Effect of Gamma Irradiation on Cell Wall Polysaccharide Model Systems
Tropical fruits such as banana, pspaya, pinoappb, and mango
an important aommoraial fruits which servo not only as tablo
fruits, but alw as raw matorials in u w m l
fruit processing
industries for produaing among othorr noatar, juiao, jam, and
fruit Claks.
Truits p r o d w t h n ia Indoaosk inamasad from 4.53 million
tons in 1989 to 5.62 million tons in 1993. I t was also nportod
t h a t produativity of tropical fruits incroasod ltom 7.53 t o n l h a in
1988 to 12.13 too/ha in 1993 (Winasno, 1991). I U a n p is on0 of
t h e oxotia tropbaal fruits which could also k promotod as non-oil
aommodities to inaroaso aounlry dovisa. Export v a l w of mango
h m Indonosir to various importing a o u n t r h s in tho pllprld
s h m d a n inorrase from 579 thousand U8D in 1990
to 85'7
thowand UdlD in 1992 (Punwadaria et aL, 1998).
I t has k e n well understood t h a t changos in b u i t t o x t u n ,
flavour and a p p a r a n a o after harvesting k a o m o w r y important
quality standard critoria for evaluation of fresh fruits in tho
market. Fruits a m commonly hannstod a t a maturo stagr after
which softening takes place inside the fruit and continues even
after harumsting if the conditions are suitable.
sa
Textural changes induced by tissue 8ofYening of fruit i
result of the break down of cell -11
polymer net work either due
to biochemical change8 or actions of hydrolytic endogenow
ensymes which convert the insoluble constituent8 into soluble
and edible ones (Biale, 1960; Pilnilc and Vosagon, 1970; Bulme,
1970); Pilnik and Voragen, 1984, and Pilnik, 1984) but l e u
intensive studies on irradiated cell wall polysaccharides induce
depolymmrhation (Diohl st rl., 1978; and Kon, 1978). Lazan and
Ali (1993)reported that a n extensive softening in mango appear8
to be related to more extensive modification of the wall pectins.
BXost fruits contain large amount of water, as well as solutes
which could be readily utilized by microorganisms, insects, and
other pests. This situation makes the fruits prone to spoilage,
specially under storage condition which prevail in most tropical
countries. Furthermore, such economically important tropical
fruits normally have a limited postharvest life of about 2 weeks.
There are some technological approaches which might only
be effective in improving the shelf-life of particular fruits, i.e.,
.
s t o r a p in a modified atmomphere (MA) /control atmosphere
storage (CAS), or in a modified atmosphere pachaging (MAP)
(Punwadaria, 1995),and coating (IYLAC) treatments, storage at low
temperature
, hot
water dipping, and dipping in calcium salt
a8
reported by Jasim el al. (1968). Ionlting radiation using gamma
rays, X-ny. and h i g h a n e r w electron beams can also be used for
such purposes (Batin, 1993).
Gamma irradiation
a8 a
preservation technique 1.widely w e d
to extend the storaw life of some fruit. and vegetables for
dimerent purposes such as sprout inhibition of bulb. and tubers
p t s u y a m a and Umeda, 1983), delaying post hwvest
ripening
and senescence of fruits (Akamine and Xoy, 1983), insect
disinfestation for quarantine purposes (Tilton and Burditt, Jr.,
1983), and reducing microbial loads by single treatment of low
Irradiation dose or in combination with hot-water dipping (May,
1983).
Purar*nto and Maha (1986) reported that gamma irradiation
induced t i u u n sofbning in fresh mango was due to the
wlubilltation of protopactin into ~ l u b l epectin. %ince inwluble
pectic materials maintain cellular adhesion in plant tissues, the
destruction of protopectin by ionizing radiation appears to be the
cause of radiation induced softening of fresh fruit. which is
mostly considered as a n advetme effect. Alteration of ptobpectin
in the intercellular areas allows the tissue cell. to separate
resulting in loosing of texture.
Adverse physicochembal changes occuring in irradiated
fresh fruits and vegwtabbs using high radiation doses are surface
and flesh darkening as a result of enzymatic browning, and
immediate softening due to the degradation of long chain
polysaaahuidos p u r r a y , 1990; Clarke, 1961;Yasia rt d.,1987;
and ajoberg, 1987).The extent of changes in different structures
and chemical constituents of irradiated fruits and vegetables
depends o n several factors such as radiosensitivity of each
individual product, temperature of the environment and the
absorbed h a d t a t i o n dose through the materials (Bomogyi and
Romany, 1964).
The ionizing gamma radiation degrades a t b a s t the m a o r
polymers in the primary cell wall of plant materialm (pectic
substances, hemicelluloses and cellulose), (Clarke, 1968; and
Maxi* and Sommer, 1968)although the mechanism has not been
clearly explained (IYlc. Ardb and Hehemias, 1966; Rouse and
Denisson, 1968;Eolli-Donini and t3tronaiwl0, 1969; Eahandi et
uf., 1970;and Diohl r t rf., 1978).
The d e w a t i o n of the cell-wall polysaccharides such as in
pectin induced by gamma irradiation is duo to hydrolysis by
random attack of the glycosidic linkages within the polymers
leading to smaller units having the same general structure
(Korkr et rl., 1956; Ma.Ardb et uf., 1966; Maxi0 and &mmer,
1968;g i n n e r and Kerb+ 1960; and Clarke et 81, 1961)causod
by free radicals generated by irradiation (Murray, 1990).Likewise
in the immediate softening process is not attributable to
accelerate enrcymatic hydrolysis of the m l l component8 F
e
and mmmer, 1968).Irradiation of pectic substances derived from
apple probably resulting in random hydrolytic cleavage of the
galactutonan backbone yielding fragments of lower molecular
weight (@joberg,1987).
Fresh fruits such as mango is normally exposed to irradiation
as aonstituontm aooxisting mqjor and minor aomponents of
complex fruit systems. Rowaver, it is extremely difficult to
eluaidate the degradation mechanism occurs in irradiated mango
fruit induced softening in d t u Pattern and composition of the
radiolytic
products
polysaccharides
are
and
degree
strongly
of
sensitivity
depended
on
the
of
these
particular
environments, e.g., type of cell wall, physical orientation of the
plysaccharides molecules, and other artifacts.
It is estimated that some changes observed in a n irradiated
isolated cell wall polysaccharides as individual model systems
might also give similar pattern when the irradiated complex fruit
systems containing such plysaccharides h observed.
Different speculation on the study of depradation mechanism
of Irradiated fruits and w g e t a b k s proposed t h a t free radical
species are probably responsible for pectin depolymeritation (Kon,
1978; and Urbain, 1986), and the limit degradation of pectins
caused by radiation was explained by competitive 0%.scavengers
present in apples t8joberg, 1987).
Due to the complexicity in structure of mango fruit
, it
h
difficult to obtain an explicit data on the degradation mechanism
of the irradiated fruit induced softening. The studies were aimed
to obtain some information on degradation products of irradiation
of isolated cell wall polysaccharides
in
mango
fruit, and
irradiated polyraccharides model systems, The effect of radiation
on these various polysaccharides is estimated by direct and
indirect effect reactions through different .ample preparations In
order to elucidate t h e k degradation mechanism.
HYPOTHESIS
Immediate softening in gmmma irradiated mango fruit is
mainly due to hydrolysis process occur in the primary cell wall
polysaccharides vla a specific pattern through preferable attacked
of free rudbalr to the cell m l l components, resulting new
h g m e n h with lower molecular weight.
A. Irradiation
or F r e s h F r u i t s md Vemtables
Ionking radiation applied as a cold preservation methods
-ems
to be a n alternative for fruits and vegetables in order to
reduce their l o u e s and to improve their quality. a m p l e
conditions (degree of ripening, water content, uniformity in
she, etc,), and radiation parameters (dose, presence and
absence of oxygen, temperature, etc.) are of important
-tor8
in the irradiation of fruits and vegetables.
The chemical mechanisms of depolymerizrition reaction in
irradiated polyoaccharides can be invastis[ated in two my.,
i,e., by evaluating the structure of the radiation induced-
radicals through interpretation of different physical methods
suah as Electron 19pinIParamagnetic Resonance (EElR) spectra,
and by a n exact structure analysl. of the radb1y.l. products.
Irradiation
may
induce
severe
depolymeriaatbn
of
macromolecules resulting in structure loss which must be
considered aa a n adverse effect. Various studies on the enect
of (Lamma irradiation on physicochemical changes of fruits
and vegetabbs have h e n reported by many investi(Lators.
Studies on irradiated
fresh mango both
using single
irradiation or hot water dipping, and combination treatment
b e m e n irradiation a t 0.78 kOy and hot water dipping for I
min. at SSOC revealed that the combination treatment was the
best in prolonging the shelf-life of fresh mango for 3 weeks a t
31°C and then another 1 .crmek at room tempomtun (29-30%)
to allow the fruits into normal riponing without affecting w m e
chemical
characteristics,
and
the
organoleptic
quality
(Purrvanto and Maha, 1987). Other study has revealed, that
irradiated mango with doses up to 0.75 kOy did not influence
significantly to the amount of glucose, sucrose, fruotore and
total sugars but aftor storag. the irradiated mango showrd
more changuts of those mlues than in the control (aingh,
1990). It is also reporkd that immediakly aftor irradiation the
wmkr solubk protin MIW e f fresh auam@a
u high as in
other fruits but during storaw its rate of increaso was lower in
the untroakd samples. A contrary result was obtained in
insoluble poctin lfaction aftor storap. The irradiated k c t i o n
d e c r e a n d but the rate of decrease was Iomhr than in the
unirmdhted mango.
Btudy on radiolytic changes occuring in man-
revealed
t h a t irradiation of carbohydrate component produces carbonyl
compounds. Using models of carbohydrate derived from manthrough different preparations, i.e., solution, juice, pulp and
whole fresh mango, the finding result showed that the
individual component of the Itradiated food was mutually
protected. Other experimental work on sugar solution used as
model system indicated that radiolytk products of D-arabinohe--2
ulose (D-glucmne) was present, but this type of
radiolytk products wtu not found in the irradiated fiesh
mango (Delincee, 1989).
Physicothemical analyses of irradiated frozen de-seeded
kiwi pulp during storage at -18 OC has been i n v r a t i a t e d (Lodga
eC d.,
1988).The result revealed t h a t soluble solid fraction, pH
and titratabk auidity did not ahange aignifioantly during
storago up to 7 -aka
and o w n up to 6 months froesing
storage the total poutin remained eonstant a t values of 0.S0.6%. Iioward and Buesuher (1989) conaluded that softening
in uuuumber piukles caused by irradiation waa primarily
auociated with t h e changes in pectic substances and w m e
changes of galactom in the cell-wall.
8omoWi and Romany (1964) reported t h a t irradiated pear8
and p a c h o s a t 18.30C with t h e doses ranging &om 3.6 up to 9
kQy
tho
a clear indication on irradiation induced .oftoning of
fruit
tbuo.
Protopoctin
was
signifbaantly
redwod
immediately after gamma irradiation, then incnaaed aftor 4
day storago in comparison to tho untroakd samples. Poctin
v a l w incroasod twico of t h a t in t h e aontrol but poctate only
s h m d a slight incnaso. After storago unirradiakd pars wore
softer than the irradiated ones at moderat. d o u , but at a
higher radiation d o u more t i u u e damage was obsonnd than
in t h e irradiakd aamplos. Boftoning of pram and p a a h o s wore
loas in irradiation of samplms under IT2 atmoaphoro. Other
result waa obtained from irradiated pectin of 1%concentration
in solute state either under lT2 or oxygen atmosphere. Under
t h o w two atmospheric oonditions during irradiation, reducing
powar and viscosity were decreasing immediately. They '
postulated t h a t a n
increase in methylestemse activity may
alw contribute to the initial pectin degradation in irradiated
fruits. Similar investiwtor also studied on t h e behavior of
irradiated of fresh apple. It is reported t h a t it would not be
pouible to differen tiate between unirradiated and irradiated
samples on the basis of changes in molecular weight of pectin.
The degree of esterification, however, was decreased by
irradiation
treatment
at
low
doses.
Furthermore
they
concluded that t h e depolymerizatbn in the molecular chains
could give a n effect on the vfscority of apple pectin, and the
activity of p e c t i n m e t h y l e s t e r observed in f r d b increased
directly after irradiation but the activity tended to decrease
after 4 days of sorage.
Degradation of cellulose embedded in the apple fruit cell-11
by irradiation would probably contribute to a loss of
texture but i t seems unlikely that this compound is the
primary structural components being dewadad (Foamr C at.,
1980).In irradiated apple the threshold dose for degradation of
pectin and cellulose
mms similar to softening of the tissue
(Chute, 1968).
Immediate increased in water soluble pectin was found in
irradiated fresh apple but the pectic substances in irradiated
samples had similarity to those control after storagu (Clarke,
1968). ajoberg (1987) reported that pectin content and
molecular
weight of
fresh
apple
were
not
signifkantiy
influenced by irradiation with doses up to 10 kGy.
Clarke (1961)mentioned t h a t texture is not a n important
factor in t h e quality justification of irradiated fresh apple with
the d a m up to 1 kQy and under storage condition at 3OC. I t is,
however, during storago total pectic substances in t h e t h s h
apple
decnased
significantly
in
both
unirradiatod
and
irradiated samples. The same result was obtained in t h e
h a d i a t e d pectin fractions at the same d o u .
A study o n irradiated carrot has k e n rrp0ct.d
by Echandi
et uf,(1970).
Irradiation with the dose of 10 kGy did not alter
total exttaatable pectin nor on total calcium contont. Other
result on t h e irradiated aarrot as conducted by Urnail et al.
(1977)rewaled t h a t water wluble fkaction and anhydrouronic
acrid aontent showed a n inoreased upon irradiation while
ammonium oxalate h e t i o n was both increase aftar irradiation
and during storaga, but deoreand In HCl extrwtable fraotion.
Reduction on t h e degree of esterifkation was duo to the
aativation of endogonous poctin methyl-esteraw
irradiated
nfiigorated
cucumber pickles and
(PME) in
the pactin
wlubillaation was the most notable response to irradiation
(Howard and Buescher, 1989). Irradiated pickles at 1 kGy
showed a reduction of alkali and non extractable pectin but a
marked increase in water soluble and chelating agent wluble
pectin.
The activities of PO, PME, and Cellulase in irradiated
m a t u n groan tomato wcu domanstrated (Yasia et ad, 1987).
Using a viscosity measurement seemed t h a t the activities of
PQ. and PXE were not influenced directly after radiation up to
8 kOy. After 3 days storage PE activity showed a n increase a t
the dose of 0.8 kOy, as occured In unirndiated sample, but the
activity of PC) in the irradiated samples higher than 1kOy was
suppreued
during storage.
Cellulase
activity showed
a
significant increase dlrectly after irradiation but i t was not
affected by stoiap time. Result on irradiated tomato revealed
t h a t a reduction in the ratio of protopectin to total pectin, and
in t h e polyuronides extracted
from all
fractions,
were
indicated but hexamethaphosphate wluble pectin s h m d an
increase while water wluble pectin remained constant.
Other authors reported that in tomatoes and potatoes pectic
substances
are
responsible
for
the
changes
in
total
polysaccharides due to irradiation or stwagxi time before
irradiation pea* and Jona, 1980).
B. &lmbilib of Primup Cell Wall Indmcrd 8oISaninp in
Prrsh F r u i t s
Primary cell wall polysaccharides i. the major constituent
which is responsible for the integrity of plant tissue due to
their function as cellular adhesion.
The three major components of cell-wall material in
fruits and vegetables are pectic substances, hemicellulose
and
cellulose.
The
pectin
as
a
complex cell wall
polysaccharides member has a backbone which concists of
1 regions, i.e., "smooth" regions which mainly contain of a-
D-1,4- mlao turonan, and ramified regions introdwed
as
interupted sideohains which are attached to a-L-(1---->a)linked
rhamnwe
residues present
in
the
backbone
interupting the galacturonosyl residues (Schols, 1995).
Pectic substances is alw responsible for the integrity and
aohosiwnou of plant t i u w s (Davidok rt af., 1990).
Bolubilbtion of pectic substances related to the softaning
proceu in some tropical fruits such as bananas, manw,
oran*
and apple has k e n studied by Nwanokoarri rt rl
(1994). The result revealed that b comparison to the other
selected tropical fruits mango showed the highest pectin
(1.8%) and
methovl
contenta
and
the
purity
of
anhydromlacturonic acid content of 8 1 . 6 O h .
8 k h n i n g process in mature fruits is due to the b r e a k d m of
the cell wall structure in the fruit pulp by the activities of
endogenous ensymes resulting in conversion of inwluble
wall-bound protopectin of high molecular w i g h t into m t e r
soluble pectin. From many studies it has become c h a r that
pectin is not a homopolysaccharides but that neutral s u m
an part of the p a t i n molecules (Vrie* et .I.,
1982).
Pectic substance8 as non-starch polysaccharides and its
endogenous enrymes play the main impoatant role in the
proceu of wftening and in their contribution to the
metabolic pool of the cell (Pllnik and Voragen in Hulme,
1970; and Pilnik and Rombouts, 1981). Polygalacturonam
-
(Pa),the en=yme responsible for d e m i n g the (I----*)
linked ~ l a c t u r o n i cacid residues, and P-galactosidase are cell
mll hydrolases who playa a n important role in tropical fruit
softening. f3-galactoaidase may function as galactanam and
may act as a pectin debranching enayme (Latan and Ali,
.
1993) Other reseachers also reported that in addition to
pectinases the aativities of other enzymes such as cellularas
and b e m b e l l u l a u s in the cell wall might contribute to the
softening pooceu of fresh fruits. Further study on the
reducing viscocity caused by ensymatk degradation was
obwrvrd by b n e n a t h et rl. (1987).The study showmd that a
commercial enzyme, Ultrazym 100, was the most efGectiva
enzyme in reducing the viscosity of mango pulp.
2. BIuebuIlc~lcbmnges e t e d b RPlt so-ing
u d
rIpnb#
&ftening,
increasing svmetneu, aroma, and colour
changes are among the most striking phenomena related to
ripening of fruits. The changes might include respiration
rate, metabolism pathwmys, and individual constituents.
Polysaccharldes a# important carbohydrate constituents
of many foodstuf?~, are complex
macromolecules as
condensation polymer of high molecular weight compcming
from single monosaccharide units. One unit b connected to
t h e adjacent monosaccharide unit by a glycosidic linkage,
such as at C-1 and C 4 position, as oxygen bridge by the
elimination of water between the hemiacetal hydrorryl
group of one unit and availabb hydroxyl moup of another
(Aspinall, 1983; Pilnib, 1984; Belitz and Cirosch, 1987, and
Davidok
oC rl., 1990).
Fleshy fruits,
mostly its edible portion,
composes
mainly of parcrnchyme cells and larw intercellular air
spaces (King J r , and Bolin, 1989). Parenchyme cell. in the
pulp mainly contain primary cell walls defined as nonstarch polyraccharidea, The polyseccharides consisting predominantly by pectic substances which responsible for
tissue wftening, hemicelluloses, and cellulose microfibril..
The latter are firmly fixed in a matrix of pectic substances
and hemicellulosic polysaccharides {Aspinall, 1983; and
John and Dey, 1986). It k obvious that textural changes
induced softening of the fleshy fruits during ripening is
essentially caused by the conversion of high molecular
weight insoluble wall-bound protopectin into water soluble
pectin (Hulme, 1970; PilnUr and Rombouts, 1981; Davideh
eta/., 1990; and Murray, 1990).
Biochemical changes in some fruit. such as apricot and
mango a t different ripening stage have been carried out by
Sharaf ef rl. (1989).
It was reported that the percentage and
total
soluble
carbohydrates
increased
gradually
by
increasing extent of ripening. A t in their immature stage,
glucose and sucrose content were highest in apricot and
man*,
respectively. In ripe and over ripe stages, fructose
war. t h e predominant sugar in aprlcot and whilst in mango
I. glucose. The minimum value of glucose and srylose were
reached at over ripe stage in apricot. In mango fructose
tended to increase while glucose and sucrose decreased
significantly.
The Phllltppine maage, Ceuabao variety, showed some
changes in both physical and chemical characteristics
during ripening. The most significant chemical changes
obaenmd a t ripening stage indicated a decrease in starch
content but increased significantly in total soluble solids. At
harvest time the pectin content was 0.37-0.83 mg percent
and. it increased significantly in the ripening stage about
twiae higher than that in unripe stagxi @Coraet
1979).
Other study on the physico chemical changes of kinnow
mandarine orange and pineapple juice concentrates during
storaw in diffmrrnt t a m p r a t u r e raportad by bandhu ot aL
(1985).They found that pectin content decreased during
storage and the losses increase with increasing temperature
in thou, mamples.
AIR spectrophotometry in certain circumstances can be
applied optimally to estimate total pectic substances, water
soluble and in.oluble fractions, and methoxyl content of
alcohol insoluble solid prepared from
fresh peach, apricot,
and apple with a better result in comparison to the use o t
ahemiaal analysis as carrhd out by Polesello et al. (1990).
It was a h reported by BImpton et rf. (1984) that a
quantitatiw determination on pectin content of w m e
tropical
fruits
using
Ca-pectate
method
gave
more
representative data than that of methanol precipitation.
Methoxyl content of fruits mostly increases with increasing
content of pectin.
el rl. (19953 roported that mango (16mngiRrs
#!hputra
jndlca var. grdong) can be classified in t o thok taste b a u d
on sucrose and malic acid concentration as determined by
t h e Rear Infra Red Diffuse Reflectance at the wavelength of
1900-1975 nm and calibrated by HPLC.
C. Cell WIll Polv.acchuides, and T h e i r Similar
Sarrm
Building Bloeka
Pectic substances are complex colloidal polymaecharid-
es
consisting
residues,
of
in which
a-D-l,4-linked
(~alacturonic acid'
the carboxylic acid
groups are
partially esterifled by methyl groups and partially or
completely neutmlhed by bases (Pilnik and Voragen in
Hulme, 1970). Degradation of pectic substances may
ybld
rhamnogalacturonans
branched /
(slightly
rhamnogalacturonan
I
and
and
highly
111,
homoqalacturonan,
arab-inan.,
galactans
and
arabinogalactans (Aspinall, 1983 ; Pilnik, 1984; and John
and
Dey,
1986). Pectic
substances
protopectia, pectinic acid, pectinate.,
may
include
pectin(s), pectater
and pectic acid (Pilnik, 1970;and Davidek el d.,1990).
Pectins are subdivided according to their degree of
esterification and designation of the percent of carborryl
groups esterifkid with methanol. Pectin with DE > 5 W
are high m e t h o w l pectins and pectins with DE <
5Wo
are classified as low methoxyl pectins. Molecular weight
distribution of commercial high methoxyl pectins h a s
k e n analymd by Brigand et a& (1990)and many other
investillaton
using
s
exclusion
chromatopphy
coupled with low angle laser light scattering method..
It
is found that the pectins have molecular weight in the
range of 150,000-280,000 Dalton (Da). The araban and
gahctan chains are present as free polysacchartdes
found in the lower molecular weight region. It seems
t h a t degree of esterification remains constant all along
the
molecular
weight
distribution.
Intermolecular
distribution of the degree of esterification is narrow but
neutral s u m and acetyl content were less stable.
Pectins can be extracted by a variety of mild extractants
such as alkali, hot water, ammonium oxalate solution,
we&
acid, and chalating agent (Vries et af., 5981;
ahalom et d,1983;Bartlay and Knee, 1982;Baig et a/.,
1982;Simpson et nl., 1984;Renard et nl., 1990;Braudo
et rl., 1992; and &hols,
hydrolysed
into
smaller
199s). Pectins c a n also be
fragments.
The
resulting
different types of saccharides depend upon the attacked
point of t h e molecular chain by various degmdative
ensymes such cu pectinames, pectin methylesterase, and
pectin lyase (Pilnik and Rombouh, 1981;John and Dey,
1986;and Sreenath et sl., 1987).
Pectate is particuIarly found in
between
t h e middle lamella
adjacent cells. The nativm pectin play8 a n
important role in the consistency of fruits and vegetables
and in t h e textural
changes during ripening, storage,
HemicelluIoses occur covalently linked to the pectic
material but noncovalently bonded to cellulose fibrils.
They are not extractable in cold water, but extractable
In alkali solution.
Hemicellulose in the primary cell wall composes of
thmm m 3 w groups of polysaccharidms, Lo., wlurs which
is
a
mqlor
component
in
hemicellulose
fractions,
consisting of backbone polymers of fb-l,4-linked Dwlopyranose chains. Hemicelluloses have a n e t negative
charge due to the presence of a -Dglucuronic acid
residues a t tho sfdo chain (Fry, 1988) ; mannrrr.gfucomannu as drrivativrs of 6-l,4,-linked manno-
pytanose and B-l,4-linked glucopymnose units with'
various portions of glucose and mannose; and the
grlrctans.rv~bogakct~ns,
highly branchmd dorivativos of
1,3and
1,6-linked D-galactopyranose
polymem
with
arabinose side chains (Aspinall, 1983; Pilnik,19S9; and
John and Dey,
1986). Qalactans,
arabinogalactans,
arabinans,
xylans
and
glucomannas
are
important
representatives of hemicellulose.
Cellulose is composed of long chains of
D-glucopyranose residues which
fibrils by hydrogen bonds. Aggre-tes
p-1, 4- linked
are held together in
of these fibrils are
the basis of the rigid structure of plant cell -11
and it
may associates with other polymers such as pectin,
hemicellulose, lignin and protein (Stevens, 1975).
A n o r g a n b d aggregates of cellulose molecules in plant
cell wall 1.reffered to as microfibrills. These microfibrills
are composed of crystalline and amorphous regions.
Cellulose is a linear polymer, consisting of D-glucose in
%1,4
linkage
(Aspinall,
1983; Beldman,
1986; and
Davidek ef al., 1990). Cellulose can be hydrolymd by
strong acid yielding only D-glucose. Partial hydroly.is,
however, produces the reducing diuacharide cellubiose
(Voragen et sl., 1983; and Murray, 1990). The individual
chains of glucan are held together in fibril. by hydrogen
bonds between the hydroxyl groups at C-6 and the
glyoolridic -gens
of the adjacent chains. A methylation
prooeu followed by hydrolysis s t e m of cellulose yields
only 3,3,6,-&is-methyl@uco.e
without
branch
point.
Cellulose is insoluble in water and it is not related
structurally to hemicellulose p o n e r , 1988; and Beldman,
1986).
The delgadation of cellulose is catalyzed by cellulases
acting a8 B - l , 4 - ~ ~ c a n ~ ~ c a n o h y d r W
o ll a
dm
~ a n , 1986)
resulting in cell-dbintegration of pericarp in t h e ripening
staw
of fruits but these encymes does not directly
contribute t o the ripening (John and Dey, 1986).
As individual polysaccharides alginates occcur in all
brown algae as a skeletal component of their cell walls.
Algae are extracted with alkalies and then precipitated
from t h e extract by acid or Ca- salts. Alginates are linear
copolymers consisting of the following structural units:
Mannuronb
acid-Mannutonic
acid;
Guluronic
GuIuronQ acid; and Mannuronic acid-Guluronic acid.
acid-
Alginates designated as alginic acid, a polymer similar
to cellulose (Stevens, 1975) is a heteropolyuwcharides
compoms of varying and alternating sequences building
blocks of 8-D-mannuronio acid and a -L guluronic acids,
joined by the I------linkages
.
The -(I--->4)
glycaaidk linkages between a -L-gnluronic
acid molecules leads to a d u a l orientation similar to
that in galacturonan and equally suitable for an egg box
type of chain association with ca-ions (Pilnlb, 1984). The
ratio of the two s u q a n (mannuronic/guluronlc acids) is
generally 1.S with some deviation
with source. The
gelling properties of alginah depending upon the amount
of guluronic acid in the main chain. Partial hydrolysis of
alginate
produces
some
fragments
whbh
consist
predominantly of mannuronic or guluronic acid, and also
fragments where the two uronic acid residues alternate in
1:l ratio.
Distribution of uronate residues in a l d n a t e chains is
related to alginate gelling properties. This statement mu
approved
after
using
an
experimental
model
of
enrichment of p-D-mannuronic acid and depletion of a -Lguluronic acid In solution fraction as conducted by
(iarnoarek and Qiarncarek (1993);and Btokke et rl. (1993).
Their report is basically concerned about the binding
strength
between low methoxyl pectin and alginate
mixed gel.
The differences between low methoxyl pectin and
alginate appear on their heat stability under vwbw pH
conditions. Under low pH and neubal conditions aldnates
are more heat stable.
Oat.
and Ledward (1990) have
studied the effect of heat on alginates. They revealed that
samples with high level of mannuronic acid residue were
far less stable against heat.
Alginates are water soluble in the form of alkali,
magnesium, amonia or amine salt..
The viscosity of
alginate solution is influenced by molecular weight and
the counter ion of the salt. In the absence of di and
trivalent cation or in the presence of a chelating agent,
the viscosity is low. However, with a rise in multivalent
oation levels, e.g., calcium, there is a parallel rise in
viscosity. Freezing and thawing of Na-aldnate wlution
.
containing Ca++ ions can increase thelr viscosity. The
viscosity k unaffected within a pH range of 4.5-10. It rise8
a t a pH below 4.5, reaching a maximum a t pH 3-3.5 (Belitz
and QK#Ch, 1987).
Propybne glycol alginate is derivatisation of alginate
through a reaction of mannuronic acid with propylene
oxyde via hidrown abstraction a t C-6 of mannuronic acid
(Pilnik and Voragan, 1984).It is wlubIe down to pH 2 and,
in the presence of Ca++ ions it will form soft, elastic, l e u
brittle and syneresis-free gels (Gray and Philp, 1991).
k a b i n a n s as a member of dietary fibres
play a n
important role in fruits processing. Arabinms are also
found abundant in the biomass of c r o p as waste products.
Thi. type of homopolysaccharides present. as pentoran
sugars, mainly composed of a-L-arabinohuanoryl residues.
They are generally arranp(ed in (1---->I)
linked chains with
varying numbers of residues substituted with other a-Larabinofuranosides a t the 0-2 and/or 0-3position (Beldman
at el., 1991;Pilnilc and Voragen, 1984).
k a b i n a n s are present in the plant tissues auociated
with
pectic
arabinoxylans,
substances
as
arabinogalactans
homoglycans
and
such
aa
arabinogalactan-
proteins. It can be isolated by preferential extraction with
boiling ethanol or with lime-mter in which associated
pectic acid will be transfarmed into insolubla Ca-salt.
Highly branched arabinan can be syntheslted by the
isolation of 2,3,5,-tri, 2,3,-di, and 2-0-methyl-Larabinose
on hydrolysis of the methylated arabinans. This type of
polysacchatides have
w m e difficulties in the isolation
process from other sugar residues, particularly if the
arabinans attach to the pectin molecules. Since pectin I.
unstable a e i n s t alkaline reagent, the purity of arabinan
obtained from such iwlation is doubtful. Other method in
iwlation of arabinan under non depadative condition haa
still to be developed. Arabinan degrading enzymes become
an essential method to elucidate structural characteristics
of arabinan (8.ldman et aJ., 1991). Iiowavor, in sugar boot
.
polysaccharides the presence of arablnans linkages in
aucrciation with pectic s u b t a n c e s can still be isolated of a
linear
1--->5 linked
ambinan
using
an
a-Larabino-
huanosidaae (Aapinall, 1983). In the concentrated apple
juice haes become t h e mqfor problem due to the effect of
high amounts of ambinan side chains. A side-branched
araban, wuter soluble amban, links at 1,3 and 1,3 position
of amban straight chains with branched points a t C1 and
C5 as glycosidic linhgam. An ondo
1,s
-maban-
is able
to break doam the side chains of arabinan resulting in a
clear solution in the concentrated apple juice (PPnUr and
Voragen, 19%
and Voragen st al., 1987).
Ioalrciug (y ) radiation, is found in the range of wave length
between 1014 and 10-10 cm, as non-particle energy radiation
whioh c a w s direct and indirect i o n h t i o n or excitation of a
permanent qar (O'Donnell and Bangster, 1970). I t has the
highest emition energy level among othera and has dimerent
ability in penetrating materials compare to a rays and $ rays.
k a cold process, gamma irradiation seems to be a promising
tachnique to substitute or to improve the existing preservation
methods. This technique can be utilized for decontamination
to reduce the food losses or as quarantine treatment of
agricultural products to protect horticultural export against
chemical fumigants by improving the environment and
hygienic quality. However, among the important benefits of
irradiation, destruction or reduction of pathogenic bacteria in
foodstuffs is of moat importance. In the long term, food
irradiation may be seen as a part of the food processing
technology to provide food which stays fresher for a longer
period.
In food irradiation, which is merely a physical process, the
ionking energy only interacts with the outer orbital electron
of t h e atoms and it does not interact with the nucleus.
Consequently, there is no radioactivity induced
in
the
irradiated foodstuffs. The changes only occur chemically in
the
food wlthout
any
nuclear
changer
(Becker,
1983).
Irradiation does not cook the foodstuffs since no heat is
produced and the food retains its initial characteristics (Satin,
1993).It mlut also be pointed out that such reactions do not
lead to the formation of aromatic ringa, condensation of
aromatic rings, and formation of heterocyclic rings (aimic,
1983;and Diehl, 1990).
Since
nutritional adequacy
and wholesomeness
of
irradiated foods are important considerations in ascertaining
feasiblity of irradiation technolow, changes in chemical
components or nutritional significance have received much
attention. However, it has been jpnerally observed that
complex foodrtuffs are leu stuceptible to irradiation induced
c h a n w s h chemical structure of the components ru expected
from studies w i n g pure
Delincee,
1989). The
compounds (Adam, 1983; and
reaction
mechanisms occuring in
irradiated foods must be fully undentood if consequences of
irradiation are to be predicted.
Radiation wurces and dosimetry technique are playing
important roles in food irradiation. There are 3 types of
radiation souroes w h k b e r n k wed, 1.0.~radionuolido8 m d
particle accelerators. Bome radionuclides emit a form of
electromagnetic wave energy called photon. Co-60or Cs- 137
provides photon, rays, with the energy sufficient for good
penetration but unable to induce radioactivity in the irradiated
foodstufls. Particle accelerators provide a beam of high energy
as charge particles. Electron beams are used for radiation
processing since the energy is limitted t o 10 million electron
volts WeV) avoiding traces of radioactivity which may be
induced at higher energy.
Several parameters need to be considered in determining
the Co-60 radiation dose received by a package, i.e., amount of
Ca-60 in the source plaque, distance between source and
&radiated
products,
the
exposure
time,
and
radiation
absorption within the packages which increases as t h e package
density and the package 11 width increase. It i. important to
minimize dose variation within each package when food is
irradiated. Such variation are inevitable becaulre the distance
to the eource varies from point to point within the package
and becauw of radiation aboorption within the package. The
ratio of maximum to minimum dose (Dm,/Dmin),
as uniformity
(U),
designated
must be kept within masonabla limits,
usually Ieu than 2. Dmin must be sullicient to achieve the
objectiw of the process. The,,D
must bo within the
toleranee of the commodity. DmMfDmin can be minimbed
partly by moving the packages around the source during the
irradiation so that position
of high
and
d
doso
is
interchanged. The energy absorbed per unit of time is called
the dosa rate.
The new unit of radiation,
Gray,
as the amount of
irradiation e n e r w that a food absorb. L measured in units. It
h noted that 1 Gray = 1 joule per-kg of abaorbed energy = 100
fads (the old radiation unit) while a t medium irradiation dose,
10 kQy, the energy is equaI to to the amount of energy
required to raise the temperature of water by 2.90C.
It is considered that gamma Irradiation is applied for
different purpc#es for premedng foodstuffm, e.g., inhibition of
w e t a b l e s sprouting is (up to 0.2 kQy), insect diminfestation
(0.2-1.0 kOy), pararrite elimination (up to 6 kGy), microbial load
reduction (O.Ob-5 IrGyI, killing of non sporing pathogens
(3-10kGy), and absolute sterilization (80 key) (Diehl, 1990;
and a t i n , 1993).
,
E. Irradiation of Carbohydrate a n d it. Derivrtives
The most important effects of ionizing
radiation on
macromolecubs are degradation and croru linking.
The
degradation process 1.merely a hydrolysis of glycosidic bounds
and
other
weak
linkages
yielding
simpler
compounds
(Charlesby, 1960; Charlesby, 1987; and aonntag, 19911.
Fundamental study on radiation chemistry of different
faodstufi is identification and measurement of various species
formed during and after irradiation. Chemistry of the s p e c k s
in the reactions with different compounds covered reaction
kinetics, mechanisms, intermediate and stable end products
(O'Donnell
and
Sangster,
1970; and
Taub,
1983). An
observation on the rate constant of transient species can be
done using puke radiolysis, EBR p a n g et
dl.,
1987) and in
combination with competition kinetics (Raff'i and Agnel, 1983).
In principle
can detect the spin unpair electrons even in a
very low concentration and the amplitude of ELSR signal is
approximately proportional
to the
total number
of the
electrons in the sample (Wertr: and Bolton, 1972).Thus, E8R 1.
,
obviously useful for detecting and characterlcing free radicals
formed in the irradiated samples.
The radiation chemistry of carbohydrate can be determined
by three m 4 o r factors, i.e., the radiation chemistry of the
simple sugar sub-units; the effect of subatltuents on the sub
units; and the fate of glycosidic liqkage p i e h l , 1990). The
splitting of glycoddic linkages in carbohydrate molecules is
not only caused by gamma irradiation but also by entymes
action or by chemical means with some differences in the
results. These differences depend on the degree of selectivity
among bond cleavages. When carbohydrate as a complex
matrix of macromolecules interact with ionizing radiation
ionlrced molecules are produced which immediately react
further to form some new products called free radicals. These
free
radicals
can
be
produced
in
several
micromolar
concentration in time periods of a few nanoseconds (Bchmxrz,
1981).These free radicals continue to react each other and to
other compounds leading to new stable chemical radiation
products. The life time of thore free radicals strongly depend
upon several factors such a s temperature and the presence of
q p n (Nawar, 1983).
Another theoretical explanation of the depolymerisation of
high molecular weight carbohydrate has been proposed by
&her= (1971). He postulated t h a t in t h e solid state the free
radicala react with the oxygan of t h e glycolliclic linkage
forming a -0-
radical. In the following step the 0 - C Iinkaagr,
next t o the hexme split off forming a positive charge at t h e
C4
atom. This positive ion roacts with tho OH* from irradiated
=tor
which is always prosont eithor as natural moisture or as
salvrnt. Basically similar to t h a t explanation has also been
suggmtod by ' h u b (1983), a m i c (1983), and Diohl (1990).
Whoa water is present in t h e samplo during irradiation,
carbohydrate react primarily with
hydrarryl radicals and
abstraat. predominantly the hydrogen of tho C-E bond. The
resulting
radicals
mechanisms which
will
react
further
through
difforont
is rrforred as the secondary effect
reaction loading to the final formation of products such as
w i d , b t o n and aldehyde depending upon tho position of CEO
bond formod attar tho reaction (&nntag, 1980; Sirnic, 1983;
Diehl, 1990, and Anonymous, 1994).
.
Wben oxygen is present during irradiation of carbohydrate,
the formation of deoxy
compounds is suppreued and the
yield of sugar acid and ketoaugars will increase with
increasing dicarbonyl sugar. The life time of free radical.
depend on several important factors such as the presence of
oxygen, pII, moisture content of substrate, and temperature
during irradiation (Simic, 1983).
The effect of gamma irradiation on degradation pathwam of
foodstuffs containing water, and its simpler carbohydrate
compounds are illustrated in 3 different figures respectively.
Figure 1 describes t h e mechanism of water radiolysi.
(Taub,
1983; and Diehl, 1990). The stable end products of water
radio1y.i~are hydrogen and hydrogen peroxide even they are
largerly consumed but the yield is quite low. During irradiation
of foodstuffs containing water, the presence of two reactive
s p a i e s , i.r., OE* is a n oxidizing agent, and a-l,,, as n d u a i n g
agent should k oonsidarrd.
H ~ -+%o+.
O
L
+
emaq
Hz0*
deprotonation/ %0+. + -0
(proton transfer reaation)
ionitation
(1)
excitation
(2)
---+H30+
+ OH- (3)
solvated proton
deexaitation (4)
=O*
7""
Ha
+ OH-
diuociation
(5)
recombination (6a)and combination reactions (6b) :
Figure
1, Reaction
mechanism
(Diehl, 1990)
of
water
radiolyslr
When glucose as a simple model for carbohydrate Is
irradiated in solute state, water radiolysk
generates OH
radicala which abstract predominantly hydrogen of C-H bonds
a t position
C-1 resulting acid, aldehyde
or ketone (Figure 2)
(Diehl, 1990).
CHOH
H
-
H
OH
-
OH
GLUCOSE
GLUCOSE R A D I C A L
GLUCONIC ACID
Through loss of CO the 6carbon sugar glucose can also be
converted to the 5-carbon sugar arabinose :
OH
H
-
C O
-
H
CHOH
OH
GLUCOSE R A D I C A L
5-CARBON R A D I C A L
ARAB I N O S E
Ring opening can lead to 5-deoxy gluconic acid :
GLUCOSE R A D I C A L
6-CARBON RADICAL
5-DEOXYGLUCONIC A C I O
Figure 2. Degradation mechanism of irradiation glucose
#amma irradiation induced degradation both as direct
and indirect effects on aliphatic polyhydric ether compound
WCHORCHOHOR') as a structure of aliphatic carbohydrate
representative is illustrated in Figure 3 (Taub, 1983).
When
an
aliphatic-polyhydric
ether
compound
representing a simpler carbohydrate is irradiated by gamma
rays a coatinuour chain reaction along the molecules will
occur through either direct or indirect effect depending on
the substrates.
Many products in the radiolyais of organic ruimtrates and
rdiolytic of aromatic systems after radiation exposure a t a
very low dose can be determined in the range of micromolar
levsl. using HPLC methods involving spectrophotometric
detection with diode array detector. (Schuler, 1993).
RCHOHCHOHOR'
3
CHOHCHOHOR')++c-ionisation
-H*
PCHOHCHOHOR~)+ A RCHOH~OHOR*
deprotonation
dissodativu electron attachment
&I m d d i c d n i w )
.rdepol~~erirrClasw
f
RCHOH~OHOR*+ H.
di.&tior
(C-H scksior)
OH.. +RCHOHCHOHOR* LR C H O H ~ O H O R+~ H&
abstraction
H.
R.]
OR.'
+RCHOHCHOHOR~ +?H
R'OH
+ RCHOH~OHORB
.b.traatiom
+ RCHOHCOOR'
/IRCHOHCHOHOR'
disproportionation
Figure 3,Direct and indirect e Kect reactions on irradiation of
aliphatic polyhydric compound ( Taub, 1983)
ESR
experiments
haw
been
carried
out
by
other
investigators in order to elucidate t h e mechanism of
production of free radicals from irradiated carbohydrate
containing starches, fruits and vegetables (Dexrosier and
Laughlin, 1986) and spices The E8R study of irradiated
popper and cinnamon rewaled that decreasing in free
radiaal concentration indicated by the reduced v a l w signal
wa. due t o the type of samples, irradiation d o n and storage
time (Budiro et af., 1994).
Effect of irradiation on the development of radiolytic
products in other
carbohydrate membars such as starch
polysaccharides deriwd from different foodstuffs such as
spbes, grains, and cereals have also been intensively
studied in many oountries.
The test which is proposed for the proof of irradiated
starch is based upon the determination of malonaldehyde
and other radiolytk compounds produced by irradiation.
The changes in the level of induced radiolytic products
ware analyzad as a function of irradiation condition (doset,
dose rate, temperature and atmosphere), starch properties
(water conten t,and pH) and post irradiation treatment or
storagn condition (Bonntag, 1980; Bonntag, 1992; Diehl,
1978; Taub, 1983, and Taub, 1984).
An experimental work using different typas of irradiated
starches such as maize (MN),amylomaim (AIM),waxy m a k e
(WM),bread wheat (B),manioc
(X), rice
(R),potato (P)and
haricot bean ('3)has been carried out by Raffia et aL (1981);
~ a f f et
i ~al. (1981); RaffiC et al. (1981), and Ftaffid et al.
(1981). Irradiation
was
conducted
under
3
different
conditions, via air, nitrogen, and oxygen. Samples were
irradiated with doses ranging from 5 to 30 kGy at 2S0C to
study radiation induced formation of radiolytb products,
and irradiation at 10-60 kGy for depolymeritation study and
kinetic law of the radicals. The parameters o b s e m d were
total
carbonyl
method),
malonaldehyde
formaldehyde
(2,4,-dinikophenylhydrcuine
derivatives
(thiobarbituric
(phenylhyd-ne
acid
test),
hydrochloride)
and
hydrogen peroxide (ammonium thiocyanide with iron
sulfate); p chromatography for formic acid (under methyl
formate
measured
form)
by
and
acetaldehyde.
pH-metry,
g;ravimetry
Total
for
acidity
was
water soluble
dextrina and reducing power was measured under back-
titration of iron [PI cyanide method. The spectra were
reuorded a t room temperature on a sprotrophotomehr
coupled with computer programme. The result shows t h a t at
water content equilibrium
the
quantities of
carbonyl
derivatirrrs radioinduced in the different starches almay.
h a w the same order of magnitude : r varies from 3.1 (total
carbonyl deriuatiws) to 4.6 (malonaldehyde) while intrinsic
vfwosity has strong correlation with the average degree of
polymerhation.
I i c h e l et a/. (1977) reported t h a t the percentage of
identified and unidentified carbonyl fraction resulting from
irradiated m a i u starh were 40 and
-,
respectively. Half
of t h e unknown fraction was linked to the radio dextrins.
Formic acid presence in t h e irradiated maim starch was
observed (100pg/g/Mrad, in oxygen) (Dauphin et el, 1974).
Research
on
the
formation
of
glycolaldehyde
in
irradiated m a h staroh has been done by Hamidi and
Dauphin (1976). They reported t h a t in irradiated m a h
starch
glycolaldehyde
owwnl.
was
found
(5.6 pg/g/Yrad,
in
Different
of sugars with low molecular weight
8
derived from irradiated maire starch h a w k e n identified
and determined w i n g ion exehange chromatography. The
samples
warn
treated
in
different
conditions,
i.e.,
irradiation atmosphere, starch properties, and storage.
Different type of sugars worn found such as one saccharide
(maltose), and
hexumes
fruatose). Pentoses
(glue-,
mannose,
-lactose,
such as arabinose, xylcne and ribora
ware a h found and also slight uncertainty amount of
tetrose/erythraee) (Berger et ul, 1973).
Studies
on
the
dihydroxyacetone
formation
(Dm) and
of
glyceraldehyde
(a),
2-hydrol~ymalonaldehyde
(E2M) in m a h a starch (amylopectin rich) using RlLI[R and gar,
chromatography method have been carried out by Rame et
nl. (1981). They obtained t h a t the concentration of t h e
radioinduced products increased in a linear manner with
the dose up to SO hay. (Lt-0.49
&gJkOy;
~.@gJkOy;
and BaMm0.63 p&g/kOy).
DXAdl.12
Tha total quantity
slightly decreased when the samples were irradiated under
nitrogen atmosphere. Storage condition
, in the presence of
o w g e n at room temperature, could reduce t h e amount of
those products rapidly particularly in the first umek. They
ooncluded that gonerally the radio induced qwntitios of O,
DEIA, and HdiX a n too low to i n w l r n health hatards. For
industrial purposes, the synergistic effect was obtained
when proton was combined with gamma radiation.
Further
experimental
wwk
on
indicated t h a t Rama eC
an important aommoraial fruits which servo not only as tablo
fruits, but alw as raw matorials in u w m l
fruit processing
industries for produaing among othorr noatar, juiao, jam, and
fruit Claks.
Truits p r o d w t h n ia Indoaosk inamasad from 4.53 million
tons in 1989 to 5.62 million tons in 1993. I t was also nportod
t h a t produativity of tropical fruits incroasod ltom 7.53 t o n l h a in
1988 to 12.13 too/ha in 1993 (Winasno, 1991). I U a n p is on0 of
t h e oxotia tropbaal fruits which could also k promotod as non-oil
aommodities to inaroaso aounlry dovisa. Export v a l w of mango
h m Indonosir to various importing a o u n t r h s in tho pllprld
s h m d a n inorrase from 579 thousand U8D in 1990
to 85'7
thowand UdlD in 1992 (Punwadaria et aL, 1998).
I t has k e n well understood t h a t changos in b u i t t o x t u n ,
flavour and a p p a r a n a o after harvesting k a o m o w r y important
quality standard critoria for evaluation of fresh fruits in tho
market. Fruits a m commonly hannstod a t a maturo stagr after
which softening takes place inside the fruit and continues even
after harumsting if the conditions are suitable.
sa
Textural changes induced by tissue 8ofYening of fruit i
result of the break down of cell -11
polymer net work either due
to biochemical change8 or actions of hydrolytic endogenow
ensymes which convert the insoluble constituent8 into soluble
and edible ones (Biale, 1960; Pilnilc and Vosagon, 1970; Bulme,
1970); Pilnik and Voragen, 1984, and Pilnik, 1984) but l e u
intensive studies on irradiated cell wall polysaccharides induce
depolymmrhation (Diohl st rl., 1978; and Kon, 1978). Lazan and
Ali (1993)reported that a n extensive softening in mango appear8
to be related to more extensive modification of the wall pectins.
BXost fruits contain large amount of water, as well as solutes
which could be readily utilized by microorganisms, insects, and
other pests. This situation makes the fruits prone to spoilage,
specially under storage condition which prevail in most tropical
countries. Furthermore, such economically important tropical
fruits normally have a limited postharvest life of about 2 weeks.
There are some technological approaches which might only
be effective in improving the shelf-life of particular fruits, i.e.,
.
s t o r a p in a modified atmomphere (MA) /control atmosphere
storage (CAS), or in a modified atmosphere pachaging (MAP)
(Punwadaria, 1995),and coating (IYLAC) treatments, storage at low
temperature
, hot
water dipping, and dipping in calcium salt
a8
reported by Jasim el al. (1968). Ionlting radiation using gamma
rays, X-ny. and h i g h a n e r w electron beams can also be used for
such purposes (Batin, 1993).
Gamma irradiation
a8 a
preservation technique 1.widely w e d
to extend the storaw life of some fruit. and vegetables for
dimerent purposes such as sprout inhibition of bulb. and tubers
p t s u y a m a and Umeda, 1983), delaying post hwvest
ripening
and senescence of fruits (Akamine and Xoy, 1983), insect
disinfestation for quarantine purposes (Tilton and Burditt, Jr.,
1983), and reducing microbial loads by single treatment of low
Irradiation dose or in combination with hot-water dipping (May,
1983).
Purar*nto and Maha (1986) reported that gamma irradiation
induced t i u u n sofbning in fresh mango was due to the
wlubilltation of protopactin into ~ l u b l epectin. %ince inwluble
pectic materials maintain cellular adhesion in plant tissues, the
destruction of protopectin by ionizing radiation appears to be the
cause of radiation induced softening of fresh fruit. which is
mostly considered as a n advetme effect. Alteration of ptobpectin
in the intercellular areas allows the tissue cell. to separate
resulting in loosing of texture.
Adverse physicochembal changes occuring in irradiated
fresh fruits and vegwtabbs using high radiation doses are surface
and flesh darkening as a result of enzymatic browning, and
immediate softening due to the degradation of long chain
polysaaahuidos p u r r a y , 1990; Clarke, 1961;Yasia rt d.,1987;
and ajoberg, 1987).The extent of changes in different structures
and chemical constituents of irradiated fruits and vegetables
depends o n several factors such as radiosensitivity of each
individual product, temperature of the environment and the
absorbed h a d t a t i o n dose through the materials (Bomogyi and
Romany, 1964).
The ionizing gamma radiation degrades a t b a s t the m a o r
polymers in the primary cell wall of plant materialm (pectic
substances, hemicelluloses and cellulose), (Clarke, 1968; and
Maxi* and Sommer, 1968)although the mechanism has not been
clearly explained (IYlc. Ardb and Hehemias, 1966; Rouse and
Denisson, 1968;Eolli-Donini and t3tronaiwl0, 1969; Eahandi et
uf., 1970;and Diohl r t rf., 1978).
The d e w a t i o n of the cell-wall polysaccharides such as in
pectin induced by gamma irradiation is duo to hydrolysis by
random attack of the glycosidic linkages within the polymers
leading to smaller units having the same general structure
(Korkr et rl., 1956; Ma.Ardb et uf., 1966; Maxi0 and &mmer,
1968;g i n n e r and Kerb+ 1960; and Clarke et 81, 1961)causod
by free radicals generated by irradiation (Murray, 1990).Likewise
in the immediate softening process is not attributable to
accelerate enrcymatic hydrolysis of the m l l component8 F
e
and mmmer, 1968).Irradiation of pectic substances derived from
apple probably resulting in random hydrolytic cleavage of the
galactutonan backbone yielding fragments of lower molecular
weight (@joberg,1987).
Fresh fruits such as mango is normally exposed to irradiation
as aonstituontm aooxisting mqjor and minor aomponents of
complex fruit systems. Rowaver, it is extremely difficult to
eluaidate the degradation mechanism occurs in irradiated mango
fruit induced softening in d t u Pattern and composition of the
radiolytic
products
polysaccharides
are
and
degree
strongly
of
sensitivity
depended
on
the
of
these
particular
environments, e.g., type of cell wall, physical orientation of the
plysaccharides molecules, and other artifacts.
It is estimated that some changes observed in a n irradiated
isolated cell wall polysaccharides as individual model systems
might also give similar pattern when the irradiated complex fruit
systems containing such plysaccharides h observed.
Different speculation on the study of depradation mechanism
of Irradiated fruits and w g e t a b k s proposed t h a t free radical
species are probably responsible for pectin depolymeritation (Kon,
1978; and Urbain, 1986), and the limit degradation of pectins
caused by radiation was explained by competitive 0%.scavengers
present in apples t8joberg, 1987).
Due to the complexicity in structure of mango fruit
, it
h
difficult to obtain an explicit data on the degradation mechanism
of the irradiated fruit induced softening. The studies were aimed
to obtain some information on degradation products of irradiation
of isolated cell wall polysaccharides
in
mango
fruit, and
irradiated polyraccharides model systems, The effect of radiation
on these various polysaccharides is estimated by direct and
indirect effect reactions through different .ample preparations In
order to elucidate t h e k degradation mechanism.
HYPOTHESIS
Immediate softening in gmmma irradiated mango fruit is
mainly due to hydrolysis process occur in the primary cell wall
polysaccharides vla a specific pattern through preferable attacked
of free rudbalr to the cell m l l components, resulting new
h g m e n h with lower molecular weight.
A. Irradiation
or F r e s h F r u i t s md Vemtables
Ionking radiation applied as a cold preservation methods
-ems
to be a n alternative for fruits and vegetables in order to
reduce their l o u e s and to improve their quality. a m p l e
conditions (degree of ripening, water content, uniformity in
she, etc,), and radiation parameters (dose, presence and
absence of oxygen, temperature, etc.) are of important
-tor8
in the irradiation of fruits and vegetables.
The chemical mechanisms of depolymerizrition reaction in
irradiated polyoaccharides can be invastis[ated in two my.,
i,e., by evaluating the structure of the radiation induced-
radicals through interpretation of different physical methods
suah as Electron 19pinIParamagnetic Resonance (EElR) spectra,
and by a n exact structure analysl. of the radb1y.l. products.
Irradiation
may
induce
severe
depolymeriaatbn
of
macromolecules resulting in structure loss which must be
considered aa a n adverse effect. Various studies on the enect
of (Lamma irradiation on physicochemical changes of fruits
and vegetabbs have h e n reported by many investi(Lators.
Studies on irradiated
fresh mango both
using single
irradiation or hot water dipping, and combination treatment
b e m e n irradiation a t 0.78 kOy and hot water dipping for I
min. at SSOC revealed that the combination treatment was the
best in prolonging the shelf-life of fresh mango for 3 weeks a t
31°C and then another 1 .crmek at room tempomtun (29-30%)
to allow the fruits into normal riponing without affecting w m e
chemical
characteristics,
and
the
organoleptic
quality
(Purrvanto and Maha, 1987). Other study has revealed, that
irradiated mango with doses up to 0.75 kOy did not influence
significantly to the amount of glucose, sucrose, fruotore and
total sugars but aftor storag. the irradiated mango showrd
more changuts of those mlues than in the control (aingh,
1990). It is also reporkd that immediakly aftor irradiation the
wmkr solubk protin MIW e f fresh auam@a
u high as in
other fruits but during storaw its rate of increaso was lower in
the untroakd samples. A contrary result was obtained in
insoluble poctin lfaction aftor storap. The irradiated k c t i o n
d e c r e a n d but the rate of decrease was Iomhr than in the
unirmdhted mango.
Btudy on radiolytic changes occuring in man-
revealed
t h a t irradiation of carbohydrate component produces carbonyl
compounds. Using models of carbohydrate derived from manthrough different preparations, i.e., solution, juice, pulp and
whole fresh mango, the finding result showed that the
individual component of the Itradiated food was mutually
protected. Other experimental work on sugar solution used as
model system indicated that radiolytk products of D-arabinohe--2
ulose (D-glucmne) was present, but this type of
radiolytk products wtu not found in the irradiated fiesh
mango (Delincee, 1989).
Physicothemical analyses of irradiated frozen de-seeded
kiwi pulp during storage at -18 OC has been i n v r a t i a t e d (Lodga
eC d.,
1988).The result revealed t h a t soluble solid fraction, pH
and titratabk auidity did not ahange aignifioantly during
storago up to 7 -aka
and o w n up to 6 months froesing
storage the total poutin remained eonstant a t values of 0.S0.6%. Iioward and Buesuher (1989) conaluded that softening
in uuuumber piukles caused by irradiation waa primarily
auociated with t h e changes in pectic substances and w m e
changes of galactom in the cell-wall.
8omoWi and Romany (1964) reported t h a t irradiated pear8
and p a c h o s a t 18.30C with t h e doses ranging &om 3.6 up to 9
kQy
tho
a clear indication on irradiation induced .oftoning of
fruit
tbuo.
Protopoctin
was
signifbaantly
redwod
immediately after gamma irradiation, then incnaaed aftor 4
day storago in comparison to tho untroakd samples. Poctin
v a l w incroasod twico of t h a t in t h e aontrol but poctate only
s h m d a slight incnaso. After storago unirradiakd pars wore
softer than the irradiated ones at moderat. d o u , but at a
higher radiation d o u more t i u u e damage was obsonnd than
in t h e irradiakd aamplos. Boftoning of pram and p a a h o s wore
loas in irradiation of samplms under IT2 atmoaphoro. Other
result waa obtained from irradiated pectin of 1%concentration
in solute state either under lT2 or oxygen atmosphere. Under
t h o w two atmospheric oonditions during irradiation, reducing
powar and viscosity were decreasing immediately. They '
postulated t h a t a n
increase in methylestemse activity may
alw contribute to the initial pectin degradation in irradiated
fruits. Similar investiwtor also studied on t h e behavior of
irradiated of fresh apple. It is reported t h a t it would not be
pouible to differen tiate between unirradiated and irradiated
samples on the basis of changes in molecular weight of pectin.
The degree of esterification, however, was decreased by
irradiation
treatment
at
low
doses.
Furthermore
they
concluded that t h e depolymerizatbn in the molecular chains
could give a n effect on the vfscority of apple pectin, and the
activity of p e c t i n m e t h y l e s t e r observed in f r d b increased
directly after irradiation but the activity tended to decrease
after 4 days of sorage.
Degradation of cellulose embedded in the apple fruit cell-11
by irradiation would probably contribute to a loss of
texture but i t seems unlikely that this compound is the
primary structural components being dewadad (Foamr C at.,
1980).In irradiated apple the threshold dose for degradation of
pectin and cellulose
mms similar to softening of the tissue
(Chute, 1968).
Immediate increased in water soluble pectin was found in
irradiated fresh apple but the pectic substances in irradiated
samples had similarity to those control after storagu (Clarke,
1968). ajoberg (1987) reported that pectin content and
molecular
weight of
fresh
apple
were
not
signifkantiy
influenced by irradiation with doses up to 10 kGy.
Clarke (1961)mentioned t h a t texture is not a n important
factor in t h e quality justification of irradiated fresh apple with
the d a m up to 1 kQy and under storage condition at 3OC. I t is,
however, during storago total pectic substances in t h e t h s h
apple
decnased
significantly
in
both
unirradiatod
and
irradiated samples. The same result was obtained in t h e
h a d i a t e d pectin fractions at the same d o u .
A study o n irradiated carrot has k e n rrp0ct.d
by Echandi
et uf,(1970).
Irradiation with the dose of 10 kGy did not alter
total exttaatable pectin nor on total calcium contont. Other
result on t h e irradiated aarrot as conducted by Urnail et al.
(1977)rewaled t h a t water wluble fkaction and anhydrouronic
acrid aontent showed a n inoreased upon irradiation while
ammonium oxalate h e t i o n was both increase aftar irradiation
and during storaga, but deoreand In HCl extrwtable fraotion.
Reduction on t h e degree of esterifkation was duo to the
aativation of endogonous poctin methyl-esteraw
irradiated
nfiigorated
cucumber pickles and
(PME) in
the pactin
wlubillaation was the most notable response to irradiation
(Howard and Buescher, 1989). Irradiated pickles at 1 kGy
showed a reduction of alkali and non extractable pectin but a
marked increase in water soluble and chelating agent wluble
pectin.
The activities of PO, PME, and Cellulase in irradiated
m a t u n groan tomato wcu domanstrated (Yasia et ad, 1987).
Using a viscosity measurement seemed t h a t the activities of
PQ. and PXE were not influenced directly after radiation up to
8 kOy. After 3 days storage PE activity showed a n increase a t
the dose of 0.8 kOy, as occured In unirndiated sample, but the
activity of PC) in the irradiated samples higher than 1kOy was
suppreued
during storage.
Cellulase
activity showed
a
significant increase dlrectly after irradiation but i t was not
affected by stoiap time. Result on irradiated tomato revealed
t h a t a reduction in the ratio of protopectin to total pectin, and
in t h e polyuronides extracted
from all
fractions,
were
indicated but hexamethaphosphate wluble pectin s h m d an
increase while water wluble pectin remained constant.
Other authors reported that in tomatoes and potatoes pectic
substances
are
responsible
for
the
changes
in
total
polysaccharides due to irradiation or stwagxi time before
irradiation pea* and Jona, 1980).
B. &lmbilib of Primup Cell Wall Indmcrd 8oISaninp in
Prrsh F r u i t s
Primary cell wall polysaccharides i. the major constituent
which is responsible for the integrity of plant tissue due to
their function as cellular adhesion.
The three major components of cell-wall material in
fruits and vegetables are pectic substances, hemicellulose
and
cellulose.
The
pectin
as
a
complex cell wall
polysaccharides member has a backbone which concists of
1 regions, i.e., "smooth" regions which mainly contain of a-
D-1,4- mlao turonan, and ramified regions introdwed
as
interupted sideohains which are attached to a-L-(1---->a)linked
rhamnwe
residues present
in
the
backbone
interupting the galacturonosyl residues (Schols, 1995).
Pectic substances is alw responsible for the integrity and
aohosiwnou of plant t i u w s (Davidok rt af., 1990).
Bolubilbtion of pectic substances related to the softaning
proceu in some tropical fruits such as bananas, manw,
oran*
and apple has k e n studied by Nwanokoarri rt rl
(1994). The result revealed that b comparison to the other
selected tropical fruits mango showed the highest pectin
(1.8%) and
methovl
contenta
and
the
purity
of
anhydromlacturonic acid content of 8 1 . 6 O h .
8 k h n i n g process in mature fruits is due to the b r e a k d m of
the cell wall structure in the fruit pulp by the activities of
endogenous ensymes resulting in conversion of inwluble
wall-bound protopectin of high molecular w i g h t into m t e r
soluble pectin. From many studies it has become c h a r that
pectin is not a homopolysaccharides but that neutral s u m
an part of the p a t i n molecules (Vrie* et .I.,
1982).
Pectic substance8 as non-starch polysaccharides and its
endogenous enrymes play the main impoatant role in the
proceu of wftening and in their contribution to the
metabolic pool of the cell (Pllnik and Voragen in Hulme,
1970; and Pilnik and Rombouts, 1981). Polygalacturonam
-
(Pa),the en=yme responsible for d e m i n g the (I----*)
linked ~ l a c t u r o n i cacid residues, and P-galactosidase are cell
mll hydrolases who playa a n important role in tropical fruit
softening. f3-galactoaidase may function as galactanam and
may act as a pectin debranching enayme (Latan and Ali,
.
1993) Other reseachers also reported that in addition to
pectinases the aativities of other enzymes such as cellularas
and b e m b e l l u l a u s in the cell wall might contribute to the
softening pooceu of fresh fruits. Further study on the
reducing viscocity caused by ensymatk degradation was
obwrvrd by b n e n a t h et rl. (1987).The study showmd that a
commercial enzyme, Ultrazym 100, was the most efGectiva
enzyme in reducing the viscosity of mango pulp.
2. BIuebuIlc~lcbmnges e t e d b RPlt so-ing
u d
rIpnb#
&ftening,
increasing svmetneu, aroma, and colour
changes are among the most striking phenomena related to
ripening of fruits. The changes might include respiration
rate, metabolism pathwmys, and individual constituents.
Polysaccharldes a# important carbohydrate constituents
of many foodstuf?~, are complex
macromolecules as
condensation polymer of high molecular weight compcming
from single monosaccharide units. One unit b connected to
t h e adjacent monosaccharide unit by a glycosidic linkage,
such as at C-1 and C 4 position, as oxygen bridge by the
elimination of water between the hemiacetal hydrorryl
group of one unit and availabb hydroxyl moup of another
(Aspinall, 1983; Pilnib, 1984; Belitz and Cirosch, 1987, and
Davidok
oC rl., 1990).
Fleshy fruits,
mostly its edible portion,
composes
mainly of parcrnchyme cells and larw intercellular air
spaces (King J r , and Bolin, 1989). Parenchyme cell. in the
pulp mainly contain primary cell walls defined as nonstarch polyraccharidea, The polyseccharides consisting predominantly by pectic substances which responsible for
tissue wftening, hemicelluloses, and cellulose microfibril..
The latter are firmly fixed in a matrix of pectic substances
and hemicellulosic polysaccharides {Aspinall, 1983; and
John and Dey, 1986). It k obvious that textural changes
induced softening of the fleshy fruits during ripening is
essentially caused by the conversion of high molecular
weight insoluble wall-bound protopectin into water soluble
pectin (Hulme, 1970; PilnUr and Rombouts, 1981; Davideh
eta/., 1990; and Murray, 1990).
Biochemical changes in some fruit. such as apricot and
mango a t different ripening stage have been carried out by
Sharaf ef rl. (1989).
It was reported that the percentage and
total
soluble
carbohydrates
increased
gradually
by
increasing extent of ripening. A t in their immature stage,
glucose and sucrose content were highest in apricot and
man*,
respectively. In ripe and over ripe stages, fructose
war. t h e predominant sugar in aprlcot and whilst in mango
I. glucose. The minimum value of glucose and srylose were
reached at over ripe stage in apricot. In mango fructose
tended to increase while glucose and sucrose decreased
significantly.
The Phllltppine maage, Ceuabao variety, showed some
changes in both physical and chemical characteristics
during ripening. The most significant chemical changes
obaenmd a t ripening stage indicated a decrease in starch
content but increased significantly in total soluble solids. At
harvest time the pectin content was 0.37-0.83 mg percent
and. it increased significantly in the ripening stage about
twiae higher than that in unripe stagxi @Coraet
1979).
Other study on the physico chemical changes of kinnow
mandarine orange and pineapple juice concentrates during
storaw in diffmrrnt t a m p r a t u r e raportad by bandhu ot aL
(1985).They found that pectin content decreased during
storage and the losses increase with increasing temperature
in thou, mamples.
AIR spectrophotometry in certain circumstances can be
applied optimally to estimate total pectic substances, water
soluble and in.oluble fractions, and methoxyl content of
alcohol insoluble solid prepared from
fresh peach, apricot,
and apple with a better result in comparison to the use o t
ahemiaal analysis as carrhd out by Polesello et al. (1990).
It was a h reported by BImpton et rf. (1984) that a
quantitatiw determination on pectin content of w m e
tropical
fruits
using
Ca-pectate
method
gave
more
representative data than that of methanol precipitation.
Methoxyl content of fruits mostly increases with increasing
content of pectin.
el rl. (19953 roported that mango (16mngiRrs
#!hputra
jndlca var. grdong) can be classified in t o thok taste b a u d
on sucrose and malic acid concentration as determined by
t h e Rear Infra Red Diffuse Reflectance at the wavelength of
1900-1975 nm and calibrated by HPLC.
C. Cell WIll Polv.acchuides, and T h e i r Similar
Sarrm
Building Bloeka
Pectic substances are complex colloidal polymaecharid-
es
consisting
residues,
of
in which
a-D-l,4-linked
(~alacturonic acid'
the carboxylic acid
groups are
partially esterifled by methyl groups and partially or
completely neutmlhed by bases (Pilnik and Voragen in
Hulme, 1970). Degradation of pectic substances may
ybld
rhamnogalacturonans
branched /
(slightly
rhamnogalacturonan
I
and
and
highly
111,
homoqalacturonan,
arab-inan.,
galactans
and
arabinogalactans (Aspinall, 1983 ; Pilnik, 1984; and John
and
Dey,
1986). Pectic
substances
protopectia, pectinic acid, pectinate.,
may
include
pectin(s), pectater
and pectic acid (Pilnik, 1970;and Davidek el d.,1990).
Pectins are subdivided according to their degree of
esterification and designation of the percent of carborryl
groups esterifkid with methanol. Pectin with DE > 5 W
are high m e t h o w l pectins and pectins with DE <
5Wo
are classified as low methoxyl pectins. Molecular weight
distribution of commercial high methoxyl pectins h a s
k e n analymd by Brigand et a& (1990)and many other
investillaton
using
s
exclusion
chromatopphy
coupled with low angle laser light scattering method..
It
is found that the pectins have molecular weight in the
range of 150,000-280,000 Dalton (Da). The araban and
gahctan chains are present as free polysacchartdes
found in the lower molecular weight region. It seems
t h a t degree of esterification remains constant all along
the
molecular
weight
distribution.
Intermolecular
distribution of the degree of esterification is narrow but
neutral s u m and acetyl content were less stable.
Pectins can be extracted by a variety of mild extractants
such as alkali, hot water, ammonium oxalate solution,
we&
acid, and chalating agent (Vries et af., 5981;
ahalom et d,1983;Bartlay and Knee, 1982;Baig et a/.,
1982;Simpson et nl., 1984;Renard et nl., 1990;Braudo
et rl., 1992; and &hols,
hydrolysed
into
smaller
199s). Pectins c a n also be
fragments.
The
resulting
different types of saccharides depend upon the attacked
point of t h e molecular chain by various degmdative
ensymes such cu pectinames, pectin methylesterase, and
pectin lyase (Pilnik and Rombouh, 1981;John and Dey,
1986;and Sreenath et sl., 1987).
Pectate is particuIarly found in
between
t h e middle lamella
adjacent cells. The nativm pectin play8 a n
important role in the consistency of fruits and vegetables
and in t h e textural
changes during ripening, storage,
HemicelluIoses occur covalently linked to the pectic
material but noncovalently bonded to cellulose fibrils.
They are not extractable in cold water, but extractable
In alkali solution.
Hemicellulose in the primary cell wall composes of
thmm m 3 w groups of polysaccharidms, Lo., wlurs which
is
a
mqlor
component
in
hemicellulose
fractions,
consisting of backbone polymers of fb-l,4-linked Dwlopyranose chains. Hemicelluloses have a n e t negative
charge due to the presence of a -Dglucuronic acid
residues a t tho sfdo chain (Fry, 1988) ; mannrrr.gfucomannu as drrivativrs of 6-l,4,-linked manno-
pytanose and B-l,4-linked glucopymnose units with'
various portions of glucose and mannose; and the
grlrctans.rv~bogakct~ns,
highly branchmd dorivativos of
1,3and
1,6-linked D-galactopyranose
polymem
with
arabinose side chains (Aspinall, 1983; Pilnik,19S9; and
John and Dey,
1986). Qalactans,
arabinogalactans,
arabinans,
xylans
and
glucomannas
are
important
representatives of hemicellulose.
Cellulose is composed of long chains of
D-glucopyranose residues which
fibrils by hydrogen bonds. Aggre-tes
p-1, 4- linked
are held together in
of these fibrils are
the basis of the rigid structure of plant cell -11
and it
may associates with other polymers such as pectin,
hemicellulose, lignin and protein (Stevens, 1975).
A n o r g a n b d aggregates of cellulose molecules in plant
cell wall 1.reffered to as microfibrills. These microfibrills
are composed of crystalline and amorphous regions.
Cellulose is a linear polymer, consisting of D-glucose in
%1,4
linkage
(Aspinall,
1983; Beldman,
1986; and
Davidek ef al., 1990). Cellulose can be hydrolymd by
strong acid yielding only D-glucose. Partial hydroly.is,
however, produces the reducing diuacharide cellubiose
(Voragen et sl., 1983; and Murray, 1990). The individual
chains of glucan are held together in fibril. by hydrogen
bonds between the hydroxyl groups at C-6 and the
glyoolridic -gens
of the adjacent chains. A methylation
prooeu followed by hydrolysis s t e m of cellulose yields
only 3,3,6,-&is-methyl@uco.e
without
branch
point.
Cellulose is insoluble in water and it is not related
structurally to hemicellulose p o n e r , 1988; and Beldman,
1986).
The delgadation of cellulose is catalyzed by cellulases
acting a8 B - l , 4 - ~ ~ c a n ~ ~ c a n o h y d r W
o ll a
dm
~ a n , 1986)
resulting in cell-dbintegration of pericarp in t h e ripening
staw
of fruits but these encymes does not directly
contribute t o the ripening (John and Dey, 1986).
As individual polysaccharides alginates occcur in all
brown algae as a skeletal component of their cell walls.
Algae are extracted with alkalies and then precipitated
from t h e extract by acid or Ca- salts. Alginates are linear
copolymers consisting of the following structural units:
Mannuronb
acid-Mannutonic
acid;
Guluronic
GuIuronQ acid; and Mannuronic acid-Guluronic acid.
acid-
Alginates designated as alginic acid, a polymer similar
to cellulose (Stevens, 1975) is a heteropolyuwcharides
compoms of varying and alternating sequences building
blocks of 8-D-mannuronio acid and a -L guluronic acids,
joined by the I------linkages
.
The -(I--->4)
glycaaidk linkages between a -L-gnluronic
acid molecules leads to a d u a l orientation similar to
that in galacturonan and equally suitable for an egg box
type of chain association with ca-ions (Pilnlb, 1984). The
ratio of the two s u q a n (mannuronic/guluronlc acids) is
generally 1.S with some deviation
with source. The
gelling properties of alginah depending upon the amount
of guluronic acid in the main chain. Partial hydrolysis of
alginate
produces
some
fragments
whbh
consist
predominantly of mannuronic or guluronic acid, and also
fragments where the two uronic acid residues alternate in
1:l ratio.
Distribution of uronate residues in a l d n a t e chains is
related to alginate gelling properties. This statement mu
approved
after
using
an
experimental
model
of
enrichment of p-D-mannuronic acid and depletion of a -Lguluronic acid In solution fraction as conducted by
(iarnoarek and Qiarncarek (1993);and Btokke et rl. (1993).
Their report is basically concerned about the binding
strength
between low methoxyl pectin and alginate
mixed gel.
The differences between low methoxyl pectin and
alginate appear on their heat stability under vwbw pH
conditions. Under low pH and neubal conditions aldnates
are more heat stable.
Oat.
and Ledward (1990) have
studied the effect of heat on alginates. They revealed that
samples with high level of mannuronic acid residue were
far less stable against heat.
Alginates are water soluble in the form of alkali,
magnesium, amonia or amine salt..
The viscosity of
alginate solution is influenced by molecular weight and
the counter ion of the salt. In the absence of di and
trivalent cation or in the presence of a chelating agent,
the viscosity is low. However, with a rise in multivalent
oation levels, e.g., calcium, there is a parallel rise in
viscosity. Freezing and thawing of Na-aldnate wlution
.
containing Ca++ ions can increase thelr viscosity. The
viscosity k unaffected within a pH range of 4.5-10. It rise8
a t a pH below 4.5, reaching a maximum a t pH 3-3.5 (Belitz
and QK#Ch, 1987).
Propybne glycol alginate is derivatisation of alginate
through a reaction of mannuronic acid with propylene
oxyde via hidrown abstraction a t C-6 of mannuronic acid
(Pilnik and Voragan, 1984).It is wlubIe down to pH 2 and,
in the presence of Ca++ ions it will form soft, elastic, l e u
brittle and syneresis-free gels (Gray and Philp, 1991).
k a b i n a n s as a member of dietary fibres
play a n
important role in fruits processing. Arabinms are also
found abundant in the biomass of c r o p as waste products.
Thi. type of homopolysaccharides present. as pentoran
sugars, mainly composed of a-L-arabinohuanoryl residues.
They are generally arranp(ed in (1---->I)
linked chains with
varying numbers of residues substituted with other a-Larabinofuranosides a t the 0-2 and/or 0-3position (Beldman
at el., 1991;Pilnilc and Voragen, 1984).
k a b i n a n s are present in the plant tissues auociated
with
pectic
arabinoxylans,
substances
as
arabinogalactans
homoglycans
and
such
aa
arabinogalactan-
proteins. It can be isolated by preferential extraction with
boiling ethanol or with lime-mter in which associated
pectic acid will be transfarmed into insolubla Ca-salt.
Highly branched arabinan can be syntheslted by the
isolation of 2,3,5,-tri, 2,3,-di, and 2-0-methyl-Larabinose
on hydrolysis of the methylated arabinans. This type of
polysacchatides have
w m e difficulties in the isolation
process from other sugar residues, particularly if the
arabinans attach to the pectin molecules. Since pectin I.
unstable a e i n s t alkaline reagent, the purity of arabinan
obtained from such iwlation is doubtful. Other method in
iwlation of arabinan under non depadative condition haa
still to be developed. Arabinan degrading enzymes become
an essential method to elucidate structural characteristics
of arabinan (8.ldman et aJ., 1991). Iiowavor, in sugar boot
.
polysaccharides the presence of arablnans linkages in
aucrciation with pectic s u b t a n c e s can still be isolated of a
linear
1--->5 linked
ambinan
using
an
a-Larabino-
huanosidaae (Aapinall, 1983). In the concentrated apple
juice haes become t h e mqfor problem due to the effect of
high amounts of ambinan side chains. A side-branched
araban, wuter soluble amban, links at 1,3 and 1,3 position
of amban straight chains with branched points a t C1 and
C5 as glycosidic linhgam. An ondo
1,s
-maban-
is able
to break doam the side chains of arabinan resulting in a
clear solution in the concentrated apple juice (PPnUr and
Voragen, 19%
and Voragen st al., 1987).
Ioalrciug (y ) radiation, is found in the range of wave length
between 1014 and 10-10 cm, as non-particle energy radiation
whioh c a w s direct and indirect i o n h t i o n or excitation of a
permanent qar (O'Donnell and Bangster, 1970). I t has the
highest emition energy level among othera and has dimerent
ability in penetrating materials compare to a rays and $ rays.
k a cold process, gamma irradiation seems to be a promising
tachnique to substitute or to improve the existing preservation
methods. This technique can be utilized for decontamination
to reduce the food losses or as quarantine treatment of
agricultural products to protect horticultural export against
chemical fumigants by improving the environment and
hygienic quality. However, among the important benefits of
irradiation, destruction or reduction of pathogenic bacteria in
foodstuffs is of moat importance. In the long term, food
irradiation may be seen as a part of the food processing
technology to provide food which stays fresher for a longer
period.
In food irradiation, which is merely a physical process, the
ionking energy only interacts with the outer orbital electron
of t h e atoms and it does not interact with the nucleus.
Consequently, there is no radioactivity induced
in
the
irradiated foodstuffs. The changes only occur chemically in
the
food wlthout
any
nuclear
changer
(Becker,
1983).
Irradiation does not cook the foodstuffs since no heat is
produced and the food retains its initial characteristics (Satin,
1993).It mlut also be pointed out that such reactions do not
lead to the formation of aromatic ringa, condensation of
aromatic rings, and formation of heterocyclic rings (aimic,
1983;and Diehl, 1990).
Since
nutritional adequacy
and wholesomeness
of
irradiated foods are important considerations in ascertaining
feasiblity of irradiation technolow, changes in chemical
components or nutritional significance have received much
attention. However, it has been jpnerally observed that
complex foodrtuffs are leu stuceptible to irradiation induced
c h a n w s h chemical structure of the components ru expected
from studies w i n g pure
Delincee,
1989). The
compounds (Adam, 1983; and
reaction
mechanisms occuring in
irradiated foods must be fully undentood if consequences of
irradiation are to be predicted.
Radiation wurces and dosimetry technique are playing
important roles in food irradiation. There are 3 types of
radiation souroes w h k b e r n k wed, 1.0.~radionuolido8 m d
particle accelerators. Bome radionuclides emit a form of
electromagnetic wave energy called photon. Co-60or Cs- 137
provides photon, rays, with the energy sufficient for good
penetration but unable to induce radioactivity in the irradiated
foodstufls. Particle accelerators provide a beam of high energy
as charge particles. Electron beams are used for radiation
processing since the energy is limitted t o 10 million electron
volts WeV) avoiding traces of radioactivity which may be
induced at higher energy.
Several parameters need to be considered in determining
the Co-60 radiation dose received by a package, i.e., amount of
Ca-60 in the source plaque, distance between source and
&radiated
products,
the
exposure
time,
and
radiation
absorption within the packages which increases as t h e package
density and the package 11 width increase. It i. important to
minimize dose variation within each package when food is
irradiated. Such variation are inevitable becaulre the distance
to the eource varies from point to point within the package
and becauw of radiation aboorption within the package. The
ratio of maximum to minimum dose (Dm,/Dmin),
as uniformity
(U),
designated
must be kept within masonabla limits,
usually Ieu than 2. Dmin must be sullicient to achieve the
objectiw of the process. The,,D
must bo within the
toleranee of the commodity. DmMfDmin can be minimbed
partly by moving the packages around the source during the
irradiation so that position
of high
and
d
doso
is
interchanged. The energy absorbed per unit of time is called
the dosa rate.
The new unit of radiation,
Gray,
as the amount of
irradiation e n e r w that a food absorb. L measured in units. It
h noted that 1 Gray = 1 joule per-kg of abaorbed energy = 100
fads (the old radiation unit) while a t medium irradiation dose,
10 kQy, the energy is equaI to to the amount of energy
required to raise the temperature of water by 2.90C.
It is considered that gamma Irradiation is applied for
different purpc#es for premedng foodstuffm, e.g., inhibition of
w e t a b l e s sprouting is (up to 0.2 kQy), insect diminfestation
(0.2-1.0 kOy), pararrite elimination (up to 6 kGy), microbial load
reduction (O.Ob-5 IrGyI, killing of non sporing pathogens
(3-10kGy), and absolute sterilization (80 key) (Diehl, 1990;
and a t i n , 1993).
,
E. Irradiation of Carbohydrate a n d it. Derivrtives
The most important effects of ionizing
radiation on
macromolecubs are degradation and croru linking.
The
degradation process 1.merely a hydrolysis of glycosidic bounds
and
other
weak
linkages
yielding
simpler
compounds
(Charlesby, 1960; Charlesby, 1987; and aonntag, 19911.
Fundamental study on radiation chemistry of different
faodstufi is identification and measurement of various species
formed during and after irradiation. Chemistry of the s p e c k s
in the reactions with different compounds covered reaction
kinetics, mechanisms, intermediate and stable end products
(O'Donnell
and
Sangster,
1970; and
Taub,
1983). An
observation on the rate constant of transient species can be
done using puke radiolysis, EBR p a n g et
dl.,
1987) and in
combination with competition kinetics (Raff'i and Agnel, 1983).
In principle
can detect the spin unpair electrons even in a
very low concentration and the amplitude of ELSR signal is
approximately proportional
to the
total number
of the
electrons in the sample (Wertr: and Bolton, 1972).Thus, E8R 1.
,
obviously useful for detecting and characterlcing free radicals
formed in the irradiated samples.
The radiation chemistry of carbohydrate can be determined
by three m 4 o r factors, i.e., the radiation chemistry of the
simple sugar sub-units; the effect of subatltuents on the sub
units; and the fate of glycosidic liqkage p i e h l , 1990). The
splitting of glycoddic linkages in carbohydrate molecules is
not only caused by gamma irradiation but also by entymes
action or by chemical means with some differences in the
results. These differences depend on the degree of selectivity
among bond cleavages. When carbohydrate as a complex
matrix of macromolecules interact with ionizing radiation
ionlrced molecules are produced which immediately react
further to form some new products called free radicals. These
free
radicals
can
be
produced
in
several
micromolar
concentration in time periods of a few nanoseconds (Bchmxrz,
1981).These free radicals continue to react each other and to
other compounds leading to new stable chemical radiation
products. The life time of thore free radicals strongly depend
upon several factors such a s temperature and the presence of
q p n (Nawar, 1983).
Another theoretical explanation of the depolymerisation of
high molecular weight carbohydrate has been proposed by
&her= (1971). He postulated t h a t in t h e solid state the free
radicala react with the oxygan of t h e glycolliclic linkage
forming a -0-
radical. In the following step the 0 - C Iinkaagr,
next t o the hexme split off forming a positive charge at t h e
C4
atom. This positive ion roacts with tho OH* from irradiated
=tor
which is always prosont eithor as natural moisture or as
salvrnt. Basically similar to t h a t explanation has also been
suggmtod by ' h u b (1983), a m i c (1983), and Diohl (1990).
Whoa water is present in t h e samplo during irradiation,
carbohydrate react primarily with
hydrarryl radicals and
abstraat. predominantly the hydrogen of tho C-E bond. The
resulting
radicals
mechanisms which
will
react
further
through
difforont
is rrforred as the secondary effect
reaction loading to the final formation of products such as
w i d , b t o n and aldehyde depending upon tho position of CEO
bond formod attar tho reaction (&nntag, 1980; Sirnic, 1983;
Diehl, 1990, and Anonymous, 1994).
.
Wben oxygen is present during irradiation of carbohydrate,
the formation of deoxy
compounds is suppreued and the
yield of sugar acid and ketoaugars will increase with
increasing dicarbonyl sugar. The life time of free radical.
depend on several important factors such as the presence of
oxygen, pII, moisture content of substrate, and temperature
during irradiation (Simic, 1983).
The effect of gamma irradiation on degradation pathwam of
foodstuffs containing water, and its simpler carbohydrate
compounds are illustrated in 3 different figures respectively.
Figure 1 describes t h e mechanism of water radiolysi.
(Taub,
1983; and Diehl, 1990). The stable end products of water
radio1y.i~are hydrogen and hydrogen peroxide even they are
largerly consumed but the yield is quite low. During irradiation
of foodstuffs containing water, the presence of two reactive
s p a i e s , i.r., OE* is a n oxidizing agent, and a-l,,, as n d u a i n g
agent should k oonsidarrd.
H ~ -+%o+.
O
L
+
emaq
Hz0*
deprotonation/ %0+. + -0
(proton transfer reaation)
ionitation
(1)
excitation
(2)
---+H30+
+ OH- (3)
solvated proton
deexaitation (4)
=O*
7""
Ha
+ OH-
diuociation
(5)
recombination (6a)and combination reactions (6b) :
Figure
1, Reaction
mechanism
(Diehl, 1990)
of
water
radiolyslr
When glucose as a simple model for carbohydrate Is
irradiated in solute state, water radiolysk
generates OH
radicala which abstract predominantly hydrogen of C-H bonds
a t position
C-1 resulting acid, aldehyde
or ketone (Figure 2)
(Diehl, 1990).
CHOH
H
-
H
OH
-
OH
GLUCOSE
GLUCOSE R A D I C A L
GLUCONIC ACID
Through loss of CO the 6carbon sugar glucose can also be
converted to the 5-carbon sugar arabinose :
OH
H
-
C O
-
H
CHOH
OH
GLUCOSE R A D I C A L
5-CARBON R A D I C A L
ARAB I N O S E
Ring opening can lead to 5-deoxy gluconic acid :
GLUCOSE R A D I C A L
6-CARBON RADICAL
5-DEOXYGLUCONIC A C I O
Figure 2. Degradation mechanism of irradiation glucose
#amma irradiation induced degradation both as direct
and indirect effects on aliphatic polyhydric ether compound
WCHORCHOHOR') as a structure of aliphatic carbohydrate
representative is illustrated in Figure 3 (Taub, 1983).
When
an
aliphatic-polyhydric
ether
compound
representing a simpler carbohydrate is irradiated by gamma
rays a coatinuour chain reaction along the molecules will
occur through either direct or indirect effect depending on
the substrates.
Many products in the radiolyais of organic ruimtrates and
rdiolytic of aromatic systems after radiation exposure a t a
very low dose can be determined in the range of micromolar
levsl. using HPLC methods involving spectrophotometric
detection with diode array detector. (Schuler, 1993).
RCHOHCHOHOR'
3
CHOHCHOHOR')++c-ionisation
-H*
PCHOHCHOHOR~)+ A RCHOH~OHOR*
deprotonation
dissodativu electron attachment
&I m d d i c d n i w )
.rdepol~~erirrClasw
f
RCHOH~OHOR*+ H.
di.&tior
(C-H scksior)
OH.. +RCHOHCHOHOR* LR C H O H ~ O H O R+~ H&
abstraction
H.
R.]
OR.'
+RCHOHCHOHOR~ +?H
R'OH
+ RCHOH~OHORB
.b.traatiom
+ RCHOHCOOR'
/IRCHOHCHOHOR'
disproportionation
Figure 3,Direct and indirect e Kect reactions on irradiation of
aliphatic polyhydric compound ( Taub, 1983)
ESR
experiments
haw
been
carried
out
by
other
investigators in order to elucidate t h e mechanism of
production of free radicals from irradiated carbohydrate
containing starches, fruits and vegetables (Dexrosier and
Laughlin, 1986) and spices The E8R study of irradiated
popper and cinnamon rewaled that decreasing in free
radiaal concentration indicated by the reduced v a l w signal
wa. due t o the type of samples, irradiation d o n and storage
time (Budiro et af., 1994).
Effect of irradiation on the development of radiolytic
products in other
carbohydrate membars such as starch
polysaccharides deriwd from different foodstuffs such as
spbes, grains, and cereals have also been intensively
studied in many oountries.
The test which is proposed for the proof of irradiated
starch is based upon the determination of malonaldehyde
and other radiolytk compounds produced by irradiation.
The changes in the level of induced radiolytic products
ware analyzad as a function of irradiation condition (doset,
dose rate, temperature and atmosphere), starch properties
(water conten t,and pH) and post irradiation treatment or
storagn condition (Bonntag, 1980; Bonntag, 1992; Diehl,
1978; Taub, 1983, and Taub, 1984).
An experimental work using different typas of irradiated
starches such as maize (MN),amylomaim (AIM),waxy m a k e
(WM),bread wheat (B),manioc
(X), rice
(R),potato (P)and
haricot bean ('3)has been carried out by Raffia et aL (1981);
~ a f f et
i ~al. (1981); RaffiC et al. (1981), and Ftaffid et al.
(1981). Irradiation
was
conducted
under
3
different
conditions, via air, nitrogen, and oxygen. Samples were
irradiated with doses ranging from 5 to 30 kGy at 2S0C to
study radiation induced formation of radiolytb products,
and irradiation at 10-60 kGy for depolymeritation study and
kinetic law of the radicals. The parameters o b s e m d were
total
carbonyl
method),
malonaldehyde
formaldehyde
(2,4,-dinikophenylhydrcuine
derivatives
(thiobarbituric
(phenylhyd-ne
acid
test),
hydrochloride)
and
hydrogen peroxide (ammonium thiocyanide with iron
sulfate); p chromatography for formic acid (under methyl
formate
measured
form)
by
and
acetaldehyde.
pH-metry,
g;ravimetry
Total
for
acidity
was
water soluble
dextrina and reducing power was measured under back-
titration of iron [PI cyanide method. The spectra were
reuorded a t room temperature on a sprotrophotomehr
coupled with computer programme. The result shows t h a t at
water content equilibrium
the
quantities of
carbonyl
derivatirrrs radioinduced in the different starches almay.
h a w the same order of magnitude : r varies from 3.1 (total
carbonyl deriuatiws) to 4.6 (malonaldehyde) while intrinsic
vfwosity has strong correlation with the average degree of
polymerhation.
I i c h e l et a/. (1977) reported t h a t the percentage of
identified and unidentified carbonyl fraction resulting from
irradiated m a i u starh were 40 and
-,
respectively. Half
of t h e unknown fraction was linked to the radio dextrins.
Formic acid presence in t h e irradiated maim starch was
observed (100pg/g/Mrad, in oxygen) (Dauphin et el, 1974).
Research
on
the
formation
of
glycolaldehyde
in
irradiated m a h staroh has been done by Hamidi and
Dauphin (1976). They reported t h a t in irradiated m a h
starch
glycolaldehyde
owwnl.
was
found
(5.6 pg/g/Yrad,
in
Different
of sugars with low molecular weight
8
derived from irradiated maire starch h a w k e n identified
and determined w i n g ion exehange chromatography. The
samples
warn
treated
in
different
conditions,
i.e.,
irradiation atmosphere, starch properties, and storage.
Different type of sugars worn found such as one saccharide
(maltose), and
hexumes
fruatose). Pentoses
(glue-,
mannose,
-lactose,
such as arabinose, xylcne and ribora
ware a h found and also slight uncertainty amount of
tetrose/erythraee) (Berger et ul, 1973).
Studies
on
the
dihydroxyacetone
formation
(Dm) and
of
glyceraldehyde
(a),
2-hydrol~ymalonaldehyde
(E2M) in m a h a starch (amylopectin rich) using RlLI[R and gar,
chromatography method have been carried out by Rame et
nl. (1981). They obtained t h a t the concentration of t h e
radioinduced products increased in a linear manner with
the dose up to SO hay. (Lt-0.49
&gJkOy;
~.@gJkOy;
and BaMm0.63 p&g/kOy).
DXAdl.12
Tha total quantity
slightly decreased when the samples were irradiated under
nitrogen atmosphere. Storage condition
, in the presence of
o w g e n at room temperature, could reduce t h e amount of
those products rapidly particularly in the first umek. They
ooncluded that gonerally the radio induced qwntitios of O,
DEIA, and HdiX a n too low to i n w l r n health hatards. For
industrial purposes, the synergistic effect was obtained
when proton was combined with gamma radiation.
Further
experimental
wwk
on
indicated t h a t Rama eC