Effect of technological processes and storage on flavonoids content ...

Eur Food Res Technol (2007) 225:905–912 DOI 10.1007/s00217-006-0500-0

ORIGINAL PAPER

Effect of technological processes and storage on flavonoids content and total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities of citrus juices Enrique Sentandreu · José L. Navarro · José M. Sendra

Received: 7 September 2006 / Revised: 7 September 2006 / Accepted: 8 October 2006 / Published online: 16 November 2006 C Springer-Verlag 2006

Abstract Juices from the mandarin Clemenules (Citrus clementina Hort. ex Tan.), the tangor Ortanique (Citrus reticulata Blanco × Citrus sinensis Osb.) and the sweet orange Valencia Late (Citrus sinensis) have been industrially squeezed, pasteurized, concentrated and stored under refrigeration (4 ◦ C) and at room temperature (20 ◦ C). After each process, the flavanone-7-O-glycosides (FGs) and fully methoxylated flavones (FMFs) contents as well as total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities were determined and compared with those from the corresponding fresh hand-squeezed juices. Neither industrial-squeezing, nor pasteurization or concentration significantly affected FGs and FMFs contents and antiradical activities of assayed juices. Storage caused a slight decrease of the FMFs contents but a significant reduction of both soluble hesperidin contents and cumulative fast-kinetics antiradical activities in all assayed juices. These decreases were dependent on storage temperature. Characteristic values of the varietal characterization parameters, which are derived from the FMFs contents and antiradical activities of fresh hand-squeezed juices, held valid for industrially squeezed, pasteurized and concentrated juices. After storage, however, only the FMFs-derived varietal characterization parameters and cumulative slowkinetics antiradical activity remained valid for the resulting juices. Keywords Citrus juices . Industrial processes . Storage . Antiradical activity . Flavanone-7-O-glycosides . Fully methoxylated flavones E. Sentandreu · J. L. Navarro · J. M. Sendra () Instituto de Agroqu´ımica y Tecnolog´ıa de Alimentos, P.O. Box 73, 46100 Burjassot, Valencia, Spain e-mail: [email protected]

Abbreviations AA: Ascorbic acid . ABTS•+ : 2,2-Azinobis-(3-ethylbenzothiazoline)6-sulphonate . CMN: Clemenules cultivar . DID: Didymin . DPPH• : 2,2-Diphenyl-1-picrylhydrazyl . FAA: Fast-kinetics antiradical activity (mmol l−1 , molar equivalents of AA) . FG: Flavanone-7-O-glycoside . FMF: Fully methoxylated flavone . GOS: Hexamethyl-O-gossypetin . HEP: Heptamethoxyflavone . HES: Hesperidin . IS1 : Internal standard for FGs (o-coumaric acid) . IS2 : Internal standard for FMFs (flavone) . k1 : Average rate constant of cumulative FAA (l mol−1 min−1 ) . k2 : Average rate constant of cumulative SAA (l mol−1 min−1 ) . LDL: Low-density lipoprotein . NAR: Narirutin . NOB: Nobiletin . ORT: Ortanique cultivar . QUE: Hexamethyl-O-quercetagetin . SAA: Slow-kinetics antiradical activity (mmol l−1 , molar equivalents of AA) . SCU: Tetramethyl-O-scutellarein . TAA: Total antiradical activity of juice (mmol l−1 , molar equivalents of AA) . VAL: Valencia Late cultivar . y: Time-dependent concentration of DPPH• (µmol l−1 ) . y1 : Asymptotic value of the DPPH• concentration (µmol l−1 ) due solely to the cumulative FAA of juice . y2 : Asymptotic value of the DPPH• concentration (µmol l−1 ) due solely to the cumulative SAA of juice . y0 : Initial concentration of DPPH• (µmol l−1 ) . ys : Experimental asymptotic value of the DPPH• concentration (µmol l−1 ) due to TAA . ρ 1 : Adjustable parameter ( = k1 /σ 1 , l mol−1 min−1 ) . ρ 2 : Adjustable parameter ( = k2 /σ 2 , l mol−1 min−1 ) . σ 1 : Average stoichiometric constant of cumulative FAA . σ 2 : Average stoichiometric constant of cumulative SAA Introduction Fresh hand-squeezed sweet orange and mandarin juices are very pleasant non-alcoholic beverages whose consumption is Springer

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strongly recommended due to their known bioactive healthpromoting components. Citrus juices contain vitamin C as well as flavonoids (including anthocyanins in juices from pigmented citrus varieties), carotenoids, hydroxycinnamic acid derivatives, etc., which exhibit antioxidant and antiradical activities. Hence, citrus juice intake affects both vitamin C concentration and biomarkers of antioxidant status in human blood [1], thus being directly associated with the decrease of lipid peroxidation [2] and LDL levels [3] and with the risk of suffering coronary heart diseases [4] and some types of cancer [5–7]. Moreover, they can act as health-promoters against age-related degenerative diseases [8] and even to increase the concentration of chemotherapics into carcinogenic cells [9]. Contrary to fresh hand-squeezed juices, commercial citrus juices are typically obtained using industrial-squeezers and subsequently they are subjected to a series of industrial processes (pasteurization, concentration, etc.) and to storage. Consequently, several attempts have been made to determine the effect of these processes and storage on the bioactive components of citrus juices. Most of these studies have been focused on sweet orange juice, since it is by far the most important commercial citrus juice, and thus relevant bibliography about other citrus juices, such as mandarin juices and hybrids thereof, is very scarce. It is known that, as a general rule, industrial-squeezing incorporates additional amounts of both FGs and FMFs (from the peel oil) than hand-squeezing to the resulting juices [10], but let practically unchanged the ascorbic acid (AA) content and total antiradical activity (TAA) [11]. Conventional pasteurization (up to 95 ◦ C for 30 s) of orange juice has a negligible affect on its FGs, FMFs, hydroxycinnamic acid derivatives [11] and AA contents as well as on its TAA [12]. Storage, on the contrary, significantly decreases AA and soluble hesperidin (HES) contents as well as TAA of the juice, and these decreases are proportional to storage temperature [12–14]. The absolute decrease of AA during storage, however, seems to depend on the presence and concentration of other components, particularly polyphenolic compounds, which would protect ascorbic acid from oxidation [15]. In a recent work, Sendra et al. [16] studied the reduction kinetics (in methanol) of the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH• ) by the antiradicals found in citrus juices. According to their experimental kinetics, these antiradicals were grouped into three general groups: fast-kinetics (i.e., ascorbic acid), fast + slow-kinetics (i.e., chlorogenic acid) and slow-kinetics (i.e., citrus FGs). It was found that the TAA of a citrus juice results from the sum of two distinguishable contributors: the cumulative fast-kinetics (FAA) and the cumulative slow-kinetics (SAA) antiradical activities. Sentandreu et al. [17] determined the FGs and FMFs contents of a series of fresh hand-squeezed mandarin juices and found that by using the concentration Springer

Eur Food Res Technol (2007) 225:905–912

quotients tetramethyl-O-scutellarein/nobiletin (SCU/NOB) and heptamethoxyflavone/nobiletin (HEP/NOB) as parameters, all the assayed juices could be characterized and significantly ( < 0.05) discriminated. Afterwards, Sentandreu et al. [18], using the DPPH• assay, demonstrated that in these fresh hand-squeezed mandarin juices, cumulative FAA is the major contributor to TAA, AA is the major contributor to cumulative FAA and there is no a significant correlation between the total FGs content and cumulative SAA. Moreover, it was found that cumulative FAA and cumulative SAA are also useful parameters to characterize and significantly ( < 0.05) discriminate between all the assayed mandarin juices. In this work, juices from the mandarin Clemenules (Citrus clementina Hort. ex Tan.), the tangor Ortanique (Citrus reticulata Blanco × Citrus sinensis Osb.) and the sweet orange Valencia Late (Citrus sinensis) have been industrially squeezed, pasteurized, concentrated and stored under refrigeration (4 ◦ C) and at room temperature (20 ◦ C). After each process, the FGs and FMFs contents as well as TAA, cumulative FAA and cumulative SAA of the resulting juices were determined and compared with those from the corresponding fresh hand-squeezed juices. Results indicate that neither industrial-squeezing, nor pasteurization or concentration have a significant influence on the FGs and FMFs concentrations and antiradical activities of the juices. On the contrary, storages under refrigeration and especially at room temperature strongly decrease both soluble HES concentration and cumulative FAA of the juices, but let unchanged their cumulative SAA.

Materials and methods Assayed cultivars and analysed samples Effect of technological processes and storage on the FGs and FMFs contents of juices To determine the effect of technological processes and storage on the FGs and FMFs contents of juices, sample CMN-2, belonging to the Clemenules cultivar, sample ORT-4, belonging to the Ortanique cultivar, and sample VAL, belonging to the Valencia Late cultivar, were selected since their FGs and FMFs contents in fresh hand-squeezed juices are already known [17]. Data relative to these samples are given in Table 1. Samples of juices were obtained using the industrial extractor Excel (Luzyssa, El Puig, Valencia, Spain) and aliquots were pasteurized at the optimal conditions of 85 ◦ C for 10 s [19] using a plate heat exchanger. The pasteurized juices were aseptically poured into water steam sterilized twist-off glass bottles, avoiding the formation of headspaces, and immediately stored under refrigeration (4 ◦ C) and at ambient temperature (20 ◦ C) until their analyses.

Eur Food Res Technol (2007) 225:905–912 Table 1 Code, harvest time, maturity indexes and ◦ Brix of assayed juices

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Sample

Code

Harvest time

Maturity index (◦ Brix/acid)

◦

Clemenules Ortanique Valencia Late Clemenules Ortanique

CMN-2 ORT-4 VAL CMN ORT

10/12/2003 29/04/2004 20/05/2004 21/12/2004 15/3/2005

16.4 13.5 9.6 13.3 8.3

11.0 12.7 10.6 11.5 13.8

Effect of technological processes and storage on TAA, cumulative FAA and cumulative SAA of juices To determine the effect of technological processes and storage on the antiradical activities of juices, one sample of the clementine Clemenules (CMN) and one sample of the mandarin hybrid (tangor) Ortanique (ORT) were selected. Data relative to both cultivars are given in Table 1. Both handsqueezed and industrially squeezed juices were obtained and industrial juices were immediately pasteurized as indicated previously. One aliquot of each pasteurized juice was bottled, as indicated previously, and stored under refrigeration (4 ◦ C) and at room temperature (20 ◦ C) up to its analysis; the other aliquot was immediately concentrated at 25–30 ◦ C up to 53 ◦ Brix (CMN) and 52 ◦ Brix (ORT), using a Luwa (L020 type) agitated film (scrapped surface) concentrator. The concentrates were bottled and stored under freezing ( − 25 ◦ C) up to their analyses that were carried out, after redilution to their original ◦ Brix, within the next 2 days. Reagents and standards HPLC-grade acetonitrile (ACN) and tetrahydrofuran (THF) were purchased from Panreac (Panreac Qu´ımica, S.A., Barcelona, Spain). Methanol (spectrophotometric grade) and DPPH• (95% purity) were from Sigma (Sigma–Aldrich Co., St. Louis, MO, USA). Water used was Milli-Q grade (Millipore Ibérica, Barcelona, Spain). o-Coumaric acid (IS1 ) and flavone (IS2 ), to be used as internal standards, and the filter aid Celite 545 were purchased from Fluka (Fluka Chemie GmbH, Switzerland). C-18 Sep-Pack cartridges were obtained from Waters (Waters Co., Milford, MA, USA). Narirutin (NAR), hesperidin (HES), didymin (DID), sinensetin (SIN) and tetramethyl-O-scutellarein (SCU) were purchased from Extrasynthèse (Extrasynthèse, Genay, France). Isosinensetin (ISOSIN), hexamethyl-Ogossypetin (GOS), tetramethyl-O-isoscutellarein (ISOSCU), hexamethyl-O-quercetagetin (QUE), nobiletin (NOB), heptamethoxyflavone (HEP) and tangeretin (TAN) were available [10] in our laboratory. Determination of the FGs and FMFs contents in juices Isolation, separation and quantification of the FGs and FMFs in the assayed juices were carried out following the method-

Brix

ology described by Sentandreu et al. [17]. Briefly, 60 ml of juice were filtered under vacuum after addition of Celite 545 as filter aid. Then, 30 ml of filtered juice ware passed through an activated C-18 Sep-Pack cartridge and the retained FGs and FMFs were selectively eluted with 5 ml of water/ACN (4:6, v/v) and collected. To the collected eluate, 0.5 ml of a water/ACN (4:6, v/v) solution containing accurately known weights of o-coumaric acid (IS1 , as internal standard for the FGs) and flavone (IS2 , as internal standard for the FMFs) was added. The mixture was filtered and analysed by gradient HPLC, using a reverse-phase C-18, 5 µm, 250 mm × 4.6 mm (Luna II, Phenomenex, Torrance, USA) and water/THF (98.75:1.25, v/v), adjusted to pH 3.5 with H3 PO4 , and ACN/THF (98.75:1.25, v/v) as eluants. Samples were analysed in duplicate. Determination of TAA, cumulative FAA and cumulative SAA of juices Determination of the antiradical activities of the assayed juices was carried out following the methodology described by Sendra et al. [16]. Briefly, 10 ml of juice was centrifuged at 13,000 rpm for 5 min at room temperature and the supernatant was decanted and filtered through Whatman filter paper #1. Then, a volume (between 10 and 70 µl) of filtered juice was added to a quartz spectrophotometric cuvette (3.5 ml capacity and 1 cm path-length) containing an appropriate volume of DPPH• in methanol to yield a final volume of 3 ml (the final concentration of DPPH• was around 100 µmol l−1 ). The cuvette was end-capped immediately, shaken by hand and placed in the spectrophotometer, set at a wavelength of 515 nm, for analysis. Samples were analysed in duplicate. Each set of experimental data points (curve), corresponding to each volume of juice added, was fitted using the simplified global kinetic equation: y − ys =

y2 (y0 − y2 ) y0 − y1 + 1 + ρ1 y0 t y2 − y0 (1 − eρ2 y2 t )

where y (µmol l−1 ) is the time-dependent concentration of DPPH• , t is the time (min), y0 is the initial concentration of DPPH• , y1 is the asymptotic value of DPPH• that would be reached due solely to the cumulative FAA of the juice, y2 is the asymptotic value of DPPH• that would be reached due solely to the cumulative SAA of the juice, ys is the Springer

908 Table 2 Effect of technological processes and storage (months) on the FGs contents (mean ± SD) (mg l−1 ) in assayed juices


Sample CMN-2 Fresh hand-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 1 2 3 6 Ambient (20 ◦ C) 1 2 3 ORT-4 Fresh hand-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 1 2 3 Ambient (20 ◦ C) 1 2 VAL Fresh hand-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 1 2 3 Ambient (20 ◦ C) 1 2

experimental asymptotic value of DPPH• due to the TAA of the juice, and ρi = ki /σi (i = 1, 2), where ki and σ i are the average rate and stoichiometric constants of cumulative FAA (i = 1) and cumulative SAA (i = 2). From the fittings, TAA, cumulative FAA and cumulative SAA were determined, all them expressed as mmol l−1 molar equivalents of AA.

NAR

HES

DID

5.15 ± 0.01 10.30 ± 0.07

19.14 ± 0.11 21.00 ± 0.27

0.354 ± 0.021 0.635 ± 0.030

9.89 ± 0.11 10.01 ± 0.10 9.39 ± 0.09 8.96 ± 0.06

13.15 ± 0.15 10.88 ± 0.01 10.50 ± 0.20 7.88 ± 0.08

0.432 0.454 0.428 0.332

10.82 ± 0.07 9.23 ± 0.12 8.13 ± 0.08

11.97 ± 0.05 8.77 ± 0.18 6.72 ± 0.15

0.467 ± 0.026 0.338 ± 0.009 0.429 ± 0.013

38.09 ± 0.43 45.63 ± 0.96

139.12 ± 1.12 170.36 ± 3.13

16.76 ± 0.16 18.19 ± 0.52

36.18 ± 1.03 35.94 ± 1.35 36.81 ± 2.03

45.38 ± 1.52 40.17 ± 0.86 35.77 ± 1.25

7.47 ± 0.42 7.26 ± 0.25 6.69 ± 0.13

35.92 ± 0.55 34.01 ± 0.88

42.57 ± 1.32 35.22 ± 1.11

7.54 ± 0.88 6.77 ± 0.36

14.92 ± 0.02 15.63 ± 0.38

181.13 ± 0.17 173.18 ± 2.11

6.60 ± 0.02 6.51 ± 0.10

11.56 ± 0.37 12.28 ± 0.33 11.75 ± 0.18

56.98 ± 1.65 55.60 ± 1.24 40.93 ± 0.82

2.95 ± 0.11 3.09 ± 0.10 2.50 ± 0.13

11.79 ± 0.19 12.21 ± 0.32

51.33 ± 0.48 49.98 ± 1.14

2.83 ± 0.01 2.86 ± 0.01

± ± ± ±

0.021 0.007 0.052 0.013

of this cultivar. In any case, these results indicate that a mild pressure during industrial-squeezing, thus avoiding a significant loss of sensory quality, followed by a normal pasteurization have minor effects on the concentration of these components in the resulting juice. Effect of storage on the FGs content of juices

Results and discussion Effect of technological processes on the FGs content of juices Table 2 gives the NAR, HES and DID concentrations in both fresh hand-squeezed and industrial-pasteurized juices from samples CMN-2, ORT-4 and VAL. As it can be observed, for CMN-2 and VAL cultivars, the FGs contents in their fresh hand-squeezed and corresponding industrialpasteurized juices were similar. For ORT-4 cultivar, however, the FGs contents were somewhat greater in industrialpasteurized juice, probably due to the larger peel oil content Springer

Table 2 gives the time evolution of NAR, HES and DID concentrations during storage under refrigeration (4 ◦ C) and at ambient temperature (20 ◦ C) of industrial-pasteurized juice from CMN-2, ORT-4 and VAL. As it can be observed, for all assayed cultivars, the NAR and DID concentrations decayed very slowly during both storage conditions whilst the HES concentration, on the contrary, decayed much more rapidly, mainly at room temperature. This differential behaviour of HES vs. NAR and DID during storage of citrus juices was already described [11, 20]. It is known that hesperidin, which is very insoluble in water, stands in intact citrus fruit juices as an unstable oversaturated solution. Immediately after squeezing, a portion of solubilized hesperidin begins to crystallize


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as white crystals, which are incorporated into the insoluble fraction (cloud) of the juice [21, 22].

Effect of storage on TAA, cumulative FAA and cumulative SAA of juices

Effect of technological processes on the FMFs content of juices

Table 4 gives the time evolution of TAA, cumulative FAA and cumulative SAA of industrial-pasteurized juices from CMN and ORT stored under refrigeration (4 ◦ C) as well as from ORT stored at room temperature (20 ◦ C). As it could be expected, TAA gradually decreased during storage time and the amount of this decrease was dependent on storage temperature. Interestingly, the decrease of TAA was exclusively due to an equivalent decrease of cumulative FAA, since cumulative SAA remained practically constant in all cases. It seems, therefore, that relatively short storage times only significantly affects those juice components responsible of cumulative FAA (components belonging to fast-kinetics or fast + slow-kinetics groups), but apparently does not affect those juice components which are the main responsible of cumulative SAA (although citrus FGs belong to the slowkinetics group, its total concentration is almost uncorrelated with SAA in citrus juices). Data in Table 4 confirm that the initial cumulative FAA of a CMN juice (3.073 mmol l−1 molar equivalents of AA) is greater by far than that of a ORT juice (1.804 mmol l−1 molar equivalents of AA), although in both juices the relative contribution of AA to this activity ( ≈ 93%) is almost the same [18]. After 30 days of refrigerated storage, the absolute losses of cumulative FAA in CMN and ORT juices were 0.510 and 0.834 mmol l−1 molar equivalents of AA, respectively. It seems thus that a constant relative contribution of AA, the higher the initial cumulative FAA of a citrus juice, the smaller the loss of this activity during storage. This result was quite surprising since one would expect that two similar juices stored at exactly the same conditions would loss approximately the same absolute amounts of cumulative FAA (or TAA) after the same storage time. Miller and Rice-Evans [15] found similar results when they determined the initial and final TAA, using the ABTS•+ assay, as well as the initial and final AA concentrations, using the ferrozine assay, in ready-to-drink (16.7% extract) and fortified (20% extract) blackcurrant beverages stored at 37 ◦ C for 24 h. During this storage period, ready-to-drink blackcurrant lost 1014 µmol l−1 TEAC (20%) of TAA and 1751 µmol l−1 (47%) of AA, whilst fortified blackcurrant lost 759 µmol l−1 TEAC (10%) of TAA and 502 µmol l−1 (9%) of AA. Although these data suggest that some other antiradicals present in blackcurrant (which evidently must also exhibit fast-kinetics antiradical activity) protect ascorbic acid from oxidation, there persists the question about the different experimentally determined absolute losses of TAA (or FAA), 1014 and 759 µmol l−1 TEAC, for ready-to-drink and fortified blackcurrant beverages, respectively. Probably, this discrepancy indicates that neither the DPPH• (in methanol) nor the ABTS•+ (in water) assays

Table 3 gives the FMFs concentrations in both fresh handsqueezed and industrial-pasteurized juices from CMN-2, ORT-4 and VAL. As it can be seen, and similarly to FGs, the concentrations of these components were very similar in fresh hand-squeezed and corresponding industrialpasteurized juices from CMN-2 and VAL cultivars but somewhat greater in the pasteurized juice from ORT-4, as it was expected. Table 3 also gives the values of the varietal characterization parameters [17], that is, the quotients SCU/NOB and HEP/NOB, for all assayed juices. Since the values of these parameters in both fresh hand-squeezed and corresponding industrial-pasteurized juices were practically identical in all cases, it seems reasonable to assume that they hold valid for pasteurized juices. Effect of storage on the FMFs content of juices The time evolutions of FMFs concentrations during storage under refrigeration (4 ◦ C) and at room temperature (20 ◦ C) in industrial-pasteurized juices from CMN-2, ORT-4 and VAL cultivars are given in Table 3. As it can be observed, for all assayed cultivars, there was a slow and monotonic decrease of concentrations during storage, although somewhat more marked in the case of storage at room temperature. Table 3 also gives the time evolution of the quotients SCU/NOB and HEP/NOB during storage under refrigeration and at room temperature for industrial-pasteurized juices from all the assayed cultivars. Since within each cultivar, the value of these quotients remained practically unchanged during both types of storage, it seems reasonable to assume that storage temperature during a limited period of time does not affect their validity as varietal characterization parameters. Effect of technological processes on TAA, cumulative FAA and cumulative SAA of juices Table 4 gives the values of TAA, cumulative FAA and cumulative SAA of fresh hand-squeezed, fresh industrialsqueezed, industrial-pasteurized and industrial-pasteurized plus concentrated juices from CMN and ORT cultivars. As it can be seen, there were almost no differences between the values of the antiradical activities within the juices belonging to the same cultivar. Consequently, it seems reasonable to conclude that neither industrial-squeezing, nor pasteurization or concentration significantly affect the antiradical activities of juices.

Springer

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a From

reference [17].

CMN-2 Fresh hand-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 1 2 3 6 Ambient (20 ◦ C) 1 2 3 ORT-4 Fresh hand-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 1 2 3 Ambient (20 ◦ C) 1 2 VAL Fresh hand-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 1 2 3 Ambient (20 ◦ C) 1 2

Sample

0.065 ± 0.003 0.064 ± 0.002 0.063 ± 0.003 0.449 ± 0.001 0.767 ± 0.012 0.645 ± 0.008 0.639 ± 0.002 0.633 ± 0.006 0.610 ± 0.010 0.543 ± 0.002

0.324 ± 0.017 0.329 ± 0.004 0.323 ± 0.006 0.285 ± 0.004 0.250 ± 0.009

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.014 ± 0.001 0.017 ± 0.008 0.010 ± 0.001 < 0.01 < 0.01 < 0.01 < 0.01

0.151 ± 0.002 0.143 ± 0.002

0.181 ± 0.001 0.174 ± 0.003 0.164 ± 0.001

0.148 ± 0.001 0.131 ± 0.002

0.018 ± 0.001 0.016 ± 0.005

0.011 ± 0.001 < 0.01 < 0.01

0.017 ± 0.005 < 0.01

0.381 ± 0.005 0.321 ± 0.003

0.002 0.002 0.001 0.002

0.018 ± 0.003 0.016 ± 0.005 < 0.01

± ± ± ±

0.065 0.065 0.064 0.062

< 0.01 < 0.01 < 0.01 < 0.01

0.018 ± 0.001 0.016 ± 0.003 < 0.01 < 0.01

0.065 ± 0.003 0.066 ± 0.001

SIN

0.033 ± 0.001

GOS 0.018 ± 0.004 < 0.01

< 0.01

ISOSIN

± ± ± ± 0.003 0.001 0.002 0.002

0.074 ± 0.001 0.079 ± 0.001 0.079 ± 0.002

< 0.01 < 0.01 < 0.01 0.071 ± 0.003 0.064 ± 0.007

0.091 ± 0.003 0.079 ± 0.001

< 0.01 < 0.01

< 0.01 < 0.01

0.033 ± 0.001 0.031 ± 0.004

0.034 ± 0.002 0.031 ± 0.002 0.031 ± 0.003

0.023 ± 0.001 0.050 ± 0.001

0.083 ± 0.002 0.068 ± 0.010 0.078 ± 0.003

0.077 0.075 0.075 0.076

0.078 ± 0.002 0.086 ± 0.006

QUE

0.104 ± 0.001 0.079 ± 0.002

0.131 ± 0.001 0.134 ± 0.004 0.116 ± 0.002

0.112 ± 0.001 0.187 ± 0.002

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01

ISOSCU

± ± ± ± 0.002 0.006 0.005 0.006

0.381 ± 0.011 0.334 ± 0.009

0.438 ± 0.004 0.441 ± 0.001 0.444 ± 0.003

0.522 ± 0.001 0.448 ± 0.006

1.631 ± 0.054 1.361 ± 0.002

1.914 ± 0.052 1.881 ± 0.032 1.757 ± 0.005

1.506 ± 0.001 2.426 ± 0.100

0.234 ± 0.009 0.218 ± 0.027 0.209 ± 0.003

0.242 0.239 0.230 0.221

0.276 ± 0.009 0.259 ± 0.006

NOB

± ± ± ± 0.002 0.002 0.001 0.003

0.100 ± 0.005 0.087 ± 0.003

0.114 ± 0.001 0.114 ± 0.002 0.111 ± 0.002

0.149 ± 0.003 0.124 ± 0.002

0.846 ± 0.021 0.751 ± 0.037

0.918 ± 0.011 0.923 ± 0.015 0.900 ± 0.008

0.734 ± 0.002 1.290 ± 0.021

0.044 ± 0.002 0.038 ± 0.001 0.043 ± 0.005

0.045 0.043 0.044 0.041

0.057 ± 0.002 0.054 ± 0.003

SCU

± ± ± ± 0.001 0.005 0.005 0.004

0.127 ± 0.002 0.114 ± 0.004

0.144 ± 0.001 0.147 ± 0.001 0.144 ± 0.004

0.177 ± 0.001 0.149 ± 0.001

0.193 ± 0.006 0.165 ± 0.003

0.226 ± 0.001 0.222 ± 0.009 0.211 ± 0.004

0.175 ± 0.001 0.299 ± 0.002

0.372 ± 0.028 0.336 ± 0.034 0.326 ± 0.002

0.366 0.362 0.350 0.338

0.397 ± 0.011 0.409 ± 0.002

HEP

± ± ± ± 0.001 0.001 0.003 0.002

0.047 ± 0.001 0.046 ± 0.003

0.057 ± 0.006 0.056 ± 0.004 0.054 ± 0.003

0.078 ± 0.002 0.063 ± 0.002

0.521 ± 0.001 0.415 ± 0.006

0.682 ± 0.002 0.667 ± 0.003 0.575 ± 0.005

0.682 ± 0.007 1.000 ± 0.010

0.040 ± 0.004 0.033 ± 0.001 0.035 ± 0.002

0.047 0.043 0.042 0.037

0.063 ± 0.004 0.052 ± 0.002

TAN

0.262 0.261

0.261 0.259 0.251

0.519 0.551 – 0.285 0.276

0.479 0.491 0.512

0.186 0.175 0.205 0.496–0.450 0.487 0.532

0.186 0.182 0.190 0.186

0.222–0.176 0.206 0.207

SCU/NOB

0.334 0.342

0.329 0.334 0.326

0.119 0.121 – 0.340 0.332

0.118 0.118 0.120

1.591 1.539 1.561 0.206–0.044 0.116 0.123

1.511 1.518 1.523 1.527

1.472–1.310 1.439 1.579

HEP/NOB

Table 3 Effect of technological processes and storage (months) on the FMFs content (mean ± SD) (mg l−1 ) of assayed juices; characteristic intervals of the quotients SCU/NOB and HEP/NOB from fresh hand-squeezed juices are shown in bolda

910 Eur Food Res Technol (2007) 225:905–912

Eur Food Res Technol (2007) 225:905–912 Table 4 Effect of industrial processes and storage (days) on total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activitiesa (mean ± SD) of assayed juices; characteristic intervals from fresh hand-squeezed juices are shown in boldb

Expressed as mmol l−1 molar equivalents of ascorbic acid.

a

b

From reference [18].

Process CMN Fresh hand-squeezed Fresh industrial-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 10 20 30 Industrial concentrated ORT Fresh hand-squeezed Fresh industrial-squeezed Industrial-pasteurized Refrigeration (4 ◦ C) 10 20 30 Ambient (20 ◦ C) 3 5 7 9 12 17 22 29 Industrial concentrated

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Total

Cumulative fast-kinetics

Cumulative slow-kinetics

3.33 ± 0.05 3.36 ± 0.02 3.35 ± 0.05

2.80–2.46 3.05 ± 0.01 3.05 ± 0.03 3.07 ± 0.01

0.32–0.22 0.28 ± 0.04 0.31 ± 0.02 0.28 ± 0.04

2.06 ± 0.04 2.12 ± 0.04 2.13 ± 0.06

2.85 ± 0.03 2.77 ± 0.01 2.56 ± 0.04 3.04 ± 0.01 1.68–1.34 1.75 ± 0.05 1.82 ± 0.01 1.80 ± 0.02

0.26 ± 0.02 0.27 ± 0.01 0.30 ± 0.04 0.26 ± 0.04 0.37–0.27 0.31 ± 0.03 0.34 ± 0.01 0.34 ± 0.02

1.93 ± 0.06 1.57 ± 0.04 1.34 ± 0.04

1.62 ± 0.04 1.23 ± 0.04 0.97 ± 0.02

0.32 ± 0.02 0.35 ± 0.01 0.36 ± 0.01

3.11 3.05 2.56 3.30

1.98 1.84 1.64 1.61 1.40 1.25 1.05 1.03 2.08

are adequate methodologies to determine true antiradical activity against oxygen radicals in water. In other words, relative rate and stoichiometric constants of AA vs. the other antiradical components, determined using the DPPH• (in methanol) or the ABTS•+ (in water) assays, are not good estimations of their true values against oxygen radicals in water. Consequently, additional experiments including comparison of the DPPH• assay with other assays based upon the reaction of oxygen containing radicals, such as ORAC or TOSC, are needed to clarify this discrepancy. In fact, it is well known that the antiradical activity of some compounds can be very sensitive to experimental conditions. For instance, against DPPH• and in protic solvents, compounds having a free p-catechol group exhibit two consecutive and apparently different antiradical activities: the first with a fast-rate kinetic constant and the second with a solvent-dependent pseudo-slow-rate kinetic constant [16], this latter due to a slow-kinetics adduct formation between the corresponding quinone and the solvent to regenerate the free p-catechol group [23]. Hence, when in aprotic solvents such as acetone, the adduct formation and regeneration of the free p-catechol group is not possible and the pseudo-slow-kinetics antiradical activity vanishes [23]. Results of this work indicate that neither mild industrialsqueezing, nor pasteurization or concentration have a sig-

± 0.01 ± 0.01 ± 0.01 ± 0.05

± ± ± ± ± ± ± ± ±

0.04 0.01 0.04 0.02 0.03 0.01 0.02 0.06 0.02

1.64 1.52 1.32 1.28 1.05 0.87 0.67 0.65 1.73

± ± ± ± ± ± ± ± ±

0.01 0.03 0.02 0.01 0.03 0.03 0.05 0.05 0.04

0.34 0.32 0.32 0.33 0.35 0.38 0.37 0.38 0.35

± ± ± ± ± ± ± ± ±

0.02 0.02 0.02 0.02 0.01 0.03 0.02 0.02 0.01

nificant effect on the FGs and FMFs contents, TAA, cumulative FAA and cumulative SAA of the juices. Storage, on the contrary, slightly decreases the FMFs content of the juices and has no effect on the FMFs-based discriminant parameters, but strongly decreases both soluble HES content and, more important, TAA of juices. The decrease of TAA was solely due to an equivalent decrease of cumulative FAA, since cumulative SAA remained practically constant in all cases. Hence, although the discriminant power of this latter antiradical activity is relatively small, it could be used at least to discriminate between a limited numbers of juices. Acknowledgements This research was supported by the Ministerio de Educación y Ciencia (Spain), project AGL2002-01172ALI, and AGROALIMED (Conseller´ıa de Agricultura, Pesca i Alimentació, Generalitat Valenciana, Spain).

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