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11 (2011) 1260 1266
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Changes of Some Chemical Substances and Antioxidant

Capacity of Mandarin Orange Segments during Can
Processing
Zhang Fengmei1, Hu Liangbin2, Xu Guihua2 * Chen Quanxian1
1Foreign Language Department Henan Institute of Science and Technology Xinxiang, 453003, China
2: School of Food Science Henan Institute of Science and Technology Xinxiang, 453003, China
guihuaxu@zju.edu.cn

Abstract

Changes of ascorbic acid (AA), flavanone glycosides (FGs), phenolic acids and antioxidant capacity which was
expressed as ascorbic acid equivalent capacity (AEAC) of total antioxidant capacity (TAC) determined by ferric
reducing antioxidant power (FRAP) assay and radical scavenging capacity (RSC) determined by 2, 2-diphenyl-1-
picrylhydrazyl (DPPH) assay, of mandarin orange segments during can processing were investigated. The effects of
four key processing procedures were studied, namely blanching, HCl, alkaline and canning treatment. As a result,
after canning, the total FGs content of mandarin segments decreased from 502.39 mg/kg to 254.27 mg/kg, and the
total phenolic acids from 57.51 mg/kg to 44.20 mg/kg. The similar decreasing tendency was also true to the AA
content of mandarin segments (from 251.87 mg/kg to 186.98 mg/kg), TAC (from 664.48 mg/kg to 391.95 mg/kg),
and inhibition of DPPH radical (from 34.85% to 27.31%). To be concluded, small proportions of phenolic acids and
AA were lost, and the loss of FGs and TAC was about fifty percent. However, in view of the considerable part of
phenolic compounds and AA existing in the syrup portion; the loss was not as large as that. Therefore, we suggested
that the majority of AA, phenolic compounds (FGs and phenolic acids) and antioxidant capacity of mandarin
segments were retained after can processing, and mandarin cans could serve as a substitute when fresh mandarin
fruits are not available.

Keywords: Citrus fruits; Mandarin can; can processing; Phenolic acids; Flavanone glycosides; Total antioxidant capacity

1. Introduction

Lots of epidemiological studies have confirmed that high consumptions of fruits and vegetables were
related with the lower accident of various dangerous illnesses, such as cancer and cardiovascular diseases
[1]
. Among enormous kinds of fruits, citrus fruits, with good flavors and many healthy properties, are
welcomed and consumed widely. Recent studies indicated that besides vitamin C and carotenoids, phenolic

1878-0296 2011 Published by Elsevier Ltd. Open access under CC BY-NC-ND license.
Selection and/or peer-review under responsibility of the Intelligent Information Technology Application Research Association.
doi:10.1016/j.proenv.2011.12.189
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Environmental SciencesSciences 11000000
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compounds which including flavonoids and phenolic acids, were also abundant in citrus fruits and might
play an important role in organism [2].
Although sweet orange (C. sinensis) dominated in the world market. Comparatively, mandarin orange
(C. reticulate) was the leading one in China, and it covered over almost two thirds of the whole production
of citrus fruits. Traditionally, the majority of mandarin fruits were consumed freshly, while in recent years,
and some mandarin fruits were processed to can and exported mainly to USA, Japan, and EU, etc. The
storage of mandarin fruits can be prolonged largely after canning; nevertheless, its nutritional value might
change during processing, too.
Many papers had been published on the antioxidant capacity of citrus fruits, and AA and phenolic
compounds were deemed playing an important role [3, 4]. However, there is no report about the effect of can
processing on the antioxidant capacity mandarin fruits. In this study, the changes of AA, phenolic
compounds and antioxidant capacity of mandarin fruits during can processing were measured and
discussed.

2. Materials and methods

2.1 Chemicals

Standards of p-hydroxybenzoic, vanillic, p-coumaric, caffeic, ferulic, sinapic, narirutin, hesperidin, 2, 2-

diphenyl-1-picrylhydrazyl radical (DPPH), and TPTZ (2, 4, 6-tris (2-pyridyl)-s-triazine) were purchased
from Sigma. All other chemicals used were analytical grade.

2.2 Fruits materials

Satsuma mandarin fruits for processing were collected from a local farm in Ningbo city, and after about
one month storage, they were processed in a local mandarin cannery of Huayu Ltd Xiangshan county,
Ningbo city, Zhejiang province.

2.3 Processing procedures

Mandarin fruits selecting and grading blanching (100C for about 1 min) peeling
segmenting HCl treatment (0.5% for about 45 min) alkaline treatment (0.37% for about 20 min)
water washing manual choosing and trimming filling and weighting filling of sucrose syrup
sealed processing (85C for 25 min) cooling product

2.4 Sample preparation and extraction of phenolics

Unprocessed mandarin fruits and heat treated mandarin fruits were peeled and sliced by hands and
slurred by an electronic mixer, while the samples after HCl and alkaline treatment were slurred directly.
Several tins of mandarin can were opened and the segments were separated from the syrup, and the
segments of can were slurred too. Water content was determined according to GB/T 8858-1988, and total
carotenoid was measured according to GB/T 12291-1990.
1g fruit sample was extracted with 9 mL of 80% methanol for 30 min at room temperature. After
centrifugation at 5000 rpm for 10 min, the supernatant was taken out for the analysis of FGs, and the
evaluation of antioxidant capacity by FRAP assay and DPPH assay.
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2.5 FGs content

The contents of narirutin and hesperidin were determined according to a method described elsewhere
with some modification [5]. 10 L extract was injected into a HPLC system, and it was filtered through a
millipore membrane (0.22 m) before injection. The analysis utilized a Diamonsil C18 column (2504.6
mm i.d.) using methanol: water: acetic acid (37:59:4) (v/v/v) as the mobile phase at a flow rate of 1.0
mL/min at 25 C oven temperature, and the eluent was monitored at 283 nm for quantification of FGs.
Identification of the FGs was accomplished by comparing the retention times of peaks in samples to those
of FG standards. Calculation of FGs concentration (expressed as mg/g of DW) was carried out by an
external standard method using calibration curves.

2.6 Phenolic acids content

Phenolic acids were isolated from the extract according to some previously described methods with
some modification [6]. 2 g sample was firstly diluted to 5mL with ddH20, and then it was treated by
alkaline hydrolysis (5mL of 4 M NaOH, which contained 1% ascorbic acid and 10mM EDTA) for 4 h
under a nitrogen atmosphere at room temperature. After acidification to pH 2 using 6 M HCl, phenolic
acids were extracted from the hydrolysate 5 times with diethyl ether-ethyl acetate (1: l) (v/v) at a solvent to
water phase ratio of 1: l. The ether-ethyl acetate extracts were dehydrated with anhydrous sodium sulfate,
filtered, and evaporated to dryness under vacuum at 30C. The dry residues were dissolved into 5 mL
methanol for HPLC analysis.
Phenolic acids of HPLC analyses were carried out on an Alliance 2695 separations module (Waters)
linked simultaneously to a PDA 2996 (Waters). Prepared phenolic acid solution was filtered through a
millipore membrane (0.45 m) before injection, and 10 L was injected on the reversed phase column
((2504.6 mm i.d.). The column thermostat was set at 40 C. Solvent A consisted of 4% acetic acid, and
solvent B consisted of methanol (A: B=20:80) at a flow rate of 1 mL/min, which was in accordance with
Subba Rao [6] with minor revision. After each run the column was washed with 100% methanol and
equilibrated to initial conditions for 15 min. The PDA detector was set scanning range from 210 to 400 nm
with resolution of 1.2 nm. Phenolic acids were identified by the retention time and the UV-Vis spectra of
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standards. Quantification of phenolic acids was carried out by an external standard method using
calibration curves, and concentration of phenolic acids was expressed as g/g of dry weight (DW).

2.7 AA content

10 g samples were homogenized with 15 ml of 5% metaphosphoric acid. The homogenate was filtered
with four layers of cheesecloth and the residue was treated with 10 ml of 5% metaphosphoric acid for two
successive extractions. The filtrate was combined and centrifuged at 4000 g for 10 min. The supernatant
was collected and made up to 50 ml, and then it was immediately filtered through a millipore membrane
(0.45m). Ascorbic acid was analyzed by using liquid chromatography on an RP-Phase with UV detection
according to [8]. The separation was performed on a Diamonsil C18 column (2504.6 mm i.d.) using 0.1%
(w/v) oxalic acid as the mobile phase at a flow-rate of 1.0 mL/min at 25C oven temperature and the eluent
was monitored at 243 nm. The ascorbic acid contents are expressed here in mg/kg FW.

2.8 FRAP assay

The ferric reducing ability of each standard solution was measured according to a modified protocol
developed in [9].

2.9 DPPH free radical-scavenging assay

The DPPH free radical-scavenging activity of fruits was measured using the method described in [10]
with some modification. A 0.1mmol/L solution of DPPH in methanol was prepared. An aliquot of 0.2mL
of sample was added to 2.8 mL of this solution and kept in the dark for 30 min. The ability of scavenging
the DPPH radical was calculated with the following equation:
%Inhibition = [(A0-A1)/A0] 100
Where A0 is the absorbance of the control, A1 is the absorbance in the presence of sample.

2.10 Statistics

All samples were prepared and analyzed in triplicate. To verify the statistical significance of all
parameters, the values of means S.D were calculated. To compare several groups, analysis of variance
(ANOVA) was used. The Pearson correlation coefficient (R) and P-value were used to show correlations
and their significance (SPSS for Windows, Release 11.5.0 (June 2002, SPSS Inc)). Probability value of
p<0.05 and p<0.01 was adopted as the criteria for significant differences.

3. Results and discussion

Narirutin and hesperidin, two major FGs in mandarin fruits, were measured by HPLC. Six phenolic
acids, including four cinnamic acids (caffeic, p-coumaric, ferulic, and sinapic) and two benzoic acids ( p-
hydroxybenzoic and vanillic), were measured by HPLC-PDA with ferulic dominating in the phenolic acids
(account for over two thirds of total phenolic acids). AA was determined by HPLC, and TAC being
expressed as AEAC which was determined by FRAP assay, and the contribution of AA to TAC was
calculated feasibly, together with the inhibition of DPPH radical was adopted to express the radical
scavenging capacity (RSC). The changes of FGs, phenolic acids, AA and antioxidant capacity of mandarin
fruits during can processing were shown in Table 1.
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Four key processing procedures were investigated, which were heat, HCl, alkaline and canning
treatment. After the blanching procedure, the content of narirutin, total FGs and most of phenolic acids
increased to some extent. The result was accordance with the report of Lo Scalzo [11]. Meanwhile, content
of AA decreased from 251.87 to 240.58 mg/kg, while TAC determined by FRAP decreased from 664.68
mg/kg (AEAC) to 560.11 mg/kg (AEAC), whilst the contribution of AA to TAC increased from 37.89% to
42.95%. However, the radical scavenging capacity (RSC) determined by DPPH assay increased from
34.85% to 36.52%, which was inversed with the FRAP assay. This result was supported by the report of
Dewanto, who found that after blanching, the total antioxidant activity of tomato, which was measured by
total oxyradical scavenging capacity (TOSC) assay increased significantly [12], and also thermal processing
at 115 C for 25 min significantly elevated the total antioxidant activity (determined by TOSC assay) of
sweet corn [13]. Similarly, Jeong reported that heat treatment could increase the antioxidant activity of citrus
peel [14]. It seemed that different methods adopted to measure the antioxidant capacity may have conflicting
results, which also appeared in the result of Lo Scalzo [11]. We supposed that there might be other
photochemicals, such as carotenids, with antioxidant capacity which was not involved in this study or other
mechanism responsible for the above divergences.
After the procedure of HCl treatment, about half of narirutin and one third of hesperidin were lost.
However, the loss of phenolic acids was not so great (Table 1). AA content decreased from 240.58 mg/kg
to 227.32 mg/kg, and AEAC of TAC decreased from 560.11 mg/kg to 484.65 mg/kg, whilst the
contribution of AA to TAC increased from 42.95% to 46.90%. The result indicated that the procedure of
HCl treatment had relatively limited impact on the content of phenolic compounds and AA of mandarin
fruits, however, the content of FGs and TAC decreased considerably,

Table 2 Correlation coefficients for FRAP, DPPH, vitamin C, total FGs and total phenolic acids (n=6)

FRAP DPPH Vitamin C Total FGs Total phenolic acid

DPPH 0.952 (**)

Vitamin C 0.972 (**) 0.989 (**)
Total FGs 0.954 (**) 0.915 (*) 0.910 (*)
Total phenolic acids 0.949 (**) 0.997 (**) 0.981 (**) 0.912 (*)
Total carotenoid 0.913 (**) 0.949 (**) 0.969 (**) 0.819 (*) 0.933 (**)

* Correlation is significant at the 0.05 level (2-tailed).

** Correlation is significant at the 0.01 level (2-tailed).

After the procedure of alkaline treatment, it was observed that the loss of FGs and phenolic acids were
minimal. AA content decreased from 227.32 mg/kg to 197.67 mg/kg, and AEAC of TAC decreased from
484.65 mg/kg to 425.64 mg/kg, while the contribution of AA to TAC remained almost unchanged. It
seemed that alkaline treatment had more inverse impact on AA than HCl treatment since considerable
amount of AA was lost in this step.
The can product was divided to two parts, the mandarin segments (filling) and syrup. It was found that
considerable FGs, phenolic acids and AA were appeared in the syrup part. After the sterilization step, the
total FGs content decreased from 315.12 mg/kg to 254.27 mg/kg (content of narirutin from 65.19 mg/kg to
48.23 mg/kg, and content of hesperidin from 249.93 mg/kg to 206.03 mg/kg), and total phenolic acids
decreased from 48.34 mg/kg to 44.20 mg/kg. Meanwhile, the decreasing tendency was also found in AA
content (from 197.67 mg/kg to 186.98 mg/kg) and TAC (from 425.36 mg/kg to 391.95 mg/kg (AEAC)),
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and the contribution of AA to TAC remained stable. The results indicated that sterilization procedure had
little effect on the AA, TAC and phenolic compounds of the mandarin segments, given that considerable
amount of AA and phenolic compounds (had been) or was moved to syrup portion.
It was noted that the syrup of can products contained considerable content of FGs (106.42 mg/kg),
phenolic acids (20.09 mg/kg), AA (76.10 mg/kg) and TAC (142.39 mg/kg), which accounted for about one
third of the content of these compounds in mandarin segments. And we supposed that their contents would
still increase as storage time consuming, since their contents were lower than that of mandarin can
segments. Similarly, Chaovanalikit reported that during canning, about half of the anthocyanins and
polyphenolics leached from the fruits into the syrup with little total loss [15]. Therefore, from the nutritional
view, we suggested that mandarin can syrup could be consumed for its rather high content of nutrients and
high TAC.
The DPPH assay was performed following each step of sample treatment to monitor the change of RSC
of mandarin segments. The result showed that the RSC increased after blanching procedure, afterwards,
RSC decreased after the rest three procedures, however, the change was rather small compared with TAC
determined by FRAP assay. The results indicated that if only one assay was performed, for example, FRAP
assay, it might cause some misunderstanding on the change of antioxidant capacity of mandarin segments.
Correlation coefficients of FRAP, DPPH, vitamin C, total FGs and total phenolic acids were shown in
Table 2. Apparently, correlations between all cases were significant (P<0.05). Therefore, we suggested that
AA and phenolic compounds (FGs and phenolic acids) all played important roles to TAC of mandarin
fruits. In conclusion, after can processing, the total FGs content of mandarin segments decreased from
502.39 mg/kg to 254.27 mg/kg (narirutin from 116.67 mg/kg to 48.23mg/kg, and hesperidin from 385.27
mg/kg to 206.03 mg/kg), and the total phenolic acids decreased from 57.51 mg/kg to 44.20 mg/kg. The
loss of total phenolic acids was not as large as that of FGs, which was possibly due to that considerable
phenolic acids occurred as bound forms [16], which were not water soluble. The AA content of mandarin
segments decreased from 251.87 mg/kg to 186.98 mg/kg, and TAC from 664.48 mg/kg to 391.95 mg/kg,
and inhibition of DPPH radical from 34.85% to 27.31%.
To sum up, after can processing, small proportions of phenolic acids and AA were lost, and about half
of FGs and TAC were lost. However, in view of that considerable portions of phenolic compounds and AA
existing in the syrup portion, so the loss was not so great, and if the result of DPPH assay was taken into
consideration, the loss of antioxidant capacity was also not as large as that of TAC determined by FRAP
assay. Finally, we suggested that the majority of AA, phenolic compounds (FGs and phenolic acids) and
antioxidant capacity of mandarin segments were retained after can processing, and mandarin cans could
serve as a substitute when fresh mandarin fruits are not available.

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