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International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064

Index Copernicus Value (2013): 6.14 | Impact Factor (2015): 6.391

Volume 5 Issue 5, May 2016

www.ijsr.net Licensed Under Creative Commons Attribution CC BY

Extraction, Identification and Utilization of

Pigments Extracted from Citrus Wastes

Shukla P.S.1, Gawande S.S.

2, Thorat A.V.

3

1,2 Research Scholar, Department of Food Process Engineering of Vaugh School of Agriculture, Engineering and Technology, Sam

Higginbottom Institute of Agriculture, Technology and Sciences (Deemed-To-Be-University)

3 Assistant Professor, K. K. Wagh college of Food Technology, Nashik

Abstract: Food processing wastes may impose heavy burden on factories and cause enormous environmental problems. Citrus wastes

typically are about 45-50% of the weight of citrus original and the percentage of waste to 30-50% for vegetables and fruits in general.

Natural color plays a significant role in determining the degree of consumer acceptance of the product. In addition, carotenoids (vitamin

A precursor) have high nutritional values which are important for human nutrition. The efficiency of different organic solvents such as

acetone 85%, hexane, petroleum ether, ethyl acetate and ethanol 90% in the extraction of pigments from citrus peel was studied. Ethyl

acetate is the best solvent in extracting carotenoids from citrus peel, followed by ethanol 90%. HPLC was used to identify the extracted

pigments and their components. The extracted natural pigments were mixed with different carriers such as starch, lactose, dextrin,

arabic gum, and it was noted that lactose is the best one, followed by starch compared with different tested carriers. It was also found

that Alpha-tocopherol was relatively more stable than Butylated hydroxy toluene (BHT) antioxidant (an artificial compound). Natural

extracted pigments were used in food product (e.g. jelly) evaluations and gave better values for the color, flavor and taste compared to

commercial samples with artificial additives.

Keywords: Citrus wastes, Carotenoids, HPLC, Organic solvents, Artificial additives

1. Introduction

Citrus is a common term and genus (Citrus) of flowering

plants in the Rue family, Rutaceae. Citrus fruits are non-

climacteric and respiration slowly declines and the

production and release of ethylene is gradual.

India ranks sixth in the production of citrus fruits in the

world. It is of particular interest because of its high content

of vitamin

C and refreshing juice. Citrus wastes typically are about 45-

50% of the weight of citrus original and the percentage of

waste to 30-50% for vegetables and fruits in general. Natural

color plays a significant role in determining the degree of

consumer acceptance of the product. In addition, carotenoids

(vitamin A precursor) have high nutritional values which are

important for human nutrition. Francis and Isaksen (1988)

stated that carotenoids play a very important role for

extraction as for protection of health against cancer,

cardiovascular and eye disease and as anti-oxidant.

Natural extracted pigments are being used in food product

(e.g. jelly) evaluations and gave the better values for the

color, flavor and taste compared to commercial samples with

artificial additives. Citrus by-products and wastes such as

molasses, concentrated citrus peel juice, and pulp wash can

be subjected to fermentation, pectolytic treatment and

extraction with alcohol to obtain natural beverage clouds

and also to improve cloud strength and stability. Most of the

citrus fruits grown worldwide are oranges of various

varieties. More than half of these are processed and have

their juice extracted. Orange, one of the famous citrus fruits,

is commonly consumed in world. About 50% of the

processed oranges turn out in the form of orange peels.

The citrus processing industry yearly generates tons of

residues, including peel and segment membranes, from the

extraction of citrus juice in industrial plants. The

management of these wastes, which produce odor and soil

pollution, represents a major problem for the food industry.

Orange peel contains soluble sugars and pectin as the main

components. the orange peel is in fact constituted by soluble

sugars, 16.9 % wt; starch, 3.75 % wt; fiber (cellulose, 9.21

% wt; hemicelluloses, 10.5 % wt; lignin 0.84 % wt; and

pectin, 42.5 % wt), ashes, 3.50 % wt; fats, 1.95 % wt; and

proteins, 6.50 % wt. The total sugar content of orange peel

varies between 29 and 44 %, soluble and insoluble

carbohydrates being the most abundant and economically

interesting constituents of this residue. Approximately 50 %

of the dry weight of orange is soluble in alcohol , and

soluble sugars are the major components also of this

fraction. Glucose, fructose and sucrose are the main sugars,

although xylose can also be found in small quantities in

orange peel. Insoluble polysaccharides in orange peel are

composed of pectin, cellulose and hemicelluloses. Pectin

and hemicelluloses are rich in galacturonic acid, arabinose

and galactose, but they also contain small amounts of

xylose, glucose, and perhaps rhamnose . Glucose is the

dominant sugar in the cellulosic fraction, which also

contains some quantities of xylose and arabinose, traces of

galactose and uronic acids, and in some instances mannose.

On the other hand, lignin seems to be absent in these tissues.

Consequently, a mixture of cellulases and pectinases is

needed to complete the conversion of all polysaccharides to

monosaccharides . However the outer layer usually called,

flavedo in comparison with the inner layer of the peels

usually called albedo contains considerable amounts of the

natural pigments.

Carotenoids play important roles in human nutrition through

their provitamin A activity, but also by acting as

antioxidants, for prevention of age-related macular

degeneration or skin protection against UV radiation. The

antioxidant capacity of carotenoids was proved for pure

Paper ID: NOV163451 840

http://en.wikipedia.org/wiki/Genus

http://en.wikipedia.org/wiki/Rue

http://en.wikipedia.org/wiki/Rutaceae

http://en.wikipedia.org/wiki/Climacteric_(botany)





compounds as well as for plant extracts and food. Recent

studies investigate the profile of carotenoids in some Italian

grape varieties and the effect of lightening and irrigation on

carotenoids concentration.The main objective of this

research work is extraction, determination and identification

of natural colorants from different citrus wastes (citrus peel).

Also to study the effects of pH and oxygen on the stability

of extracted pigments and determination of the specific

carriers for the extracted pigments using solid matrices

(starch, dextrin, arabic gum and flour).

The feasibility of using produced pigments in manufacturing

of food product (Jelly). Rousseff et.al., (1992) employed

liquid- liquid extraction sample preparation with non-

aqueous reverse phase HPLC and photo-diode array

detection to separate and quantify lycopene, beta- carotene,

phytoene and phytofluene in newer red grapefruit cultivars.

These cultivars contained appreciably higher concentrations

of both visible and colorless carotenoids when compared to

the Ruby red cultivar. Lycopene and beta-carotene were

found in highest concentrations in the Star Ruby cultivar.

Mean lycopene (all trans) levels were Star Ruby (33

microg/g), Ray (21 microg/g), and Flame (7.9 microg/g).

Lycopene levels in the comparison Ruby Red grapefruit

were 2.9(interior) and 1.6microg/g (Indian River).Mean beta

carotene levels in decreasing order were Star Ruby (9.6

microg/g), Flame (8.6 microg/g) and Ray (7.0microg/g). The

comparison Ruby Red Fruits were both 4.2microg/g. These

cultivars could serve as important dietary sources of beta-

carotene. The natural colors of carrots to improve the color

of children candy. Six treatments were investigated; three of

them were prepared using fresh yellow carrot after

blanching in water, blanching by steam and without

blanching. Each sample of these three treatments was dried

and grounded to a fine powder, which was added to the

candy in 5%. The other three treatments were prepared using

fresh red carrot, which was dried and grounded to a fine

powder. Two of them were prepared by extracting the

pigment from red carrot powder (10 grams /each) using 0.8

and 1.0% citric acid respectively. Meanwhile, the last

treatment was prepared by adding 10 grams of powder to

500g candy. The candy samples analyzed directly after

production and the organoleptic evaluation of different

samples indicated that the yellow and red carrot could be

good natural colorant for candy (Hanaa et.al., 1993)

Lee et.al. (2001) applied high-performance liquid

chromatography with photodiode array detection for the

separation and characterization of carotenoids from a red-

fleshed navel orange (Cara Cara). Carotenoid pigments were

extracted using hexane/acetone/ethanol and saponified using

10% methanolic potassium hydroxide. More than 29

carotenoid pigments were separated within 60 min using a

ternary gradient (75% acetonitrile/25% methanol,

methyl tert-butyl ether, and water) elution on a

C30 reversed-phase column. The presence of lycopene (3.9

± 1.1 ppm) and a relatively large percentage of β-carotene

were distinct differences in the pigment profile of this red

navel orange juice as compared to the profile of standard

navel orange juice. The juice color in the red navel was

much deeper orange than that from navel orange; hue angle

ranged from 84.1 to 89.4 for red navel compared to 98.2 to

100.5 for standard navel.

Negi and Jayaprakasha (2001) extracted Citrus

paradisi peels with hexane, chloroform, acetone and

methanol using a Soxhlet extractor for 8 h each. Thin layer

chromatography (TLC) of hexane and chloroform extracts

showed three spots with different concentrations; hence both

the extracts were mixed and fractionated into alcohol soluble

and insoluble fractions. Naringin was isolated from acetone

and methanol extracts by column chromatography and its

structure was confirmed by 1H and

13C NMR spectroscopic

studies. Antibacterial activity of these fractions was

evaluated against different bacteria. The alcohol soluble

fraction was found to possess maximum activity followed by

hexane extract. Extracts were more effective against Gram-

positive than Gram-negative bacteria.

2. Materials and Methods

This chapter deals with the materials and methods involve

during this work. The investigation and analysis was

conducted at the Department of Food Process Engineering

of Vaugh School of Agriculture, Engineering and

Technology, Sam Higginbottom Institute of Agriculture,

Technology and Sciences (Deemed-To-Be-University). The

following chart shows actual process of jelly making using

extracted pigments.

Figure 1: Jelly Making Using Extracted Pigments

Procurement of raw materials

Fresh citrus fruit varieties i.e. Sour orange (Citrus

aurantium) and Grape fruit (Citrus paradisi) were purchased

from local market of Allahabad. At the time of fruit selection

care was taken that fruits should be tender and free from

damage.

Experimental plan

Based upon reported literature, the experimental plan was

devised. Fruits were cleaned, washed and peeled. The peels

were treated with antioxidants and dried. After drying, the

carotenoid pigments were extracted and identified by HPLC.

Further, the stability of pigments was analyzed by studying

the effect of pH, oxidation and adsorption of concentrated

pigments on solid supports. Finally the jelly was prepared

using the extracted pigments and its sensory analysis was

done. Experimental variables / parameters and there levels

and descriptions are given in following table:-






Table 1: Experimental Plan

Sr. No. Parameters/

Variables

Levels Description

1.

2.

3.

4.

Product

Raw materials

Ingredients

Treatments

1

2

3

4

Jelly prepared with the

extracted pigments.

Peels of two citrus varieties:

Sour orange and Grapefruit

Water, Sugar, Pectin, Citric

acid, Extracted pigments

from Sour orange and

Grape fruit peels.

Extraction and

identification of carotenoid

pigments, Studying the

effect of pH and oxygen on

the pigments, Selection of a

suitable carrier,

Organoleptic evaluation of

the prepared jelly from the

extracted pigments.

2.1 Equipment Used

Electric grinder, Oven, Rotary evaporator,

Spectrophotometer MODEL NO.305 (Range: 340-960 nm),

SHIATS used for the measurement of transmittance.

2.2 Methods

2.2.1 Sample preparation

The outer layer called flavedo of two mature citrus varieties

i.e. Sour orange and grape fruit were prepared as follows:

Each sample was divided into three parts; the first part was

treated with 0.1% of α-tocopherol (w/w) as natural

antioxidant. The second part was treated with 0.1% of

butylated hydroxy toluene (BHT, w/w) as artificial

antioxidant. The third part was untreated sample (as control).

The samples were dried in oven at 45˚C for 72 hours and

ground into fine powders in an electric grinder. Three

replicates per sample were taken for further analysis.

Figure 1: Drying of peels in hot air oven

Figure 2: Powder obtained from dried peels

2.2.2 Extraction of natural pigments

Colorants were extracted from citrus samples using high-

performance liquid chromatography. A known volume of

some solvents (30ml of each ) (Acetone, Petroleum ether,

Hexane, Ethanol 90%, Ethyl acetate, Methylene chloride)

was mixed with a known weight of dry samples(0.5 gm.) in

dark bottles and kept overnight and then filtered on paper

Whatman No.1. The filtrate was made upto a known volume

(100ml.) by the same solvent. The absorbance was measured

by a spectrophotometer, Model no. 305(Range:-340-960nm)

at 440, 644 and 662 nm.

2.2.3 Identification of natural pigments

Carotenoid pigments extracted from citrus samples were

identified by High Performance Liquid Chromatography

(HPLC) apparatus in the following ways;-

2.2.3.1 Chromatography

The HPLC system comprised of a dual piston solvent

delivery pump. The column system consisted of a 10 mm, 5

pm ODS2 metal-free guard column, with a 100 X 4.6 mm, 5

pm 0DS2 metal-free column connected to a 250 x 4.6 mm, 5

mm analytical modified by the replacement of metal frits

with ‘bio-compatible’ Teflon frits. Column connections

were made with PEEK (poly ether ether ketone) tubing. The

column temperature was maintained at 22.5˚+0.1˚C using a

column water jacket, with a thermostatically controlled

circulating water bath. The mobile phase consisted of

acetonitrile, methanol, dichloromethane

(MeCN:MeOH:DCM) 75 : 20 : 5 v/v/v, containing 0.1%

butylated hydroxytoluene (BHT) as an antioxidant and

0.05% triethylamine (TEA). The methanol contained 0.05 M

ammonium acetate. The prepared mobile phase was filtered

through a 0.5 pm PTFE membrane filter and degassed using

ultrasonic agitation. The solvent reservoir was equipped

with a helium degassing facility. The flow rate was 1.5

ml/min. Samples were injected via a syringe loading sample

injector fitted with a 50 µl loop. Peak responses were

measured at 450 nm using a variable wavelength UV/Vis

monitor with an output to a chromatographic data handling

system which permitted manual manipulation of peak

integration. A diode array detector was coupled to the

UV/Vis monitor to assess peak homogeneity and confirm

spectral identity of carotenoids.

2.2.3.2 Sample extraction

Duplicate 10 g aliquots of the ground sample were placed in

conical flasks together with 1 g solid magnesium carbonate






(Fisons) to neutralize any organic acids. Fifty ml of

tetrahydrofuran and methanol (1: 1 v/v THF : MeOH) were

added along with an internal standard(/3-apo-8’-carotenal or

echinenone) appropriate for the type of sample. Carotenoids

were extracted from the food matrix by homogenizing for 1

min using an ultra-turrax homogenizer The resulting

suspension was filtered through a glass fibre filter pad in a

buchner funnel under vacuum. The flask and homogenizer

were washed with 50 ml THF/MeOH and the washing

filtered. The filter pad was washed with two further 50 ml

aliquots of THF : MeOH. The combined THF : MeOH

filtrates were transferred to a separating funnel. Fifty ml

petroleum ether (40-60’ fraction, containing 0.1% BHT) and

50 ml 10% sodium chloride solution were added and mixed

by careful shaking. The lower THF/MeOH/aqueous phase

was drawn off and the upper petroleum ether phase was

transferred to a 250 ml evaporating flask. The

THF/MeOH/aqueous phase was extracted two more times

with 50 ml aliquots of petroleum ether. The petroleum ether

phases were combined in the flask and evaporated at 35°C in

a rotary evaporator to near dryness. Ten to fifteen ml of

petroleum ether was added and the residue redissolved by

ultrasonic agitation, transferred to a 25 ml evaporating flask

and evaporated just to dryness. The residue was redissolved,

by ultrasonic agitation, to a final volume of 5 ml in DCM. If

necessary this extract was saponified as described below,

otherwise it was diluted with the mobile phase to a suitable

concentration for HPLC analysis and filtered through a

0.45ppm PVDF syringe filter. All manipulations were

carried under gold fluorescent lighting.

2.2.3.3 Saponification

Four ml of the DCM extract from the extraction procedure

was saponified with an equal volume of loo/o potassium

hydroxide in MeOH (under nitrogen, in the dark) for 1 h at

room temperature. The carotenoids were extracted from the

KOH/methanolic phase by careful shaking with 20 ml

petroleum ether (40-60” fraction, containing 0.1% BHT),

and 20 ml 10% sodium chloride solution in a separating

funnel. The lower KOH/MeOH/aqueous phase was removed

to another separating funnel and was extracted two more

times with 20 ml aliquots of petroleum ether. The petroleum

ether phases were combined in a separating funnel and

washed with water until washings were neutral to pH paper.

The petroleum ether phase was transferred to a 100 ml

round-bottomed flask and dried in a rotary evaporator at

35°C. Ten to fifteen ml of petroleum ether were added and

the residue redissolved by ultrasonic agitation, transferred to

a 25 ml evaporating flask and evaporated just to dryness.

The residue was redissolved by ultrasonic agitation in 4 ml

DCM, diluted with mobile phase to a suitable concentration

for HPLC analysis and filtered through a 0.4 pm PVDF

syringe filter. All manipulations were carried out under gold

fluorescent lighting.

2.2.3.4 Internal standards

An internal standard was used to assess losses during the

extraction procedures. One of two standards was used

depending on the carotenoid profile of the sample. P-apo-

8’carotenal was used where possible. Both standards showed

similar recoveries during the extraction procedure.

2.2.3.5 Preparation of standard carotenoid solutions and

calibration

Lutein, α-carotene, β-carotene and β-apo-8’-carotenal were

dissolved in chloroform and made to volume with hexane to

give a final solvent ratio of 1 : 9 v/v. Echinenone and β-

cryptoxanthin were dissolved in 1 : 1 v/v

chloroform/hexane. Zeaxanthin was dissolved in

chloroform. All solvents contained 0.1% BHT. All solutions

were stored in air-tight screw-topped brown bottles.

Under nitrogen at -18°C. Prior to measuring absorbance, the

stock solutions were brought to room temperature and

filtered through a 0.45 pm PVDF syringe filter. An aliquot

of the solution was evaporated under nitrogen and diluted in

the appropriate solvent to give an approximate absorbance

reading of 0.5 AU and the exact absorbance measured using

a spectrophotometer equipped with a wavelength scanner to

assess the spectrum. The concentrations were calculated

using the appropriate extinction coefficients. Individual

working solutions of around 0.5-1.0 µg/ml were prepared

from stock solutions by evaporating an aliquot under

nitrogen and making to volume with mobile phase and their

‘purity’ assessed by HPLC analysis. ‘Purity’ of a carotenoid

was expressed as the peak area of that carotenoid as a

percentage of the total area of the chromatogram. The

concentration calculated from the absorbance reading was

corrected accordingly. The concentration of the stock

standard solutions and their purity were measured each time

a new working standard was prepared. A mixed working

standard solution was prepared, in mobile phase, from

individual stock solutions.

2.2.4 Stability of pigments

2.2.4.1 Effect of pH

Degradation of carotenoids extracted from citrus samples

caused by different pH values were measured. The retention

of carotenoids extracted from Sour orange and Grape fruit,

after seven days of refrigeration at 4˚C using different buffer

solution with pH values ranged from 2 to 9 was calculated.

Samples were prepared by dissolving appropriate amounts

of pigment in a 0.1M citric-phosphate buffer at a specific pH

value. Buffer solutions with pH values of 2.0 and 9.0 were

prepared by the addition of 1N HCL or 1N NaOH,

respectively. Percent pigment retention was calculated in

terms of:

% Pigment Retention= A2/A1 *100

Where: A1 is the initial light absorption value and A2 is the

light absorption value after 7 days of storage at 4°C.

2.2.4.2 Effect of oxidation

Samples of the carotenoids pigment solution were divided

into groups, the first group was exposed to aeration for 4

hours, and the other was exposed to nitrogen gas for 4 h,

then absorbance measurements were read by

spectrophotometer.

2.2.4.3 The adsorption of concentrated pigments on solid

supports

The collected extracts of carotenoids were concentrated by

removal of solvent in a rotary vacuum evaporator at 40˚C.

The concentrated pigments were adsorbed to solid matrices

(starch, dextrin, flour, lactose and arabic gum) in different






ratios (1:1, 1:2, 1:3 and 1:4 carrier/pigment) and the

mixtures were dried in oven at 40˚C for 24 hours.

Plate 3: Pigment powders

2.2.5 Jelly Preparation

Jelly was prepared in the laboratory in the laboratory.

2.2.6 Organoleptic evaluation of jelly Samples of jelly were subjected to organoleptic evaluation.

The analysts were asked to evaluate color (10), clarity (10),

flavor (10), taste (10), texture (10), shape (10) and overall

acceptability (10).

2.2.7 Statistical analysis of the experimental results The results were analyzed by Two Way Anova by using

Statistical 7.0 software. Significant differences (p<0.05)

were determined by Duncans multiple range test.

3. Results and Discussion

3.1 Extraction of natural pigments

Carotenoid pigments content of Sour orange and Grape

fruits are shown in Table 4.1. The tabulated results showed

that, ethyl acetate was the most efficient solvent in extract of

carotenoid pigments from Sour orange and Grape. In Sour

orange samples, the total carotenoids was 5.35,4.23, 3.56,

4.55, and 3.00 mg/L for ethyl acetate, acetone 85%,

petroleum ether, n-hexane and ethanol, while in Grape fruit

samples, carotenoids were, 1.18, 1.23, 1.05, 0.45 and 0.31

mg/L for ethanol, respectively.

Therefore, it could be concluded that, the higher the

extraction yield of carotenoid pigments in Sour orange

referred to such samples could be considered as a good

source of carotenoids comparing to grape fruit.

Table 4.1: Effect of some solvents on extraction of

pigments (mg/L) Citrus

peels

Solvents Chlorophyll

A

Chlorophyll

B

Total

carotenoids

Sour

orange

Ethyl acetate 0.15 0.12 5.35

Acetone 85% 0.15 0.12 4.23

Petroleum ether 0.06 0.29 3.56

n-hexane 0.11 0.03 4.55

Ethanol 90% 0.02 0.31 3.00

Grape

fruit

Ethyl acetate 0.08 0.03 1.18

Acetone 85% 0.17 0.11 1.23

Petroleum ether 0.23 0.02 1.05

n-hexane 0.14 0.23 0.45

Ethanol 90% 0.08 0.18 0.31

Fig.4.1.1 Effect of some solvents on extraction of

pigments from Sour orange peels

Fig.4.1.2 Effect of some solvents on extraction of

pigments from Grapefruit peels

3.1.1 Determination and identification of carotenoids

The technique of HPLC has been widely applied to

investigate the composition of carotenoids extracted with

ethyl acetate method. HPLC is characterized by short

analysis time, high resolution, good reproducibility, and

little structural modification.

Data in Table 4.1.1 shows the carotenoid pigments

composition of Sour orange and Grape fruits, which were

separated based on their functional groups into nine

fractions for the Sour orange and six fractions for Grape

fruit.






Table 4.1.1: Identification of carotenoid pigments of Sour

orange and Grape fruit. Types of Citrus peels Modified carotenoids %

Sour orange

Capsorubin 38.67

Zeaxanthin 3.90

Crytptoxanthin 4.49

Unidentified 0.23

Unidentified 0.52

Lutein 2.98

Canthaxanthin 4.90

Capsanthin 33.90

β-carotene 4.20

Grape fruit

Antheraxanthin 3.95

Violaxanthin 35.75

Lutein 3.92

Zeaxanthin 45.22

Cryptoxanthin 3.50

β-carotene 4.92

Figure 4.1.1: Identification of carotenoid pigments from

Sour Orange peels

Figure 4.1.2: Identification of carotenoid pigments from

Grapefruit peels

3.2 Stability of Pigments

3.2.1 The effect of pH value

The retention of carotenoids extracted from Sour orange and

Grape fruit, after seven days of refrigeration at 4˚C using

different buffer solution with pH values ranged from 2to 9

was calculated and the results are presented in Table 4.2.1.

The tabulated results showed that, increasing in pH value

increased retention of carotenoids (97.13%) until pH 7.0.

However, the retention percentage was increased from 45.36

and 39.53 at pH 2.0 to 97.13 and 96.43 at pH 7.0 for Sour

orange and Grape fruit, respectively. Then the retention

percentage was decreased to 79.36 and 77.76 at pH 9.0 for

the same samples, respectively.

Table 4.2.1: The effect of pH value on the stability of

carotenoids extracted from Sourorange pH

value

Sour orange peels Grape Fruit Peels

Retention

(%)

Degradation

(%)

Retention

(%)

Degradation

(%)

2 45.36 54.32 39.53 60.53

3 55.02 45.33 52.66 47.12

4 71.33 28.36 70.12 29.87

5 82.25 17.82 80.54 19.32

6 93.31 6.11 96.51 3.56

7 97.13 2.77 96.43 3.45

8 86.54 13.16 78.21 22.32

9 79.36 20.11 77.76 22.21

Figure 4.2.1: Effect of pH on the pigments extracted from

Sour Orange peels

Figure 4.2.2: Effect of pH on the pigments extracted from

Grapefruit peels

3.2.2 The effect of oxygen on carotenoids stability The results presented in Table 4.2.2 showed the effect of

oxygen on stability of carotenoids extracted from Sour

orange and Grape fruits treated with antioxidant (α-

tocopherol as natural antioxidant and BHT as synthetic

antioxidant) in solution of pH 7.0 after exposing to air for 4h

in comparison with that kept under nitrogen gas, then

absorption spectra was measured.

Concerning the data of untreated samples showed in Table

4.2.2, the retention rate of carotenoid extracted from Sour

orange was 89.90% followed 86.23% (Grape fruit) when

these samples were exposed to air for 4 h. Meanwhile, this

retention rate of Sour orange and Grape fruits carotenoids

was 95.62 and 98.87 % respectively, under nitrogen

conditions. In other words, the degree of degradation in Sour

orange carotenoids was lower than that of the Grape fruit;

meanwhile there was no pronounced decrease in the content

of these pigments when kept under untreated conditions.

Regarding to retention rate of carotenoids extracted from

Sour orange treated with α-tocopherol and BHT presented in






Table 4.2.2. The rate increases from 90.20% (control

sample) to 98.26% (α-tocopherol) and 93.11% (BHT), after

exposing to air for 4 h. Meanwhile, this retained reached to

100% under nitrogen gas conditions for either α-tocopherol

treatment or BHT treatment.

The retention rate of carotenoids from Grape fruit treated

with α-tocopherol and BHT presented in Table 4.2.2. After

exposing to air for 4 h, it was increased from 88.83%

(control) to 93.22% and93.11% respectively. On the other

hand, the retention rate of Grape fruit carotenoids recorded

maximum value (100%) when nitrogen gas was used for

both carotenoids treated with α-tocopherol treatment or

BHT. However, it can be concluded that α-tocopherol

treatment had the highest desirable effect on the carotenoid

stability followed by BHT treatment then control samples

for Sour orange and Grape fruits.

Table 4.2.2: Effect on the stability of carotenoids extracted

from Sour orange and Grape fruit.

Citrus Peels

Exposure to

air for 4 h

Under nitrogen

gas for 4 h

Retained

%

Degradation

%

Retained

%

Degradation

%

Sour orange

(Control)

90.20 9.80 95.62 4.38

Sour orange (α-

tocopherol)

98.26 1.74 99.87 0.13

Sour orange

(BHT)

95.34 4.66 99.64 00.36

Grape fruit

(Control)

89.53 10.47 98.87 1.13

Grape fruit (α-

tocopherol)

92.80 7.20 100 0.00

Grape fruit

(BHT)

92.90 8.10 99.82 00.18

Figure 4.2.1: Effect on the stability of carotenoids extracted

from Sour orange

Figure 4.2.2: Effect on the stability of carotenoids extracted

from Grapefruit

3.3 Adsorption of colorants

Trials were made to select a suitable carrier for coating the

carotenoid to keep and store it in a way by which it could be

easily used. The data obtained for the adsorption of

carotenoids extracted from Sour orange as a natural food

(1:1, 2:1, 3:1, 4:1 pigment: carrier w/w) are given in Table

4.3. These results indicated that, lactose adsorbent had the

highest carotenoid concentrations (1550, 1900, 2150 and

2500 mg/100 gm) at all studied ratios, respectively, followed

by starch. Also, from the same table it could be noticed that

Arabic gum had the lowest carotenoid concentrations at all

studied ratios. Lactose was the most effective adsorbent

coated carrier material for red colorant extracted from Sour

orange followed by starch. On the other hand, it could be

observed from Table 7 that the adsorption of carotenoids

extracted from Grape fruit recorded the maximum

concentrations when starch was used as carrier, while the

minimum concentrations were recorded with Arabic gum.

Generally, the obtained results proved that, the best carrier

for yellow pigment extracted from Grape fruit was the

starch, while the worst carrier was the Arabic gum.

Table 4.3: Color concentration (mg/100gm carrier) of

extracted carotenoids from Sour orange and Grape fruits

with different carriers at different ratios (w/w) Pigment/

carrier (w/w)

Applied Carriers

Lactose Starch Flour Dextrin Arabic gum

Sour orange mg/100gm

1:1 1550 950 856 750 82.28

2:1 1900 1200 850 910 127.76

3:1 2150 1700 1260 1350 435.25

4:1 2500 2110 1450 1480 623.9

Grape fruit mg/100gm

1:1 400 375 370 320 84.8

2:1 780 760 690 550 122.4

3:1 1000 1200 985 728 163.8

4:1 1244 1550 1340 936 195.2

3.4 Organoleptic evaluation of jelly

Sensory properties of jelly with different ratios (0.0066,

0.0133 and 0.0266%) of carotenoid pigments extracted from

Sour orange peels were evaluated. Organoleptic

characteristics of jelly samples were determined in respect to






commercial jelly sample as control samples and the results

were tabulated in Table 4.4. From the noticed data, it could

be noticed that, addition of carotenoids by 0.0066% led to

insignificant increase in recorded color value comparing to

control sample. On the other hand, only the third addition

ratio had significant decrease in respect to control sample.

Concerning jelly taste results (Table 4.4), it could be observe

that, only the first addition ratio of sour orange peels

carotenoids had significant improvement in jelly taste

comparing to control sample. A significant disorder in jelly

shape was observed at all studied ratios comparing with

control sample. However, the lowest disorder was recorded

for jelly sample prepared by addition of 0.0066% sour

orange peel carotenoids.

For instance, insignificant reduction in overall acceptability

was achieved for jelly sample prepared with the first

addition ratio, while the two other studied ratios led to

decrease the recorded value significantly from 17.41 in

control sample to 15.08 and 13.58 for the samples prepared

by the second and third addition ratios, respectively.

Generally, from the previous results, it could be noticed that

no significant in most sensory characteristics of jelly sample

prepared only by addition of 0.0066% of sour orange peels

pigments was accrued. On the other hand, there were no

significant differences between synthetic colored sample and

natural colored samples for taste, texture and bleeding, when

was carried out sensory evaluation of glazing jelly products.

Figure 4.4.1: Sensory evaluation of the prepared jelly from

extracted pigments

Figure 4.4.2: Score of control sample

4. Conclusion

Natural colors (carotenoids) play very important role in

determining the acceptability of the food for consumers.

Furthermore, some components of the carotenoids are

precursors of vitamin A which is very important in human

nutrition and food colorants, beside its anticancer properties.

However, from the above mentioned results it can be

concluded that:

The higher extraction yield of carotenoid pigments from

Sour orange referred to such samples which could be

considered as a good source of carotenoids comparing to

Grape fruit. Ethyl acetate was the most efficient solvent in

extracting natural pigments from Sour orange, while, that of

Grape fruit were ethyl alcohol. The highest carotenoid

extracted from Sour orange was 97.13 at pH 7.0, while, the

highest for the carotenoid extracted from Grape fruit was

96.43. Using nitrogen gas had losses approximately in

carotenoid content for all studied samples either treated or

untreated with antioxidants. The Sour orange is the best

source of carotenoids due to its very rich carotenoid content

and heat tolerance followed by Grape fruit. The antioxidants

treatments (either by α-tocopherol or BHT) relatively

enhanced the carotenoids stability, but the highest effect was

observed for the Sour orange. Lactose was the best carrier

for the dispersion of Sour orange carotenoid, while, starch

was more effective adsorbent coated carriers for pigments

extracted from Grape fruit. Regarding to the overall

acceptability of jelly samples, it could be showed that, slight

insignificant reduction in overall acceptability was achieved

for sample prepared with the first addition ratio 0.0066%.

However, no significant in most sensory characteristics of

jelly sample prepared only by addition of 0.0066%

comparing to control.

References

[1] Francis GW, Isaksen M (1988) Thin-Layer

Chromatography of Carotenoids with Tertiary Alcohol-

Petroleum Ether Solutions as Developing Solvents.

Journal of Food Science 53: 979-980.

[2] Hanaa A, Sidky M, Abou-Zaid A, Hemmet I (1993)

Study on the use of natural color in candy, 149-153, In:

Natural colors for the food industry, conference and

exhibition, Ismailia.

[3] H. Morais, C. Ramos, E. Forgasc, T. Cserháti, N.

Matos, V. Almeida, J. Oliveira (2001) Stability of

anthocyanins extracted from grape skins. Journal of

Agriculture and Food Chemistry. (56) 173-175.

[4] Lee HS, Castle WS, Coates GA (2001) High-

performance liquid chromatography for the

characterization of carotenoids in the new sweet orange

(Earlygold) grown in Florida, USA. 913(1-2) 371-377.

[5] Negi, P.S.Jayaprakasha and G.K. (2011) Anti-bacterial

activity of grapefruit (Citrus paradisi) peel extracts.

European Food Research and Technology.213 484-487.

[6] Rousseff R.L., Sadler G D, Putnam T J, Davis JE (1992)

Determination of beta-carotene and other hydrocarbon

carotenoids in red grapefruit cultivars. Journal of

Agricultural and Food Chemistry. 40(1) 47-51.


http://link.springer.com/search?facet-creator=%22H.+Morais%22

http://link.springer.com/search?facet-creator=%22C.+Ramos%22

http://link.springer.com/search?facet-creator=%22E.+Forgasc%22

http://link.springer.com/search?facet-creator=%22T.+Cserh%C3%A1ti%22

http://link.springer.com/search?facet-creator=%22N.+Matos%22

http://link.springer.com/search?facet-creator=%22N.+Matos%22

http://link.springer.com/search?facet-creator=%22V.+Almeida%22

http://link.springer.com/search?facet-creator=%22J.+Oliveira%22

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