. This could be the reason that the amount of gallic acid was very nominal on the HPLC chromatograms.

Flavanones

Flavanone is the major flavonoids found in citrus fruits, especially in pummelo. Flavanones are characterised by the presence of a chiral centre at the 2-position and by the absence of the double bond between the 2- and 3-positions. Flavanones and their glycosides were generally detected at 280 nm and 290 nm (Bogdanov 1989). Three flavanones; naringin, hesperidin and narirutin were identified in both varieties. However, studies done by Sun et al. (2013) and Robards et al. (1997) on pummelo showed that only naringin and tangeretin were present. Whereas, grapefruit compounds studied by Robards et al. (1997) showed hesperidin and narirutin were present, the exact compounds found in this study on both varieties. These findings could suggest that Ledang and Tambun could have had grapefruit hybrid, hence, the presence of naringin, hesperidin and narirutin in both varieties of pummelo fruit juice.

Table 3 and Fig. 1 show that, the dominant neohesperidosyl flavanones, naringin has the most abundant phenols in all samples. Naringin may be instrumental in inhibiting cancer-causing compounds and thus may have potential chemotherapeutic value (Nicoli et al. 1997). Naringin was found with 131 and 267.6 mg/L respectively in non-enzymatically treated Ledang and Tambun variety. Naringin has beneficial antioxidant property but it is also known to give a bitter aftertaste to the juice which may affect the marketability of the juice. The positive observation of the enzymatic clarification was the reduced amount of naringin in the enzymatically-treated juice for both varieties. Naringin was reduced to 128.8 (1.6 %) and 266.0 (0.59 %) in Ledang and Tambun respectively. The decrease may not be significant but the potential for the enzyme to be exploited to minimize the bitterness that pummelo is known for, is important.

Hesperidin, the second most abundant rutinosyl flavanones found in the Ledang variety, is known to be tasteless and therefore does not contribute to the taste of pummelo juice (Kelebek et al. 2009). Hesperidin occurs in the intact fruit in the open-ring soluble chalcone form, but ring closure occurs following juice extraction leading to less soluble hesperidin. It slowly precipitates as fine crystals during juice storage. Hesperidin was found to increase (3.51 %) in Ledang variety after the enzymatic treatment but decreased insignificantly (0.37 %) in Tambun variety. The literature reports (Prasad et al. 2005) that hesperidin level in orange juice depends on the extraction method, technological treatment and storage. Narirutin was found in contrast to hesperidin level in Ledang and Tambun variety. Narirutin was shown to increase in Tambun (0.28 %) and decreased in Ledang by 6.67 %. According to an earlier study by Kelebek et al. (2009), narirutin-to-hesperidin ratio was proposed for quality control of orange juices. It is theorized that, narirutin-to-hesperidin ratio can also be used to gauge the juice clarity. Since hesperidin is insoluble, it can be found in juices in the form of a fine suspension. If the amount of hesperidin is higher, narirutin-to-hesperidin ratio will be lower, thus, lucidity of the juice is expected to be lower and a clearer juice will be produced. In this study, Ledang was recorded to have a ratio of 0.84 and Tambun at 1.33 in comparison to the value of 0.76 and 1.36 when enzymatically-treated. The ratio would indicate that Tambun variety will produce a slightly cloudier juice than Ledang variety, which in this case further proved that Ledang clarifies better with enzymatic treatment. The results corresponded with the initial clarity values found using the spectrophotometer method (Table 1) which showed Ledang and Tambun had 0.36 and 0.49 absorbance at 660 nm, respectively.

With respect to the phenol levels, it was worth noting that the level of total phenols in no case decreased significantly (p >0.05) by the alternative treatments. The finding of the measured levels of total phenols and chromatographs were dissimilar in the control and experimentally treated samples (Fig. 2), could indicate the influence of the different enzymatic treatment most likely reflected in the size differences and perhaps the shape of the clarity-causing molecules and not in their total content (Pinelo et al. 2010). The concentration of the total phenol determined by Folin-Ciocalteu method was higher than the concentration obtained by HPLC method. Rapisarda et al. (1999) claimed the spectrophotometric method overestimates the polyphenolic content as compared to chromatographic method. This can be explained by the lack of sensitivity of Folic-Ciocalteu reagent, which reacts not only with phenols but also with other reducing compounds such as amino acids, sugars and vitamin C.

Fig. 2
figure 2

Total phenolic contents (GAE mg/L) of pummelo fruitT juice with different methods of analysis. *FC – Folin Ciocalteu reagent method. HPLC – High performance liquid chromatogram method. ‡Results were expressed as mean ± SEM;n = 3. ‡‡The means within the column followed by different letters were significantly different at p <0.05

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Report by Aturki et al. (2004) has conclusively shown close relationship between phenolic contents and antioxidative activity of fruit juice. Since the chemical composition and structures of active extract components are important factors governing the efficacy of natural antioxidants, the antioxidant activity of an extract could not be explained on the basis of their phenolic contents alone. Xu et al. (2008) reported that the antioxidant activity, not phenolic compounds, was the major contributor of the total antioxidant capacity of citrus juice. It seemed that some factors such as different citrus variety, maturity, material preparation and analyzing methods might cause the divergence.

The lack of significant results of enzymatic clarification treatment towards both varieties of pummelo fruit juice could be due to; (1) The temperature used for the enzymatic treatment was not in the peak region of pectinase activity, which was reported at 50 °C (Sandri et al. 2011 & Sun et al. 2007). The authors further noted that pectinase activity could vary with temperature depending on the size of the pectin chain and its ramification and methylation degree. The temperature chosen for the enzymatic treatment was based on the minimized effect on the juice characteristics and thus proved that at this point, the effect of pectinase was not debilitating enough to the natural content of phenolic. (2) The enzymatic clarification treatment could also be related to other particular features of juice such as the level of pectin, polyphenol and protein which, warrants further experimental works. Sandri et al. (2011) further supports that other factors, such as the juice chemical composition and the presence of other enzymes apart from pectinases during the enzymatic clarification treatment, could interfere significantly on the clarification process.

Conclusion

In the present study, the antioxidant contents from enzymatically clarified pummelo fruit juice from two Malaysian varieties were evaluated against non-enzymatically treated of the same varieties. Tambun variety was shown to possess high phenolic and flavonoid content, thus a higher antioxidant capacity can be anticipated, due to the amount of naringin found in single strength and enzymatically treated juice. Naringin, the most dominant flavanone and the main contributor of antioxidant property however, decreased twofold in Ledang variety. Although theoretically Tambun would have made a better choice for juice development for the total phenolics it possessed, however, considerations have to be made to suit consumer’s taste bud and the enzymatic clarification conditions (temperature and enzyme concentration) needed to market the juice.