pg. 1

Nutritional potential and stability of

Opuntia elatior Mill.

fruit juice

M.Sc. DISSERTATION

Submitted to

Department of Botany

Faculty of Science

THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA

IN PARTIAL FULFILLMENT FOR THE AWARD OF THE DEGREE IN

MASTER OF SCIENCE

SUBMITTED BY – GUIDED BY –

Ms. Mital Rajnikant Bhatt Dr. Padmanabhi Shankar Nagar

pg. 2

Faculty of Science

The Maharaja Sayajirao University of Baroda

VADODARA-390 002, Gujarat, (INDIA)

Date: 04 – 05 – 2012

CERTIFICATE

This is to certify that the dissertation work submitted

here by Ms. Mital Rajnikant Bhatt entitled “Nutritional

potential and stability of Opuntia elatior Mill. fruit

juice” was carried out under my direct supervision in my

laboratory. She has completed her work satisfactorily

and has submitted it as a part of fulfillment for the

award of Degree of Master of Science in Botany.

Prof. Arun Arya Dr. P. S. Nagar

Head of the Department Guide

pg. 3

ACKNOWLEDGEMENTS

First of all I would acknowledge my guide Dr Padamnabhi S.

Nagar. Sir introduced me with the plant Opuntia elatior Mill.;

moreover, giving this idea of studying nutritional potential

and its stability of fruit juice. His suggestions have shaped

nearly every aspect of this research. I may not sufficiently

express my gratitude for his guidance.

Special thanks to Dr. Arun Arya sir Head of the Dept.,

Botany, for providing with excellent laboratory facilities.

During this work, my colleagues and seniors shared their

special knowledge, volunteered their co-operation, thoughtful

suggestions and criticisms, which have greatly influenced this

topic and I extend my sincere thanks to all of them esp. Ms.

Poonam Manglorkar who was always there to assist me when

I was muddled in any analytical or technical task. I am also

thankful to Ms. Gagandeep Kaur Bhambra who was always

ready in assisting me for measuring pH and other technical

tasks.

To friends and family, I remain grateful, especially to my

parents who helped, encouraged, and supported me during my

research work. I want to express my deep appreciation to my

respected father, Shri Rajnikant Bhatt who himself went to

collect Opuntia elatior Mill. fruits from the field and send me

in given time via State Transport Bus services.

In addition to these I would also like to acknowledge Mr. S. B.

Chauhan (Asst. Librarian, Faculty of Technology, M.S.U.) who

was helpful in providing me with the BIS testing procedures.

pg. 4

Table of contents

TOPICS Page no.

1 INTRODUCTION 5

2 OBJECTIVES 9

3 REVIEW OF LITERATURE 10

4 METHODOLOGY 16

5 RESULTS AND DISCUSSION 29

6 CONCLUSION 44

7 REFERENCES 45

pg. 5

Introduction

Opuntia is a large genus of succulent shrubs, native of the new world, now

widely grown in the warmer parts of the world, on account of their unique

appearance and attractive flowers. They are commonly known as Prickly pears,

because of their edible fruits. The prickly pears are said to have been

accidentally introduced into India and other eastern countries by early European

travellers, who used to carry these plants for use as vegetable to prevent scurvy

during their long voyages. In India, as well as in other countries, they spread

with rapidity and soon become noxious weeds, monopolizing large areas of

forest and cultivated lands (As per anonymous, 2001).

HABIT:

As per Bentham and Hooker (1862):

Angiospermae

Dicotyledonae

Polypetalae

Calyciflorae

Ficoidales

Cactaceae

Opuntia elatior Mill.

pg. 6

Current systematic position is as placed under Caryophyllales:

Systematic position as per current classification APG (2011)

Opuntia elatior Mill. was first time identified by Miller in 1880.

Origin: This plant is native of the Caribbean, Central America and northern

South America, but now naturalized in many part of the world like

Mediterranean, Australia, Africa and India.

Distribution: Opuntia elatior Mill. is found in western India (The Wealth of

India, 2001). Gujarat, Rajasthan, Madhya Pradesh, Maharashtra, Andhara

Pradesh, Tamilnadu and in small pockets across the country in arid and semi-

arid regions.

Synonyms: O. nigricans Haw.; O. burgeriana; Cactus tuna var. elatior; C.

elatior

pg. 7

VERNACULAR NAMES (Kirtikar and Basu, 1999):

Arabic Jhakawoon

Bengal Negphana, Phenimama

Burma Kalzaw, Shasounglitwa

Canarese

(Kannada;

South India)

Chappatigalli, Dabbugalli, Mullugalli, Nagadali, Papasakalli,

Papasukattale, Sivaramakalli

Deccan Chappal, Chappalsend, Nagphansi

English Prickly pear, Slipper Thorn

French Raquette

Gujarati Chorhathalo, Zhorhatheylo, Phafda thor

Hindi Haththathoira, Nagphana, Nagphani

Malayalam Nagamullu, Nagatali, Palakakkalli

Marathi Chapal, Nagaphana Samar

Porbander Hathalo

Portugese Palmatoria d’inferno

Sanskrit

Bahudugdhika, Bahushala, Dondavrikshaka, Guda, Gula,

Kandarohaka, Kandashakha, Krishnakhara, Kubshadruma,

Mahavriksha, Nagadru, Nagaphana, Netrari, Nistrinshapatrika,

Samantadugdha, Shakhakanta, Shihunda, Sihunds, Sinhatunda,

Snuha, Snuhi, Snuka, Snusha, Sudha, Vajra, Vajradruma,

Vajrakantaka, Vajri, Vidara, Visvasakara

Sinhalese

(Sri Lanka) Kodugaha

Tamil

Kalli, Manjarnagadali, Mullukkalli, Nagadali, Nagakkalli,

Palagaikkalli, Pattanadugalli, Sappattu, Sappattukkalli,

Sapattumul

Telegu Nagadali, Nagajemudu, Nagamullu

Urdu Nagaphani, Thuar

DESCRIPTION: Subarborescent or shrubby, 3 meter high or more. Leaves

7.5 mm long, subulate, recurved, reddish at the tips. Joints variable in size,

about 18-30 cm in height by 10-18 cm in width, obovate or elliptic, rather thin,

not undulate, dull bluish green. Areoles bearing about 4-5 cm increasing up to

10 cm, rather slender straight prickles which are grey and opaque except when

quite young, the largest 3-5 cm. long; glochidia inconspicuous, almost hidden

amongst woolly hairs, rusty-brown. Flowers 5 cm. across, yellow or orange.

Perianth rotate, the outer segments short, ovate, acute, red in the centre, yellow

at the edges, the inner spathulate, acute. Stamens a little shorter than the

pg. 8

perianth. Style exceeding the stamens; stigmas 6 in number. Berry pyriform,

angular or more or less warty, bearing tufts of glochidia and occasionally a few

prickles, reddish purple when ripe (Kirtikar and Basu, 1999).

Popularity of Opuntia elatior Mill. over time

Plots of numbers of papers mentioning Opuntia elatior Mill. (filled column

histogram and left hand axis scale) and line of best fit, 1926 to 2006 (complete

line, with equation and % variation accounted for, in box on the left hand side);

Plots of a proportional micro index, derived from numbers of papers mentioning

Opuntia elatior Mill. as a proportion (scaled by multiplying by one million) of

the total number of papers published for that year (broken line frequency

polygon and right hand scale) and line of best fit, 1926 to 2006 (broken line,

with equation and % variation accounted for, in broken line box on the right

hand side)

(Fernandez A., 1999; Hoenigsberg HF et al. 1991; Iyer SR, Williamson D, 1991; Pande PC,

Kandpal MM, Joshi GC, 1987; Pande PC, Joshi GC, 1985; Benado M et al., 1984; Pande PC,

Joshi GC, 1984; Sebastian MK, Bhandari MM, 1984; Bhatia DS, Malik CP, 1977; Shabbir

M, Zaman A, 1968; Ganguly AK, Govindachari TR, Mohamed PA, 1965; Tiagi YD, 1961;

Burns W, 1941; Hummelinck PW, 1940; Deshpande VG, 1935)

pg. 9

Objectives of study :

Collection of fruits

Extraction of juice

Preservation of juice with natural preservatives

Studying the stability of juice by means of measuring pH

Study seed viability

Extraction and physiochemical characterisation of oil from the seeds

Saponification value

Iodine value

Acid value

Fatty acid composition (GLC)

Phytochemical characterization of fruit juice

Betalains

Carbohydrates

Flavonoids

Phenolic acids

pg. 10

Review of literature TRADITIONAL USES OF OPUNTIA SPECIES

o The flowers cure bronchitis and asthma. The fruit is considered a

refrigerant, and is said to be useful in gonorrhea. The baked fruit is said to

be given in whooping cough and syrup of the fruit is said to increase the

secretion of bile and control spasmodic cough and expectoration (Kirtikar

and Basu, 1999; The Wealth of India, 2001).

o In addition to food, Indian fig is used to treat whooping cough, diabetes,

prostate problems, rheumatism, nosebleed, and in dentistry in central

Mexico (Duke and Vasquez, 1994). Sicilians (Resident of Sicily, Italy)

use the fruits as Mexicans do, boiling the juice into syrup and also

producing a jam. A tea is made from the flowers and drunk for kidney

problems. Dried flowers are also ground into a paste and applied to the

skin for measles (Galt and Galt, 1978). The Sicilians do not eat the stem

joints, however, which Mexicans call nopales and nopalitos. Instead, stem

joints are fed to livestock on occasion because of their high water content

(Barbera et al., 1992).

o Many species of cactus are found growing either as wild plants in arid

and semiarid regions of India or an ornamental plant in urban homes and

gardens. Generally, these species are used as live fences to protect

agricultural fields from human and animal encroachments with few

exceptions; there has been no attempt to cultivate this plant as a

horticultural or fodder crop in India.

o In countries such as Mexico, USA, Spain, Italy and northern Africa,

where the crop is commonly known, it already forms an integral part of

the people’s dietary requirement. In addition to the excellent quality and

favor of the fresh fruit, the young phylloclades serve both as a vegetable

and salad dish and the immature fruit is used to make mock gherkins

(Gurbachan singh, 2003).

o Although traditionally appreciated for its pharmacological properties by

the Native Americans, cactus pear is still hardly recognized because of

insufficient scientific information (Feugang et al., 2006).

The plant grows in arid and semi-arid region of India (Bole & Pathak 1988).

pg. 11

The nutritional potential of the species Opuntia ficus-indica has been studied:

1. Brain-Derived Neurotrophic Factors (Western blotting) enhances long-

term memory. J.: Progress in Neuro – Psychopharmacology & Biological

Psychiatry, 2010

2. Wound healing and inflammatory activity. J.: Fitoterapia, 2001

3. Antiulcerogenic and antioxidant. J.: Journal of Agriculture and Food

chemistry, 2003

4. Anti- diabetic. J.: Food science and Biotechnology, 2011

5. Cancer prevention. J.: Nutrition journal, 2005

As per the latest reference of Moreno 2008, following parameters

were studied:

Sr.no. Parameters

Opuntia elatior Mill.

Venezuela, South America

(Moreno, 2008)

1 Moisture 88.17 ± 0.04 %

2 Acidity 0.04 ± 0.06 %

3 pH 5.20 ± 0.01

4 Total carotenoids 3.234 ± 0.010 mg/ 100 g

5 Betalains 6.93 ± 0.01 mg/ 100 ml

6 Vitamin –C 15.09 ± 0.08 mg/ 100 g

7 Protein 0.83 ± 0.05 %

8 Fats 0.30 ± 0.06 %

9 Calcium 50.66 ± 1.52 mg/ 100 g

10 Phosphate 8.88 ± 0.21 mg/ 100 g

11 Crude fibre 2.38 ± 0.13

12 Ash 0.31 ± 0.05

pg. 12

FRUIT:

The cactus pear fruit is an oval, elongated berry with a thick pericarp and a juicy pulp

and many hard seeds. The large variability in percentage of chemical composition

depends on cultivar, cultural practices, fecundated and aborted seed number, fruit

load, lighting period, elimate and harvesting season. The ripe fruits of Opuntia spp.

are 30 – 220 g in weight contain pulp (43–67%), seeds (2–10%) and peel (33–55%).

The pH range of the pulp is 5.3 – 7.1. The fairly high sugar content and low acidity of

the fruit make it very sweet and delicious (Piga, 2004; Moßhammer et al., 2006). The

prickly pear may be divided into three fractions: peel, pulp and seed contain chief

chemical constituents as summarized in table 2.6.

Table 2.6: Chief chemical constituents in fruits of Opuntia spp.*

Parameters Peel Pulp Seed

% of fresh

Weight 33 – 55 43 – 67 2 – 10

Color green, orange,

red, purple

white, yellow – orange, red,

purple Not Available

Mineral Potassium &

Calcium

Potassium, Calcium &

Magnesium Potassium & Calcium

Vitamin Vitamin E (in

oil) Vitamin C Not Available

Amino acid Not Available Proline & Taurine Not Available

Sugar Glucose Glucose & Fructose Not Available

Hydrocolloids Cellulose &

Pectin

Pectin, Complext mixture of

rhamnogalacturonan and at

least 50% nonpectic

substances

Cellulose, Arabinans,

Rhamnogalacturonans

Organic acids Not Available Citric acid Not Available

Lipid

γ – linolenic

acid & α –

linolenic acid

Linoleic acid, Palmitic acid, Linoleic acid, Palmitic

acid, Oleic acid

Sterols β – sitosterol,

Campesterol β – sitosterol, Campesterol

β – sitosterol,

Campesterol

Phenolic Not Available Quercetin, Kaempferol,

Isorhamnetin Not Available

Pigments Betacyanin, Betaxanthins Not Available

*According to (Moßhammer et al., 2006; Kossori et al., 1998)

pg. 13

ETHANOPHARMACOLOGICAL ACTION

Opuntia species has been used by humans for thousands of years. Besides

being consumed as food or beverages, most portions of the plants have been

used as medicine and in modern times have also been prepared as juice,

jam, flour, frozen fruit, juice concentrate, and spray-dried juice powder

(Smith, 1967; Stintzing & Carle, 2005, 2006; Feugang et al., 2006;).

A remarkable number of cacti are used by indigenous people of the New

World for healing. According to Parmar and Kaushal (1982), Kirtikar &

Basu (1999) and Patil et al. (2008), the plant is bitter, laxative; stomachic,

carminative, antipyretic. Cures biliousness, burning, leucoderma, urinary

complains, tumours, loss of consciousness, piles, inflammations, anaemia,

ulcers, respiratory disorders like asthma and the enlargement of the spleen.

Medically related used of some species are discussed here.

The Shoshoni make a poultice from the inner part of the stem of Opuntia

basilaris and apply it to cuts and wounds for pain (Moerman, 1998).

Grenand et al. (1987) report that Opuntia cochenillifera is widely used in

Mexico and Central America as an antifungal agent.

People throughout Asia employ Opuntia dillenii for a variety of purposes.

In India, it is used to treat sores, pimples, even syphilis (Jain and

Tarafder, 1970).

Some species are effective in reducing the adverse consequences of adult-

onset or insulin-independent diabetes. This may result from the presence of

saponins in these species. The extracts of O. ficus-indica were effective in

treating abdominal cancer (Cruse, 1973).

1. Park et al. (2001) studied the various fractionation of the methanol extract

of stems of Opuntia ficus-indica Mill. for anti-inflammatory action using

adjuvant-induced pouch granuloma model in mice and identified β-

sitosterol as an active anti-inflammatory compound.

2. Lyophilized aqueous extract (100–400 mg/kg, i.p.) of the fruits of Opuntia

dillenii (Ker-Gawl) Haw was evaluated for analgesic activity using writhing

and hot plate test in mice and rat, respectively and also anti-inflammatory

activity using carrageenan-induced paw edema in rats, the results exhibited

dose dependent action (Loro et al., 1999).

pg. 14

3. The prickly pear cactus stems have been used traditionally to treat diabetes

in Mexico (Domínguez López, 1995). Nowadays, Opuntia species is

amongst the majority of products recommended by Italian herbalists that

may be efficacious in reducing glycemia (Cicero et al., 2004).

4. The antioxidative action is one of many mechanisms by which fruit and

vegetable substances might exert their beneficial health effects. The presence

of several antioxidants (ascorbic acid, carotenoids, reduced glutathione,

cysteine, taurine and flavonoids such as quercetin, kaempferol and

isorhamnetin) has been detected in the fruits and vegetables of different

varieties of cactus prickly pear. More recently, the antioxidant properties of

the most frequent cactus pear betalains (betanin and indicaxanthin) have

been revealed (Tesoriere et al., 2002, 2003, 2004, 2005, 2005a; Stintzing et

al., 2005). Kuti (2004) investigated antioxidant compounds in extracts from

four Opuntia species (O. ficus-indica, O. lindheimeri, O. streptacantha, O.

stricta var. stricta) fruit.

5. In Sicily folk medicine, Opuntia ficus-indica (L.) Mill. cladodes are used for

the treatment of gastric ulcer and cicatrisant action.

6. An interesting study by Ahmad et al. (1996) demonstrated that

administration of a cactus stem extract (Opuntia streptacantha) to mice,

horses, and humans inhibits intracellular replication of a number of DNA-

and RNA-viruses such as Herpes simplex virus Type 2, Equine herpes virus,

pseudorabies virus, influenza virus, respiratory syncitial disease virus and

HIV-1. An inactivation of extra-cellular viruses was also reported by the

same authors. However, the active inhibitory component(s) of the cactus

extract used in this study was not investigated, and as of yet, no further study

dealt with this specific topic. Mtambo et al. (1999) evaluated the efficacy of

the crude extract of Opuntia vulgaris against Newcastle virus disease in

domestic fowl in Tanzania.

7. Galati et al. (2002) studied the diuretic activity of Opuntia ficus-indica (L.)

Mill. waste matter in rat. The results show that O. ficus-indica cladode, fruit

and flower infusions significantly increase diuresis. This effect is more

marked with the fruit infusion and it is particularly significant during the

chronic treatment. The fruit infusion shows also antiuric effect.

pg. 15

8. Prickly pear is traditionally used by Pima Indians as a dietary nutrient

against diabetes mellitus. Wolfram et al. (2003) examined the effect of daily

consumption of 250g in 8 healthy volunteers and 8 patients with mild

familial heterozygous hypercholesterolemia on various parameters of platelet

function. Beside its action on lipids and lipoproteins, prickly pear

consumption significantly reduced the platelet proteins (platelet factor 4 and

β-thromboglobulin), ADP-induced platelet aggregation and improved

platelet sensitivity (against PGI1 and PGE2) in volunteers as well as in

patients. Prickly pear may induce at least part of its beneficial actions on the

cardiovascular system via decreasing platelet activity and thereby improving

haemostatic balance.

9. A methanolic extract from O. dillenii Haw. defatted with chloroform and

petroleum ether exerted antispermatogenic effects in animal tests on rats.

According to (Gupta et al., 2002), the flavone derivatives vitexin and

myricetin were found to be the active principles.

10. In traditional medicine extracts of polysaccharide-containing plants are

widely employed for the treatment of skin and epithelium wounds and of

mucous membrane irritation. The extracts of Opuntia ficus-indica cladodes

are used in folk medicine for their antiulcer and wound-healing activities.

Trombetta et al. (2006) described the wound-healing potential of two

lyophilized polysaccharide extracts obtained from O. ficus-indica (L.)

cladodes applied on large full-thickness wounds in the rat.

Cacti have long been considered an important nutritional source in Latin

America (bread of the poor) among which Opuntia has gained highest

economic importance worldwide. It is cultivated in several countries such

as Mexico, Argentina, Brazil, Tunisia, Italy, Israel and China. Both fruit

and stems have been regarded to be safe for food consumption. The

constantly increasing demand for nutraceuticals is paralleled by a more

pronounced request for natural ingredients and health-promoting foods.

The multiple functional properties of cactus pear fit well this trend.

Recent data revealed the high content of some chemical constituents,

which can give added value to this fruit on a nutritional and technological

functionality basis. High levels of betalains, taurine, calcium, magnesium,

pg. 16

and antioxidants are noteworthy (Piga, 2004; Stintzing & Carle, 2005;

Feugang et al., 2006).

The Opuntia spp. cladodes and fruits serve as a source of varied number

of phytoconstituents mainly sugar, phenolics and pigments. Total

betalains are well reported with their qualitative and quantitative

analytical methods. Though various analytical methods are reported, but

still some focus is required towards HPTLC with marker’s evidence.

Although the reported evidences provide the effectiveness of Opuntia

spp., but active constituents, bioavailability, pharmacokinetics and

physiological pathways for various biological actions are not well known

with sufficient detail or confidence. Ethnopharmacological actions may

be due to presence of phenolics and pigments. Still more attention is

required towards the development of simple, feasible and cost effective

pharmaceutical preparations of Opuntia spp. cladodes and fruit juice as

well as the ethnopharmacological approach, if combined with mechanism

of action, biochemical and physiological methods, would provide useful

pharmacological leads.

pg. 17

Methodology

1. EXTRACTION OF JUICE

pg. 18

Biomass of fruit

After extraction, juice and pulp were weighed on the same digital balance.

The juice was stored at cold temperature (4 – 6°C) in refrigerator.

Weight of fruit - 7,600 kg.

Juice extracted - 4,525 kg.

Pulp + seed - 2,100 kg.

Seeds were then removed from the pulp and were dried at room

temperature and both were further processed.

75 ml juice was taken in two amber glass bottles; one was placed

under room temperature, and other was placed under cold temperature

(4 – 6°C).

pH was taken regularly till 15 days – no preservative was added.

Initially the pH of the juice was 6.3

2. PRESERVATION OF JUICE

The extracted juice was filtered and then poured in amber glass bottle under

laminar flow to prevent any microbial attack. The juice was preserved using

different natural preservatives at different concentrations.In each bottle 75ml

of juice was poured.

The preservatives used are as follows:

a) Glycyrrhiza glabra extract(20%)

b) Tartaric acid

c) Steviol (95%)

d) Curcumin (95%)

e) Piperin (95%) and

f) Sodium benzoate (synthetic for comparative study)

pg. 19

Piperine

Sodium Benzoate

Curcumin

Steviol

For each sample preservative were taken in different concentrations as

follows:

a) 0.2 g/ 70 ml,

b) 0.4 g/ 70 ml,

c) 0.6 g/ 70 ml,

d) 0.8 g/ 70 ml, and

e) 1 g/ 70 ml,

pg. 20

3. STABILITY OF JUICE BY MEASURING PH

Stability of the juice was studied by taking the pH at regular interval of 10 days.

(Handy pH meter HI98107)

4. PROCEDURE OF MOISTURE CONTENT AND TOTAL SOLIDS

As per Anonymous 1989:

Fruit pulp 10g was taken and placed in a tarred evaporating dish and dried at

105°C in an oven at constant weight.

The moisture content and total solids were determined using following

equation:

intial weight - dried weight% Moisture content = 100

initial weight

dried weight% Total solids = 100

initial weight

5. SEED VIABILITY

The seed viability was determined by tetrazolium method (ISTA 2003). The

seeds were bisected longitudinally to expose embryo and then soaked in

0.1% solution of 2,3,5-triphenyl tetrazolium chloride for 72 hrs in dark at 25

± 1ºC in incubator. The seeds with red stained embryos were considered as

viable.25 seeds were taken.

(Reagents: 0.1 gm tetrazolium in 100ml distilled water)

PHYTOCHEMICAL ANALYSIS QF JUICE

1. BETALAINS Betalains were measured by taking Optical Density at 536 nm by taking beet

root juice as a standard.

Dilution was done because of the high concentration of Betalains. By measuring

the O.D. one can check whether there is degradation of Betalains or not over a

period of time.

For every concentrations of each preservative at cold temperature, three

different dilutions were made.

For 1% dilution – 0.1ml juice + 9.9ml water

For 2% dilution – 0.2ml juice + 9.8ml water

For 5% dilution – 0.5ml juice + 9.5ml water

pg. 21

2. QUANTITATIVE ANALYSIS FOR CARBOHYDRATES:

1 ml juice

+ Stock solution

9 ml distilled water

1. Take 0.1 ml stock solution, make it 10 ml with distilled water

2. Add 4 ml Anthrone reagent to test tube containing 1 ml standard glucose

solution and in test tube containing test solution

3. Place the test tubes at 5°C for 10 min.

4. Boil 5 min. on boiling water bath

5. Cool at room temperature for 15 min.

6. Check absorbance of coloured solution at 620 nm against reagent blank

7. Spectrometric response was compared to standard curve of glucose and

total sugar was expressed as g/100 ml of glucose

Anthrone reagent: 0.1gm Anthrone + 50ml conc. H2SO4

Standard glucose solution: 0.1gm glucose + 100 ml distilled water

3. QUALITATIVE ANALYSIS FOR CARBOHYDRATES:

The aqueous fraction remaining after the separation of aglycones was

neutralized by the addition of anhydrous Na2Co3 was concentrated to

dryness and was banded on Whatman No. 1 paper and chromatogram was

developed in BAW as solvent system.

Solvent system

B : A : W

4 : 1 : 5

40 10 50

Butanol - 40 ml

Acetic acid - 10 ml

Water - 50 ml

100 ml

pg. 22

Spray-reagent

AnilineHydrogen-pthalate

4. FLAVONOIDS

The procedures followed in the present work for the extraction, isolation and

identification of flavonoids are described below.

50ml of juice was hydrolysed in a water-bath for one hour using 7% HCl.

This hydrolysate was extracted with diethyl ether/solvent ether, whereby the

aglycones got separated into ether fraction (fraction A).

An ether fraction A was concentrated analysed for aglycones using standard

procedures. The concentrated extract was banded on Whatman No. 1 paper.

The solvent was 30% glacial acetic acid. The developed chromatograms

were dried in air and visibly colour compounds were marked out. These

chromatograms were observed under ultra-violet light (360 nm) and the

bands were marked out. The marked bands of the compounds were cut out

from unsprayed chromatograms and were eluted with spectroscopic grade

methanol. The UV absorption spectra of these compunds were recorded in

methanol using ‘Perkin-Elmer Lambda 25 UV/Vis’ spectrophotometer.

5. PHENOLIC ACIDS

Analysis of phenolic acids in the combined ether fraction was carried out by

two-dimensional ascending paper chromatography. Toluene: acetic acid:

water (6:7:3 v/v/v, upper organic layer) in the first direction and sodium

formate: formic acid: water (10:1:200 w/v/v) in the second direction were

used as irrigating solvents. The sprays used to locate the compounds on the

chromatograms were diazotized p-nitroaniline or diazotized dulphanilic acid

and a 10% Na2CO3 overspray.

Diazotization: 0.7 gms of p-nitroaniline/sulphanilic acid was dissolved in

9ml of HCl and the volume made upto 100 ml. 5ml of 1% NaNO2 was taken

in a volumetric flask and kept in ice till the temperature was below 4oC. The

diazotized sprays were prepared by adding 4ml of p-nitroaniline/sulphanilic

pg. 23

acid stock solution to the cooled NaNO2 solution. The volume was made up

to 100ml with ice-cold water.

The various phenolic acids present in the extract were identified based on the

specific colour reactions they produce with the spray reagents and the

relative Rf values in the different solvent system.

6. EXTRACTION OF OIL FROM SEEDS

40g of dry finely powdered sample is taken in an extraction thimble of a

Soxhlet’s apparatus and is extracted by light petroleum for 6 – 8 hours. The

thimble is taken out dried and the contents are finely ground in a mortar.

The material is again transferred to the thimble and extraction for one hour

more. After cooling, the solvent is distilled off. Thus oil is extracted. From

this the percentage amount of oil is calculated. (Harborne J., 1984)

7. DETERMINATION OF SAPONIFICATION VALUE

The material is saponified by refluxing with a known excess of alcoholic

potassium hydroxide solution. The alkali consumed for saponification is

determined by titrating the excess alkali with standard hydrochloric acid.

REAGENTS

Alcoholic Potassium Hydroxide Solution

Aldehyde-Free Rectijied Spirit

Phenolphthalein Indicator Solution

Standard Hydrochloric Acid – 0.5 N

PROCEDURE

Melt the sample, if it is not already liquid, and filter through a filter paper to

remove ‘any impurities and the last traces of moisture. Make sure that the

sample is completely dry. Mix the sample thoroughly, and weigh accurately by

difference about 1.5 to 2.0 g of the sample in a conical flask. Add 25 ml of the

alcoholic potassium hydroxide solution and connect the reflux air condenser to

the flask. Heat the flask on a water-bath or an electric hot-plate for not more

than one hour. Boil gently but steadily until the sample is completely saponified

as indicated by absence of any oily matter and appearance of clear solution.

After the flask and condenser have cooled somewhat, wash down the inside of

the condenser with about 10 ml of hot ethyl alcohol neutral to phenolphthalein.

Add about one milliliter of phenolphthalein indicator solution, and titrate with

pg. 24

standard hydrochloric acid. Prepare and conduct a blank determination at the

same time.

CALCULATION:

56.1Saponification value =

B S N

W

where

B = volume in ml of standard hydrochloric acid required for the blank,

S = volume in ml of standard hydrochloric acid required for the sample,

N = normality of the standard hydrochloric acid, and

W = weight in g of the material taken for the test.

8. DETERMINATION OF IODINE VALUE ( WIJS ) The material is treated, in carbon tetrachloride medium, with a known excess of

iodine monochloride solution in glacial acetic acid (Wijs solution). The excess

of iodine monochloride is treated with potassium iodide and the liberated iodine

estimated by titration with sodium thiosulphate solution.

REAGENTS

Potassium Dichronrate

Concentrated Hydrochloric Acid

Potassium Iodide Solution

Starch Solution

Standard Sodium Thiosulphate Solution

Iodine Crystals

Acetic Acid

Chlorine Gas (Dry)

Iodine Trichloride

Iodine A4onochloride

Wijs Iodine Afonochloride Solution

Carbon Tetrachloride or Chloroform

PROCEDURE

Melt the sample if it is not already completely liquid, and filter through a filter

paper to remove any impurities and the last traces of moisture. Make sure that

the sample as well as the glass apparatus used is absolutely clean and dry.

Weigh accurately, by difference, an appropriate quantity of the oil or fat

between the limits indicated in co1 2 and 3 of Table 3, into a clean dry 500-ml

iodine flask or well ground glass-stoppered bottle to which 25 ml of carbon

tetrachloride have been added, and agitate to dissolve the contents. The weight

of the sample shall be such that there is an excess of 50 to 60 percent of Wijs

pg. 25

solution over that actually needed. Add 25 ml of the Wijs solution and replace

the glass stopper after wetting with potassium iodide solution; swirl for intimate

mixing, and allow standing in the dark for 30 minutes in the case of non-drying

and semi-drying oils and one hour in the case of drying oils. Carry out a blank

test simultaneously under similar experimental conditions. After standing, add

15 ml of potassium iodide solution and 100 ml of water, rinsing in the stopper

also, and titrate the liberated iodine with standard sodium thiosulphate solution,

swirling the contents of the bottle continuously to avoid any local excess until

the color of the solution is straw yellow. Add one millilitre of the starch solution

and continue the titration until the blue color formed disappears after thorough

shaking with the stopper on.

CALCULATION:

12.69Iodine value =

B S N

W

where

B = Volume in ml of standard sodium thiosulphate solution required for

the blank,

S = Volume in ml of standard sodium thiosulphate solution required for

the sample,

N= normality of the standard sodium thiosulphate solution,

and

W = weight in g of the material taken for the test.

pg. 26

9. DETERMINATION OF ACID VALUE

The acid value is determined by directly titrating the material in an alcoholic

medium with aqueous sodium or potassium hydroxide solution. Free fatty acid

is calculated as oleic, lauric, ricinoleic or palmitic acids.

REAGENTS

Ethyl Alcohol

Phenolphthalcin Indicator Solution

Sandard Aqueous Potassium Hydroxide or Sodium Hydroxide

Solutions - 0.1 N or 0.5 N

PROCEDURE

Mix the oil or melted fat thoroughly before weighing. Weigh accurately a

suitable quantity of the cooled oil or fat in a 200-ml conical flask. The weight of

the oil or fat taken for the test and the strength of the alkali used for the titration

shall be such that the volume of alkali required for the titration does not exceed

10 ml. Add 50 to 100 ml of freshly neutralized hot ethyl alcohol, and about one

milliliter of phenolphthalein indicator solution. Boil the mixture for about five

minutes and titrate while as hot as possible with standard aqueous alkali

solution, shaking vigorously during titration.

CALCULATION:

56.1Acid value =

V N

W

where

V = volume in ml of standard potassium hydroxide or sodium

hydroxide solution used,

N = normality of standard potassium hydroxide or sodium

hydroxide solution, and

W = weight in g of the material taken for the test.

10. DETERMINATION OF UNSAPONIFIABLE MATTER

The material is completely saponified with alcoholic potassium hydroxide

solution and extracted with petroleum ether. The petroleum ether extract is

washed with aqueous alcohol and then again with water. The washed ether

extract is evaporated and the residue weighed. Unsaponifiabie matter is this

pg. 27

residue minus the fatty acid present in it, which is determined by titration with

sodium hydroxide solution in alcoholic medium.

REAGENTS

Alcoholic Potassium Hydroxide Solution

Ethyl Alcohol

Phenolphthalein Indicator Solution

Petroleum Ether

Aqueous Alcohol

Standard Sodium Hydroxide Solution

Acetone

PROCEDURE

Weigh accurately about 5 g of the well-mixed sample into the flask. Add

50 ml of alcoholic potassium hydroxide solution. Roil gently but steadily

under a reflux condenser for one hour or until the saponification is complete.

Wash the condenser with about 10 ml of ethyl alcohol. Cool the mixture and

transfer it to a separating funnel. Complete the transfer by washing the flask

first with some ethyl alcohol and then with cold water. Altogether, add 50ml

of water to the separating funnel followed by an addition of 50 ml of

petroleum ether. Insert the stopper and shake vigorously for at least ‘one

minute and allow to settle until both the layers are clear. Transfer the lower

layer containing the soap solution to another separating funnel, and repeat

the ether extraction at least six times more using 50 ml of petroleum ether for

each extraction. If any emulsion is formed, add a small quantity of ethyl

alcohol or alcoholic potassium hydroxide solution.

Collect all the ether extracts in a separating funnel: Wash the combined

extracts in the funnel three times with 25-ml portions of aqueous alcohol

shaking vigorously and drawing off the alcohol-water layer after each

washing. Again wash the ether layer successively with 20-ml portions of

water until the wash-water no longer turns pink on addition of a few drops of

phenolphthalein indicator solution. Do not remove any of the ether layers.

Transfer the ether layer to a tared flask containing a few pieces of pumice

stone, and evaporate to dryness on a water-bath under a gentle stream of

clean dry air. To remove the last traces of ether, place the flask in an air-

oven at 80 to 90°C for about one hour. To remove the last traces of moisture,

add a few millilitres of acetone and pass a gentle stream of clean dry air over

the surface of the material or evacuate using a water vacuum pump at about

50°C for about 15 minutes. Cool in a desiccator and weigh. Repeat the

evacuating, cooling and weighing until a constant weight is obtained.

After weighing, take up the residue in 50 ml of warm neutral ethyl

alcohol, containing a few drops of phenolphthalein indicator solution and

titrate with standard sodium hydroxide solution.

pg. 28

CALCULATION:

1. Weight in g of the fatty acids in the extract (as oleic acid) = B = O-282

V.N

where

V = volume in ml of standard sodium hydroxide solution, and

N = normality of standard sodium hydroxide solution.

2. Unsaponifiable matter, percent by weight 100 A B

W

where

A = weight in g of the residue,

B = weight in g of the fatty acids in the extract,

and

W = weight in g of the material taken for the teat.

11. ANALYSIS BY GAS LIQUID CHROMATOGRAPHY

REAGENTS

Methanolic Sodium Hydroxide Solution

Methanol

Petroleum Ether

Sodium Sulphate

Sulphuric Acid

Hydrochloric Acid

PROCEDURE

Weighing –

Conditioning of the Column – Disconnect the carrier gas inlet from the column

to the detector. Raise the temperature of the thermostatically controlled oven to

185°C during the course of 4 hours with a constant carrier gas flow of 30 to 40

ml/min. Keep the column at this temperature for at least 16 hours. The column

might shrink during this process. If this be the case, add some fresh column

packing and repeat the conditioning procedure for an additional 16 hours.

Finally, heat for 2 hours at 195°C at the same carrier gas flow. Occasionally

columns are conditioned even for 48 hours to obtain a satisfactory base line.

Gas Flow - Normally a flow rate of 40 to 60 ml/min is chosen in order to obtain

optimum separation. When flame ionization detector (FID) is used the flow rate

of hydrogen gas shall be about half the rate of the carrier gas; the rate of air will

be 5 to 10 times the rate of hydrogen.

Most of the information given above is provided in the manual supplied along

with the gas chromatographic apparatus.

pg. 29

Determination of Optimum Working Conditions - The following variables arc

involved in choosing the working conditions:

(a) Column length and diameter,

(b) Amount and type of stationary phase & and solid support,

(c) Temperature of the column,

(d) Rate of flow of the carrier gas,

(e) Resolution required,

(f) Sample size, and

(g) Time of analysis.

CALCULATION:

Choice of the method of calculation - The manner in which peak area are

calculated depends on the accuracy which is required for analysis. Among the

several methods available, the following are widely used:

a) Triangulation,

b) Planimetry,

c) Disc Integrator, and

d) Electronic integrator.

Calculation and correction factors - For accurate determinations, it is necessary

to apply calibration factors in order to convert the ratios of the peak areas into

mass ratios. This can be done using a test mixture of methyl ester of fatty acids.

The components of the mixture shall have a high purity (about 99.0 percent).

The mixture is diluted with chloroform and is injected on to the column. From

the gas chromatographic chart, peak areas of different substances are calculated

and expressed in percentages. Known compositions of the fatty acid mixture are

used for calculation of the correction factors.

Calculation of the composition of the sample - This may be done by totaling up

the corrected areas of individual peaks and expressing them as percentages.

Often an internal standard, a fatty acid ester not originally present in the sample

to be examined which gives a peak not too far removed but well separated from

the other components, is used. For this purpose methyl heptadecanoate is useful.

The amount of internal standard shall be such that the area of its peak appearing

in the chromatogram is approximately equal to that of the major peak in the

chromatogram. For all practical purposes the corrected area percent composition

obtained from the gas chromatography can be regarded as percent by mass.

pg. 30

RESULTS AND DISCUSSION

1. Effect of preservative on pH of juice

1. Curcumin

a) Room temperature

There was a steady increase in the pH from 5.8 to 7.4 at 0.2 and 0.4

concentration of curcumin while at 0.6 to 1 concentration the pH was

more consistent between 5.7 and 5.8. - (Fig 5.a.2)

b) Cold temperature:

There was continuous decrease in the pH of the juice, within 1st 10

days the pH drops directly from 6.2 to 6.0 and than pH drops to 5.8 in

next 6 weeks - (Fig 5.a.1)

pg. 31

2. Glycyrrhizin

a) Room temperature:

Consistency in pH (5.8) was observed at 0.2gm concentrations in the initial 2

months while pH increases from 5.8 to 6.1 in next 15 days while at

concentration 0.4 to 1 increase in the pH of the juice. - (Fig 5.b.2)

b) Cold temperature

The pH steadily decreases from 6.2 to 5.7 initially in eight weeks. And in

last 1 month the pH of the juice becomes stable 5.7. - (Fig 5.b.1)

3. Steviol

a) Room temperature

In initial 5 weeks the pH of the juice was stable 5.8 in all concentration,

while over a period of time pH of the juice increases from 5.8 to 6.7 due to

some microbial growth. - (Fig 5.c.2)

b) Cold temperature

The pH is steadily decreased from 6.2 to 5.7 at all concentration while theere

was consistency in pH in the last 5 weeks. (Fig 5.c.1)

pg. 32

4. Sodium benzoate

a) Room temperature

There was consistency in pH (5.7) at 0.2 concentration after 3 weeks while

in other concentration from 0.4 to 1 the pH of the juice was steadily

decreasing. (Fig 5.d.2)

b) Cold temperature

There was consistent decrease in the pH of juice over a period of time. (Fig

5.d.1)

5. Piperine

a) Room temperature

At initial 8 weeks the pH(5.8) is constant in all the concentrations. Over a

period of time increase in pH from 5.8 to 7.4 is observed. (Fig 5.e.2)

b) Cold temperature

There was steady decrease in pH from 6.1 to 5.7 in all concentrations and in

later 6 weeks there was consistency in pH of the juice. (Fig 5.e.1)

pg. 33

pg. 34

6. Tartaric acid

a) Room temperature: There was steady increase in pH from 5.7 to 6.2 in

0.2 and 0.4 concentration while in 0.6 to 1 concentration while in 0.6 to

1 there was steady decrease in pH is observed. (Fig 5.f.2)

b) Cold temperature:

There was steady decrease in the pH of the juice. In 0.2 and 0.4

concentration after 6 weeks pH becomes stable. (Fig 5.f.1)

pg. 35

400.0 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700.0

0.000

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.200

nm

A

536.91

537.13

The above figure represents Spectrographic Analysis of Betalains

pg. 36

2. Effect of different concentration of preservative

on Betalains 1. Sodium benzoate

The concentration of betalains decreases steadily with the increase in the

concentration of preservative. More efficient preservation of betalains is

observed at 0.2gm concentration. (Fig 5.g)

2. Tartaric acid

The betalains are more efficiently preserved at lower concentration

(0.2gm). There is steady decrease in the amount of betalains with increase

in the concentration of the preservative showing similarity with that of

sodium benzoate (the recommended preservative). (Fig 5.h)

3. Steviol

At 0.4gm concentration of preservative more efficient preservation of

betalains was observed while below and above 0.4gm there is decrease in

the concentration of betalains. (Fig 5.i)

4. Glycyrrhizin

The amount of betalains increase with increase in concentration of the

preservative and the betalain preservation was more efficient at 0.6gm

concentration. (Fig 5.j)

pg. 37

5. Piperine

The preservation of betalains was more efficient with the increase in

concentration of the preservative. Amount of betalain is maximum at

1gm concentration. (Fig 5.k)

6. Curcumin

The more efficient preservation of betalains is observed at 0.4 and 0.8

concentration. (Fig 5.l)

pg. 38

Comparative analysis of betalains

As per the observations we may understand that in Opuntia elatior Mill. the betalain

content was half compared to Beta vulgaris; however, the betalain content was more than

Opuntia ficus indica. Fig. 5.m

Optical density measured at 536 nm

Opuntia ficus-indica O. elatior Beta vulgaris

0.05 g 0.0271 0.0690 0.1690

0.100 g 0.0482 0.0950 0.1802

0.250 g 0.0835 0.2015 0.4816

0.500 g 0.1893 0.4046 0.8325

1 g 0.3399 0.7440 1.5298

Figure no. 5.m

pg. 39

Preservatives pH taken at COLD temperature

27 – 12 – 2012

pH taken at COLD temperature

09 – 02 – 2012

Sodium

benzoate

Piperine

Tartaric acid

Steviol

Glycyrrhizin

Curcumin

Plate 1. Effect of preservatives on colour of the juice at cold temperature

pg. 40

Preservatives pH taken at COLD temperature

14 – 03 – 2012

pH taken at COLD temperature

28 – 04 – 2012

Sodium

benzoate

Piperine

Tartaric acid

Steviol

Glycyrrhizin

Curcumin

Plate 2. Effect of preservatives on colour of the juice at cold temperature

pg. 41

Preservatives pH taken at ROOM temperature

27 – 12 – 2012

pH taken at ROOM temperature

09 – 02 – 2012

Sodium

benzoate

Piperine

Tartaric acid

Steviol

Glycyrrhizin

Curcumin

Plate 3. Effect of preservatives on colour of the juice at room temperature

pg. 42

Preservatives pH taken at ROOM temperature

14 – 03 – 2012

pH taken at ROOM temperature

06 – 04 – 2012

Sodium

benzoate

Piperine

Tartaric acid

Steviol

Glycyrrhizin

Curcumin

0.2 and 0.4 conc.

Plate 4. Effect of preservatives on colour of the juice at room temperature

pg. 43

3. Oil analysis

The oil content in mature seeds of Opuntia elatior varied from between 10 to

15%. The oil composition of the seed is presented in Table1. The acid value is

1.64 and iodine value is 110.95 which was higher compared to Opuntia ficus

indica .The oil from mature seeds contains linoleic acid (65.81%) as the major

fatty acid, followed by Oleic (16.8%) and Palmitic (12.18%) acids. The

minor acids were Stearic (3.47%), Eicosadienoic (1.66%) acids. The total

unsaturated acids were upto 84.35 %. The ratio of saturated to unsaturated

acids was 15.65:84.35 (Fig. 5.n).

TABLE – 1

PHYSICOCHEMICAL CHARACTERISTICS OF THE TWO SPECIES OF OPUNTIA SEED OIL

Characteristics Species

Opuntia elatior Mill. O. ficus indica

Acid value 1.64 --

Iodine value 110.95 101.5 ± 1.0

Saponification value 191.85 169.0 ± 0.1

Unsaponifiable matter 2.65 --

Fatty acid profile report

Palmitic acid (16:0) 12.18% 9.32 ± 0.19

Stearic acid (18:0) 3.47% 3.11 ± 0.04

Oleic acid (18:1) 16.88% 16.8 ± 0.47

Linoleic acid (18:2) 65.81% 70.3 ± 0.60

Eicosadienoic acid (20:2) 1.66% --

pg. 44

Seed oil analysis of Opuntia elatior Mill.

Method File: FATTY ACID CAPILLARY.MET

Detector: FID. System: GC

Type of Analysis: Percent on Area and Height

Pk. Width Peak Thrsh. Area Rej. Height Rej. Time Scale

4 15 10 10 60.0

Figure: 5.n

No. R.I. Ht. Area Ht.

%

Area

%

Pk

Ty

Peak

Width

1 27.45 0 284618 0.0000 12.1838 BB 0.115 C16:0

2 32.63 0 81077 0.0000 3.4707 BB 0.169 C18:0

3 33.34 0 394289 0.0000 16.8786 BB 0.150 C18:1n9c

4 34.90 5155 1537341 100.0000 65.8100 BB 0.202 C18:2n6c

5 39.18 0 38706 0.0000 1.6569 BB 0.085 C20:2

5e+03 2336031 100.0000 100.0000

pg. 45

4. QUALITATIVE ANALYSIS FOR CARBOHYDRATES:

By paper chromatography it was analysed that in Opuntia elatior SUCROSE and

GLUCOSE is present.

Carbohydrate composition of

Opuntia ficus indica

Carbohydrate composition of

Opuntia elatior Glucose Glucose

Galactose Absent

Xylose Absent

Arabinose Absent

Sucrose Sucrose

Six standards used are

1. Glucose

2. Galactose

3. Ribose

4. Arabinose

5. Sucrose

6. Xylose

5. QUANTITATIVE ANALYSIS FOR CARBOHYDRATES:

Figure: 5.o

O.D. in 0.1ml of sample was 0.7267

pg. 46

6. FLAVANOIDS

Absent

7. PHENOLIC ACIDS

VANILLIC ACID

SYRINGIC ACID

PROTOCATECHURIC ACID WERE IDENTIFIED BY 2D PAPER

CHROMATOGRAPHY

8. SEED VIABILITY

Out of the 25 seeds taken, 16 were viable (Pink) and 9 non-viable

(colourless)

64% VIABILITY

9. MOISTURE CONTENT

37.80%

10. TOTAL SOLIDS

62.20%

pg. 47

Conclusion During preservation at cold temperature there was a continuous decrease in pH

and over a period of time, stability in pH was observed. While in case of room

temperature there is an increase in pH, but it becomes stable after some time;

except for Sodium Benzoate in which there was a decrease in pH. At room

temperature pH was not stable probably due to microbial growth as indicated by

false smell of juice. In some of the samples of Curcumin, pH becomes alkaline

probably due to microbial growth. The shortcoming was the ‘alcoholic smell’

emanating from the drinks due to fermentation as the days of the experiment

progressed. This was mainly in the juices which were kept at room temperature.

Lower concentration of Sodium Benzoate and Tartaric acid preserve Betalains

more efficiently as compared to higher concentration and stability of pH was

also observed for both the preservative; while in case of Steviol, Glycyrrhizin,

Piperine and Curcumin, Betalains were more efficiently preserved at higher

concentration of preservatives. Preservation of Betalains was recorded

maximum at 0.2gm Tartaric Acid, 0.4gm Steviol, and 0.8gm Curcumin.

Betalain concentration in Opuntia elatior when compared with Beta vulgaris

was found to be almost half in Opuntia. Also the concentration in Opuntia ficus

indica was relatively lower than that of Opuntia elatior.

In addition Carbohydrates determined by paper chromatography for Opuntia

elatior showed the presence of Glucose and Sucrose while Galactose, Ribose,

Arabinose and Xylose were absent; which were recorded to be present in

Opuntia ficus indica.

Flavanoids were not recorded while vannilic, syringic and protocatechuric

phenolic acids were recorded in Opuntia.

The oil analysis showed that the Iodine value and saponification value in

Opuntia elatior was higher than Opuntia ficus indica. The Palmitic acid

concentration in Opuntia elatior was 12.18% while in Oputia ficus indica it is

9.32±0.19. Eicosadienoic acid was found to be present only in Opuntia elatior,

and was not reported from Opuntia ficus indica.

In brief the fruit of Opuntia was found to be useful nutritionally while the

seeds were found to be useful edible oil.

pg. 48

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243rd ACS National Meeting & Exposition, San Diego, CA, United

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African Journal of Microbiology Research (2012), 6(6), 1345-1353.

Applied Biochemistry and Microbiology (2012), 48(2), 151-158.

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Australian Journal of Basic and Applied Sciences (2011), 5(10), 356-

377.

BioResources (2012), 7(1), 1232-1237, 6 pp.

Environmental Science & Technology Ahead of Print.

Environmental Toxicology and Pharmacology (2011), 32(3), 406-416.

Food and Chemical Toxicology (2012), 50(3-4), 895-902.

Food Research International (2011), 44(7), 2169-2174.

Food Science and Biotechnology (2011), 20(5), 1283-1288.

Industrial Crops and Products (2012), 37(1), 342-346.

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International Journal of Chemical Sciences (2011), 9(4), 1987-1992.

Journal of Agricultural and Food Chemistry (2011), 59(13), 7054-

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Journal of Food and Nutrition Research (2011), 50(4), 221-228.

Journal of Innate Immunity (2012), 4(2), 176-186.

Journal of Medicinal Plants Research (2011), 5(18), 4519-4524.

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