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Sudan A cadem y o f Sciences

Atomic Energy Council

Effect o f gamma irradiation on the storage and

vitam in C concentration o f Allium cepa onion samples

By:

Mervet Ahmed Mohammed Saleh

A thesis submitted to Sudan Academy o f Sciences in partial fulfillment o f

the requirements for die degree o f Master o f Nuclear Science and

T echnology

Supervisor

Dr. Mohammed Elimam Ahmed

2013

I ^ U U

Examination Committee

Supervisor Dr. M ohammed Elim am Ahm ed1 11 ■\•

•f | a ̂ . .--♦** •Internal Exam Prof. Farouk Habbani 1 ̂•!

External Exam Dr. Siddig talha * i -• 3---------

* /

Date of Exam: 9/12/2013

Dedication

To my parents

My husband o '

My Twins

Myfamily

and all friends

With love

I

Acknowledgement

It is far beyond to express my pleasure, my grateful thanks and deep

appreciation to my Supervisor Dr. Mohammed Elimam. My grateful and deep

thanks to my colleagues cooperative work during the whole period of the

project .Also I would like to thank Mr. Ammar Mohammed Alamin. Ms.

Khalda Awad Albadwai and the the staff of the Radiation Technology

Institute. Special to thank Mr. Hassan Alzen. Finally, my deep thanks are due

to my parents for their encouragement during this work.

u

ABSTRACT

This study was done to investigate the effect of gamma irradiation on storage,

germination and vitamin C concentration of local onion { A l l i u m c e p a ) . 5 onion

samples were irradiated with 5 different radiation doses (0.1. 0.2. 0.3. 0.4. and 0.5

kGray) using cobalt- 60 irradiator (Nor din) compared to non irradiated samples as

controls. The irradiated and control onions were stored at room temperature for three

months. The results of this study showed that the non irradiated samples were either

deteriorated or grown while all the irradiated samples w7ere not. Regarding the

concentration of vitamin C it was clear that it decreased with the dose increase from

30.53 to 14.44 mg/ lOOg. This study concluded that the irradiation is very effective in

prevention of spoilage, elongation of germination period and decrease of vitamin C

concentration.

m

الغصة

ت ي ر ج سة هذه أ را د ى ال ت عل ن عينا ل م ص د و الب ت ق م خد ست ة ا ي ن ق ظ ت حف م ال خدا ست ة با ع ش ما ا قا

ر ص ن ع ن ا ع ب ث م ن ت- ا وبن 6 ك 0 )Nodin( ت و م ة ت رن متا ن ال ي ت ب عينا ش ال ها تم ال ع عي ش ت

)XO. 1 ,0 .2 , 0 .3 . 0 .4 and 0 .5 kGray ى ع لم الت ع ش ك ت ة ونل رف ع م ع تأثير ل شعا لا ى ا عل

رة ن فت زي خ ت ٠٠ الت لانبا ز ا كي ر ن وت مي تا ي ى ف ك س د وذل ع ها ب ن زي خ ة ت د م ه ل لاث ى اثعهر ث ة ف ج ر د

رة را ه. ح رف غ ت ان صل و ة ت س را د ى ال ن ال ت ا عينا ر ال مععة غي مث ث ال ها حد ي ن ق عف ت ت نبا د وا ع ى ب ض م

رة ما 5 وجيزة زمنية فت ت ا عينا ش ال ها تم ال ع شعي ت ت عا ر ج غة ب ختل د م ق ظ ف ح و ه ل ن ها يححعثة لم ا في

ن عف ت لا و ت نبا لا ا ن ا ز ا ركي ن ت مي تا ي ى ف ص س ة ع٠ يتناق د ا ت زي عا ر ج ة ال عي عا ش لا ن ا 3 م 0 .5 و 3

ى 1 حت 4 .4 ما4 را ج م 100 ١مل را . ج

Contents

Dedication

A ckn o w led g m en t.......................................................................................................................................................... i i

Abstract............................................................................................................................. iii

Arabic Abstract................................................................................................................. iv

Contents.............................................................................................................................. v

List of Table...................................................................................................................... vi

List of Figure....................................................................................................................vii

CHAPTER ONE: Introduction & Literature Review..................................................... 1

1.1-Types of radiation........................................................................................................1

1.2 General overview about Radiation Technology....................................................... 2

1.3 Principles of Food Irradiation....................................................................................3

1.3.1 Radiation sources used in food processing............................................................ 3

1.4 Radiation dose for food irradiation...........................................................................4

1.5 Techniques used for food irradiation........................................................................ 5

1.5.1 Electron beam irradiation....................................................................................... 5

1.5.2 X-ray Irradiation.......................................................................................................5

1.5.3 Gamma radiation technique.....................................................................................5

1.6 Food Irradiation Equipment...................................................................................... 5

1.7 Alluim cepa, Classification, species, anatomy, vitality............................................6

1.7.1 Vitamin C, Structure, function deficiency

1.7.2 Absorption, transport, and disposal

v

1.7.3 Physiological function in mammals....................................................... 10

1.7.3.1 Collagen, carnitine, and tyrosine synthesis, and microsomalmetabolism................................................................................................. 10

1.7.3.2 Antioxidant......................................................................................11

1.7.3.3 Pro-oxidant.......................................................................................11

1.7.3.4 Immune system................................................................................. 12

1.7.3.5 Antihistamine....................................................................................12

1.8 Daily requirements..................................................................................12

1.9 Deficiency................................................................................................. 13

1.10 Effect of radiation on allium cepa and vitamin C

1.11 Effects of ionizing radiation................................

1.11.1 Chemical effects of food irradiation.................

1.11.2 Biological effect of food irradiation.................

1.12 Factors affecting the efficacy of food irradiation

1.13 Applications of Food Irradiation..........................

1.13.1 Reduction of pathogenic microorganisms.......

1.13.2 Decontamination...............................................

1.13.3 Extension of shelf-life.......................................

1.13.4 Disinfestations...................................................

1.13.5 Other potential appl ications...............................

1.14 Safety of Irradiated Food....................................

1.14.1 Radiological safety.............................................

1.14.2 Microbiological safety.......................................

1.14.3 Toxicological safety...........................................

1.14.3.1Toxicity studies in animals..............................

1.14.3.2 Human clinical studies....................................

1.14.3.3 Chemical toxicological studies......................

.15

.16

.16

17

17

.18

.18

.18

.19

.19

.19

.20

.20

.20

.21

.21

vi

1.15 Nutritional adequacy................................................................................23

1.16 Previous studies: Effect of radiation.......................................................24

1.17 Objectives................................................................................................. 25

CHAPTER TOW: Materials and methods..................................................... 26

2.1- Study subjects............................................................................................ 26

2.2- Study design.............................................................................................. 26

2.3 Irradiation................................................................................................... 26

2.4 Methods...................................................................................................... 26

2.4.1 Preparation............................................................................................... 26

2.4.2 Assay iodine solution.............................................................................. 26

2.4.3 Calibration of onion juice............................

2.4.4: measurement of Vitamin C.........................

CHAPTER THREE: Results and discussion.......

3.1: Effect of storage on germination and rotting:

3.1.1: Control group:..............................................

3.1.2 The irradiated group.....................................

.26

.27

.28

28

.28

29

3.2 concenteration of vitamin c

3.3 discussion..................................................................

CHAPTER FOUR: Conclusion and Recommendations

4.1 Conclusion.................................................................

4.2 Recommendations.....................................................

References........................................................................

■*» 3H

vu

-List of T ables

Table 3.1: The average volumes of titration and Concentration of Vitamin C .......... 3!

List of FiguresFig3.1 Storage effect on the control (unirradiated) samples.........................................28

Fig 3.2: The effect of the radiation doses (0.5 KGy) on the onion samples and storage

for three months..............................................................................................................29

Fig 3.3: The effect of the radiation doses (0.1 KGy) on the onion samples and

storage for three months................................................................................................. 30

Fig 3.3: change Vitamin C concentration of the irradiated onion Samples

versus the dose ................................................................................ 32

vm

CHAPTER ONE

Introduction and Literature Review

1.1-Types o f radiation

There are two types of radiation: ionizing radiation is energy that is carried by any of

several types of particles and rays (electromagnetic radiation) given off by radioactive

material, X-ray machines, and nuclear reactions. This energy can knock electrons out of

molecules with which they interact, thus creating ions. Non-ionizing radiation, such as

that emitted by a laser, is different because it does not create ions when it interacts with

matter but dissipates energy generally in the form of heat. The three main types of

ionizing radiation are alpha particles, beta particles, and gamma rays (Ionizing

Radiation, 2005).

An alpha particle consists of two protons and two neutrons and is identical to the

nucleus of a helium atom. Because of its relatively large mass and charge, an alpha

particle produces ions in a much localized area. An alpha particle loses some of its

energy each time it produces an ion (its positive charge pulls electrons away from atoms

in its path), finally acquiring two electrons from an atom at the end of its path to

become a complete helium atom. An alpha particle has a short range (several

centimeters) in air and cannot penetrate the outer layer of skin (Ionizing Radiation.

2005).

Beta particles can be either negative (negatron) or positive (positron). Negatrons are

identical to electrons and originate in the nucleus of an atom that undergoes radioactive

decay by changing a neutron into a proton. The only difference between a negative beta

particle (negatron) and an electron is the ancestry. A beta particle originates in the

nucleus whereas an electron is external to the nucleus. Unless otherwise specified, the

term “beta particle” generally refers to a negatron. A positron is emitted from an atom

that decays by changing a proton into a neutron. Beta particles are smaller and more

penetrating than alpha particles, but their range in tissue is still quite limited. When its

energy is spent, a negatron attaches itself to an atom and becomes an ordinary electron,

while a positron collides with an ambient electron and the two particles annihilate each

other, producing two gamma rays. When a negatron passes close to the nucleus of an

atom, the strong attractive Coulomb force causes the beta particle to deviate sharply and1

lose energy at a rate proportional to the square of the acceleration. I his energy

manifests itself as photons termed Bremsstrahlung. The amount of beta energy

converted into photons is directly proportional to the energy of the beta particle. This

effect is only significant for high-energy beta particles generally passing through \ery

dense materials such as lead, i.e., those with higher atomic numbers and so more

protons in the nucleus (Ionizing Radiation, 2005).

Gamma rays are electromagnetic radiation given off by an atom as a means of releasing

excess energy. They are bundles (quanta) of energy that have no charge or mass and can

travel long distances through air (up to several hundred meters), body tissue, and other

materials. A gamma ray can pass through a body without hitting anything, or it may hit

an atom and give that atom all or part of its energy. This normally knocks an electron

out of the atom, ionizing it. This electron then uses the energy it receives from the

gamma ray to create additional ions by knocking electrons out of other atoms. Because

a gamma ray is pure energy, it no longer exists once it loses all its energy. The

capability of a gamma ray to do damage is a function of its energy, w here the distance

between ionizing events is large on the scale of the nucleus of a cell. Ionizing radiation

is a type of energy similar to radio and television waves, micro waxes, and infrared

radiation (Dionfsio et al 2009).

Gamma rays and X-rays are short wavelength radiations of the electromagnetic

spectrum. Gamma rays are emitted by radioisotopes such as Cobalt-60 and Caesium-

137 while electrons and X-rays are generated by gaseous discharge using electricity.

Gamma rays are a part of the electromagnetic spectrum (Dionisio et al 2009).

Cobalt-60 is produced in a nuclear reactor via neutron bombardment of highly refined

cobalt-59 (59Co) pellets, while cesium-137 is produced as a result of uranium fission.

Both cobalt-60 and cesium-137 emit highly penetrating gamma rays that can be used to

treat food in bulk or in its final packaging. Cobalt-60 is, at present, the radioisotope

most extensively employed for gamma irradiation of food (Dionfsio et al 2009).

1.2 General overview of Radiation Technology

Ionizing radiation has been widely used in industrial processes, especially in the

sterilization of medicals, pharmaceuticals, and cosmetic products, and in food

processing. Similar to other techniques of food processing, irradiation can induce

2

certain alterations that can modify both the chemical composition and the nutritional

value of foods. These changes depend on the food composition, the irradiation dose and

factors such as temperature and presence or absence of oxygen in the irradiating

environment (Motaa, et al 2010).

The sensitivity of vitamins to radiation is unpredictable and food vitamin losses during

the irradiation are often substantial (Melgar, 1999).

1.3 Principles o f food irradiation:

1.3.1 Radiation sources used in food processingAccording to the Codex General Standard for Irradiated Foods, ionizing radiations

recommended for use in food processing can be categorized as follow (Safety of

irradiated Food 2009).

(I) Isotopic sources: Gamma rays produced from the radioisotopes cobait-60 (*’ Co)

and cesium-137 (137Cs)

(II) Machine sources: Electron beams and X-ray can be generated using machines.

A major advantage of machine-sourced ionising radiation is that no radioactive

substance is involved in the whole processing system. Powered by electricity, electron-

beam machines use linear accelerators to produce accelerating electron beams to near

the speed of light. The high-energy electron beams have limited penetration power and

are suitable only for foods of relatively shallow depth (Stewart. 2001).

Electron beams can be converted into various energies of X-rays by the bombardment

with a metallic target. Although X-rays have been shown to be more penetrating than

gamma rays from cobalt-60 and cesium-137, the efficiency of conversion from

electrons to X-rays is generally less than 10% and this has hindered the use of machine

sourced radiation so far Internationally (Consultative Group on Food Irradiation 1999).

Food irradiation is the process of exposing food, either prepackaged or in bulk, to

controlled levels of ionizing radiation. However, the high energy produced by ionizing

radiation allows it to penetrate deeply into food, killing microorganisms without

significantly raising the food’s temperature. Depending on the dose of radiation used,

rapidly growing cells (such as those in foodbome pathogens, spoilage microorganisms,

insects, parasites, and plant tissues) are deactivated or killed. As a result, irradiation is

used for a variety of purposes, such as reducing or eliminating foodborne pathogens,

disinfesting food, and extending product shelf life. However, not all foods are suitable

for irradiation. For example, some fruits are very sensitive to radiation and their skins3

are damaged, and other foods (such as cucumbers, grapes, and some tomatoes) turn

mushy (Bliley 2000).

According to the International Atomic Energy Agency (IAEA), more than 50 countries

have approved the use of irradiation for about 50 different types of food, and 33 are

using the technology commercially. The positive list of irradiated products varies

between countries but is often limited to spices, herbs, seasonings, some fresh or dried

fruits and vegetables, seafood, meat and meat products, poultry and egg products.

Despite the fact that irradiation has been used for decades for food disinfection and

satisfying quarantine requirements in trade, there is considerable debate on the issue of

health concerns over the consumption of irradiated food. These include concerns over

the toxicity of the chemicals generated and the change in nutritional quality of food

products after irradiation (Safety of irradiated Food. 2009).

The irradiation process involves passing of food through a radiation field allow ing the

food to absorb desired radiation energy. The food itself never comes in contact w ith the

radioactive material. Gamma rays, X-rays, and electrons prescribed for radiation

processing of food do not induce any radioactivity in foods. In comparison to other food

processing and preservation methods the nutritional value is least affected by irradiation

(Neal, 2009).

1.4 Radiation dose for food irradiation

The radiation energy absorbed by the food is measured using a unit called

the Gray (Gy) (Melgar, 1999).Low doses of radiation (0.15 k Gray’) can arrest the sprouting of potatoes and onions.

The process consists of exposing potatoes or onions to gamma rays in a shielded room

for a specified duration. Then they are brought into and taken out of the room by

conveyors or carriers. As a result, storage losses of tubers and bulbs due to sprouting,

and their dehydration can be reduced substantially. Low-dose applications (less than

one k Gy) also lead to the disinfestations of insects in stored grain, pulses, and food

products, and the destruction of parasites in meat and meat products.

A medium dose ( \ -10 k Gvf eliminates microbes in fresh fruits, meat, and poultry

products, destroys food pathogens in meat, and helps in the hvgienization of spices and

herbs.

4

A high dose (above 10 k Gv) produces shelf-stable foods without resort to refrigeration,

and the sterilization of food for special requirements (Kader 1986).

1.5 Techniques used for food irradiation:There are three techniques that are used for food irradiation: Electron beam irradiation,

gamma radiation, and X-ray irradiation. Each varies in exposure time needed to treat the

food, penetrative ability of the radiation, and safety to workers (Neal, 2009).

1.5.1 Electron beam irradiation uses an electron gun to bombard the food with high

energy electrons. The treatment time is very short, but electrons do not penetrate deeply

into the food. Accordingly, electron beam irradiation is not a appropriate method for

food irradiation (Neal, 2009).

1.5.2 X -ray irradiation: an electron gun produces an electron beam which strikes a

metal target, producing the X-rays. Although the treatment time is longer than that of

the gamma radiation technique, the penetration is just as deep. The spread of the beam

is controllable, increasing the safety of the workers, and without the use of a radiation

source, the machinery can be turned off unless in operation. Workers need only use

heavy concrete shielding to protect themselves when the machine is in use. As X-ray

irradiation uses an electron gun just like electron beam irradiation, the two techniques

can be used in the same facility (Neal, 2009).

1.5.3 G am m a irradiation technique: usually employ either a cobalt-60 or cesium-137

radioactive source. It is considered as the most applicable technique for food irradiation.

This technique normally requires ashort time due to the efficiency of gamma rays to

penetrate materials. From softly point of view', the radioactive source is kept under

water when the machine is off, w'hile during the irradiation the workers need to remain

behind thick concrete barriers (Neal, 2009).

1.6 Food Irradiation EquipmentGamma irradiation produced by cobalt-60 was used as the energy sources to provide

ionising radiation for the process. The common features of all commercially irradiation

facilities are the irradiation room and a system to transport the food into and out of the

room. The major structural difference between irradiation plant to any other industrial

building is the concrete shielding (usually 1.5-1.8 meters thick) surrounding the

irradiation room, which ensures that ionizing radiation does not escape to the outside of

5

the room. As in any gamma irradiator, the radionuclide source continuousi> emits

radiation and when not being used to treat food, the radiation source is stored in a water

pool (around six meters in depth). Known as one of the best shields against radiation

energy, water absorbs the radiation energy and protects workers from exposure if they

must enter the room (Benkeblia and Khali M 1996).

The transport system employed in the food irradiation facility is a rail system which

conveys the food products through the irradiation chamber for irradiation treatment. B\

controlling the time and the energy of the irradiation source, specific dose of ionizing

radiation is delivered to the food products to achieve specific purpose.

In China, industrial food irradiation facilities must be licensed, regulated and inspected

by national radiological safety and health authorities. Reference was made to irradiation

standards and codes of practice (Codex Alimentarius Commission 1983).

established by other authorities. The IAEA and FAO have jointly de\eloped the food

Irradiation Facilities Database which provides a list of authorised food irradiation

facilities by country for public reference (FAO/IAEA Programme).

1.7Alluim cepa, classification, species, anatomy, vitality

Onions (Allium cepa L.) are one of the world’s oldest cultivated vegetables and are the

second most important horticultural crop after tomatoes in Portugal. Onions contain

high levels of flavonoids, a major class of non-nutrient antioxidants. The major classes

of flavonoids present in onions are flavonols (quercetin glycosides) and anthocyanins

(cyaniding glycosides). These compounds are effective scavengers of free radicals that

are thought to induce DNA damage and tumour promotion. Flavonoids. in general, also

have preventive effects on a number of degenerative pathologies such as cardiovascular

and neurological diseases, and other dysfunctions related to oxidative stresses. Plant

phenolic compounds are synthesized via the phenylpropanoid pathway and play a role

in plant defense mechanisms against biotic and abiotic stresses. The induction of

phenylpropanoid metabolism can be achieved artificially by treatments with elicitors or

exposure to specific stress conditions. Interest in the role of antioxidants in human

health lead to an effort to evaluate antioxidant properties of fruits and vegetables and to

determine whether these properties can be maintained or improved through crop

breeding, cultural practices, post-harvest storage and emergent technologies such as

6

UV-C, Several studies have addressed the changes in the phenolic compounds during

storage of different fruits and vegetables (Motaa, 1999).

Onions are natural parts of the daily diet for most of the world’s population. Common

yellow onion (Allium cepa L.) is a crop of great economic importance grown all over

the world. Onion phenol compounds, particularly flavonols, are known to be potent free

radical scavengers and antioxidants; they are considered to be protective against

cardiovascular diseases and to contribute in the prevention of colorectal cancers in

humans. In addition, when processing onion it is important to consider not only the

consumer’s perception and onion safety and quality but also onion nutritional attributes.

Therefore, it was of our interest to analyze how a processing technology affects onion

nutritional nronerties. Hieh-nressure onion processing treatments new

onion products with similar organoleptic properties of fresh onion that additionally

could offer potential human health benefits (Motaa, 1999).

1.7.1 V itam in C, structure, function deficiency

Vitamins are organic compounds that are indispensable in very small amounts in the

diet. Vitamins are unstable in foods. Processing and cooking conditions cause vitamin

loss. Vitamin losses: Retention during heat treatment and continual changes expressed

by mathematical models (Lesvkova, et al 2006).

The name vitamin C refers to the L-enantiomer of ascorbic acid and its oxidized forms;

the opposite D-enantiomer has no physiological significance. L-Ascorbic acid is a weak

7

sugar acid structurally related to glucose. In biological systems, ascorbic acid can be

found only at low pH, but in neutral solutions above pH 5 is predominantly founci in the

ionized form, ascorbate ( http.7/en.wikipedia.org/wiki/Vitamin C. 2012).

The biological role of L-ascorbate is to act as a reducing agent, donating electrons to

various enzymatic and non-enzymatic reactions. The one- and two-electron oxidized

forms of vitamin C, semidehydroascorbic acid and Dehydroascorbic acid, respectively,

can be reduced by the body by glutathione and NADPH-dependent enzym atic

mechanisms. The presence of glutathione in cells and extracellular fluids helps maintain

ascorbate in a reduced state ( http://en.wikiDedia.org/wiki/Vitamin C. 2012).

The vast majority of animals and plants are able to synthesize vitamin C. through a

sequence of enzyme-driven steps, which convert monosaccharides to v itamin C. In

plants, this is accomplished through the conversion of mannose or galactose to ascorbic

acid. In some animals, glucose needed to produce ascorbate in the liver (in mammals

and perching birds) is extracted from glycogen; ascorbate synthesis is a glycogenoiysis-

dependent process. In reptiles and birds the biosynthesis is carried out in the kidneys

('http://en.wikipedia.org/wiki/Vitamin C. 2012).

Among the animals that have lost the ability to synthesise vitamin C are simians and

tarsiers, which together make up one of two major primate suborders, the anthropoidea.

also called haplorrhini. This group includes humans. The other more primitive primates

(strepsirrhini) have the ability to make vitamin C. Synthesis does not occur in a number

of species (perhaps all species) in the small rodent family caviidae that includes guinea

pigs and capybaras, but occurs in other rodents (rats and mice do not need vitamin C in

their diet, for example). A number of species of passerine birds also do not synthesise,

but not all of them and those that don't are not clearly related; there is a theory that the

ability was lost separately a number of times in birds. All tested families of bats,

including major insect and fruit-eating bat families, cannot synthesise vitamin C

(http://en.wikipedia.org/wiki/Vitamin C. 2012).

1.7.2 Absorption, transport, and disposal«

Ascorbic acid is absorbed in the body by both active transpo *t and simple diffusion.

Sodium-Dependent Active Transport—Sodium-Ascorbate Co-Transporters (SVCTs)8

and Hexose transporters (GLUTs)—are the two transporters required for absorption.

SVCT1 and SVCT2 import the reduced form of ascorbate across plasma membrane.

GLUT1 and GLUT3 are the two glucose transporters, and transfer only dehydroascorbic

acid form of Vitamin C. Although dehydroascorbic acid is absorbed in higher rate than

ascorbate, the amount of dehydroascorbic acid found in plasma and tissues under

normal conditions is low, as cells rapidly reduce dehydroascorbic acid to ascorbate.

Thus, SVCTs appear to be the predominant system for vitamin C transport in the body.

SVCT2 is involved in vitamin C transport in almost every tissue, the notable exception

being red blood cells, which lose SVCT proteins during maturation. "SVCT2 knockout"

animals genetically engineered to lack this functional gene die shortly after birth,

suggesting that SVCT2-mediated vitamin C transport is necessary for life (Savini. et al

2008).

With regular intake the absorption rate varies between 70 to 95%. However, the degree

of absorption decreases as intake increases. At high intake (1.25g). fractional human

absorption of ascorbic acid may be as low as 33%; at low intake (<200 mg) the

absorption rate can reach up to 98%. Ascorbate concentrations over renal re-absorption

threshold pass freely into the urine and are excreted. At high dietary doses

(corresponding to several hundred mg/day in humans) ascorbate is accumulated in the

body until the plasma levels reach the renal resorption threshold, which is about 1.5

mg/dL in men and 1.3 mg/dL in women. Concentrations in the plasma larger than this

value (thought to represent body saturation) are rapidly excreted in the urine with a half-

life of about 30 minutes. Concentrations less than this threshold amount are actively*

retained by the kidneys, and the excretion half-life for the remainder of the \ itamin C

store in the body thus increases greatly, with the half-life lengthening as the body stores

are depleted. This half-life rises until it is as long as 83 days by the onset of the first

symptoms of scurvy (Packer, 1997).

Although the body's maximal store of vitamin C is largely determined by the renal

threshold for blood, there are many tissues that maintain vitamin C concentrations far

higher than in blood. Biological tissues that accumulate over 100 times the level in

blood plasma of vitamin C are the adrenal glands, pituitary, vhymus. corpus luteurn. and

retina. Those with 10 to 50 times the concentration p r e s e n t m flood p l a s m a i n c l u d e the

brain, spleen, lung, testicle, lymph nodes, liver, thyroid, small intestinal mucosa.9

leukocytes, pancreas, kidney and salivary glands. Ascorbic acid can be oxidized (broken

down) in the human body by the enzyme L-ascorbate oxidase. Ascorbate that is not

directly excreted in the urine as a result of body saturation or destroyed in other bod>

metabolism is oxidized by this enzyme and removed (Packer. 1997).

1.7.3 Physiological function in m am m als

In humans, vitamin C is essential to a healthy diet as well as being a highly effective

antioxidant, acting to lessen oxidative stress; a substrate for ascorbate peroxidase in

plants (APX is plant specific enzyme); and an enzyme cofactor for the biosynthesis of

many important biochemicals. Vitamin C acts as an electron donor for important

enzymes (Packer, 1997).

1.7.3.1 Collagen, carnitine, and tyrosine synthesis, and m icrosom al m etabolism

Ascorbic acid performs numerous physiological functions in the human body. These

functions include the synthesis of collagen, carnitine, and neurotransmitters: the

synthesis and catabolism of tyrosine; and the metabolism of microsomc. During

biosynthesis ascorbate acts as a reducing agent, donating electrons and preventing

oxidation to keep iron and copper atoms in their reduced states (McGregor and

Biesalski 2006).

Vitamin C acts as an electron donor for eight different enzymes:

• T h re e e n zym e s participate in collagen hydroxylation. Th ese reactions add hydroxyl

groups to th e am in o acids proline or lysine in the collagen m o lecu le via prolyl

hydroxylase and lysyl hydroxylase, both requiring vitam in C as a cofactor.

H ydroxylation allow s th e collagen m olecule to assu m e its trip le helix structure, and

th u s v ita m in C is essential to th e d e ve lo p m e n t and m ain te n an ce of scar tissu e, blood

ve sse ls, an d cartilage.

• Tw o e n zym e s are n ecessary for syn th esis of carnitine. C arn itin e is essen tial for the

tran sp o rt of fatty acid s into m ito ch o n d ria for A TP generatio n.

• T h e rem ain in g th re e en zym es have th e follow ing functio ns i 1 co m m o n , but h ave other

fu n ctio n s as w ell:

10

• D o p a m in e beta hydro xylase participates in th e b io syn th esis of n o rep in ep h rin e

from d o p am in e.

• A n o th e r e n zym e ad d s a m id e groups to p ep tid e h o rm o n e s, greatly increasing

th e ir stability.

One modulates tyrosine metabolism (McGregor and Biesalski 2006).

1.7.3.2 A ntioxidant

Ascorbic acid is well known for its antioxidant activity, acting as a reducing agent to

reverse oxidation in liquids. When there are more free radicals (reactive oxygen species.

ROS) in the human body than antioxidants, the condition is called oxidative stress, and

has an impact on cardiovascular disease, hypertension, chronic inflammatory diseases,

diabetes as well as on critically ill patients and individuals with severe burns.

Individuals experiencing oxidative stress have ascorbate blood levels lower than 45

pmol/L, compared to healthy individual who range between 61.4-80 umolL (Kelly

1998)

It is not yet certain whether vitamin C and antioxidants in general prevent oxidative

stress-related diseases and promote health. Clinical studies regarding the effects of

vitamin C supplementation on lipoproteins and cholesterol have found that vitamin C

supplementation does not improve disease markers in the blood. Vitamin C may

contribute to decreased risk of cardiovascular disease and strokes through a smailW

reduction in systolic blood pressure, and was also found to both increase ascorbic acid

levels and reduce levels of resistin serum, another likely determinant of oxidativ e stress

and cardiovascular risk. However, so far there is no consensus that vitamin intake has

an impact on cardiovascular risks in general, and an array of studies found negative

results. Meta-analysis of a large number of studies on antioxidants, including vitamin C

supplementation, found no relationship between vitamin C and mortality (Preedy et al

2010).

11

1.7.3.3 Pro-oxidant

Ascorbic acid behaves not only as an antioxidant but also as a pro-oxidant. Ascorbic«s

acid has been shown to reduce transition metals, such as cupric ions (Cu“~). to cuprous1 + l i ^ -

(Cu ), and ferric ions (Fe ) to ferrous (Fe‘~) during conversion from ascorbate to

dehydroascorbate i n v i t r o . This reaction can generate superoxide and other ROS.

However, in the body, free transition elements are unlikely to be present while iron and

copper are bound to diverse proteins and the intravenous use of vitamin C does not

appear to increase pro-oxidant activity. Thus, ascorbate as a pro-oxidant is unlikely to

convert metals to create ROS i n v i v o . However, vitamin C supplementation has been

associated with increased DNA damage in the lymphocytes of healthy volunteers

(Preedyetal2010).

1.7.3.4 Im m une system

Vitamin C is found in high concentrations in immune cells, and is consumed quickly

during infections. It is not certain how vitamin C interacts with the immune system: it

has been hypothesized to modulate the activities of phagocytes, the production of

cytokines and lymphocytes, and the number of cell adhesion molecules in monocy tes

(Johnston 1992).

1.7.3.5 A ntihistam ine

Vitamin C is a natural antihistamine. It both prevents histamine release and increases

the detoxification of histamine. A 1992 study found that taking 2 grams vitamin C daily

lowered blood histamine levels 38 percent in healthy adults in just one week. It has also

been noted that low concentrations of serum vitamin C has been correlated with

increased serum histamine levels (UN 2013).

1.8 vitamin C: Daily requirements

The North American Dietary Reference Intake recommends 90 milligrams per day and

no more than 2 grams (2,000 milligrams) per day of . Other related vitamin C species

sharing the same inability to produce vitamin C and requiring exogenous vitamin ('

consume 20 to 80 times this reference intake. There is continuing debate within the

12

scientific community over the best dose schedule (the amount and frequency of intake)

of vitamin C for maintaining optimal health in humans. A balanced diet without

supplementation usually contains enough vitamin C to prevent scurvy in an average

healthy adult, while those who are pregnant, smoke tobacco, or are under stress require

slightly more. However, the amount of vitamin C necessary to prevent scurvy is less

than the amount required for optimal health, as there are a number of other chronic

diseases whose risk are increased by a low vitamin C intake, including cancer, heart

disease, and cataracts. A 1999 review suggested a dose of 90-100 mg Vitamin C daily

is required to optimally protect against these diseases, in contrast to the lower 45 mg

daily required to prevent scurvy (Khaw, et al 2001)

High doses (thousands of milligrams) may result in diarrhea in healthy adults, as a result«

of the osmotic water-retaining effect of the unabsorbed portion in the gastrointestinal

tract (similar to cathartic osmotic laxatives). Proponents of orthomolecular cia.m the

onset of diarrhea to be an indication of w'here the body's true vitamin C requirement

lies, though this has not been clinically verified (Khaw, et al 2001).

1.9 Deficiency

Scurvy is an avitaminosis resulting from lack of vitamin C, since without this v itamin,

the synthesised collagen is too unstable to perform its function. Scurvy leads to the

formation of brown spots on the skin, spongy gums, and bleeding from all mucous

membranes. The spots are most abundant on the thighs and legs, and a person with the

ailment looks pale, feels depressed, and is partially immobilized. In advanced scurvy

there are open, suppurating wounds and loss of teeth and, eventually, death. The human

body can store only a certain amount of vitamin C, and so the body stores are depleted

if fresh supplies are not consumed. The time frame for onset of symptoms of scurvy in

unstressed adults switched to a completely vitamin C free diet, however, may range

from one month to more than six months, depending on previous loading of vitamin C

( Hemila, 2007).

It has been shown that smokers who have diets poor in vitamin C are at a higher risk of

lung-borne diseases than those smokers who have higher concentrations of v itamin C in

the blood. Nobel prize winner Linus Pauling and G. C. Willis have asserted that chronic

13

long term low blood levels of vitamin C ("chronic scurvy") is a cause of atherosclerosis

( Hem ilS, 2007).

Western societies generally consume far more than sufficient Vitamin C to prevent

scurvy. In 2004, a Canadian Community health survey reported that Canadians of i 9

years and above have intakes of vitamin C from food of 133 mg. d for males and -20

mg/d for females; these are higher than the RDA recommendations. Notable human

dietary studies of experimentally induced scurvy have been conducted on conscientious

objectors during WW II in Britain, and on Iowa state prisoners in the late 1960s. These

studies both found that all obvious symptoms of scurvy previously induced b> an

experimental scorbutic diet with extremely low vitamin C content could be complete!)

reversed by additional vitamin C supplementation of only 10 mg a day. In these

experiments, there was no clinical difference noted between men given 70 mg v itamin

C per day (which produced blood level of vitamin C of about 0.55 mg/dl. about 1 3 of

tissue saturation levels), and those given 10 mg per day. Men in the prison stud)

developed the first signs of scurvy about 4 weeks after starting the vitamin C free diet,

whereas in the British study, six to eight months were required, possibly due to the pre-

loading of this group with a 70 mg/day supplement for six weeks before the scorbutic

diet was fed (Hem ila, 2007).

Men in both studies on a diet devoid, or nearly devoid, of vitamin C had blood lev els of

vitamin C too low to be accurately measured when they developed signs of scurvy, and

in the Iowa study, at this time were estimated (by labeled vitamin C dilution) to have a

body pool of less than 300 mg, with daily turnover of only 2.5 mg/day. implying a

instantaneous half-life of 83 days by this time (elimination constant of 4 months)

(Barbosa, et al 2009).

Moderately higher blood levels of vitamin C measured in health) persons have been

found to be prospectively correlated with decreased risk of cardiovascular disease and

ischaemic heart disease, and an increase life expectancy. The same stud) found an

inverse relationship between blood vitamin C lev els and cancer risk in men. but not in

women. An increase in blood level of 20 micromol/L if vitamin C ( about 0.35 mg dl ..

and representing a theoretical additional 50 grams of fruii and vegetables per day) was

found epidemiologically to reduce the all-cause risk of mortality, four \ears alter

14

measuring it, by about 20%. However, because this was not an intervention studv.

causation could not be proven, and vitamin C blood levels acting as a proxy marker for

other differences between the groups could not be ruled out. However, the lour-vear

long and prospective nature of the study did rule out proxy effect from any v itamin C

lowering effects of immediately terminal illness, or near-end-of-life poor health

(Barbosa, et al 2009).

Studies with much higher doses of vitamin C, usually between 200 and 6000 mg/day,

for the treatment of infections and wounds have shown inconsistent results.

Combinations of antioxidants seem to improve wound healing (Barbosa, et al 2009).

1.10 Effect of radiation on allium cepa and vitamin C

Generally, results show that antioxidant activity and concentration of phenolics often

increases during storage although a few studies report constant or decreasing levels

during storage (Melgar, 1999).

Vitamins, different types of vitamins have varied sensitivity to irradiation and to some

other food processing methods. The sensitivity of the vitamins to irradiation depends on

the complexity of the food system and the solubility of the vitamins in water or fat.

Irradiation of vitamins in pure solution results in considerable destruction of these

compounds thus some reports in literature have overestimated the losses (Melgar.

1999).

For example, vitamin B1 (thiamin) in aqueous solution showed 50% loss after

irradiation at 0.5 kGy, while irradiation of dried whole egg at that dose caused less than

5% destruction of the same vitamin. This is due to the mutually protective action of

various food constituents on each other. Vitamin losses can be minimized by irradiating

the food in frozen form or by packaging it in an inert atmosphere such as under

nitrogen. Four vitamins are recognized as being highly sensitive to irradiation: Bl. C

(ascorbic acid), a (retinol) and E (tocopherol). However, Bl is even more sensitive to

heat than to irradiation. It has been demonstrated that pork and beef sterilized by

irradiation retain much more vitamin Bl than canned meat sterilized thermally (Melgar.

1999).

Seemingly conflicting results of low versus high losses of vitamin C for some irradiated

foods may be attributed to differences in analytical approaches used by researchers.

15

Some have measured only ascorbic acid, while others have measured total ascorbic

acid, a mixture of ascorbic acid and dehydroascorbic acid. Both acids have vitamin C

biological activity and are easily transformed from one to the other. If on!\ ascorbic

acid were measured, any apparent reduction in vitamin C level would be exaggerated.

Research has shown that the natural differences in total vitamin C content of four

varieties of strawberry are much greater than the reduction which occurs on irradiation.

With, for example, potatoes it has been demonstrated that although irradiation does

reduce vitamin C content, cooking and storage also have a significant effect. The benefit

of irradiating potatoes is to inhibit sprouting during storage. Following six months of

storage the vitamin C content of irradiated and unirradiated potatoes have been shown

to be similar. Since the optimal dose for irradiation treatment of fruit and vegetables, is

generally below 2 kGy, effects on vitamin C at higher doses are irrelevant (Melgar.

1999).

The stability of vitamin C (ascorbic acid) in onions irradiated with three different doses

of y rays and stored at two different temperatures was studied. Gamma radiation of

bulbs with 0.10; 0.15 and 0.31 KGy causes losses of 10%, 13% and 20% of v itamin C

contents respectively. During storage vitamin C decreased over 12 weeks in both

control and irradiated bulbs and at both temperatures (Benkeblia, N and Khali. M.

1996).

After 12 weeks and till the end of storage period, vitamin C content increased in each

share but the final content was lower than the initial. In all cases, no differences were«

noted in evolution of vitamin C at each temperature and in both untreated and treated

bulbs during storage (Benkeblia, N and Khali, M. 1996).

1.11 Effects of ionizing radiatioWhen ionizing radiation passes through matter such as food, the energy is absorbed and

leads to the ionization or excitations of the atoms and molecules of the food

constituents, which in turn, results in the chemical and biological changes known to

occur when food is irradiated (Ionizing Radiation 2005).

1.11.1 Chemical effects of food irradiation.The chemical effects of irradiation results from breakdown of the excited molecules and

ions and their reaction with neighboring molecules, giving a cascade of reactions The

primary reactions include isomerisation and dissociation within molecules and reactions

with neighbouring species to produce series of new products including the highly16

reactive free radicals. Usually the free radicals generated in food on irradiation have a

short lifetime. However, in dried or frozen foods containing hard component such as

bone, the free radicals will have limited mobility and therefore, persist for a longer

period of time (Grandison AS. 2006).

Another important chemical reaction resulted from ionizing irradiation is water

radiolysis. Hydroxyl radicals and hydrogen peroxide generated upon the irradiation of

water molecules are highly reactive and readily react with most aromatic compounds,

carboxylic acids, ketones, aldehydes, and thiols. These chemical changes are important

in terms of their effects on the elimination of living food contaminants in foods.

However, undesirable side effects, such as off-flavour, will be inevitable for certain

food commodities if condition of irradiation is not well controlled (Safety of irradiated

Food. 2009).

1.11.2 Biological effect of food irradiationThe major purpose of irradiating food is to cause changes in living ceils. I hese can

either be the contaminating organisms to reduce pathogenic microorganisms or cells of

the living foods to achieve better quality. The biological effect of ionising radiation is

inversely related to the size and complexity of the organism. The exact mechanism of

action on cells is not fully understood. However, the chemical changes described in the

previous paragraphs are known to alter cell membrane structure, reduce enzyme

activity, reduce nucleic acid synthesis, affect energy metabolism through

phosphorylation and reduce compositional changes in cellular DNA (Grandison. AS.

2006).

The DNA damage may be due to direct but random strikes of the ionising radiation that

causes the formation of lesions on either both or one of the DNA strands. Double strand

lesions are almost invariably lethal (Dickson JS. 2001).

This direct effect on DNA predominates under dry conditions, such as when dry spores

are irradiated. Alternatively, the radiations may produce free radicals from other

molecules, especially water, which diffuse towards and cause damage to the DNA (Joint

FAO/IAEA/WHO, 1999).

1.12 Factors affecting the efficacy of food irradiationThe efficacy of ionising radiation for microorganism inactivation depends mainly on the

dose of use and the level of resistance of the contaminating organisms. Radiation

resistance varies widely among different species of bacte.ia, yeasts and moulds.17

Bacterial spores are generally more resistant than vegetative cells, which is at (east

partly due to their lower moisture content. Yeast is as resistant as the radiation-tolerant

bacterial strains. Viruses are highly radiation resistant (Joint FAO/IAEA/WHO 19991.

Other factors such as temperature, pH, presence of oxygen and solute concentration

have also been shown to correlate with the amount of radio lytic products formed during

irradiation which in turn affect the ultimate effectiveness of ionising radiation (Stewart

ES, 2001).

1.13 Applications of food irradiation1.13.1 Reduction of pathogenic microorganismsSince irradiation does not substantially raise the temperature of food under irradiation, it

is of particular importance for the control of food-borne illnesses in seafood, fresh

produces, and frozen meat products. Ionising radiation has been shown to reduce the

number of disease-causing bacteria such as L i s t e r i a m o n o c y t o g e n e s . E s c h e r i c h i a c a l l

0157:H7, S a l m o n e l l a , C l o s t r i d i u m b o t u l i n u m , V i b r i o p a r a h a e m o l y t i c u s . e t c . in various

food commodities and allow food to be irradiated in its final packaging. However,

irradiation alone may not be sufficient to reduce the number of food poisoning

outbreaks, it is essential to adhere to good manufacturing practice to prevent subsequent

contamination during processing (Safety of irradiated Food. 2009).

1.13.2 DecontaminationSpices, herbs and vegetable seasonings are valued for their distinctive flavors, colors

and aromas. However, they are often contaminated with microorganisms because of the

environment and processing conditions under which they are produced (Safety of

irradiated Food, 2009).

Until the early 1980s, most spices and herbs were fumigated, usually w ith sterilising

gases such as ethylene oxide to destroy contaminating microorganisms. However, the

use of ethylene oxide has been banned in a number of countries due to its proven

carcinogenicity. Irradiation has since emerged as an alternative and widely used in the

food industry for the decontamination of dried food ingredients (Farkas J. Radiation

2001).

In addition to the improvement of hygienic quality of various foods, irradiation has also

been used as a method for decontaminating medicinal herbs (Farkas J. 1998).

18

1.13.3 Extension of shelf-lifeThe shelf-life o f many fruits and vegetables, meat, poultry, fish and seafood can be

considerably prolonged by treatment with irradiation (Benkeblia, N and Khali, M.

1996).

Depending on the dose of ionising energy applied, irradiation produces virtually no or

minor organoleptic changes to food under irradiation that make it particularly important

for the control of postharvest quality of fresh produces (Niemira BA and Fan X. 2006).

By modifying the normal biological changes associated with ripening, maturation,

sprouting, and aging (World Health Organization 1988).

Exposure to a low dose of radiation has been demonstrated to slow down the ripening

of bananas, mangoes and papaya, control fungal rot in strawberries and inhibit sprouting

in potato tubers, onion bulbs, yams and other sprouting plant foods (Thomas, P. 2001).

1.13.4 DisinfestationsThe major problem encountered in preservation of grains and grain products is insect

infestation. Irradiation has been shown to be an effective pest control method for these

commodities and a good alternative to methyl bromide, the most widely used fumigant

for insect control, which is being phased out due to its ozone depleting properties.

Disinfestations is aimed at preventing losses caused by insects in store grains, pulses,

flour, cereals, coffee beans, fresh and dried fruits, dried nuts, and other dried food

products including dried fish. It is worth mentioning that proper packaging of irradiated

products is required for preventing reinfestation of insects (Ahmed, M. 2001).

1.13.5 Other potential applicationsBesides the sanitary purposes, irradiation has been studied to reduce or eliminate

undesirable or toxic materials including, food allergens (Lee JW, 2001).

Carcinogenic volatile N-nitrosamines and biogenic amines (Byun MW, 2000)

On the other hand, irradiation has been shown to enhance colour of low-nitrite meat

products32 and low-salt fermented foods (Byun MW, 2001).

In addition, ionizing radiation can be used to destroy chlorophyll b in vegetable oil

resulting in protection of oil from photooxidation and elimination of undesirable colour

change in oil processing industry (Byun MW,et al. 2006).

19

1.14 Safety o f irradiated food

1.14.1 Radiological safetyIrradiation process involves passing the food through a radiation field at a set speed to

control the amount of energy or dose absorbed by the food. Under controlled conditions,

the food itself should never come into direct contact with the radiation source (Thomas.

P. 2001). On other hand, workers should follow all international radiation safety

precautions.

At high energy levels, ionising radiation can make certain constituents of food become

radioactive (World Health Organization 1988).

Studies showed that induced radioactivity was detected in ground beef or beef ashes

irradiated with X-rays produced by 7.5 MeV electrons. However, the induced activity

was found to be significantly lower than the natural radioactivity in food.

Corresponding annual dose is several orders of magnitude lower than the environmental

background. The risk to individuals from intake of food irradiated with X-rays

generated by electrons with nominal energy as high as 7.5 MeV is trivial.36 26 Studies

carried out by IAEA showed that increase in radiation background dose from

consumption of food irradiated to an average dose below 60kGy with gamma-rays from

cobalt-60 or cesium-137, with 10 MeV electrons, or with X-rays produced by electron

beams with energy below 5 MeV are insignificant, and best characterized as zero

(World Health Organization 1988).

Based on the experimental findings of WHO, FAO and IAEA, Codex has set out the

maximum absorbed dose delivered to a food should not exceed 1 OkGv and the energy

level of X-rays and electrons generated from machine sources operated at or below 5

MeV and 10 MeV respectively, in part, to prevent induced radioactivity in the irradiated

food (Melgar, 1999).

1.14.2 Microbiological safetyTwo concerns that have been raised regarding the irradiation of microorganisms present

in food are the effect of the reduction in the natural micro flora on surviving pathogens

and the potential for the development of radiation resistant mutants.

Ionizing radiation significantly reduces the populations of indigenous micro flora in

foods. There is concern that these “clean’' foods w'ould allow a nore rapid outgrowth of

20

bacteria o f public health concern, since the lower populations of indigenous micro flora

would have less of an antagonistic effect on the pathogenic bacteria (Jay JM. 1995).

It has also been hypothesised that irradiated foods would be more amenable to the

growth of foodbome pathogens if the food was contaminated after irradiation.39

However, studies in irradiated chicken and ground beef has illustrated that the growth

rates of either salmonellae (chicken and beef) or E s c h e r i c h i a c o l i 0157:H7 (beef) were

the same in both nonirradiated and irradiated meats suggesting that the indigenous

microflora in these products does not normally influence the growth parameters of these

bacteria (Dickson JS and Olson DG. 1999).

The concern with radiation mutations is significant because ionising radiation has been

known for years to induce mutations (Muller, 1928).

Induction o f radiation-resistant microbial populations occurs when cultures are

experimentally exposed to repeated cycles of radiation (Davies and Sinskey 1973).

Mutations developed in bacteria and other organisms can result in greater, less, or

similar levels of virulence or pathogen city from parent organisms. Although it remains

a theoretical risk, there was no report of the induction of novel pathogens attributable to

food irradiation. Ingram (M and Farkas J. 1977)

Bacteria that undergo radiation-induced mutations are more susceptible to

environmental stresses, so that a radiation-resistant mutant would be more sensitive to

heating than would its nonradiation-resistant parent strain (Joint FAO/IAEA/WHO

1999).

1.14.3 Toxicological safety1.14.3.1 Toxicity studies in animals.The possible toxicological effects of consuming irradiated foods have been extensively

studied since the 1950s.46 Feeding trails involved a variety of laboratory diets and food

components given to human and different species of animals including rats, mice, dogs,

quails, hamsters, chickens, pigs and monkeys have been conducted to assess the

toxicological safety of irradiated foods (IAEA 2007).

Animal feeding trails conducted include lifetime and multi-generation studies to

determine if any changes in growth, blood chemistry, histopathology, or reproduction

occurred that might be attributable to consumption of different types of irradiated foods.

Data from many of these studies were evaluated by the Joint FAO/IAEA/WHO Expert

Committee on the Wholesomeness of Irradiated Food (JECFI). In 1980, JEFCI

21

concluded that “Irradiation of any food commodity up to an overall average Jose of io

kGy introduces no toxicological hazard; hence, toxicological testing of food so treated

is no longer required” (FAO/IAEA/WHO 1981).

The safety of irradiated food was also supported by recent study with laboratory diets

that had been sterilized by irradiation. Several generations of animals fed diets

irradiated with doses ranging from 25 to 50 kGy, which is considerably higher than dose

used for human foods, suffered no mutagenic, teratogenic and oncogenic ill effect

attributed to the consumption of irradiated diets (Kava, 2007).

1.14.3.2 Human clinical studiesThere have been relatively few trails performed on humans, the majority being carried

out by the US Army. The subjects were assessed by clinical examination and for cardiac

performance, haematological, hepatic and renal function. All studies have been short

term. No clinical abnormalities were discovered up to one year following the trials

(Fielding, 2007).

One of the best known human feeding trails is that performed in 1975 where 15

malnourished children in India were fed a diet containing irradiated wheat at dose of

0.75 kGy. Increase in the frequency of polyploidy and number of abnormal cells were

observed during the course of the trial. When the irradiated diet was discontinued, the

abnormal cells reverted to a basal level. The author attributed these observations to the

consumption of the irradiated food (Bhaskaram, and Sadasivan. 1975).

However, when the report was examined more closely, it was found that only 100 cells

from each of the five children in each group w ere counted. The sample number w as too

small upon which to base any conclusion (Benkeblia, N and Khali, M. 1996).

A number of concerns regarding the impact of irradiated food on health have been

raised. Among these was the criticism of the design and execution of a number of i n

v i t r o studies into toxicological safety. These studies used food juices, extracts and

digests in mutagenic studies using cells of mammalian, bacterial and v egetable origin

and largely produced negative effects.

Some possible chromosome changes and cytotoxic effects were reported but. as food

contains many compounds that may interfere with the tests, the result were not deem

significant (Benkeblia, N and Khali, M. 1996).

There was also concern that when the WHO published its report on the wholesotneness

of foods irradiated at doses of above 10 kGy, five peer reviewed publications, ali of

22

which were feeding trails reporting toxicological effects of irradiated food, were

disregarded (Joint FAO/IAEA/WHO 1981).

It also has been pointed out that all the animal studies were of much too short duration

to demonstrate carcinogenicity of irradiated food, which usually takes several decades

(Tritsch, 2000).

1.14.3.3 Chemical toxicological studiesThe presence of several compounds, most notably 2-alkylcyclobutanones and furan has

generated some concerns about the safety of irradiated foods.

2 - A l k y l c y c l o b u t a n o n e s . Irradiation of fat-containing food generates a family of

molecules, namely 2-alkycycIobutanones (2-ACBs), that result from the radiation

induced breakage of triglycerides. 52 The 2-ACBs have been found exclusively in

irradiated fat-containing food, and have until now never been detected in non-irradiated

foods treated by other food processes.53, 54 Thus, these compounds were considered to

be unique markers for food irradiation. In irradiated foods, level of 2-ACBs generated is

proportional to the fat content and absorbed dose. Depending on the dose absorbed, the

concentration of 2-ACBs in irradiated food ranged from 0.2 to 2 _g/g of fat.

Previous study feeding rats daily with a solution of highly pure solution of 2-ACBs and

injected with a known carcinogen azoxymethane (AOM) showed that the total number

of tumours in the colon was threefold higher in the 2-ACB-treated rats than in the AOM

controls six months after injection. Medium and larger tumours were detected only in

animals treated with 2-ACB and AOM. This demonstrated that 2-ACBs found

exclusively in irradiated dietary fats may promote colon carcinogenesis in animals

treated with a chemical carcinogen. It does also suggest that the 2-ACBs alone do not

initiate colon carcinogenesis. However, it is worth noting that the amount of 2-ACBs

consumed was much higher in this study than that a human would consume in a diet

containing irradiated food.

1.15 Nutritional adequacyFood processing and preparation methods in general tend to result in some loss of

nutrients, and food irradiation is no exception. Nutritional changes in food attributable

to irradiation are similar to those results from cooking, canning, pasteurising, blanching

and other forms of heat processing Irradiation-induced changes in nutritional value

depend on a number of factors: radiation dose, the type of fo< d, the temperature and

atmosphere in which irradiation is performed, packaging and storage time. In general.23

macronutrients (protein, lipid and carbohydrate) quality does not suffer due to

irradiation8 and minerals have also been shown to remain stable (Diehl 1995).

However, loss of vitamins during irradiation is an obvious concern and has been

studied in detail in a variety of foods.

Different types of vitamin have varied sensitivity to irradiation. Some vitamins such as

riboflavin, niacin, and vitamin D, are fairly resistant to irradiation but vitamins A. Bl

(thiamine), E and K are relatively sensitive. Their sensitivities depend on the

complexity of the food, whether the vitamins are soluble in water or fat. and the

atmosphere in which irradiation occurs. In general, the effects of irradiation on

nutritional value of foods are insignificant for low dose (up to 1 kGy) but may be

greater at medium doses (1-lOkGy) if food is irradiated in the presence of air. At high

doses (above 10 kGy), losses of sensitive vitamins such as thiamine may be significant.

Vitamin losses can be mitigated by protective actions, for example, using low

temperatures and air exclusion during processing and storage. As irradiated foods are

normally consumed as part of a mixed diet and the process will have little impact on the

total intake of specific nutrients (Joint FAO/IAEA/WHO 1981)

1.16 Previous studies: Effect of radiationExtensive scientific studies have shown that irradiation has very little effect on the main

nutrients such as proteins, carbohydrates, fats, and minerals. Vitamins show varied

sensitivity to food processing methods including irradiation. For example, vitamin C

and Bl (thiamine) are equally sensitive to irradiation as well as to heat processing.

Vitamin A, E, C, K, and Bl in foods are relatively sensitive to radiation, while

riboflavin, niacin, and vitamin D are much more stable.«

Several investigations have been carried out throughout the world on the application of

ionizing radiation for sprout inhibition of onions grown under varying agroclimatic

conditions. Results have shown that treated bulbs could be stored for several months

without heavy spoilage .Storage conditions are important factors in determining the

storage behavior of onions .Onions are generally consumed for their flavours but their

nutritive value has been appreciated only recently .Ascorbic acid constitutes a major

vitamin in the bulb and its degradation occurs during several treatments such as heating, freezing .Molco and Padova , have shown that the content of vitamin C in onions irradiated at 0.07 KGy and stored at ambient temperature was essentially the same as that of untreated bulbs one day after irradiation and during the next 5 months storage period. Murray, found that onions

24

treated with 0.02 to 0.06 KGy in the presence of air resulted in some conversion of ascorbic acid to dehydroascorbic acid without significantly affecting the nutritional v alue. The purpose of this study is to elucidate the rate of vitamin C destruction as a function of both irradiation doses and temperatures during long term storage (Benkeblia. N and Khali. M. 1996).

1.17 Objectives

General objective:-Investigate the effect of radiation on storage and vitamin C concentration of A l l i u m

c e p a onion

Specific objectives:-

1. Apply different irradiation doses on the onion.

2. Measure the content of vitamin C before and after each irradiation dose.

25

CHAPTER TWO

Materials and Method

2.1- Study subjects:

12 onions were involved in this study and they were divided to six groups each group

contains two onions.

2.2- Study design:

The design of this research is pre post test, true experimental research design.

2.3 Irradiation

The onion samples were exposed to different radiation doses using the Co-60 irradiator

located at Soba, Sudan atomic Energy Commission. The doses were selected to be

within 0.1-0.5 kGy. This selection criterion depends on the fact that, the lowest dose

where the vitamin C is still stable is 0.1 kGy after which the brakeage of vitamin C can

be started. The control group was kept away from radiation.

2.4 Methods

2.4.1 Preparation

Preparation of standard solutions of iodine (0.05 Molar) and solution of ascorbic acid

(vitamin C) concentration of 0.1 grams per 100 ml of distilled water

2.4.2Assay iodine solution

100 ml of solution of ascorbic acid was withdrawn; 2 ml of evidence starch were added

and titrated against iodine solution (0.05 Molar) to endpoint (dark blue color). The

titration was repeated twice.

2.4.3 Calibration of onion juice

Onion was peeled and then cut up into small pieces, 100 grams were weighted and

mixed by a mixer, nominated by refinery in 250 ml beaker, solution was transferred to

250 ml volumetric flask then filled up with distilled water to the mark, 50 ml of filtrate

was pipette by burette and unloaded in the calibrated flask, 2 ml of the starch were

26

added and then titrated against the iodine solution to endpoint (dark blue color), the

experiment was repeated three times.

2.4.4: measurement of Vitamin C

Vitamin C concentration was measured using the titration method as shown in the

following equations:

I2+2e'—2T (1)

C6H80 6 —»C6H606+2H++2e- (2)

l2+C6H80 6-£ r + C6H60 6+ 2H+ (3)

From equation (3), the I2. C6H6O6 ratio was found to be 1:1

Mox V oX/X=MreV re/Y

Where:

Mox: morality of Iodine Vox: Volume of

Iodine

X: number of Iodine mole Mrc: Ascorbic Acid

Vre: Volume of Ascorbic Acid Y: number of Ascorbic Acid mole

The table (3.1) shows vitamin Contents in control and irradiated onions. Fresh onions contained 98 mg of vitamin C. Without treatment the control sample started germination and putrefaction after approximately three month. On the other hand, the shelf life of the treated onion was extended regardless of the radiation dose, Also the percentage of .vitamin C decreased according to the dose of radiation

The concentration of Vitamin C was calculated using the titration method

Equation of calculation

Weight = no of moles from the titration * molecular weight

The concentration percentages of vitamin C in the irradiated onions (different doses) as

well as the control group that have not been irradiated were presented in table 3.1

27

CHAPTER THREE

R esults and Discussion

3.1: Effect of storage on germination and rotting:

3.1.1: Control group:

The control group was stored for three months to study the germination and rotting

according to the storage period. Figure 3.1 shows images o f onion samples o f the

control group; Figures 3.1a and 3.1b show onion germination while figures 3.1c and

3 .Id show onion rotting. Both, germination and rotting appeared on the third week of

storage.

Fig 3.1 Images o f onions showing storage effect on the control (un-irradiated) samples:

(a) and (b) show onion germination while (c) and (d) show onion rotting28

3 .1 .2 : T h e i r r a d i a t e d g r o u p :

T h e irrad ia ted o n io n s w e re s to red u n d er th e sam e c o n d itio n s : th e re w as neither

g e rm in a tio n n o r ro o tin g a f te r th re e m o n th s o f s to ra g e fo r th e d iffe ren t doses ol

irrad ia tio n . H en ce , it is m o st like ly th a t th e irrad ia tio n p re v e n ts g e rm in a tio n and rotting

fo r a t least th re e m o n th s a s sh o w n in fig u res 3 .2 a and 3 .2 b .

% j \ r♦ ♦ ♦ «

tV

K* * ♦

♦♦ ♦ ♦ % ♦

• • • • !r fnr V * ■

" v

*

• •

%

p . ’.4 h «**?•©,

* * • * *

♦♦ ♦ ♦♦ ♦ ♦A V* * t

%i

.3--* *•

. ( •

♦ + \

« r .* jy -. . r

• • ♦ ♦ ♦ + ♦ ♦ + + ♦

e & 'a jv K if* : . s c . ,.*5 t j W 'T '.-A vt»* -*r-t* . - * - _ £ y . a . 1

-\H* i j . : v :.* -♦ ^ *

*

V.

* * V ♦ « •

: . . * . v . ' - r . t

. . . _ -*v. - t r * •. - *•.. a r fc i-r« C x « * rs 5 j:

♦ %«*♦ *4 ♦ ♦ «

F7g 3.2a: An image of onion sample irradiated with a dose of 0.5 kGy and stored forthree months showing no effects of germination or rotting.

29

2

*t

Fig 3.2b: An image o f onion sample irradiated with a dose o f 0.1 kGy and storedfor

three months showing no effects of germination or rotting. .

30

3 .2 : C o n c e n t r a t io n s o f V ita m in C :

V ita m in C c o n ce n tra tio n s w e re fo u n d to b e in a rev e rse re la tio n sh ip w ith th e rad ia tio n

d o s e as sh o w n in ta b le 3.1 an d f ig u re 3 .3 . T he h ig h e s t c o n c e n tra tio n o f v itam in C w as

fo u n d in th e c o n tro l g ro u p ( 9 8 .3 6 m g o f th e v itam in in e ac h 100 g m o f th e o n io n ) w h ile

th e lo w es t c o n c e n tra tio n w a s fo u n d in th e sam p le g ro u p irrad ia ted w ith th e h ig h e s t d o se

: 0 .5 k G y (1 4 .4 4 m g o f th e v ita m in in e ac h 100 g m o f th e o n io n ).

T ab le 3 .1 : T h e a v e ra g e v o lu m e s o f titra tio n fo r th e v a r io u s sam p le g ro u p s an d the

c o rre sp o n d in g C o n c e n tra tio n o f V itam in C

S a m p le 1 T i t r a t e v o lu m e 1 V ita m in C C o n c e n t r a t io n in

M l(m g/lO O g)

C o n tro l- sam p le 2 .45 9 8 .3 6

g ro u p

G ro u p 1 (0.1 k G y) 1.2 3 0 .53

G ro u p 2 (0 .2 kG y) 1.0 2 0 .4 5

G ro u p 3 (0 .3 k G y) 0.8 1 8 .3

G ro u p 4 (0 .4 kG y) 0 .8 16.4

G ro u p 5 (0 .5 k G y) 0 .9 14.44

31

Vita

min

C c

once

ntra

tion

mg/

100g

m

F ig u re 3 .3 sh o w s th e c h a n g e o f v itam in C c o n c e n tra tio n o f th e irrad ia ted o n io n sam p le s

w ith th e d o se . It is o b v io u s th a t th e re is an in v erse re la tio n b e tw ee n d o se s an d v itam in C

c o n c e n tra tio n i.e. a s d o se in c rea se s v itam in C c o n c e n tra tio n d e c re a se s .

Dose in kGy

Fig 3.3: Change of vitamin C concentration o f the irradiated onion samples as

irradiation dose increases.

32

3.3: Discussion

In o u r s tu d y th e irrad ia tio n o f o n io n d ec rea sed th e c o n c e n tra tio n o f v itam in C . H o w ev er.

G h o d s e t a l re p o r te d im m ed ia te lo ss in v itam in C p ro p o rtio n a l to th e d o se o f io n iz in g

rad ia tio n .

L e w is an d M a th u r re p o rte d th a t th e re w a s no s ig n ific an t d iffe ren c e im m e d ia te ly a f te r

irrad ia tio n o f b u lb s .

In th e p re s e n t s tu d y , it h a s b een n o ticed th a t th e c o n tro l g ro u p (n o n irrad ia ted sam p les)

is d e te r io ra te d o r g ro w n w h ile a ll irrad ia ted sam p les w e re n o t. T h is in d ica te s th a t, th e

ir ra d ia tio n is a n e ffec tiv e to o l a g a in s t d e te rio ra tio n .

V ita m in C h a s b e e n q u an tif ied a f te r th re e m o n th s o f s to rag e fo r all sam p les ; th e resu lts

sh o w e d th a t, th e v itam in C c o n c e n tra tio n is d ec rea sed w ith in crease th e ra d ia tio n d o se .

T h e m o s t a p p ro p ria te d o se a t w h ich th e o n io n sam p les w e re fo u n d to have h ig h v itam in

C c o n te n t an d s till n o t d e te rio ra te d is 0.1 k G y . T h e re s t o f th e irrad ia ted g ro u p s sh o w ed

s ta b ility in th e ir sh ap es b u t w ith lo w er v ita m in C co n ten t.

33

CHAPTER FOUR

Conclusion and Recommendations

4.1: Conclusion

G am m a rad ia tio n c a u se s a n o tab le d am ag e on v itam in C o f fresh o n io n b u lb s b u t th e

ch an g e s w h ic h o c c u rre d d u rin g s to ra g e o f irrad ia ted b u lb s w ere s im ila r to th o se o f the

n o n irra d ia te d b u lb s a t b o th tem p era tu re s : 18°C a n d 4°C . It seem s th a t s tab ility an d

e v o lu tio n o f th e v itam in C d u rin g s to rag e in o n io n s d o es n o t d ep en d o n th e tre a tm e n t

a n d /o r s to ra g e co n d itio n s b u t o n o th e r fac to rs such as; re a c tio n k in e tic s and w a te r

a c tiv ity .

G a m m a irra d ia tio n is v e ry e ffec tiv e in in h ib itin g th e m ic ro b ia l g ro w th an d g e rm in a tio n .

I t w a s fo u n d th a t th e m o st a p p ro p ria te d o se a t w h ich th e o n io n sam p les w ere fo u n d to

h a v e h ig h v ita m in C co n te n t an d still n o t d e te rio ra ted is 0.1 kG v .

4.2: Recommendations:

M o re re se a rch is re co m m en d ed to d e tec t th e n u trien ts c o n te n ts o f th e irrad ia ted o n io n to

se le c t th e b e s t d o se th a t ex ten d s th e sh e lf-life o f th e o n io n w ith o u t big loss in n u tritio n a l

v a lu e o f th e o n io n .

M ic ro b io lo g ic a l s tu d ies sh o u ld be co n d u c ted on th e irrad ia ted o n io n s to s tu d y th e

su itab le d o se fo r p rev en tin g ro ttin g .

34

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