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The Journal of Food Technology. Photon 106 (2014) 248-251
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Original Research Article. ISJN: 3852-1875: Impact Index: 3.43
The Journal of Food Technology
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Studies on determination of bitterness in sweet orange juice by HPLC
Narwade R.R., Sakhale B.K.* Gaikwad S.S.
Food Technology Division, Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada
University, Aurangabad, Maharashtra, India
Narwade R.R., Sakhale B.K.* Gaikwad S.S. receives
International Food Technology Award-2014
Article history:
Received: 17 May, 2014
Accepted: 22 May, 2014
Available online: 24 November, 2014
Keywords:
Sweet orange,
bitterness.
Pretreatments,
RP-HPLC,
Limonin,
Abbreviations:
SU: Standard Uncertainty
Corresponding Author:
Sakhale B.K.*
Assistant Professor
Email: [email protected]
Narwade R.R.
Student
Gaikwad S.S.
Senior Research Fellow
Abstract
The investigation was conducted for determination
of limonin which is responsible for bitterness in
sweet orange juice by HPLC method. The cleaned
and washed sweet orange fruits were peeled and
subjected to the pre-treatment of acid and alkali
using various concentrations of NaOH and HCl. The
treatments T1, T2 and T3 were carried out by using
0.5N, 1.0N and 1.5N concentrations of NaOH and
0.2N, 0.4N and 0.8N concentrations of HCl
respectively followed by plain water wash. The
control sample was used with no pre-treatment.
Then juice was extracted and analyzed for
determination of limonin content by using Reversed
Phase HPLC system equipped with UV detector
and C18 column at 214 nm. The limonin content
detected in control was highest and treatment T1
shown the negligible amount of limonin content.
Citation:
Narwade R.R., Sakhale B.K., Gaikwad S.S., 2014. Studies
on determination of bitterness in sweet orange juice by
HPLC. The Journal of Food Technology. Photon 106, 248251
All Rights Reserved with Photon.
Photon Ignitor: ISJN38521875D706424112014
1. Introduction
Sweet orange is the important citrus fruit crop
grown throughout the world; it contributes 71 per
cent of the total citrus fruit production. Fresh juice
of sweet orange is refreshing, thirst quenching and
energizing drink that improves health and
nutritional requirements. (Siddiqui et al., 2013).The
principal bitterness compounds in the grapefruit
and orange juices are naringin and limonin. Major
classes of compounds, responsible for the bitterness
of citrus juices, are namely the limonoids and
flavonoids (Mozaffar et al., 2000).
1.1 Delayed Bitterness in Citrus Juices
Oranges in general do not taste bitter if eaten fresh
or if juice is squeezed from the fruit and consumed
immediately. However, the juice becomes bitter
within a few hours after juicing at room
temperature or overnight if stored in a refrigerator.
This gradual development of bitterness, or delayed
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bitterness, in navel orange juices is caused by the
formation of limonin from a tasteless precursor.
Maier and Beverly finally identified limonoate Aring lactone as the precursor of limonin in citrus
fruit. A ring-closing reaction proceeds under acidic
conditions below pH 6.5 and is accelerated by the
enzyme, limonin D-ring lactone hydrolase. During
pasteurization and/or evaporation of the juice, heat
catalyzes the esterification reaction (Hasegawa et
al., 2000). Limonin bitterness can be removed by
treatment of ion exchange and adsorbent resins
(Kola et al., 2010). Bitterness can also be reduced
by b-cyclodextrin polymer in tangerine citrus
reticulatablanco (Mongkolkul et al., 2006).
Hydrolysis of limonin for debittering of kinnow
mandarin juice can be achievd by Rhodococcus
fascians. (Marwaha et al., 1994). Strains of
coynebaterium fascians can be used to reduce
limonoid bitterness in citrus products (Hasegawa,
248
1984). Reduction of limonin bitterness in navel
orange juice serum can be attained with bacterial
cells immobilized in acrylamide gel (Hasegawa et
al., 1982).
1.2 Analysis of citrus orange juice by HPLC
High
performance
liquid
chromatography
technique with an UV photodiode-array detector
was used to analyze flavonoids of an extract of
Citrus species (Bilbao et al., 2007). Quantitation of
limonin D-ring lactone hydrolase and limonoate
dehydrogenase activities can be studied by highperformance liquid chromatography. In this study
ion-pairing reversed-phase HPLC has been used to
separate and determine different derivatives of
limonin (Merino et al., 1997). The method can be
developed for the quantization of the bitter
component limonin in grapefruit juice and other
citrus juices (Van et al., 1988).
2. Materials and Methods
2.1 Determination of limonin by HPLC
The sweet orange fruits of cv. Nucellar, glass
bottles of 200 ml capacity with airtight lid, sodium
hydroxide, hydrochloric acid and acetonitrile
(HPLC grade), limonene (standard sample) were
used in this study.
The equipment like Reversed Phase HPLC with a
chromatographic system (Jasco, USA) consisting
of quaternary solvent delivery pump (PU-2089), a
degasser, an auto- injector, column oven and UV
detector (UV-2075) with chrompass software and
column temperature 300C; Hand juicer and Hand
held digital refractometer (Atago, Tokyo Japan)
were used.
2.2 Chemical analysis of sweet orange
Total soluble solids were determined by using hand
held digital refractometer and the values are
expressed in degree brix. The acidity of the juice
was calculated by the titration method as given By
(Ranganna, 2004).
2.2.1 Fruit Treatment
The freshly harvested sweet orange fruits were
washed with plain H2O, peeled and then pretreated
with various concentrations of acid and alkali in
order to get the debittered juice. The fruits were
randomly selected for the treatment, numbers of
fruits in each treatment were 4 in number, and total
treatment time for each treatment was of 20
minutes. The treatment details are given below in
Table 1.
The fruits were washed and peels were removed
manually. Then the fruits with mesocarp were kept
in 0.5 N alkaline solution (NaOH) for 10 minutes.
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Table 1: Pre-treatments to the peeled sweet orange
PreDetails
treatment
T0
Control
T1
Peeled fruits were treated with 0.5 N
NaOH and 0.2 N HCl
T2
Peeled fruits were treated with 1.0 N
NaOH and 0.4 NCl
T3
Peeled fruits were treated with 1.5 N
NaOH and 0.8 N HCl
Then fruits were washed in the gentle stream of
water and the loosened mesocarp was removed till
the juice sacs were seen. Then these alkali treated
fruits were kept in acid solution (HCl) of various
concentrations for 10 minutes and then washed
under the gentle stream of the water. The treated
fruits then subjected to extract the juice. The pretreatment details of peeled sweet orange fruits are
given in Table.1 above. Then juice was extracted
and was used for further analysis.
2.1.2 Analysis of Sweet Orange juice for
limonin content by RP-HPLC Method
Prior to injection into the HPLC, the juice was
heated to 90o C for 5 minutes with 40% aqueous
acetonitrile (1:1 dilusion), in order to dissolve any
precipitated limonin, and close the ring on any
Limonoate A-Ring Lactone present in the sample.
The samples were then filtered through a 0.45 µ
filter (aqueous syringe filter). The filtered samples
were injected into the HPLC column, and 37%
acetonitrile (i. e. the mobile phase) was passed
through the column. The flow rate of the mobile
phase was 1 ml/min. and the effluent was
monitored at 214 nm. The total analysis time was
approximately of 12 minutes. The concentration of
limonin in each of the samples was quantitatively
measured by comparing the peak area with
standard limonin (Mozaffar et al., 2000).
3. Results and Discussion
The present study was undertaken in order to know
the effect of various pre-treatments of sweet orange
fruits on limonin content of juice by using HPLC
system.
3.1 Chemical analysis of sweet orange juice
Total Soluble Solids (TSS), Titrable acidity and
percentage juice yield was calculated. The results
obtained are presented in Table 2.
The peeled fruits were washed thoroughly after
each pre-treatment process. Therefore, no
significant changes were found in the natural flora
of the sweet orange fruits used and TSS, titrable
acidity and percentage juice yield. The similar
results were also obtained by (Marzouk and
Moufida, 2003).
249
Table 2: Juice yield, TSS and Acidity of Sweet orange
juice
TitrableAcidity
Treatment Juice
TSS
(%)
yield
(oBrix)
(%)
T0
36.19
10.06
0.24
T1
35.08
10.42
0.25
T2
36.03
10.75
0.23
T3
34.88
10.55
0.22
*Each value is the average of tree determinations
3.2 Limonin Content of the pre-treated Sweet
Orange juices
The peaks obtained by HPLC analysis are as
following in which the limonin peak is designated
by ‘L’ as given below, The limonin content of the
sweet orange juice determined by RP-HPLC is
presented in terms of graphs generated by HPLC.
The limonin content in ppm is calculated and is as
given in Table no. 3.
Figure 3: Limonin peak of T1 juice sample
Treatment 2
Figure 4: Limonin peak for T2 juice Sample
The figure-1 represents the HPLC graph of
standard limonin sample and the largest peak
represents the limonin peak at retention time of
6.60 min. and 12.5 ppm of limonin. The further
sample graphs are compared to this to determine
the limonin content.
Figure 1: Limonin peak of standard sample of limonin
Treatment 3
Figure 5: Limonin peak for T3 juiceSample
Control sample
Figure 2: Limonin peak of control juice sample
Treatment 1
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Table 3: Limonin content of the pre-treated, control and
standard limonin sample
Sample
Retention Time in Limonin
Analyzed
min.
content
in
ppm
Standard
6.60
12.50
limonin
T0 juice
6.50
7.03
T1 juice
6.40
0.62
T2 juice
T3 juice
-
Figure-2 represents the graph of control sample
which is not pre-treated. In this the limonin content
is 7.03 ppm which responsible for bitterness in
sweet orange juice. The retention time was 6.50
250
min. similar results were obtained by (Marzauk and
Maufida, 2003).
Figure-3 represents the graph of pre-treated sweet
oranges as given by T1. The limonin content is 0.62
ppm at retention time of 6.40 min. The limonin
content is low as compared to the control sample
but pre-treatment is in these concentrations is not
enough to remove the complete limonin content.
Similar results were obtained by (Mozaffar et al.,
2000).
Figure-4 and figure-5 shows the peak of pretreatments T2 and T3 respectively which doesn’t
show the limonin peak at all. Thus the pretreatments in concentrations as given by T2 and T3
are able to remove the limonin content completely.
Similar results were obtained by (Mozaffar et al.,
2000).
Hasegawa S., Berhow M.A., Manners G.D., 2000. Citrus
Limonoid Research: An Overview. ACS Symposium
Series: American Chemical Society:Washington, DC
Hasegawa, 1984. Strains of Coynebaterium fascians and
use thereof to reduce limonoid bitterness in citrus
products. United States Patent No. 4447456
Hasegawa S., Patel M.N., Snyder R.C., 1982. Reduction
of Limonin Bitterness in Navel Orange Juice Serum with
Bacterial Cells Immobilized in Acrylamide Gel. Journal
of Agricultural and Food Chemistry, 30(3), 509–511.
Kola O., Kaya C., Duran H., Altan A., 2010. Removal of
Limonin Bitterness by Treatment of Ion Exchange and
Adsorbent Resins. Journal of Food Science and
Biotechnology, 19(2), 411-416.
Research Highlights
Marwaha S.S., Puri M., Bhullar M.K., Kothari R.M.,
1994. Optimization of parameters for hydrolysis of
limonin for debittering of kinnow mandarin juice by
Rhodococcus fascians. Enzyme and microbial
Biotechnology, 16, 723-725.
The effect of 0.4N HCL and 1.0N NaOH was
optimum and best to reduce the bitterness in sweet
orange juice
Marzouk B., Moufida S., 2003. Biochemical
characterization of blood orange, sweet orange, lemon,
bergamot and bitter orange. Journal of Phytochemistry
62, 1283–1289.
Reversed phase HPLC system with C18 column and
UV detector is effective to determine the limonin
content in sweet orange.
Limitations
Limonine content in orange juice is varied
according to the variety, season so it cannot be
specified and determined with the same
combination of acid and alkali so the research can
be carried out on trial and error basis only.
Recommendations
It is highly recommended to carry out the research
using the same variety and under controlled
environmental conditions for betterment and
improvement of that variety.
Competing Interests
The authors declare that they have no competing
interests.
References
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