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Volume 5 • Issue 7 • 1000349
J Food Process Technol
ISSN: 2157-7110 JFPT, an open access journal
Open Access
Research Article
Food
Processing & Technology
Singh and Immanuel, J Food Process Technol 2014, 5:7
http://dx.doi.org/10.4172/2157-7110.1000349
*Corresponding author: Soma Singh, Food Process Engineering, Sam
Higginbottom Institute of Agricultural Engineering and Technology, India, Tel: 91
532 2684281; E-mail: somasingh200@gmail.com
Received June 15, 2014; Accepted August 01, 2014; Published August 11, 2014
Citation: Singh S, Immanuel G (2014) Extraction of Antioxidants from Fruit Peels
and its Utilization in Paneer. J Food Process Technol 5: 349. doi:10.4172/2157-
7110.1000349
Copyright: © 2014 Singh S, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Abstract
Natural antioxidants have gained considerable interest in recent years for their role in preventing the auto oxidation of fats, oils
and fat containing food products. In the present study, peels of pomegranate, lemon and orange were used as sources of natural
antioxidants. Among the three extracts pomegranate exhibited a high percentage of antioxidant activity and phenolic content of
92.7%, 249.41 mg/g in comparison to lemon and orange peel extract. Maximum total phenolic content was found in lemon extract
(0.9 mg/g). Paneer samples prepared by addition of natural antioxidant extracts from these peels where subjected to sensory
studies which showed that the extracts at the level of 2% was acceptable and had greater ability to prevent peroxide formation.
The ability to prevent peroxide formation in paneer sample was in the order of pomegranate peel> lemon peel> orange peel.
Synthetic antioxidants generally used in food products like BHT and BHA are toxic and may cause health hazards. Fruit
peels like pomegranate, lemon and orange are normally wasted during fruit processing thus a proper waste utilization of these
peels were done. Natural antioxidants from these peels were extracted and then utilized in paneer to increase their shelf life
by preventing peroxide formation. Thus these natural antioxidants could be added to any food product containing fat and oil to
increase their shelf life by preventing rancidity.
Extraction of Antioxidants from Fruit Peels and its Utilization in Paneer
Soma Singh
1
* and Genitha Immanuel
2
1
Food Process Engineering, Sam Higginbottom Institute of Agricultural Engineering and Technology, India
2
Department of Food Process Engineering, Vaugh School of Agricultural Engineering and Technology, Sam Higginbottom Institute of Agricultural Engineering and
Technology, Allahabad-211007, India
Keywords: Antioxidant activity; Total phenolic content; Total
avonoid content
Introduction
Fruits and vegetable processing in India generates substantial
quantities of waste. It had been previously reported that these wastes
and by-products of fruits are an abundant source of antioxidant
polyphenols [1]. ese peels and pomace are a source of sugars,
minerals and organic acids, dietary bers and phenolics which have
a wide range of actions which includes antioxidants, antimutagenic,
cardio preventive, antibacterial and antiviral activities [2]. Use of waste
as a source of polyphenols and antioxidants may have considerable
economic benet to food processors. erefore a cheap, ecient and
environmentally sound utilization of these wastes is needed.
Paneer is an important Indian traditional coagulated dairy product
that provides sound nutrition, variety, safety, novelty of avour, texture,
portability and protability to consumers. It is an acid coagulated dairy
product, which is similar to western cottage cheese and Tofu (Soy
paneer). In India, paneer production has been largely conned to small
non-organized sectors. It is estimated that 1% of the country’s total
milk production is converted into paneer and the annual production is
estimates at 150, 000 tonnes.
Antioxidants are the chemical substances that reduce or prevent
oxidation and have the ability to counteract the damaging eects of free
radicals in tissues, and thus are believed to protect against cancer, heart
disease and several other diseases. ey scavenge radicals by inhibiting
initiation and breaking of chain reaction, suppressing formation of free
radicals by binding to the metal ions, reducing hydrogen peroxide, and
quenching superoxide and singlet oxygen [3].
Antioxidants are used as food additives to guard against food
detoriation. ese are added to food products like oil, bread, cookies,
biscuits and dairy products like sandesh, paneer etc. to enhance their
shelf life by preventing lipid peroxidation and protecting from oxidative
damage. Exposure to oxygen and sunlight are the two main factors in
the oxidation of food, so food is preserved by keeping in the dark and
sealing it in containers or even coating it in wax, as with cucumbers.
ese antioxidants are an especially important class of preservatives
because like bacterial or fungal spoilage, oxidation reactions also occur
relatively rapidly in frozen or refrigerated food causing their spoilage.
e present study was done to explore with the objective to extract
antioxidants in the form of phenols and avonoids from fruit peels
like pomegranate, lemon and orange peels and to determine their
antioxidant activity. e extracts were further studied for the eect of
antioxidants on the peroxide Value of paneer.
Materials and Methods
Materials
Raw materials: Fresh peels of pomegranate, lemon and orange and
milk were procured from local market, Allahabad, India.
Chemicals: e dierent chemicals like Ethanol, Methanol,
Petroleum Ether (Central Drug House Private Limited, India); DPPH
(Diphenyl picryl hydrazyl) from (Sigma Chemicals), Folin-Ciocalteu
Reagent (MERCK Specialties Private Limited) gallic acid, quercetin,
sodium carbonate and ascorbic acid, chloroform, glacial acetic acid,
citric acid, potassium iodide, aluminium chloride and potassium
acetate of (MERCK Specialties Private Limited, India) were used during
the investigation
Experimental procedure
Extraction of antioxidant from peels of pomegranate, lemon and
orange: e dried powders of peels were extracted by cold percolation
Citation: Singh S, Immanuel G (2014) Extraction of Antioxidants from Fruit Peels and its Utilization in Paneer. J Food Process Technol 5: 349.
doi:10.4172/2157-7110.1000349
Page 2 of 5
Volume 5 • Issue 7 • 1000349
J Food Process Technol
ISSN: 2157-7110 JFPT, an open access journal
method [4] using ethanol as a solvent. 10 g of the dried powder was
taken in 100 ml of ethanol in conical ask, plugged with cotton wool
and then kept in an orbital shaker at 120 rpm for 24 h. Aer 24 h the
extract was ltered through Whatman lter paper No.41 for removal of
peel particles and concentrated under vacuum at 40°C. e dry extract
was stored at 4°C.
Determination of extraction yield: e residues obtained aer
ltration were weighed to obtain the extraction yield.
Extraction yield (%) =(weight of the residue)/(total weight of the
peel powder)×100 (1)
Determination of total phenolic content: e total phenol content
was determined according to Folin- Ciocalteu’s reagent method [5]. 0.5
ml of extract and 0.1 ml (0.5 N) Folin-Ciocalteu’s reagent was mixed and
the mixture was incubated at room temperature for 15 min. en 2.5 ml
of 20% sodium carbonate solution was added and further incubated for
30 min. at room temperature and the absorbance was measured at 760
nm. Gallic acid was used as a positive control [6]. Total phenol values
are expressed in terms of gallic acid equivalent tannic acid equivalent
for pomegranate and gallic acid equivalent for lemon and orange peel
(mg of gallic acid/g of extracted compound).
Determination of total avonoid content: e avonoid content
was determined according to aluminium chloride colorimetric method
[7]. e reaction mixture consisting in a nal volume of 3 ml, 1.0 ml
of sample (1 mg/ml) 1.0 ml methanol and 0.5 ml of (1.2%) aluminium
chloride and 0.5 ml (120 mM) potassium acetate was incubated at
room temperature for 30 min. e absorbance of all the samples
was measured at 415 nm. Quercetin was used as positive control [8].
Flavonoid content is expressed in terms of Quercetin equivalent (mg/g
of extracted compound).
Determination of DPPH Radical scavenging activity of
antioxidant extract: A modied version of the DPPH method was
used. A working DPPH solution (0.048 mg ml -1) was prepared by
making a 1 in 5 dilution of the methanolic DPPH stock solution (2.38
mg ml-1). Prior to analysis, serial dilutions of the methanolic extracts of
the samples were prepared. Diluted sample (500 µl) and DPPH working
solution (500 µl) were added to a micro-centrifuge tube. Aer vortexing,
the tubes were le in the dark for 30 min at room temperature. e
absorbance was then measured against methanol at 515 nm in 3 ml
cuvettes using a spectrophotometer. e decrease in absorbance
of a sample was calculated in comparison to a blank sample (500 µl
methanol and 500 µl DPPH). e relative decrease in absorbance was
then calculated as follows: % inhibition= 1- ((absorbance of sample –
absorbance of blank)/ absorbance of control)*100
Preparation of paneer with antioxidant extracts: In order to
obtain maximum benet from the use of antioxidant in food products,
several points are considered in their selection and use. e form of
antioxidant (powder or solution), method and time of incorporation are
particularly important for the dispersion of antioxidant and ultimately
stabilization of the product (Prevention of Food Adulteration, 1996).
In the present study the antioxidants were in solution form and they
were added in small amount (1-3%) to the paneer sample. Control was
prepared without antioxidant extract addition. e other variations
include addition of pomegranate, lemon and orange peel extracts at
dierent levels of (1%, 2% and 3%). A1, A2, A3 were samples containing
pomegranate extract at 1%, 2% and 3% respectively. B1, B2, B3 were
samples containing lemon extract at 1%, 2% and 3% respectively. C1,
C2, C3 were samples containing orange extract at 1%, 2% and 3%
respectively..
Evaluation of lipid oxidation: Paneer was prepared according to
the method given by [9]. An accelerated oxidation test was performed
to accelerate lipid oxidation in paneer sample. Before analyzing the
peroxide value paneer sample was kept in an oven maintained at 63°C
for 1 hour. Lipid oxidation was determined as change in peroxide value.
Determination of peroxide value of paneer: A clean dry boiling
tube was used to measure 3 g of paneer sample. 2.0 g of powdered
potassium iodide was added and then 20 ml of solvent (2 vol. glacial
acetic acid + 1 vol. chloroform) was added into the tube. e tube was
placed in boiling water such that the mixture boils within 30 seconds
and then allowed to boil vigorously for more than 30 seconds before it is
poured quickly into a ask containing 25 ml of water and the mixture in
the ask was titrated against with 0.01 N sodium thiosulphate solution
using starch as an indicator. e blank was performed at same time. e
experiment was repeated and calculation was done as below
Peroxide value=((S-B)×N×100)/W (2)
Where, S, B, N and W are the volume of titrant of sample, blank,
normality of sodium thiosulphate and weight of sample respectively.
Sensory analysis
e Sensory evaluation of paneer was carried out by a 10 member
semi-trained panel comprised of students and academic sta members
of the faculty who had some previous experience in sensory evaluation
of food products. e panel members were requested in measuring
the terms identifying sensory characteristics and in use of the score.
Judgments were made through rating products on a 9 point hedonic
scale with corresponding descriptive terms ranging from 9 ‘like
extremely’ to 1’dislike extremely’.
Statistical analysis
All experiment was determined 3 times and the results were
reported as mean. e data recorded during the course of investigation
were statistically analyzed by the ‘Analysis of Variance- One Way
Classication’. is technique developed by Dr. R. A. Fisher in 1923
gives an appropriate method capable of analyzing the variation of
population variance. e signicant aect of treatment was judged
with the help of ‘F’ (variance ratio). Calculated F value was compared
with the table value of F at 5% level of signicance. If calculated value
exceeded the table value the eect was considered to the signicant. e
signicance of the study was tested at 5% level.
Results and Discussion
Extraction Yield (%) of fruit peels
Table 1 shows the extraction yield of pomegranate, lemon and
orange peel
*Each value represents average of three determinations
In all these three fruit peels maximum yield of antioxidants using
ethanol as a solvent was extracted with pomegranate peel. Pomegranate
peel showed the maximum yield of 27.5% and lemon peel showed the
extraction yield of 25.8% while orange peels showed the minimum
extraction yield of 23.9%. e extraction yield of antioxidants from
Fruit peels Extraction yield (%)
Pomegranate peel 27.5
Orange peel 23.9
Lemon peel 25.8
Table 1: Extraction yield (%).
Citation: Singh S, Immanuel G (2014) Extraction of Antioxidants from Fruit Peels and its Utilization in Paneer. J Food Process Technol 5: 349.
doi:10.4172/2157-7110.1000349
Page 3 of 5
Volume 5 • Issue 7 • 1000349
J Food Process Technol
ISSN: 2157-7110 JFPT, an open access journal
fruit peels depends upon the solvent used for extraction. e extraction
yield of pomegranate was more or less similar to the work of [3] but
slightly diered due to the solvent used for extraction. e extraction
yield of orange and lemon peels were in agreement with [10] who
studied the phenolic and antioxidant activity of extracts from fruit
peel. Ethanol and water are the most widely employed solvents for
hygienic and abundance reasons, respectively. Less polar solvents such
as ethyl acetate provided slightly more active extracts than mixtures
with ethanol or methanol, or methanol alone for tamarind seed coats
[11] although ethanol and methanol extracts also presented high lipid
peroxidation-inhibiting activity, comparable to α-tocopherol.
Total phenolic content and total avonoid content
TPC was found maximum in pomegranate peel (249.41 mg/g) and
minimum in orange peel (169.56 mg/g) whereas lemon peel showed
the TPC of (211.70 mg/g). e results were in agreement with [3] who
studied the process for extraction of antioxidants from pomegranate
peel, but diered slightly this may be due to the experimental and
environmental conditions. Methanol are said to be the most suitable
solvent in the extraction of phenolic compounds due to its ability
to inhibit the reaction of PPO that causes oxidation of phenolic and
its ease of evaporation as compared to water [12]. Moure et al. [13]
explained both methanol and ethanol oered best result to extract
phenolic compound as compared to acetone. ey found that as the
polarity of the solvent is increased, higher extraction yield of total
soluble solids and total extractable polyphenolics is obtained. TFC
was found maximum in lemon peels. Lemon peels contained 0.9 mg
quercetin equivalent/g and the minimum avonoid content was found
in orange peels (0.3 mg quercetin equivalent/g) and in pomegranate
peel it was 0.6 mg quercetin equivalent/g. e total avonoid content
in pomegranate was in agreement with [14] but diered slightly in
values, this may be due to the dierence in extraction procedure
and experimental conditions. Ghafar et al. [15] also worked on the
Flavonoid content of citrus species and found avonoid content in the
range of 2.99- 22.25 mg/g in dierent cultivars of citrus species. But
they had found avonoid content as hesperidin equivalent/100 ml of
extract (Table 2).
Total antioxidant activity
DPPH radical scavenging activity assay assessed the ability of the
extract to donate hydrogen or to scavenge free radicals. DPPH radical
is a stable free radical and when it reacts with an antioxidant compound
which can donate hydrogen it is reduced to diphenylpicrylhydrazine.
Initially the solution was deep violet in color which was changed to
light yellow. e change in color was due to the reduction of DPPH
with the antioxidant compounds present in the peels of pomegranate,
lemon and orange. e reduction was determined by the decrease
in absorbance at 517 nm. Table 3 shows the % scavenging activity of
DPPH of peels of Pomegranate, lemon and orange.
Maximum antioxidant activity of 92.7% was found in pomegranate
peels and minimum antioxidant activity of 71.4% was found in orange
peels. Antioxidant activity of lemon peels was 75.9%. Scavenging
activity of peels is due to the presence of polyphenols and avonoids.
Higher antioxidant activity of pomegranate is due to the higher content
of polyphenols and avonoids. e results were more or less similar
to [16] and [17] for pomegranate peels and in agreement with [10] for
lemon and orange extract
Preparation of paneer with antioxidant extracts
Paneer was prepared to give 4 variations. Control was prepared
without antioxidant extract addition. e other variations include
addition of pomegranate, lemon and orange peel extracts at dierent
levels of (1%, 2% and 3%). A1, A2, A3 are samples containing
pomegranate extract at 1%, 2% and 3% respectively. B1, B2, B3 are
samples containing lemon extract at 1%, 2% and 3% respectively.
C1, C2, C3 are samples containing orange extract at 1%, 2% and 3%
respectively. Dierent extract samples are also shown from Plate 4.2 –
Plate 4.11. Paneer was subjected to sensory acceptability. Tables 4 and 5
shows the data of mean score of sensory acceptability of pomegranate,
lemon and orange extract paneer samples.
In order to obtain maximum benet from the use of antioxidant
in food products, several points are considered in their selection
and use. e form of antioxidant (powder or solution), method and
time of incorporation are particularly important for the dispersion of
antioxidant and ultimately stabilization of the product (Prevention of
Food Adulteration, 1954). In the present study the antioxidants were in
solution form and they were added in small amount (1-3%) to the paneer
sample. Data indicated that maximum score for overall acceptability
of pomegranate, orange and lemon peels were at the level of 1- 2% of
antioxidant extract. At the level of 3% it was not very acceptable because
of the dierence in the color and appearance however there was no
change in the taste, texture and avour in the sample.
Eect of antioxidant extracts on the peroxide value of paneer
Accelerated oxidation tests or Schall Oven tests were conducted.
Normally Schaal oven test is used for determination of oxidation of
oils but it is also used in dairy industry. Oxidative stability of milk fats
from cow’s oered naked oats- and barley-based diets were compared
in shelf-life tests using the Schaal oven test at 63°C, and peroxide value
were determined [18]. e eect of antioxidants on peroxide value of
Content
Sample TPC(mg/g) TFC(mg/g)
Pomegranate peel 249.41 0.6
Orange peel 169.56 0.3
Lemon peel 211.7 0.9
Table 2: Total phenolic content and Total Flavonoid.
Fruit peels Antioxidant activity (%)
Pomegranate peels 92.7
Orange peels 71.4
Lemon peels 75.9
Table 3: Antioxidant activities of peels of pomegranate, lemon and orange.
Sample Color Taste Texture Flavour Appearance Overall
acceptability
Control 8.32 8.43 8.5 8.52 8.58 8.56
A1 8.39 8.42 8.33 8.42 8.48 8.45
A2 8.24 8.32 8.32 8.31 8.22 8.44
A3 7.94 8.01 8.31 8.22 7 7.5
Table 4: Mean score of acceptability of paneer sample with pomegranate Extract.
Sample Color Taste Texture Flavour Appearance Overall
acceptability
Cont 8.32 8.43 8.5 8.52 8.58 8.56
B1 8.38 8.41 8.22 8.48 8.3 8
B2 8.27 8.36 8.24 8.4 8.26 8.2
B3 7.84 7.66 8.22 8.32 7.21 7.6
Table 5: Mean score of acceptability of paneer sample with Lemon extract.
Citation: Singh S, Immanuel G (2014) Extraction of Antioxidants from Fruit Peels and its Utilization in Paneer. J Food Process Technol 5: 349.
doi:10.4172/2157-7110.1000349
Page 4 of 5
Volume 5 • Issue 7 • 1000349
J Food Process Technol
ISSN: 2157-7110 JFPT, an open access journal
paneer sample over 8 days of storage is given in Tables 4 and 5, Since
only 1% and 2% level of antioxidant extracts were acceptable therefore
only there eect on peroxide value were studied.
e peroxide value of each sample increased with the storage
period. When antioxidant extracts of pomegranate, orange and lemon
peels were added at the level of 1% and 2% then there was a signicant
change in the peroxide value of paneer sample in comparison to control.
All the paneer samples containing antioxidant extracts had lower value
of peroxide value than the control samples. Initially peroxide value of
the control and all the samples was 0.09 which showed no signicant
dierence at (p ≤ 0.05) which gradually increased with the storage
time. Maximum peroxide value of the control and the samples A1, B1
and C1 was 2.56, 1.42, 1.67 and 2.23 respectively on 8th day of storage
at the level of 1% and of samples A2, B2 and C2 was 1.32., 1.47 and
2.18 respectively at the level of 2%. Tables 6 and 7 shows the eect of
antioxidant extract at the level of 1% and 2% on the peroxide value
respectively. e ability to prevent peroxide formation was higher at
the level of 2% due to the higher concentration of antioxidant extracts.
is is predicted by the lower percentage of peroxide formation in
comparison to 1%. From 2nd day to 8th day there was sucient oxidation
to cause signicant dierence in the peroxide value measured for
control and the antioxidant containing samples. ANOVA Table 8 shows
a signicant dierence in the samples at 5% level of dierence. Fcal
value (6.99) and Fcal value (6.97) was more than the Fcri Value (3.055)
which showed a signicant dierence among the peroxide values of the
samples over a period of 8 days when antioxidants were added at the
level of 1% and 2% respectively.
Ability of antioxidants in preventing peroxide formation in paneer
samples was found in the order of pomegranate extract > lemon extract
> orange extract. Pomegranate extract showed the maximum ability
in preventing the peroxide formation due to its higher antioxidant
activity. e results were in agreement with [19] who studied the
eect of synthetic antioxidants on the shelf life of paneer. ere are
not much report on the use of antioxidants from peel extract in dairy
products, however there are reports on fortication with herbal extract
to increase the avour and shelf life of dairy product. Merai et al. [20]
extracted antioxidants from Tulsi leaves and studied its eect on the
stability of Ghee. is emphasizes the importance of using antioxidants
in controlling lipid oxidation in dairy product.
Cost analysis
e fruit peels were used as waste from fruit processing industry
which was free of cost.
For each 10 ml of dried antioxidant extracts of each peel the cost is
given as follows-
• For peels – Rs. 0
• Ethanol - Rs. 275
• Chemicals used for laboratory testing – Rs.280
• Processing charge including labour cost and electric charge etc.
- Rs.17/hr
• For preparation of paneer with each antioxidant extract-
• Cost of milk was Rs. 20/liter
• Grand total in rupees= Rs. 312 excluding laboratory testing.
Conclusion
Antioxidants were extracted from fruit peels of pomegranate, lemon
and orange. Maximum antioxidant activity was found in pomegranate
followed by lemon and minimum in orange peel. It can be concluded
from the study that pomegranate peels due to its high antioxidant
activity and phenolic content may prove to be a better substitute in place
of synthetic antioxidants in extending the shelf life of food product by
preventing the peroxide formation in the product containing fat and
oil. In addition natural antioxidants are safe and impart health benet
to the consumer.
Acknowledgment
I would like to thank Sam Higginbottom Institute of Agricultural, Technology
and Sciences (Deemed – To-Be-University), Allahabad, UP, India for providing
various facilities to carryon my research.
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Sample Color Taste Texture Flavour Appearance Overall
acceptability
Control 8.32 8.43 8.5 8.52 8.58 8.56
C1 8.45 8.46 8.34 8.45 8.28 8.25
C2 8.35 8.41 8.32 8.38 8.19 8.17
C3 7.77 8.21 8.31 8.24 7.23 7.6
Table 6: Mean Score of acceptability of paneer sample with Orange extract.
Sample 0th Day 2nd Day 4th Day 6th Day 8th Day
Control 0.09 1.43 1.62 2.34 2.56
A1 0.09 0.33 0.53 0.55 1.42
B1 0.09 0.53 1.26 1.39 1.67
C1 0.09 0.63 1.92 2.38 2.23
Table 7: Effect of antioxidant extracts at the level 1% on Peroxide Value of Paneer.
Sample 0th Day 2nd Day 4th Day 6th Day 8th Day
Control 0.09 1.43 1.62 2.34 2.56
A2 0.09 0.23 0.43 0.62 1.32
B2 0.09 0.33 1.06 1.29 1.47
C2 0.09 0.41 1.23 1.48 2.18
Table 8: Effect of antioxidant extracts at the level 2% on peroxide value of Paneer.
Citation: Singh S, Immanuel G (2014) Extraction of Antioxidants from Fruit Peels and its Utilization in Paneer. J Food Process Technol 5: 349.
doi:10.4172/2157-7110.1000349
Page 5 of 5
Volume 5 • Issue 7 • 1000349
J Food Process Technol
ISSN: 2157-7110 JFPT, an open access journal
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Citation: Singh S, Immanuel G (2014) Extraction of Antioxidants from
Fruit Peels and its Utilization in Paneer. J Food Process Technol 5: 349.
doi:10.4172/2157-7110.1000349
