THE EFFECTS OF HIGH VOLTAGE CATHODE RAY IONIZING
RADIATION ON SOME OF THE PHYSICAL AND CHEMICAL
PROPERTIES OF WHEAT FLOUR PROTEIN

by
ELSIE BETH ALSUP

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Foods and Nutrition

1959

Approve aw

 

The use of ionizing radiations for insect deinfesta-
tion and sterilization of flour will not be commercially
feasible until the limitations of the process have been
thoroughly explored and the end products have been shown
to be acceptable. The purpose of the present investiga-
tion was to determine the effect of irradiation on some of
the physical and chemical prOperties of wheat flour protein.
It included a study of the baking quality and palatability of
an irradiated flour product, yields of crude gluten from
irradiated flour, and electrophoretic analyses of some of
the protein fractions of irradiated flour.

Triangle taste tests indicated that 50,000 rep was the
dosage level at which the irradiation treatment of flour
could be detected in baking powder biscuits. Taste panel
scores for color, odor, and baking characteristics of bis-P
cuits made with flour treated with 100,000 rep or less of
ionizing radiation, however, were not significantly different
from.the scores for the control biscuits. An off-odor and
darkening in color of the biscuits were readily apparent at
a dosage of 500,000 rep.

The weight of crude gluten extracted from flour was
greater from.flour exposed to dosages of 1,000,000 rep or
less than from the control flour, but no differences were
found in the volumes of the baked balls of crude gluten.
This indicates a greater water retention with the irradiated

flour. Very small yields of crude gluten at dosages of

3,000,000 rep or more indicated that definite changes had
occurred in the protein structure.

Electrophoretic analyses by the moving boundary method
were performed on acetic acid extracts of whole flour in a
citric acid-disodium.phosphate buffer pH 2.2, ionic strength
0.02h. Electrophoretic patterns obtained from the non-
irradiated flour indicated that there were at least six sep-
erate protein components present, including one which was
fast-moving and high in relative concentration, one which
was slow-moving and medium high in relative concentration,
and four relatively small components. Irradiation of the
flour with 300,000 rep produced no apparent changes in the
electrophoretic patterns obtained, but at 1,000,000 rep or
more, the relative concentrations of the two largest comr
ponents were changed--the slow-moving one becoming larger
and the faster-moving one smaller with each increase in
dosage. Also with increased radiation dosages, the peaks
of the smaller components became less distinct until only

the two main peaks were distinguishable at 10,000,000 rep.

THE EFFECTS OF HIGH VOLTAGE CATHODE RAY IONIZING
RADIATION ON SOME OF THE PHYSICAL AND CHEMICAL
PROPERTIES OF WHEAT FLOUR PROTEIN

by
ELSIE BETH ALSUP

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Foods and Nutrition

1959

TABLE OF CONTENTS

INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O O O O O O O O O 0 O O
REVIm OF LITERATURE . C . O C O C . O C C O O O O O O O O O O I O O O O O O O O O O C O 0
Use Of Ion121ng Radiations O O O O O O O O O O O O O O O O O O O O O O O O 0

Types of Radiant Energy........................
units or RadiationOOOOOOOOOOOOOOOOOOOOOOOO0....
Mechanisms of Action...........................
EerCt on InsectS..............................
'Wholesomeness of Irradiated.Foods..............
Effect on Nutritive Value of Food Proteins.....
Biochemical Effects on Proteins................
Effect of Using Irradiated Flour in Baked

ProduCtSOOOOOOOOOOOOOOOOOOOOOOCOO0.0.00.0...

Effect on Physical Properties of Flour.........
Composition of'Wheat Proteins......................

Historical...OOOOOOOOOOOOOOOOOOOOO0.00.0...O...
Protein.FraCtionSOOOOOO000......00.000.00.00...

Elements.......................................

Amino Acids....................................
Electrophoretic Studies on Wheat Proteins..........
EXPERIMENTAL PROCEDURE.................................
Baking Tests on Biscuits...........................

material-SOC...0......OOOOOOOOOOOOOOO0000.000...
Preparation.O00......0.000000000000000000000000
Evaluation of the Dough and Biscuits...........
Statistical Ana1y818000.0.0.0...000.00.00.00...

Gluten'Y161d8......................................

Preparation of Gluten..........................

SSW F} {3’53 RES-awed m m u-u

0
ho
ho

Volume Measurements............................ #2

ElOCtrophor681ao00000000000000.0000...one...coco...

Preparation of sample directly from flour......

Preparation of sample from.crude g1uten........ h
Preparation of sample from.gliadin............. h

Nitrogen determinations of the protein

GXtractSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00..

Electrophoretic procedure...................... A

#2
he

3
3
ML
5

RESULTS AND DISCUSSIONOOOOOOOOOOOOOOOOOOOOO000.000.0000 1+7
Baking Tests on Biscuits........................... h?

Organoleptic Tests.........t.................... A?
mechanical Tests..OOOOOOOOOOOOOOOOOOOO...00.0... 53

Gluten YieldSOOOOOOOOCOOOOOOOOOOOOOOOOOOOO0.00.0... 51+

weiglt of crude glutenOCCOOOOOOOOOOOCOOOOCCOOOOC 5%
Velume of baked gluten balls.......0.0.0.0000... 5

Electrophoresis.................................... 57
SUMMARY AND CONCLUSIONS................................ 69
ACKNOWLEDGMENTS........................................ 71
LIST OF REFERENCES..................................... 72
APPENDIX............................................... 79

VITA...00.00.000.000.0.0.0.0000...0.0.00.00000000000000111

ii

1.

6.

7.

9.

10.

11.

12.
13.

iii

LIST OF TABLES

Formula used in making biscuits with irradiated all-
p‘lrpose flour.00.0.0....OOOOOOOOOOOOOOOOOOO0.000.000.0000 37

Average taste panel scores (3 Judges and h replications)
for biscuits made with irradiated all-purpose flour and
tartrate baking pOWderOOOO0.0.0.000...OOOOOOOOOOOOOOOOOOO ’48

Triangle taste tests for biscuits made with irradiated
all-purpose flourOOOOOOIO0.0.0.0....OOOOOOOOOOOOOOOOOOOOO So

Triangle taste test data for biscuits made with irradiated
flour showing individual correct choices of the odd
ample by the 10 JudEOSOOOOO0.000000000000000000000000... 52

Triangle taste test data for biscuits made with irradiated
flour comparing use of tartrate and sulfate-phosphate
baking powaers...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... 53

Average compressibility, elasticity, and volume measure-
ments on dough and'biscuits (6 batches) made with
irradiated flour and tartrate baking powder.............. 53

Mean yields of crude gluten extracted from.non-
irradiated and irradiated all-purpose flour.............. 55

Mean volumes of baked gluten balls made with non-
irradiated and irradiated all-purpose flour.............. 56

Mean initial protein concentrations of acetic acid
dispersions of flour and mean final protein concentra-

tions after centrifugation and dialysis for 72 hours

against citric acid-disodium phosphate buffer, pH 2.2.... 58

Mean distribution of the protein.among the various
electroPhoretic components of untreated and irradiated
flour (ascending patterns)............................... 66

Mean electrophoretic mobilities of the protein comr
ponents of untreated and irradiated flour (ascending
pattom8)000000000000000000......OOOOOOOOOOOOOOOOOOOOOOOO 66

Odor scores for biscuits made with irradiated flour...... 83

Color of the crust scores for biscuits made with
irradiated flourOOOOOOOOOCOO...OOOOOOOOOOOOOOOOOOO0.00... 83

Color of the crumb scores for biscuits made with
irradiated flourOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOO..00.. 8h

15.
16.

17.

18.
19.

20.
21.
22.
23.

2h.
25.

26.

27.
28.
29.
30.
31.
32.

33-

3&-

iv

Flavor scores for biscuits made with irradiated flour....

Tenderness scores for biscuits made with irradiated

flour...OOOOOOOOOOCOOOOOOIOOOOOIOOOOOOOI...OOOOOOOOOOOOOO

Lightness scores for biscuits made with irradiated

flourOOOCOOOOOOOOOOOOOOOOOO0.0.0.000...00.000000000000000
Texture scores for biscuits made with irradiated flour...

Moisture scores for biscuits made with irradiated

flourOOOCOOCOOOOOOOO0.0.0....OOOOOOOOOOOOOOOOOOOOOIOOI...

General eating quality scores for biscuits made with
irradiated flour...00.0.0000...OOOOOOOOOOOOOOOOOOOOOOIOO.

Compressibility of the dough.made with irradiated flour
and tartrate baking powder (1/10 mm.)....................

Elasticity of the dough.made with irradiated flour and
tartrate baking powder (1/10 mm.)........................

Volume of the biscuits made with irradiated flour and
tartrate baking powder (1/10 mm.)........................

Analysis of variance for odor scores of biscuits.........

Analysis of variance for color of crust scores of

biscuitSCOOOOOOOOC00......0.00.00.00.0000IOOOOOOIOOOOOOO.

Analysis of variance for color of crumb scores of

biscuitSOOOOOOOOOOOOOQOOOOOO0.0.0000000000000000000000.0.

Analysis of variance for flavor scores of biscuits.......
Analysis of variance for tenderness scores of biscuits...
Analysis of variance for lightness scores of biscuits....
Analysis of variance for texture scores of biscuits......
Analysis of variance for moisture scores of biscuits.....

Analysis of variance for general eating quality scores

or biscuitSOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.0.0.0...

AnalySis of variance for compressibility of the dough
scores or biscuits....O..‘O............O.................

Analysis of variance for elasticity of the dough scores
or biscuj-tSOOOOOOOOOOOOOO0.0.000000000.000000IOOOOOOOOIOO

8h

85

86
86
87
88
89

90
91

91

91
92
92
93
93
93

93
9h

9h

35-
36.

37.

38.

39-

no.

AZ.

1&3.

Analysis of variance for volume scores of biscuits..... 9h

Yields of crude gluten.extracted from non-irradiated
and irradiated all-purpose flour...eeeeeeeeeeeeeeeeoeee 95

Volumes of baked gluten balls made with.non-irradiated
and irradiated all‘purpose f10ur....................... 96

Analysis of variance for yields of crude gluten ex-
tracted from non—irradiated and irradiated all-purpose

fIOWOOOOOOOOOOOOOOOOOOOO00.0.00...OOOOOOOOOOOOOOOOOOO. 97

Analysis of variance for volumes of baked gluten balls
made with.non-irradiated and irradiated all-purpose

flourOOCOOOOOOOOOOOOOOOOO0.000.000000000000000000000000 97

Protein concentrations of acetic acid dispersions of
flour after centrifugation and dialysis for 72 hours
against citric acid-disodium phosphate buffer, pH 2.2.. 98

Analysis of variance for protein concentrations of
acetic acid dispersions of flour....................... 98

'Distribution of protein among the various electro-

phoretic components of untreated and irradiated flour
(ascending Patterns)000000000000OOOOOOOOOOOOOOOOOOOOOOO 99

Electrophoretic mobilities of the protein components
of triplicate samples of untreated and irradiated
£10111, (ascending pattern-3)....OOOOOOOOOOOOOOOOOOOOOOOO 100

I.

II.

III.

IV.

V.

VI.

VII.

II.
III.

vi

LIST OF PLATES

Representative electrophoretic patterns of wheat
protein extracted from non-irradiated flour and
flour irradiated at four dosage levels.............. 60

Electrophoretic patterns of wheat protein extracted
from crude gluten and from gliadin from.non-
1rradiated flourOOO0.0.0.0.0....OOOOOOOOOOOOOOOOOOO. 62

Triplicate electrOphoretic patterns of wheat
protein extracted directly from untreated all-
Purpose flour...OOOOOOOOOOOOOOO0.0000000000000000000 102

Triplicate electrophoretic patterns of wheat

protein extracted directly from.flour treated with
300,000 rep ionizing radiation..................... 10h
Triplicate electrophoretic patterns of wheat protein
extracted directly from.flour treated with 1,000,000
rep 1011121118 radiationOOOOOOOOOOOOOOOOOOOOOOOOOO... 106
Triplicate electrOphoretic patterns of wheat protein
extracted directly from.flour treated with 3,000,000
rep ionizing radiation............................. 108
Triplicate electrophoretic patterns of wheat protein

extracted directly from.flour treated with
10,000,000 rep ionizing radiation.................. 110

LIST OF FORMS

score Card--Baking POWdBI‘ BISCUILS. o e e e e e e e e e e e e e e O o 80
Triangle Test--Baking Powder Biscuits............... 81
ElectrOphoresis Experiment.......................... 82

THE EFFECTS OF HIGH VOLTAGE CATHODE RAY IONIZING
RADIATION ON SOME OF THE PHYSICAL AND CHEMICAL
PROPERTIES OF WHEAT FLOUR PROTEIN

INTRODUCTION

Insect infestation and fungal growth in stored grain,
flour, meal, and cereal products result in enormous annual
losses of these foods. Studies have shown that many of these
losses may be eliminated by use of ionizing radiations which
are capable of deinfesting, pasteurizing, or sterilizing
cereal products--depending upon the dosage level applied. In
experimental work, radiation-sterilized foods have not been
found toxic, nor has any evidence of carcinogenicity appeared.
Moreover, no radioactivity is induced in foods by the beta and
gamma sources used in the radiation process. However, there
has been evidence that certain.chemical and physical properties
of wheat protein are affected by irradiation. These chemical
and physical changes may result in alteration of baking prop-
erties, palatability, and nutritional value of the wheat.

Before irradiation of wheat flour can become commercially
feasible, the limitations of the process must be thoroughly ex-
plored and the end products shown to be acceptable. There have
been several published reports concerning some of the physical
properties and the biological value of wheat protein which has
been irradiated, but there have been no reports on the actual

nature of the chemical changes that occur.

In the present study, baking tests on biscuits made from
flour irradiated at low dosage levels of 20,000 to 500,000
rep were used to determine the effect of irradiation on the
baking quality and palatability of flour products. The next
step was to determine the effect of irradiation (20,000 to
10,000,000 rep doses) on the quantitative yield of crude gluten
which.could be extracted from.the flour. Then electrophoretic
analyses were performed on some of the protein fractions of the
irradiated flour in order to follow any chemical alterations
which.may have taken place during irradiation at dosage levels
of 300,000 to 10,000,000 rep. This latter phase of the study
was not an attempt to determine the definite composition of
flour proteins but rather to make a comparative study of the
proteins from.non-irradiated flour and flour irradiated at

several dosage levels.
REVIEW OF LITERATURE
Use of Ionizing Radiations

Insect infestation in stored grains and grain products
results in at least a five percent 1083 of the annual grain
production in the United States (White, 1953). The monetary
value of the lost product combined with the cost of measures
to combat these pests brings the annual bill to $300,000,000
(Cotton, 1952).

The best methods of insect control today depend on the

use of various chemical agents which.have been found exceedingly

effective when used on standing crops and stored unprocessed
grains. Toxic chemicals cannot, however, be applied to foods
that must undergo storage, during which the attacks of insects
are costly. This has led to the consideration of radiant
energy for the protection of stored products.

T1293 9_f_Radiant Energ. X-rays are among the oldest
known artifically-produced radiations and many investigators
have studied their effect on biological materials. However,
as pointed out by Robinson (1951...), their use in food preserva-
tion is somewhat limited. Hard X-rays (produced by an accel-
erating voltage of 185 kilovolts or more) have a relatively
large penetrating power, but low efficiency of generation
which makes their use uneconomical for large scale food pres-
ervation. Soft x-rays (produced by an accelerating voltage of
100 kilovolts or less) have relatively low penetrating power
which may be controlled by the design of X-ray units, but the
over-all depth of treatment is still limited by the high ab-
sorption of the soft X-rays.

Hassett (1956) has reviewed the various forms of ionizing
radiation, other than X-rays, which can be used to kill insects.
Each form has its own characteristics, advantages, and dis-
advantages. (a) Electron accelerators, including the Van de
Graaff electrostatic generator, which produce beams of elect-
rons traveling at extremely high speeds have been found effec-
tive in insect control. Electrons penetrate only a few milli-

meters of most substances so that their use is limited to

relatively thin layers of material or to surface irradiation
of thick objects. This disadvantage is balanced somewhat by
the relatively light shielding required to protect personnel.
Another advantage is that, unlike radioactive radiation, the
electron beam can be shut off when it is not wanted. The terms
electrons, cathode rays, and beta particles are used inter-
changeably to designate the flow of electrons. (b) Radioactive
elements or radioisotopes are another very useful source of
radiation. A number of cobalt-60 units of high intensity,
rated in thousands of curies are now in use. Cobalt-60 emits
strong gamma rays, which like X-rays, penetrate deeply so that
large objects can be treated. A disadvantage of cobalt-60 is
that heavy shieldingzmust be provided to protect personnel from
the effects of this radiation. (c) Means are being sought to
develop practical ways of concentrating radioactive wastes from
the operation of nuclear reacters, or piles. Both gamma and
beta radiation can be extracted from these wastes, or the crude
waste need only be concentrated and.the total radiation used.
19.1.33}; 93. Radiation. The units which are used for bio-
logical radiation dosage are derived from.the roentgen unit
(Friedlander and Kennedy, 1989). One roentgen unit, or r unit,
is "that quantity of X or gamma radiation such that the asso-
ciated corpuscular emission per 0.001293 g (weight of 1 cc of
dry air at 0°C and 760 mm pressure) of air produces, in air,
ions carrying l esul of quantity of electricity of either sign."
This means that one r produces 1.61 x 1012 ion pairs per gram

 

1esu = electrostatic unit

of air which corresponds to the absorption of 83.8 ergs of
energy per gram.of air. The r is a unit of the total quantity
of ionization produced by gamma or X-rays, and dosage rates

for these radiations are therefore expressed in terms of
roentgens per unit time. Because of its definition, the r unit
should not be used for radiations other than.X- or gamma rays.
Another unit, the roentgen-equivalent-physical, or rep, has
been used to express ionization in tissues caused by other radi-
ation (electrons, protons, alpha particles, neutrons). How-
ever, the rep is an unofficial unit and must be defined by

each investigator since it is interpreted differently by various
workers (Friedlander and Kennedy, l9h9; Pomerantz, 1956). It is
probably most often defined as the quantity of ionization pro-
duced when 93 ergs are dissipated by the radiation per gram of
tissue. The rad is an official unit which is now commonly

used in place of the rep. It is defined as the quantity of
ionization produced when 100 ergs are dissipated by the radia-
tion per gram of tissue (Gray, 1956).

Mechanisms 2E Action. The deinfestation or sterilization
of foods by means of irradiation involves the radiation of the
foods with sufficient energy to destroy the insects or bacteria.
The killing of insects or bacteria is brought about by some
chemical structural change induced by the radiation. Chemical
changes in the food being irradiated also occur. Morgan (1955)
has estimated that these changes affect only 0.0003 percent of
all the compounds present at sterilization dosages for bacteria

and therefore would affect even smaller amounts of the compounds

present at the lower dosages necessary for killing insects.
Thus these changes are not likely to affect the nutritive
value of the macronutrients which make up the major portion
of the food but they may be recognized by alterations in odor,
taste, and color of a food. Actual studies have verified this
theory, with the result that acceptability is probably a.more
serious problem than is nutritive value of the macronutrients.
However, extensive changes may take place in some of the
vitamins, due to specific labilities.

Two main theories have been advanced to account for the
changes brought about by irradiation. The first theory (target
theory) muggested that the changes were due to direct "hits" by
the radiant energy on the molecule of the food ingredients
(Crowther, 1926; Lea, 19h0). Later, many workers came to
believe the effects of irradiation were due primarily to the
action of the radiation products of the water present (radical
or activated solvent theory) (Fricke, 1935; Johnson, 1957).
Included in these radiation products of water were atomic hy-
drogen and.hydroxy1 radicals Which are both very reactive and,
in addition, secondary products of their reactions with.dis-

solved molecular oxygen, eg., the 02H radical and H20 Thus,

2.
the radical theory indicates that the effect of rays on matter
is produced largely indirectly, the oxidative processes being
then the most important unless the irradiation is carried out in
the absence of oxygen.

Evidence for the radical theory was accumulated in investi-

gations of the influence of irradiation on aqueous solutions of

amino acids. A given dose of radiation.had more effect on a
dilute solution than on a more concentrated one (Bhatia and
Proctor, 1951; Proctor and Bhatia, 1952). This dilution
phenomenon also was observed When carotene was dissolved in
ether (Goldblith and Proctor, l9h9). Therefore, other com»
pounds must be formed by the irradiation of ether, which act
in the same way as the hydroxyl radicals and hydrogen atoms of
activated water.

In practice, probably both direct and indirect action of
the radiant energy takes place in the same system, The indirect
theory, however, offers a wider basis for chemical change
(Johnson, 1957).

Effect 22 Insects. It was not long after the discovery of
x-rays by Roentgen in 1895 that a number of investigators
reported the effect of Roentgen rays on various insects. Davey
(1917) has reviewed the very early papers that were published.
He stated that most of these studies would have been.hard to
duplicate because the physical data relating to the x-ray dos-
age was so incompletely stated. However, they could be sum-
marized by saying that x-rays may act upon an organism.in one of
three ways: (a) to produce a stimulus to growth, (b) to produce
a destructive effect which takes place only after a certain
latent period, or (c) to produce an instant destructive effect.

Even though most of the early studies indicated that ion-
izing radiations could be effective in the destruction of inp
sects, there were some negative results reported also. For ex-

ample, Hunter (1912) concluded after an extensive series of

experiments, that X-rays had no apparent effect upon the
fertility or the development of the various stages of several
insect species and thus, no practical utilization in the de-
struction of injurious species. Runner (1916), on the other
hand, found that an X-ray dosage of .50 milliampere-minutes

at 65 kilovolts and a distance of 7.5 inches could be success-
fully used in the treatment of tobacco infested with the ciga-
rette beetle (Lasioderma serricorne). He found that in treat-
ing the egg stage, heavier exposures of radiation were required
to sterilize eggs which were near the hatching point than was
required to sterilize eggs newly laid. If the embryonic
development was well advanced, the beetles were hatched from
the eggs but failed to reach the adult stage. No effect on
the length of life of adults was noted, but the adults were
rendered sterile. When larvae were treated, activity and
development ceased and eventually death occurred before the
pupae stage was reached.

More recently with the coming into existence of the2more'
powerful particle accelerators and radioactive sources, other
workers have greatly extended the knowledge of radiation treat-
ments to insects.

Hassett and Jenkins (1952) used a cobalt-60 source to
demonstrate the killing power of gamma radiation on six Species
of insects including the Attagpnus and Dermestes larvae and the
Attagenus, Lasioderma, Sitgphilus, RhyzoPertha, Tribolium, and
Dermestes adults. The 1eng¢h.of time between treatment and

death of the insects was shown to be dependent upon the radia-
tion dosage. Dosages of 322,000 r killed nearly all insects
instantly whereas dosages of 128,800 r had varying lethal
effects but killed insects of all species in from seven to 26
days.

Baker (1953) and Toboada (1953) conducted tests using
accelerated electrons which indicated that a dose of 10,000
rep would sterilize flour beetle (Tribolium.confusum) and
granary weevil (Sitophilus granarius (L.)) eggs and prevent

 

.adults from reproducing. In further studies, Baker gtflgl.
(1953. 195k) found that a dose of 500,000 rep was lethal to
100 percent of adult flour beetles immediately after treatment,
whereas a dose of 250,000 rep was lethal to 100 percent of adult
granary weevils immediately after treatment. A dose of 250,000
rep was lethal to 92 percent of adult flour beetles one week
after treatment and a dose of 100,000 rep was lethal to 82
percent of adult granary weevils one week after treatment.
Irradiation of five Species of insects by gamma, cathode,
and X-rays indicated that each type of radiation was equally
effective (Proctor, g£.§l,, 195u). The insects studied were
confused flour beetle (Tribolium confusum.(Jacq, Duval)),
yellow mealworm (Tenebrie molitor (L.)), sawtoothed grain
beetle (nggaephilus surinaminsis (L.)), lesser grain.borer
(Rhizopertha dominica (Fab.)). and the cigarette beetle
(Lasioderma serricorne (Fab.)). The differences in radiosen-

sitivities of the several insect species were relatively small.

10

The rate of death was dependent on the dose used, higher
doses causing death more rapidly. Low doses of 12,500 rep
destroyed insects in all forms within a few weeks. In
general, however, 300,000 to 600,000 rep were required to
destroy the insects immediately. With doses of approxi-
mately 50,000 rep, all the insects died within 12 days of
treatment; with doses of 100,000 rep, all the insects died
within 10 days.

In most of the work reported thus far. in this review,
irradiated insects have all been killed, regardless of the
metamorphic form treated. Very recently, two studies have
been published where more attention was given to sub-100-
percent-lethal doses in an effort to provide some clue as
to the order of death of the irradiated insects and also
some basis for comparison.among species.

One of these studies was made in the United Kingdom
(Cornwall 32 31,, 1957) where the effect of gamma-radiation
from.a cobalt-60 source was determined for 17 species of

insects including 9, granaria, Q, oryzae, R, dominica, T,

 

 

castaneum, fI_‘. confusum, Q. surinamensis, 1:, minutus, E.

 

ferrugineus, S, cerealella, E, kfihniella, E, elutella, and
T, granarium, Irradiation prolonged the duration of larval
stages; but doses greater than 20,000 rep were required to
prevent the emergence of adults from.irradiated pupae. It
was possible to sterilize 13 species with a low dose of
6,000 rep and all adults of all species were sterilized at

11

50,000 rep. A dose of 50,000 rep produced no immediate
"knockdown" effect on adults. The length of time re-
quired to kill the adults varied with the species with P..-
minutus and C_. oryzae among the most susceptible and T,

castaneum and 11. dominica the most resistant.

 

Another investigation on sub-lOO-percent-lethal doses
has been reported by Nicholas and Wiant (1959) on612 grain-
infesting pests, including the confused flour beetle (illi-
bolium confusum Duv.), rice weevil (Sitophilus 23159; (L.)),
granary weevil (Sitophilus granarius (L.)), sawtoothed grain
beetle (gyzaephilus surinamensis (L.)), cadelle beetle
(Tenebriodes mauritanicus (L.)), Angoumois grain moth (Site-
$32.59. cerealella (01iv.)), Indian meal moth (Plodia _i_r_1_1_:_e_r_-
punctella (an.)), Mediterranean flour moth, (Egestia k__d_hn_i_._-
alla gall), lesser grain borer (Rhizopertha dominica (F.)),

 

yellow mealworm (Tenebrio molitor L.), flour mite (Acarus
121.32 (L.)), and book lice (Psocoptera). The pests were
treated with l-Mevl electrons at various doses to determine
the lethality of the treatment to the metamorphic forms and
to determine sterility effects upon adults. The results
suggested differences among species, graduated resistance
within a species increasing with age of the insect and a
more marked difference between the three moths and five bee-

tles. The 1.13502 (reduction, compared with controls, in

 

lMev = million electron volts
2LD50 = dose lethal to 50 percent of the test organisms

12

number of emerged adults from the given irradiated im-
mature form) for larvae for which complete data were pre-
sented, showed a range for the moths of 5,000-8,000 rep
compared with a range of l,200-h,000 rep for the beetles.
The corresponding 99.9 percent-lethal doses were about
h0,000-60,000 rep for moths and 10,000-20,000 rep for bee-
tles.

Thus, data from these various studies indicate that a
dosage of 10,000 rep ionizing radiation is generally suffici-
ent in cereals to sterilize insect eggs and prevent the adult
insects from reproducing. However, a 25,000 to 50,000 rep
dose is recommended to provide a greater margin of assurance
of egg and insect sterility. A dose of 300,000 to 600,000
rep is necessary to destroy insects instantaneously.

Wholesomeness g£_Irradiated.Foods. Extensive work-by

 

 

Meinke (195M) and others has demonstrated that no induced
radioactivity occurs through radiation preservation of foods
with fast electrons or gamma rays below eight to 10 Mev.
Radiation-sterilized foods have not been found toxic,
nor has any evidence of carcinogenicity appeared in the long-
term animal experiments which.have been conducted during the
past few years. Swift and Co. (Poling gt_gl,, 1955) have
studied the growth, reproduction, survival and histOpathology
of rats fed beef irradiated with electrons as two-thirds of
their diet over a life span (2 years) and through three suc-

cessive generations. The Swift data provide substantial

13

evidence that treatment of beef muscle with a sterilizing
dose (2,000,000 rep) of electron radiation did not signifi-
cantly impair the wholesomeness.

The University of Michigan (Burns 25.2l3’ 1956a)has
carried out a long-term.feeding and breeding experiment with
rats fed a diet receiving h,000,000 rep of gamma radiation.
In addition to growth and reproduction performance, data on
pathology and blood cell counts were obtained on four genera-
tions of animals. The evidence collected indicated that
aside from some destruction of vitamins, the radiation-
sterilized diet was wholesome for rats.

Richardson and Brock (1958) reported that mortality
rates and general appearance of four generations of rats on
irradiated (3,000,000 rep gamma radiation) synthetic diets
gave no indication that the irradiated diet was toxic.

The Quartermaster Food and Container Institute (Donald,
1957) has conducted a test with.human volunteers in which a
100 percent diet of irradiated foods was consumed for 12 weeks
without any adverse physiological effect.

Burns‘gt El. (1956b) also have reported that there was
no evidence indicating the presence of chronic or subacute
toxicity apparent in an experiment wherein chickens were
raised and maintained for 13 months on a wet-mash diet which
had received a sterilizing dose (3,000,000 rep) of gamma radia-
tion.

Work by the Wisconsin Alumni Research.Foundation.on

concentrates of food irradiated at 500,000-10,000,000 rad
levels and fed to rats, injected subcutaneously, or paint—
ed on the skin of mice, has not revealed thus far any car-
cinogenicity (Tepley and Kline, 1956).

Effect 9_n_ Nutritive’w 9; 399g Proteins. Metta and
Johnson (1956) and Metta gt_§l, (1957) have studied the bio-
logical value of the proteins of beef, milk, peas, and Lima
beans that have been heat- and radiation-sterilized and fed
to growing rats. All four of these protein sources are
deficient in sulfur amino acids. It was found that a 3,000,000
rep gamma irradiation dose did not affect the apparent or true
digestibility of beef or of milk protein. Neither was there
an effect on the biological value of beef protein, but the
biological value of milk protein was reduced by eight per-
cent upon irradiation as compared to a reduction of six per-
cent due to heat sterilization. Pea proteins also were signi-
ficantly reduced (six percent) in biological value after ir-
radiation with 3,000,000 rep gamma-ray irradiation but only
slightly reduced by heat treatment. In contrast to these
effects, Lima bean protein was improved considerably by heat
treatment but practically unimproved by radiation processing.
It was believed that this simply indicated a greater destruc-
tive effect of heat than of radiation on the trypsin inhibitor
present in Lima beans. Cystine and.methionine seemed to be
particularly sensitive to ionizing radiation in these studies.

Metta and Johnson (1959) also have investigated the effect

15

of gamma-ray irradiation and of heat-cooking on the
nutritive value of corn protein and wheat gluten in which
the limiting amino acids are generally lysine and/or tryp-
tophan. Finely ground corn and commercial wheat gluten

were suspended in water (35 percent concentration) before
irradiation. They were then vacuumpdried and incorporated
into balanced diets to provide 10 percent protein (N x 6.25)
on a moisture-free basis. The radiation-sterilized corn and
wheat gluten were found to be completely acceptable for the
growing rat during a 20-day feeding period. Irradiation at
2,790,000 rad, like heat-cooking, did not affect either the
digestibility or the biological value of corn protein or of
wheat gluten. However, irradiation of corn at 9,300,000 rad
lowered its digestibility by five percent but did not lower
its biological value. The lysine content of wheat gluten did
not change owing to heat cooking or irradiation.

In contrast to Metta and Johnson's study (1959) where
Wheat gluten suspensions were irradiated, Melehy (1958) in-
vestigated the nutritive value of whole ground wheat irradi-
ated in the dry form. She found no significant differences
in biological values or protein efficiency ratios among rat
diets containing the ground irradiated wheat supplemented
with vitamins, minerals, and corn oil. This lack of signifi-
cance indicated that neither the availability nor the growth-
promoting value of the wheat protein was affected by irradi-
ation doses up to 1,000,000 rep cathode radiation.

16

Biochemical Effects 92 Proteins. The major effects

 

of irradiation on proteins appear to be denaturation, degrad-
ation, and polymerization. The most obvious of these effects
is denaturation. For example, upon irradiation of raw evap-
orated milk at 3,000,000 rep, coagulation and precipitation
of the milk protein occur, whereas only a darkening of the
milk occurs with.heat sterilization (Johnson, 1957).

If one molecule is inactivated per primary ionization
(Lea e_t_ 53., 19th; Setlow, 1955), it can be calculated
(Bellamy, 1955) that at 1,000,000 rep irradiation of an amino
acid of molecular weight 150, one molecule in 2000 will be
ionized (damaged) while for a protein of molecular weight
150,000, half the molecules may be altered. These alterations
are difficult to demonstrate chemically but they do show up as
changes in physical properties such as the absorption spectra,
viscosity, heat stability, and solubility (Barron 23.21,,
1952; Barron and Finkelstein, 1952). The indirect effect of
the irradiation products of the water present have been shown
to be the main cause of these alterations (Barron and Finkel-
stein, 1952). There was less alteration in the absence of 02
(necessary for 02H formation) than in its presence. However,
addition of H202 in concentrations similar to those which
would be formed by irradiation.had no effect; thus the 02H
radical must be the effective agent causing the alterations.
Low doses of irradiation.have been shown to cause proteins to

be more heat-sensitive and less soluble(Barron, 1955).

,V

17

Precipitation occurred.upon irradiation of aqueous solutions
of serum albumin with 75,000 r but not with 50,000 r.
Changes also have been observed in the viscosity
(Proctor gt_§1,, 1952; Arnow, 1935; Barron and Finklestein,
1952) and sedimentation (Barron and Finklestein, 1952) of
proteins which are indicative of degradation or splitting
and also of polymerization (crosslinking) of protein.mole-
cules. When.using high irradiation dosages of 140,000,000
rep Bellamy (1955) reported that a series of proteins showed
on chromatography marked breakdown to smaller units regard-
less of whether they were irradiated dry, in a 20 percent
aqueous solution, or in a 20 percent solution of 0.1 N Hcl.
An example of polymerization was reported by Johnson (1957)
as the formation of a faster component on sedementation of
serum.albumin irradiated with 100,000 r X-ray radiation.
The presence of this faster component was prevented or re-
duced when cysteine was added to protect the protein by re-
acting with the irradiation products of water. This protec-
tion by cysteine indicated that the polymerization might
consist of dimerization brought about by oxidation of the
sulfhydroxyl groups of the protein with the formation of a
dimer, protein-S-S-protein. Barron and.his co-workers
(Barron.g£.gl,, 1952; Barron and Finkelstein, 1952) have
observed that the sulfur linkages (-SH) and the -OH (eq.,
of tyrosine) are more susceptible to radiation rupture than

are other groups (linkages) in the protein molecule.

l8

Masking of these groups by sulfation reduced irradiation
damage to the proteins (Bellamy, 1955).

Effect 9;: Uflgg Irradiated m in Baked Products.

A previous study made at Michigan State University (Nicholas
gt_al,, 1958) showed that a dose of about 50,000 rep
(cathode rays) was the level of irradiation at which distin-
guishable taste and/or odor changes were produced in bread
made from.irradiated flour and flour milled from irradiated
wheat. Loaf volumes were not significantly different among
treatments up to 500,000 rep with bread made from irradiated
flour, but with bread made from.milled irradiated wheat,
dose levels in the range of 50,000 rep gave significantly
higher volumes than at 250,000 rep.

Brownell 23 31, (1955) at the University of Michigan
found no affect on baking, eating quality, and flavor of
cake flour, all-purpose flour and bread flour which.had
been given a dose of 20,000 rep gamma radiation. Heavier
dosages resulted in some decrease in cake and bread quality.
The baking techniques which.they used were essentially
those used in a home rather than in a commercial bakery.

Brownell g§_§1, (1955) explored the possibility that the
gluten of bread flour could be modified by radiation doses
higher than 50,000 rep in such a way as to make a softer
crumb, but found cakes made with irradiated bread flour were
progressively of poorer quality with increasing doses of
gamma radiation. Cakes made with irradiated flour had low

19

total volume, were heavier, more compact, gummier, and had
a darker yellow crumb color. The gluten appeared to lose its
binding power and the flour had a drier or more starch-like
texture in.mixing. Some flavor changes were observed at a
radiation dose of 50,000 rep and cakes made from flour given
100,000 and 150,000 rep doses were considered to have a
different flavor in which the sweetness was increased,
particularly in the crusts. Flour given 500,000 and 1,000,000
rep doses had an odor resembling extreme mustiness.
‘When.making biscuits with irradiated flour receiving
20,000 to 150,000 rep doses, Brownell and co-workers (1955)
found it necessary to add 10 co. more milk to obtain a work-
able dough. There were no marked changes in appearance of
the biscuits made with flour exposed to gamma radiation, but
there were some noticeable changes in eating quality and
flavor. The judges thought that the off-flavor of irradiated
flour might be accentuated by the presence of double-acting
baking powder. They found (a) the biscuits made with irradi-
ated flour were gummier, and (b) off-flavors were present in
samples made with flour given dosages greater than 20,000 rep.
Loaves of bread made from.bread flour given a 20,000 rep
gamma radiation dose were equal in all respects except volume,
which was slightly smaller, to those made with the non-irradi-
ated flour (Brownell g£_§l,, 1955). Loaves made with flour
given 500,000 and 1,000,000 rep had a smaller volume and

darker crumb color. Bread made from.flour given a dose of

20

1,000,000 rep was considered to have a definite off-flavor.

A study made at Kansas State University (Milner and
Yen, 1956) was concerned with breadmaking and related prop-
erties of flour milled from.normal dry wheat with gamma
radiation at levels of 125,000 to 1,000,000 rep. The irradia-
tion dosage range covered that required for the elimination of
storage fungi as well as insects. Techniques employed were
essentially those used for commercial pan bread. A 125,000
rep treatment level resulted in apparent improvement of all
loaf properties over those of the control. Higher dosages
caused some down-grading of crumb properties. However, all
of the bread-~even that from wheat treated with 1,000,000
rep-~was acceptable with no easily detectable changes in
flavor and odor. These results were in direct contrast to
those of Nicholas g§_gl, (1958) and Brownell g£_§1, (1955)
who reported detectable changes in flavor and odor at 50,000
rep and above.

Lee (1959) has reported that loaf volumes of bread baked
from two grades of flour (patent and baker's) decreased with
increasing levels (250,000, 500,000, and 1,000,000 roentgens)
of irradiation of the flours with 0060 gamma rays.

Bauman gt_§l, (1957) were interested in increase of shelf
life of cake mixes by ionizing radiation. They obtained an
appreciable reduction in.number of bacteria with a dosage
level of 50,000 rep in dry white and spice cake mixes. The
white cake mix resulted in acceptable cakes through 50,000

21

rep. Dosages of 100,000 rep or more produced off-odors

and darkening in the dry white mixes and compact structure,
low volumes, and dark colored crumb in the baked cakes.

The spice cake mix appeared to be more resistant to irradi-
ation--withstanding 100,000 rep with no signs of deteriora-
tion in the cake mix or the baked product. At 500,000 rep
the spice cake was slightly off-color, slightly off-odor,
and compact.

Effect 93 Physical Properties 2; Riggs, Lloyd, Milner,
and Finney (1957) showed that the viscosity of sols made
from irradiated dry gluten or irradiated gluten sols decreased
with doses of X-rays up to 700,000 rep which is an indication
that the wheat flour protein molecules were broken into
shorter and/or more symetrical particles. The decrease in
viscosity was greater for the irradiated sols than for the
sols prepared from irradiated dry gluten.

Milner and Yen (1956) and Milner (1957) found gamma
irradiation of wheat in a dosage range of 125,000 to
1,000,000 rep caused no immediate change in fat acidity,
protein solubility, or fluorescence of acid extracts of the
grain. However, significant effects on the flour milled
from the irradiated wheat were found. A marked and regular
increase in autolytic sugar production (maltose value), reg-
ular decrease in gluten inhibitional capacity (sedimentation

value), and a drastic reduction in starch gelatinization

22

viscosity (amylograph value) occurred with increased
radiation treatment. These workers pointed out the two
possibilities that irradiation may either activate the
beta-amylase or render the starch fraction more suscep-
tible to amylase attack, with increased production of
reducing sugars. The second explanation was favored from
consideration of the reduced amylograph values. It was
emphasized that the starch structure must have been dras-
tically disrupted by the radiation dosages applied.

Lee (1959) also obtained a regular increase in maltose
values of flours irradiated with 250,000 to 1,000,000 roent-
gens). In addition, he observed corresponding rises in the
gassing power of doughs prepared from irradiated flours. He
stated that this increased reducing sugar production.must have
arisen from some autolytic process since without incubation,
the maltose values were very low for both control and irradi-
ated flours. No rise in saccharifying power was found for
papain extracts of the irradiated flours which indicated the
absence of a possible activation of the beta-amylase in the
flour. However, autolytic production of reducing sugars
from.mdxtures of non-irradiated flour and from.irradiated
starch increased with increasing irradiation of the starch.
Likewise, rises in the saccharifying power occurred for
papain extracts of non-irradiated flour when dispersions of
irradiated starch.were used as substrate. These findings

agree with those of Milner and Yen (1956) in indicating that

23

the increases in.maltose value for the irradiated flours
were due to some modification of the flour, starch, result-
ing in an enhanced susceptibility to hydrolytic cleavage
by beta-amylase.

Pomerantz of the Quartermaster Food and Container
Institute (1956) has reported several observations in work-
ing with doughs made from irradiated flours. Dough consist-
ency, as measured by farinograph absorption, appeared to
decrease with irradiation, except that with very high dosages
an increase was apparent. Dough development time increased
regularly with increased irradiation indicating a protein
degradation. Mixing tolerance decreased at higher levels of
irradiation, indicating further damage to protein and dough
structure. Reduced elasticity and weakening of dough, as
measured by farinograph, was apparent after irradiation.

Lee (1959) found that recoveries of crude gluten from
samples of flour irradiated with 250,000 and 500,000 r were
slightly decreased over the control, while recoveries from
samples of flour irradiated with 1,000,000 roentgens were
sharply decreased. He suggested that the decrease in.crude
protein recovery was an indication of changes in the protein
which contribute to the deterioration in baking quality of
the more highly irradiated flour.

By means of crude protein analyses, Lee (1959) determined
that 1,000,000 r had very little, if any, significant effect on

the nitrogen contents of various flour fractions extracted in

water or 50 percent alcohol. However, the water-soluble
nonprotein nitrogen fraction did appear to be greater than
the control which he thought might possibly mean that at an
irradiation level of 1,000,000 r, some fragmentation of
nitrogen-containing compounds may have occurred, giving rise

to greater amounts of nonprotein nitrogen.
Composition of Wheat Proteins

Historical. Recorded studies on wheat proteins date

 

back to the announcement by Becarri, an Italian professor

of medicine, in 1728 that he had separated gluten from flour
by washing a dough with water (Bailey, 19hh). After detailing
how gluten was recovered from.a refined flour by kneading

with water to form a dough, and washing the starch and solubles
from this dough with water, Becarri proceeded to compare the
gluten with animal products. For this reason, he is considered
to have been among the first to recognize in plant material the
occurrence of a substance having characteristics in common with
those of animal protein.

After Becarri's announcement, numerous other investi-
gators of the 18th and 19th centuries worked with the proteins
of wheat but their reports were characterized by confusion in
terminology and by a lack of agreement as to the number and
nature of the individual components of gluten. A complete
literature survey of these earlier studies was prepared by

Osborne in 1907. In addition, detailed accounts of many of the

individual investigations have been given by Bailey (l9uh).

Protein Fractions. The so-called modern period of
gluten protein investigations began with the comprehensive
and systematic studies of Osborne and his associates which
extended over a period of about 15 years. The results of
these studies were published by Osborne in 1907. He
characterized the proteins of the wheat kernel on the
basis of differences in solubility. The five main fractions
were listed as: gliadin, insoluble in neutral aqueous solu-
tions, but readily soluble in neutral 70 percent alcohol;
glutenin, soluble in very dilute acid and alkaline solution,
but insoluble in dilute alcohol or neutral aqueous solutions;
leucosin, an albumin-like protein, soluble in pure water; a
globulin soluble in salt solution; and an ill-defined
“protecse” present in small quantities. Globulin, albumin,
and proteose were found chiefly in the embryo, and gliadin
and glutenin formed nearly the whole of the protein of the
endosperm, or over 80 percent of the total protein matter of
the seed.

Osborne (1907) reported that the components of gluten,
which had been obtained by washing flour with.water, were
chiefly protein, together with a little starch, fat lecithin,
and phytocholesterin. The protein substance was thought to be
an intimate mixture of two distinct individual proteins,
glutenin and gliadin, present in nearly equal amounts. The

glutenin was believed to form the nucleus to which the gliadin

26

adhered and thus the gluten proteins were bound into one
coherent elastic mass.

Sandstedt and Blish (1933) and Stockelbach gt_§l, (1938)
reported fractionation studies which indicated that gluten
was composed of three fractions, namely, gliadin, glutenine,
and an intermediate they called mesonin. The glutenin fraction
was found to be soluble in very concentrated acetic acid and
gliadin in 50-70 percent alcohol or dilute acetic acid.
Mesonin was less soluble in neutral (50-70 percent) alcohol,
but was highly soluble in dilute acetic acid.

Later studies using comparatively recent and special-
ized techniques, instruments, and criteria that were not
available to Osborne have made evident the inadequacy of his
characterization of gluten as a simple mixture of two indi-
vidual proteins. It is now thought that gluten is composed
of several, if not many, protein components and that Osborne's
"glutenin” is a derived, not a natural protein. However,
even though the true character of gluten has not been fully
ascertained, it has been convenient for purposes of identifi-
cation and discussion to retain the terms "glutenin" and
"gliadin" in referring to later studies which have been based
largely on the earlier work of Osborne. 0f the two protein
types, much.more is known about gliadin--soluble in dilute
alcohol and dilute acids-~than of glutenin, which is far less
soluble than gliadin and more indefinite as to composition

and properties.

27

Due to the presence of substantial quantities of starch,
fat, and mineral matter, which cannot be removed by the con-
ventional washing process, the term."crude gluten” should be
commonly applied to the proteinaceous material recovered by
washing a flour dough under water. Blish (l9h5) stated
that crude gluten as isolated by the washing-out procedure
contained an average water content of 65 percent, while its
dry substances contained 70-80 percent protein, 5-15 percent
residual carbohydrates (chiefly starch), 5-10 percent lipids,
and a small quantity of mineral salts. Sullivan (195A)
reported the composition of gluten to be 85 percent protein,
8.3 percent lipid, 6.0 percent starch, and 0.7 percent ash
(dry weight basis). Some variation in water content of wet
crude gluten has been noted by Blish (19A5) and a high water-
absorbing capacity is likely to be associated with desirable
physical and mechanical properties.

Elements. Osborne's (1907) elementary analysis of
gliadin and glutenin resulted in the following figures:

Gliadin Glutenin
Carbon 52.32 52.8h
Hydrogen 6. 6 6. 3
Nitrogen 17.66 l7.h9
Sulfur 1e03 1008

Since the two percentage values for nitrogen agree so closely,
5.7 serves as a factor for determining the quantity of protein

in gluten or in various protein fractions prepared from it.

Amino acids. Blish (l9h5) has assembled the known
amino acid compositions (as percentage of total protein)

of gluten, gliadin, and glutenin as follows:

Gluten Gliadin Glutenin

O O
Arginine A.3 3.2 h.7
Lysme 201 0.6 109
Histidine 2.u 2.1 1.8
Tyrosine h.2 3.1 5.1
Tryptophane 1.1 0.9 1.8
Phenylalanine 2.0 2.5 2.0
Cystine 1.9 2.3 1.7
Methionine 3.3 2.3 ---
Serlne "" 0.1 0 e7
Threonine 2.5 3.0 ---
Leucine and Isoleucine 6.0 6.0 6.0
Valine 300 3.0 100
Glutamic acid 36.0 M6.0 27.2
Aspartic acid --— 1.h 2.1
Glycine --- 1.0 1.0
Alanine 5.5 2.5 h.h
PI’Oline 11 .0 13 e 2 (4-014-
Hydroxyproline --- --- ---
Ammonia h.5 5.1 u.0

The outstanding features of gluten protein composition is
the high content of glutamic acid, especially in gliadin,
where glutamic acid constitutes nearly half of the entire
protein substance. There is also a large proline content
and relatively small amounts of the basic amino acids and

of tryptophan.
Electrophoretic Studies on.Wheat Proteins

The charge-density distribution on the surface of
colloid particles and their resulting electrokinetic be-

havior may be studied by the moving boundary method of

28

electrophoresis. By this technique, the solution of the
colloid is stratified under a suitable solvent and the be-
havior of the colloid/solvent boundary under the influence
of an electrical field is observed by suitable optical
methods.

ElectrOphoresis is one of the more recent techniques
which has been used to show the inadequacy of Osborne's
characterization of gluten as a simple mixture of two indi-
vidual proteins. However, the "glutenin" portion of the
ghuten protein has not satisfactorily lent itself to electro-
phoresis because of failure thus far to find a suitable sol-
vent in which it can be dispersed. Accordingly, most of the
studies involving this procedure have been restricted to the
more soluble "gliadin" fractions, and even in these instances
there is sometimes doubt as to whether the protein is
molecularly dispersed.

One of the first electrophoretic studies of gliadin,
using the Tiselius electrophoresis apparatus and the techniques
of Longsworth (l9h2), was reported by Schwert 23 21, (19AM).
Gliadin was prepared according to the alcohol method of
Osborne (1907) and the acetic acid peptization method of Blish
and Sandstedt (1926). Using acetate buffers at pH of 3.8 and
ionic strength of .01, Schwert gt'gl, (19hh) found no significant
difference between patterns for a gliadin prepared from.a
pure durum.flour and that prepared from a commercial mixed

flour. However, small differences observed among patterns

30

for different gliadin preparations from the same or
different flours indicated that every preparation of
gliadin was likely to show small variations in ratio of
components.

On the basis of the electrophoretic data which Schwert
gt_g1, (19MB) obtained, it was apparent that gliadin was
not an electrophoretically homogeneous material. It was
equally evident that the components of which it was con-
stituted did not migrate independently in solution under
any of the conditions studied. Pronounced asymmetry of the
patterns obtained for the ascending and descending boundaries
and variation in mobilities and relative areas of the several
fractions which were separated under various conditions were
indications of the occurrence of complex formation. Elec-
trical interaction (interionic) did not appear to account for
the complex formation since increase in ionic strength of the
systems failed to cause any noticeable decrease in the asym-
metry or complexity of the patterns. Neither time of elec-
trophoresis nor field strength influenced the mobilities of
recognizable peaks in the gliadin patterns. Changes in ionic
strength, protein concentration, nature of the buffer system
and pH caused radical changes in the contour of the patterns
obtained. Although it was found possible to obtain a dis-
tinct separation of fast and slow fractions from.the main
body of the complex, the fractions obtained were believed

to be mixtures of components themselves. Isoelectric points

31

determined on samples of these fractions showed that the
isoelectric point of the fast fraction was at pH 7 while
that of the slow fraction was at pH 5.

Laws and France (l9h8) made a comparative study of
some protein fractions of flour from various wheats by
use of the moving boundary method of electrophoresis.
They found it impractical to study the proteins in either
alcoholic buffers or in aqueous alkaline buffers with a
pH of 10.2. ‘When the electrophoretic analyses were done
in acetic acid and dialyzed against citric acid—disodium
phosphate buffer, pH 2.15, there was marked asymmetry in
the two boundaries, indicating very decided component inter-
action. The patterns showed that proteins from all flours
studied apparently undergo the same type of interactions
because all ascending boundaries were similar and all descend-
ing boundaries were similar. The patterns for protein ex-
tracted directly from the flour and for freshly washed gluten
were very nearly identical except for the slow-moving comp
ponent which was much more prominent on the patterns made
from the protein extracted directly from the flour. It was
suggested that this was probably due to the presence of part
of the water-soluble protein present in the gluten suspension
because of the method of sample preparation. The slowqmoving
"component" moved so very little that it could easily be
taken for the well-known delta and epsilon anomalous bound-

aries except for the fact that the area under the remainder

32

of the peaks only accounted for about 80 percent of the
protein present in the solution.

The electrophoretic patterns failed to reveal any
significant differences in glutens from flours milled from
three varieties of wheat which indicates that either there
was virtually no difference in the composition of the gluten
protein from poor and good quality flour, or the development
and manipulation of the gluten'during the sample preparation
altered the protein in such a manner as to destroy existing
differences. Moreover, baking tests with flour fortified
with wet and dry gluten washed from.the various flours ap-
peared to confirm.the results obtained by electrophoresis
technique, namely, that the glutens from.the flours studied
were very similar in properties.

Kondo and his co-workers in Japan studied the electro-
phoretic behavior of both wheat-glutenin and wheat-gliadin.
For the glutenin studies (Kondo and Chiba, 1951) the electro-
phoretic patterns were obtained in Kolthoff's buffer solution
(NaOH-NaZHPOu), pH of 11.0 and ionic strength of 0.16 at a
field strength of h.863 volt/emu, for 6000 to 8000 seconds.
From.the patterns obtained, it was concluded that the glu-
tenins isolated from five Species of wheat were homogeneous,
monocomponent proteinS--identica1 in electrophoretic behavior.
The mobilities of the single glutenin components ranged from
8.0 to 8.25 x 10"5 cm.2 volt'l sec.'1 for the various flours.

Even though the ascending and descending patterns agreed in

33

number and mobility of components, they were not identical
in area. The peaks produced in the ascending patterns
were much higher than those for the descending patterns.

For the studies of gliadin, Kondo and Owada (1952)
used an acetate buffer solution of pH 3.8 and ionic strength
of 0.01, at a field strength of 8.h6 volts/cm, for 6630 sec-
onds. The patterns obtained showed that the preparations
from four species of wheat were identical in electrophoretic
behavior though heterogeneous and abnormal. It was suggested
such abnormal electrophoretic patterns may depend upon the
fact that the gliadin.may disperse heterogeneously in the
buffer solution and the dispersed phase consisted of the
various gliadin particles with different hydrations, electric
charges, volume contractions and Specific viscosities.

Fidanza 23 El! (1952) reported that gliadin from.a hard
wheat did not give an electrophoretic diagram characteristic
of a pure substance in the Tiselius electrophoretic appara-
tus. They used solutions of 0.6 to 0.7 percent concentration
in a acetic acid-sodium.acetate buffer at pH 3.8.

Butler, 35 El, (19H9) partially separated the gliadin
fraction of gluten by means of a thermal fractionation
process from 70 percent alcohol. The gliadin fraction was
then examined electrophoretically in sodium.chlor1de-acetic
acid. In the order of increasing solubility, the fractions
obtained were designated as a, b, and c. Fraction a was al-

most homogeneous, while fractions b and c exhibited the same

3h

five peaks as did the whole gliadin, but in different ratios.

Mills (195h) separated gliadin into two fractions by
means of a thermal fractionation process from.70 percent
alcohol. He subjected both fractions to electrophoretic
analysis in buffer systems consisting of sodium acetate-
hydrochloric acid, glycine-sodium.chloride-hydrochloric acid,
and acetic acid-sodium chloride. In addition to these buffers,
sodium acetate-acetic acid, potassium hydrogen phthalate-hydro-
chloric acid, sodium citrate-hydrochloric acid and citric acid-
disodium phosphate were also used for fraction 2. It was found
that results over a narrow range of conditions using any of the
other buffers except the phthalate were comparable with those
of sodium acetate-hydrochloric acid buffer under similar con-
ditions. No significant difference in the patterns was ob-
served with a variation of 2.5 to 3.5 pH. Their results indi-
cated that the electrophoretic examination of gliadin is best
performed in buffers of ionic strength of 0.0h at a protein
concentration of O.h percent. At lower ionic strengths the
descending boundary became very diffuse and in some cases pre-
cipitation of protein took place. At an ionic strength of
0.0k the “enantiography” of the patterns in the two limbs was
greatly improved, although the descending limb became too
diffuse to observe towards the end of the run.

During electrophoresis of whole gliadin and fraction 2
(Mills, 195h) in sodium acetate-hydrochloric acid at values

of ionic strength of less than 0.03, patterns were produced

35

which showed a number of rapidly moving convective bound—
aries in the cathode limb at the end of 30 minutes at a field
strength of 5.5 volts/cm. At ionic strengths between about
0.03 and 0.0k, patterns were obtained in which five distinct
peaks could be discerned in the ascending limb. [Migration
was cathodic and the boundaries moved fairly rapidly under

a potential gradient of h volts/cm, The proportion of the
leading component relative to the others decreased considerably
as the protein concentration was reduced from.l.5 percent to
0.8 percent to 0.h percent. At an ionic strength of 0.0h, the
maximum solubility of the protein fell off rapidly. Using
sodium.acetate-hydrochloric acid buffer at an ionic strength
of 0.02, fraction 1 gave a pattern which showed a single,
rapidly moving boundary which partly resolved into two peaks
after 170 minutes at a field strength of 3.6 volts/cm. When
the ionic strength was increased to 0.0a it was difficult to
get sufficient protein into solution and the boundaries be-
came very diffuse at the end of the run.

Lonti, 22 21, (195k) showed gluten to consist of three
major and two minor fractions by electrophoresis of wheat
gluten in 0.015 M lactic acid-sodium.1actate buffer, pH 3.5,
containing 0.1 percent 2-mercaptoethanol. Their findings
were substantiated by the descending boundary procedure with
gluten in 0.01 M formic acid-sodium.formate buffer, pH 3.73,
with 0.1 percent 2-mercaptoethanol.

No electrophoretic studies on irradiated wheat flour were

found in the literature.

36

EXPERIMENTAL PROCEDURE

Commercial all-purpose flour (Gold Medal) to be used
in this study was irradiated with a 1,000,000 volt, reso-
nant-transformer, electron beam generator (Knowlton at El:
1953) located in the Agricultural Engineering Department,
Michigan State University. Dose measurements were based on
ionization chamber dosimetry.

The dosage levels ranged from 20,000 to 10,000,000 repl.
The lower levels used are capable of sterilizing insect eggs
and preventing adult insects from reproducing. The higher
levels used bring about instant destruction of insects and
prevent fungal growth. It was hoped that this wide range of
treatment levels would permit the observation of both physi-

cal and chemical changes in the flour protein.
Baking Tests on Biscuits

Materials. Four lO-pound lots of all-purpose flour to
be used.in.making'biscuits were exposed to varying doses of
ionizingzradiation while a fifth 20-pound lot served as a non-

irradiated control as Shown below:

Lot Treatment
1 None - control

2 20,000 rep

50,000 rep

100,000 rep

500,000 rep

 

1One rep (roentgen equivalent physical) = the absorp-
tion of 93 ergs per gram of material of unit density

37

Two types of baking powder were used:

1. Tartrate powder (Royal).
2. Sulphate-phosphate powder (Calumet).

The fat was a hydrogenated vegetable Shortening (Crisco).
Preparation. All ingredients except the milk were

 

placed in a constant temperature box (25°C) for at least
2h hours prior to use. The milk was brought to room tempera-
ture shortly before using. Each batch of biscuits were mixed
by the formula (Brownell g£_§1,, 1955) Shown in Table 1.

Table 1. Formula used in making biscuits with irradiated
all-purpose flour.

 

 

Flour 2 cups 225 gm. Shortening l/h cup 50 gm.
Baking powder 3 tsp. 12 gm. Milk 180 cc. 186 gm.
Salt 1/2 tap. 2 gm.

 

The flour, baking powder, and salt were Sifted together
once into a 2-quart mixing bowl. The fat was cut in with a
pastry blender using 60 strokes. The bowl was given a one-
sixth.turn between strokes and the sides were scraped after
the 15th, 30th, k5th, and 60th strokes. A cavity was formed
in the center of the flour mixture and the milk was added all
at once and mixed in lightly with a k-tine fork, using 30
strokes. The dough was then formed into a ball and removed
from the mixing bowl with a Spatula and placed on a thin layer
of flour on a bread board. The dough was rolled without comp
pressing it in the flour, until the sticky surface was covered.

The dough was kneaded 10 times lightly with the fingers and

38

rolled to 1/2 inch thickness with the aid of strips of wood.
Ten biscuits were cut with a 2-inch biscuit cutter. Three
to four teaSpoons of flour in addition to the amount called
for in the recipe were used on the bread board during the
kneading, rolling, and cutting of the biscuits. The bis-
cuits were baked at 218°C. (k25OF.) for 10 minutes on an
ungreased baking sheet. On removal from.the oven, the bis-
cuits were cooled on the baking sheet before evaluation.

Evaluation g§.the Dough and Biscuits. Evaluation was
accomplished by means of two panels of tasters as well as by
several mechanical tests. The tests were conducted over a
three-month period with limits of three tasting days a week
and two judgments a day at least 30 minutes apart. The bis-
cuits were tasted after they had cooled to room temperature.
Mechanical measurements on the raw dough were made immediately
after the biscuits were rolled out and on the cooked biscuits
within 1 1/2 hours after baking.

Three batches of biscuits made with untreated control
flour and flour treated at two levels of irradiation and tar-
trate baking powder were baked on each judging day until each
treatment level had been scored (1-7 range) four times for odor,
color of the crust and crumb, flavor, tenderness, lightness,
texture, moisture, and general eating quality by a panel of three
judges. Thereafter, flour batches of biscuits made with the con-
trol flour and one treated flour and two types of baking powder
were baked on each judging day. Two triangle tests were

39

then conducted each day with biscuits made with tartrate
rapid-action baking powder being used in one and biscuits
made with sulphate-phosphate double—action baking powder
being used in the other. The panel was asked to select the
odd sample and state whether they preferred it to the other
samples in the triangle tests. Samples of the two types of
score Sheets are included in the Appendix as Forms I and II.

The original taste panel consisted of three persons
who had not been informed of the irradiation treatment of
the flour and had never worked with irradiated foods. They
served as judges for both the scoring tests and triangle
tests. Additional judgments in the triangle tests were pro-
vided by a panel of seven persons who were aware of the
irradiation treatment.

Mechanical tests were made on six batches of dough and
biscuits made with tartrate baking powder and flour from
each level of irradiation. Triplicate measurements of come
pressibility and elasticity of the dough were determined
immediately after the dough.was rolled out by means of a
"Micrometer Adjustment" Penetrometer (Sold by Arthur H.
Thomas Co., Phila. Pa.). The compressibility of the dough
was recorded as the change in height of a core of dough one
inch in diameter and one-half inch in.height caused by a
load of 172 gm.acting on the surface for 15 seconds. Elas-
ticity of the dough was recorded as the change in height 15

seconds after the load had been removed from the core of

#0

dough. Specific volume of the biscuits was measured after
the biscuits had reached room temperature in a Volumemeter
(Manufactured by the National Manufacturing 00., Lincoln,
Nebr.) by the method of rapeseed displacement using five
biscuits as a unit.

Statistical Analyses. Analyses of variance were per-

 

formed on the taste panel scores for the various characteris-
tics of the biscuits and on the mechanical measurements of
the dough and biscuits. When a significant difference (F-
test) due to irradiation treatment of the flour was found
the treatment level means were compared using Studentized
multiple ranges (Duncan, 1955).

Levels of significance for the triangular taste tests
were determined from published tables for both ability to
choose the odd sample and to select the control as a prefer-

ence (Roessler §t_gl,, 19h8, 1956).
Gluten'Yields

Crude gluten yields were determined for flour samples

which had received the following doses of ionizing radiation:

 

 

L22_ Treatment Lgt’ Treatment_

1 None - control 6 None - control
2 50,000 7 300,000

g 100,000 8 1,000,000

5 500,000 10 10,000,000

Prgparation 2£_Gluten. Six replicates of crude gluten

were extracted from.samples of each lot of irradiated flour

A1

by the following method (Meiski, 1957). Twenty-four milli-
liters of tap water were added to 30 grams of flour which had
been placed in a glass bowl of an electric mixer (Kitchen Aid
Model 3-C). The flour and water dough was mixed 2 minutes at
speed 1, scraped down with a rubber Spatula; mixed 2 minutes
more at Speed 1, scraped down again; and mixed a final 1
minute at speed 1. The total mixture was scraped from.the
bowl with a rubber Spatula to Saran.wrap, which had been
sprinkled with water, and was rested for 30 minutes at a con-
stant temperature (25°C.). At the end of the rest period the
dough was scraped from.the Saran.Wrap into a glass mixing
bowl containing 750 milliliters of tap water at 25 t 1°C. and
washed at Speed 1 for 2 minutes. The wash water was then
poured through a wire Sieve. The gluten retained in the
Sieve was squeezed 10 times under running tap water to re-
move more starch. The gluten was again placed in a bowl of
water and received three more 2-minute washings at Speed 2.
The wash.water was changed at the beginning of each 2-minute
washing. The pH of the tap water was taken each day. The
gluten was squeezed 10 times under tap water after each of
the three wash periods. It was then placed on wire mesh in

a desiccator containing a saturated solution of Mg(NO3)26H20
to maintain a constant humidity of 52 percent and the dessi-
cator was placed in a constant temperature box set at 25°C.
for 30 minutes.

At the end of the draining period, the gluten was weighed,

#2

placed back in the mixing bowl and mixed 1 minute at Speed 1
and 9 additional minutes at Speed h. It was weighed again
after mixing and Shaped into a ball with 10 folding strokes.

The gluten ball was baked 15 minutes in an oven set at
232°C. (ESOQF.), transferred to an oven set at lh9°C. (300°F.)
and baked an additional 35 minutes.

'When gluten yields of less than 3 grams were obtained,
the mixing times and baking times were shortened. In such
cases mixing consisted of beating the gluten for 1 minute at
Speed 1 and h.minutes at speed A and baking was carried out
for 10 minutes at 232°C. and 20 minutes at lh9°C.

Volume Measurements. The volume of the gluten ball ob-

 

tained was determined by means of rape seed displacement in a

pint-Size plastic box.
Electrophoresis

Four 2-pound lots of all-purpose flour to be used in the
electrophoresis studies were exposed to the following doses

of ionizing radiation:

 

Lot Treatment
‘6‘ None-control
7 300,000 rep
8 1,000,000 rep
9 3,000,000 rep
10 10,000,000 rep

Preparation 22 sample directly from.flour. Twenty grams
of flour were stirred in 250 m1 of cold 0.07 N acetic acid
by stirring for 5 minutes in a‘Waring Blendor. The suspen-

sion was then centrifuged for 10 minutes at 2000 rpm. to

1+3

remove traces of starch and to destroy the foam formed
during stirring. 0f the suspension thus obtained, 100
milliliters was heated to 92°C. and held there for 2 min-
utes to destroy the proteolytic enzymes present. This
treatment does not injure the protein (Olcott gt_gl,, l9u3).
The 100 milliliters of suspension were then dialyzed against
2 liters of a citric acid-disodium phosphate buffer of pH
2.2 in the cold room.(h°C.) for approximately 72 hours in
order for the buffer and sol to reach equilibrium as Shown
by conductivity measurements. The suspension was centri-
fuged again just before being placed in the electrophoresis
cell to remove the material which salted out. This last
centrifugation was done at 12,000 rpm. for 30 minutes in a
refrigerated superspeed centrifuge (Ivan Sorvall, Inc.,
Norwalk, Conn.).

Preparation 9}; sample £39m 9_I_'_u_C_1_e_ gluten. Crude gluten
was prepared from 20 grams of non-irradiated flour as out-
lined in the previous section on Gluten Yields. The moist
crude gluten obtained was then used to prepare the electro-
phoretic sample by the same procedure as described above for
the whole flour sample.

Preparation 2£_sample fggm_gliadin. Crude gluten was
prepared by the procedure outlined in the previous section
on Gluten Yields. The gliadin was then extracted by the
method of Blish and Sandstedt (1926). Thirty grams of crude
gluten were treated with 500 milliliters of .07 N acetic acid

for 2 1/2 hours with vigorous shaking at frequent intervals.
The suspension was allowed to stand 1 1/2 hours before
siphoning off about 2/3 of the supernatant liquid. Approxi-
mately 250 milliliters of .07 N acetic acid was then added
to the residue, with stirring, and the insoluble portion
again allowed to settle before siphoning off the supernatant
liquid which was added to the extract previously withdrawn.
The total extract was then filtered water clear through a
thick mat of filter paper pulp on'a large Buchner funnel.

The clear liquid thus obtained was treated with 10 grams
of lithium chloride. There was an immediate precipitation of
gliadin, which readily collected on the stirring rod. This
was readily dissolved in 250 milliliters of 60 percent ethyl
alcohol. The gliadin was then reprecipitated by pouring into
absolute alcohol containing ether and a little LiCl. It was
redissolved in 60 percent ethyl alcohol, reprecipitated in
water containing LiCl, again redissolved in 60 percent alcohol,
and finally reprecipitated in absolute alcohol and ether.

_ The gliadin thus obtained was then suspended in .07 N
acetic acid and prepared for electrophoresis in the same manner
as used for the Samples taken directly from.flour.

Nitrogen.DeterminationS 92;the Protein Extracts. Dupli—

 

cate one milliliter aliquots of the protein extracts prepared
for the electrophoretic determinations were analyzed by the
micro—Kjeldahl method (Asso. of Off. Agr. Chemists, l9h5).

Protein content was then calculated as the product of the

LL5

nitrogen value and the factor 5.7.

Electrophoretic Procedure. An attempt was first made

 

to use a filter paper electrophoresis cell of the ridgepole
type (Spinco Model H, Specialized Instruments Corp.) with
several different buffers at various levels of pH and ionic
strength. However, none of these tests were successful since
it was not possible to get the protein to move along the
paper.

AS a consequence, the Tiselius moving boundary method
of electrophoresis was adopted since it offers one of the best
means of detecting differences in proteins despite the lack
of Suitable neutral solvents. The electrophoretic apparatus,
including ll-milliliter analytical cells, (manufactured by
the Klett Manufacturing Co.) used was located in the Agri-
cultural Biochemistry Dept., Michigan State University. The
procedure followed is presented and discussed in a manual
(Klett Mfg. Co., l9h8) which accompanied the apparatus and is
essentially as described in the literature (Abramson, g£_gl,,
19h2; Longsworth, l9h2). Runs were made at a temperature of
2.100. It required 1 1/2 hours at a field strength of 6.5 to
7.1 volts per centimeter to complete a determination in citric

acid-disodium phosphate buffer1

at pH 2.2, ionic strength
0.02h. The electrOphoretic patterns, as recorded on the photo-

graphic plates by the schlieren-scanning method, were enlarged

 

1The citric acid-disodium.phosphate buffer was prepared
by adding ho milliliters of 0.2 M NaZHPO to 1960 milliliters
of 0.1 M citric acid for each electropho etic run.

116

by projection and their outlines were traced on graph paper.
The various maxima of the gradient curve were separated by
the minimum ordinate method (Longsworth, l9h2). Then the
area of each component was measured with the aid of a pla-
nimeter (manufactured by Keuffel and Esser Co., N.Y.)--an
instrument which automatically integrates areas by tracing
their outline with the stylus of the instrument. For estimat-
ing the relative concentration of each component, the size of
its area was computed in terms of percentage of the total
area. The calculation of the electrOphoretic mobilities con-
sisted of measuring:the distance from the center of the start-
ing position of the boundary on the enlarged tracings of the
electrophoretic diagrams to the various maxima of the gradient
curve. The over-all magnification represented by the product
of the photographic and the projection magnification factors
wastaken into consideration in calculating the actual distances
in the cell. The actual distances were divided by the potential
gradient, F, and the time of electrolysis in seconds in order to
arrive at a value for the mobility in terms of centimeter"2
second'l. The potential gradient F, was computed from.the
formula F *='i/kq, where i was the current in amperes, k was
the conductivity of the system in the cell, and q was the cross-
Section of the cell in centimeterz. The relative concentrations
and mobilities reported were calculated for the ascending pat-
terns only.

A sample form for recording data necessary for the calcula-

tion of mobilities is shown in Form.3 in the Appendix.

#7

RESULTS AND DISCUSSION
Baking Tests on Biscuits

The tables containing the taste panel scores (Tables
12-20), mechanical measurement data (Tables 21-23), and
statistical analyses (Tables 2h-35) for the biscuit tests
are found in the Appendix.

Organoleptic Tests. The various characteristics which

 

were scored numerically (l to 7) will be discussed first
and the triangle tests later.

Table 2 gives the average taste panel scores for the
biscuits made with irradiated all-purpose flour and tartrate
baking powder. Each of these scores is an average of the
scores of three judges, each scoring biscuits from.four
batches identical in treatment.

The average scores for odor of the biscuits ranged from
h.2 to 5.h. Statistical analysis indicated that the mean
score for odor of the control biscuits was significantly
higher than the mean scores for the biscuits made with flour
irradiated at either 50,000 or 500,000 rep.

The color of the crust of the 500,000-rep biscuits was
noticeably darker than.that of the control, but the judges
did not seem to object to it. The average scores for the
color of the crust varied from h.6 to 5.3, but the differ-
ences were not significant. The average scores for color of

the crumb decreased Significantly in the order of increase in

k8

Table 2. Average taste panel scores (3 judges and h repli-
cations) for biscuits made with irradiated all-
purpose flour and tartrate baking powder.

 

 

Control 20,000 g50,000 100,900 500,000

8dgr t §.ha %.§ .5 b.2 %.3
Gas: : an, 621213 539° he he 3:2
Flavor 500 500 LL05 %.7 309
Tenderness (4-08 (4-08 (4.08 03 (4-06
glglgtness go; go;- geg 50%- ill-oi

e ure 0 e e e 0
Moisture 5.2 h.9 5.2 5.0 h.7
General eating

quality h.8 h.8 h.7 h.8 h.l

 

“Significantly higher than the 50,000-rep and 500,000-rep
biscuits at the .05 level.

bSignificantly'higher than the 100,000-rep and 500,000-
rep biscuits at the .01 level.

0Significantly higher than the 500,000-rep biscuits at
the .01 level.
dosage of ionizing radiation applied to the flour. These
decreases, however, were not Significant except for the highest
level of irradiation. The grayish-tan color of the crumb of
the 500,000-rep biscuits was quite noticeable compared to the
creamy-white color of the control products. The mean score
for color of the crumb of the 500,000-rep biscuits was Signi-
ficantly (.01 level) lower than the scores of the control
biscuits and biscuits from all other treatment levels. The
mean score for color of the crumb of the 100,000-rep biscuits
also was significantly (.01 level) lower than.the mean score
of the untreated control, but not of the 20,000-rep and
50,000-rep biscuits.

4’

1‘

119

There were no significant differences due to treatment
for the average scores of the other characteristics--flavor,
tenderness, lightness, texture, moisture, or general eating
quality, but several observations were noted. The average
scores for flavor tended to decrease with increase in dosage
of ionizing radiation, with a range of 5.0 for the control
products down to 3.9 for those made with flour treated at
500,000 rep. The average score for tenderness was Slightly
higher for the 100,000-rep biscuits than for any of the
others (5.3 as compared to h.6-h.8). The average scores for
lightness ranged from 11.3 to 5.1 while those for texture
varied from.a.h for the 500,000-rep biscuits to 5.h for the
50,000-rep biscuits. Average moisture scores only varied
from h.7 to 5.2. The average scores for general eating
quality were practically identical for the control biscuits
(u.8) and those made with flour given dosages of 20,000 rep,
50,000 rep, and 100,000 rep (n.8, n.7, and n.8, reSpectively),
while the average score for the 500,000-rep biscuits was
somewhat lower (h.l).

The triangle test results are shown in Table 3. The
judgments of the three panel members who were unaware of the
irradiation treatment (designated as Panel I) were small in
number, but nevertheless indicated that these judges were
able to select the odd sample a sufficient number of times at
the 50,000-rep and 500,000—rep levels of irradiation to Show

that the selection was not due to chance alone. The number

50

Table 3. Triangle taste tests for biscuits made with irradi-
ated all-purpose flour.

 

 

 

 

 

revel of
irradiation Correct selection Preference for
rep of odd sample untreated samp1e__
Panel I
20, 000 1/6 1/6
50,000 8/1lww 5/lls
100, 000 7/12 5/12
500,000 6/6wsw ----
Panel’II
20,000 11/26 9/26-::-
50,000 17/28ss l7/28sss
100,000 l7/28ww l6/28waw
500,000 lh/lhwww ----
Combined Totals fbr Pane S5I and‘II ,
20, 000 12/32 10/32
50,000 25/30www 22/ 9*ww
100, 000 amides-r. 21 Cases
500,000 20/20www ----

 

sSignificant at the .05 level.

*wSignificant at the .01 level.

*ssSignificant at the .001 level.
of correct selections of the odd sample at the 100,000-rep
level was just short of being significant. Since the darker
color of the 500,000-rep biscuits was disguised by use of a
red light, it appears that the correct selection of the odd
sample may have been due to odor alone Since Panel I found no
Significant differences in any of the other characteristics
when they were scoring the biscuits. The odor scores were
significantly lower for the 50,000-rep and 500,000-rep bis-
cuits which were the same treatment levels which were shown to

be Significant in the triangle tests. However, there is no

51

obvious explanation as to why these judges were able to
select the odd sample more often at the 50,000—rep level

than at the 100,000-rep level. The triangle test results

of the other seven judges (designated as Panel II) who

knew about the irradiation treatment indicate that these
judges were able to identify the odd sample a sufficient
number of times at all levels of irradiation of 50,000 rep
and above to show that the selection was not due to chance
alone. Thus the triangle test data from both panels indicate
that the treatment level of irradiation at which distinguish-
able changes were produced in baking powder biscuits made
with irradiated flour was approximately 50,000 rep which
agrees with the bread data of Nicholas 32 31. (1958). There
was a definite difference, however, in preference of the two
panels for the untreated product (Table 3). Although Panel I
could ascertain a difference between the control and irradia-
ted products, they did not prefer it to the irradiated product
whereas Panel II preferred the control biscuits to any of the
biscuits made from flour irradiated at the various levels.

The individual judgments of the 10 judges in the triangle
tests are shown in Table h. All judgments as to the odd
sample were correct at the 500,000-rep level of irradiation,
but there was a great deal of variation among judges in ability
to select the odd sample at the three lower levels of irradi-
ation. One judge (E.J.) who had worked with irradiated pro-

ducts previously was correct in all tests. Among the remaining

52

Table A. Triangle taste test data for biscuits made with
irradiated flour showing individual correct
choices of the odd sample by the 10 judges.

 

 

 

 

_ Love ' p
Judge 2Q,ooo *50,ooo 10o,ooo ‘Eeo,ooo
/ fan“ I M 2/2

E. s. o 2
z. .. 2 2
2,. s. 3;: 133 a: 2;.

R 2/h /ianeIII- l/h 2/2
E. .
E. a. u/u i/u u/u 2/2
B. T. o/u l/h 2/h 2/2
M. Ma. l/h 3/u 2/M 2/2
3. a a a 2;:
r: M5. ' 2/2 2/h 3/u 2/2

 

judges, two of those who did not know of the irradiation
treatment chose the odd sample just as many or more times
than the judges who were aware of the treatment.

A comparison of the triangle test data for the two
types of baking powders--tartrate, rapid-action and sulfate-
phosphate, double-action--is shown in Table 5. In contrast
to the suggestion made by Brownell g£_§l, (1955), this data
indicates that the double-action baking powder may have
tended to cover up rather than emphasize the changes brought
about by irradiation. At dosage levels of 50,000 rep and
100,000 rep, the judges correctly identified the odd sample
more often when tartrate baking powder was used than when

sulfate-phosphate powder was used.

53

Table 5. Triangle taste test data for biscuits made with
irradiated flour comparing use of tartrate and
sulfate-phosphate baking powders.

 

 

 

 

 

fieveI of Correct selection ’Preference for
irradiation of odd sample untreated sample,_
rep Tartrate S-P_' Tartrate S-P
20, 000 6/16 6/16 6/16 LL/16
So, 000 13/19.:e-x- 12/2o-x. 13/19-::-.~:~.~.:- 11 /20-::-::--::~
100, 000 lh/ZO-zws-x- 10/20 ll/ZOx-eeos lO/20ee-x-
500,000 lO/lO%%* 10/10%** ---- ----

 

*Significant at the .05 level.
*eSignificant at the .01 level.
*waSignificant at the .001 level.

Mechanical Tests. Average values for the mechanical

 

measurements on the dough and biscuits made with flour at
each level of irradiation are found in Table 6. The average

compressibility of the dough made with flour irradiated with

Table 6. Average compressibility, elasticity, and volume
measurements on dough and biscuits (6 batches)
made with irradiated flour and tartrate baking
powder.

 

 

 

 

 

mechanical ;t: Level of irradiation - re
test controI 20.000 59.005 I501805 5591050

Compressibility of
the dough (1/10 mm.) 98.5a 96.hg 96.9a 96.8a 90.2

Elasticity of the

dough (1/10 mm.) 18.8 18.2 16.7 17.5 111.9
Volume of the
biscuits (cc.) 279 275 282 275 268

 

8Significantly higher than the 500,000-rep biscuits at
the .05 level.

.O

511.

500,000 rep ionizing radiation was significantly less than
that of the doughs made from.untreated flour or flour '
irradiated at the three lower levels. The compressibility
of the other three irradiated flour doughs were practically
identical and were less than that of the control but the
difference was not significant. Mean.measurements for
elasticity or recoil of the dough varied slightly, but the
differences were not significant.

Mean volumes of the baked biscuits did not vary signifi-
cantly between the controls and those made with irradiated

flour at any of the levels used.
Gluten'Yields

Tables giving the data on yields of crude gluten
(Table 36) and volumes of the baked gluten balls (Table 37)
for each flour sample, and statistical analyses of these
data (Tables 38 and 39) may be found in the Appendix.

Weight g§_gggd2_gluten. The mean crude gluten yields
obtained by the "washing out" process from.non-irradiated
control and irradiated wheat flour are shown in Table 7.

These are the weights taken immediately after the draining
period.

Irradiation of the flour with dosages of ionizing radia-
tion up to 1,000,000 rep resulted in increased yields of crude
gluten above the yields produced from the nonirradiated

control flour. The mean crude gluten yield for control I

55

Table 7. Mean yields of crude gluten extracted from.non-
irradiated and irradiated all-purpose flour.

 

 

 

Irradiation dosage - rep Weight - grams
Control I 9.148.
50,000 10.1
100,000 10.2
250,000 10.h
500,000 10.9
Control II 9.6b
300,000 10.7
1,000,000 10.5
3,000,000 0.6°
10,000,000 0.0

 

aSignificantly less than the 250,000 rep, 300,000
rep, 500,000 rep, and 1,000,000 rep at the .01 level and the
50,000 rep and 100,000 rep at the .05 level.

bSignificantly less than the 300,000 rep and 500,000
rep at the .01 level and the 250,000 rep and 1,000,000 rep
at the .05 level.

cSignificantly less than all others at the .01 level.

was significantly less than those of all the other treat-
ment levels below 3,000,000 rep (250,000 rep, 300,000 rep,
500,000 rep, and 1,000,000 rep at the .01 level; 50,000

rep and 100,000 rep at the .05 level). The mean crude

gluten yield fer control II was significantly less than those
of the 300,000-rep and 500,000-rep samples at the .01 level
and of the 250,000-rep and 1,000,000-rep samples at the .05
level. This was in contrast to the results of Lee (1959)

who reported a decrease in recoveries of crude gluten.from
samples of flour irradiated with .25 and .50 million roentgens

over the control and sharply decreased recoveries from

56

samples of flour irradiated with 1.0 million roentgens.
However, in this study, there was a sharp decrease in crude
gbuten recovered when the flour sample had been irradiated
with 3,000,000 rep and no yield at all when the flour had
been irradiated with 10,000,000 rep. At these higher levels,
manipulation of the flour under water did not bring about
gluten.formation to any degree so that the flour was just
dispersed in the water and most or all of it went right
through the sieve.

Volume 2£_bgkgd gluten ballg, The mean volumes of the
baked gluten balls made with non-irradiated control and irra-
diated flours are shown in Table 8.

Table 8. Mean volumes of baked gluten balls made with non-
irradiated and irradiated all-purpose flour.

 

‘
*

 

Irradiation dosage - rep Volume - cc.

Control I 83.7
50,000 90.7
100,000 91.2
250,000 83.7
500,000 83.0
COHtPOl II 8902
300,000 82.8
1,000,000 95.7

3,000,000 n.7a
10,000,000 - 0.0

 

aSignificantly less than all others at the .01 level.

No significant differences due to irradiation dosage of

the flour samples was found except at the two higher dosages--

57

3,000,000 and 10,000,000 rep-~where crude gluten yields had
been small or non-existent. At the lower levels of irradi-
ation there was as much variation in the volume of gluten
balls made from flour at any one level of irradiation as
between flours at different levels of irradiation (See Appen-
dix Table 36). No logical explanation for this is known.
Thus the crude gluten yields indicate that at high
dosages of irradiation of 3,000,000 rep or more, there was
a definite indication of changes in the gluten protein of
wheat flour. Since there were no significant differences
in the volumes of the baked gluten balls at the lower levels
of irradiation, it may be that the crude gluten weights were
greater than those for the control samples due to greater
water retention rather than due to greater protein content.
In the baking tests, it was found that the biscuit doughs
made with irradiated flour were less moist in handling than
the doughs made with control flour even though the same

amount of liquid was added.
Electrophoresis

Nitrogen anlyses indicated that the all-purpose flour
used was 12.0 percent protein. Enough acetic acid was added
to the whole flour samples to produce an initial concentra-
tion of 0.91 percent protein in the dispersions prepared for
electrophoresis. After centifugation and dialysis against
citric acid-disodium phOSphate buffer, pH 2.2, for 72 hours

58

the mean concentrations of the dispersions at the various

irradiation levels ranged from.0.78 to 0.83 percent as shown

in Table 9. (Concentrations of the individual samples are

found in Appendix Table he.) These differences due to irra-

diation dosage were not significant (See Appendix Table hl).

It can be seen that large mean percentages (86.1 to 91.2)

of the protein did remain in the dispersions used for electro-

phoresis.

Table 9. Mean initial protein concentrations of acetic acid
dispersions of flour and.mean final protein con-
centrations after centrifugation and dialysis for

72 hours against citric acid-disodium.phosphate
buffer, pH 2.2.

Irradiation Protein concentration:- Z
dosage - rep n a na n .EW

 

 

None - control 0.91 0.80 88.3
300,000 0.91 0.83 91.2
1,000,000 0.91 0.82 89.7
3,000,000 0.91 0.78 86.1
10,000,000 0.91 0.79 87.2

 

At the beginning of the electrophoretic study, deter-
minations were made on the non-irradiated flour to compare
protein extracted directly from.the flour with protein ex-
tracted from.crude gluten.. The electrophoretic patterns for
protein extracted directly from.the flour (Plate I, Fig. 1)
and from.crude gluten (Plate II, Fig. 1) were very similar
except that the slow-moving component a was more prominent
on the patterns made with the protein extracted directly
from the flour. Laws and France (19h8) have reported similar

S9

EXPLANATION OF PLATE I

Representative electrophoretic patterns of

wheat protein extracted from.non-irradiated flour

and flour irradiated at four dosage levels. As-

cending boundary on left, descending on right.
Citric acid-disodium phosphate buffer pH 2.2, ionic

strength 0.02h; field strength 6.80 volts/centimeter;

temperature 2.100; time 90 minutes.

Fig.
Fig.
Fig.
Fig.
Fig.

1.
2.

Non-irradiated control flour.
300,000-rep flour.
1,000,000-rep flour.
3,000,000-rep flour.
10,000,000-rep flour.

 

60

61

EXPLANATION OF PLATE II

Electrophoretic patterns of wheat protein
extracted from crude gluten and from.gliadin from
non-irradiated flour. Ascending boundary on left,
descending on right. Citric acid-disodium phos-
phate buffer pH 2.2, ionic strength 0.02M; field
strength 6.80 volts/centimeter; temperature 2.100.;
time 90 minutes.

Fig. 1. Crude gluten

Fig. 2. Gliadin

PLATE II

 

   

Fig. 2

63

results when using the same buffer and suggested this was
probably due to the presence of part of the water-soluble
fraction in the suspension since they had found that the
main component from the water-soluble protein also had a
very low mobility. There would naturally be less water-
soluble protein present in the crude gluten suspension due
to the method of sample preparation. Since there was so
little difference in patterns between these two methods
of extraction, subsequent determinations made use of the
less time-consuming method of extracting the protein
directly from.the flour. A further reason for using the
whole flour extract was that crude gluten yields were so
very small at the higher levels of irradiation.

Ideally, electrophoretic patterns obtained from.the
ascending and the descending limbs of the apparatus should
be identical in all details, such as the number of components,
their mobility, and their relative concentration. In other
words, the two diagrams should be mirror images of each other.
This ideal case is, however, never realized in actual prac-
tice. Usually the peaks of the diagram obtained from the
ascending'boundaries are steeper than those from the descend-
ing boundaries and the mobilities, as measured in both limbs,
are also rarely identical. Longsworth and his colleagues (l9h6)
prefer the values obtained from.the descending pattern in
their work with blood sera; other workers base their con-

clusions on the ascending boundaries; and still other

6L1

investigators average the results obtained on ascending
and descending diagrams. In this study, the peaks of some
of the components were readily distinguishable in the as-
cending patterns but tended to be rounded off and merge
together in the descending patterns. Therefore, the mo-
bilities and relative concentrations were based on the as~
cending limb only. Patterns are shown for both.limbs.

Representative electrophoretic patterns for the control
flour and flour irradiated at 300,000 rep, 1,000,000 rep,
3,000,000 rep, and 10,000,000 rep are shown in Plate I.
(The patterns obtained for all three samples at each treat-
ment level may be found in the Appendix, Plates III to VII).

There was little evidence of component interaction in
this study since the patterns were nearly symmetrical in
both legs of the cell. This is in contrast to the work of
others (Schwert gt_§l,, 19hh; Laws and France, l9h8) who
reported component interaction which.was weakened somewhat
by increasing the ionic strength of the buffer.

In electrophoretic analyses of blood sera, it has been
commonly observed that there is usually an anomalous or
"false" peak at the initial starting point. This boundary
anomaly is designated as delta in the ascending limb and
epsilon in the descending limb. Likewise, with the non-
irradiated flour in this study, a slow-moving "component"
moved so very little that it could easily be taken for the

delta and epsilon anomalous boundaries. However, it is

65

believed that the first peak in these patterns indicates

a true component as reported also by Laws and France (19h8).
The area under the first peak increased as the dosage of
ionizing radiation was increased while the other main com-
ponent decreased. Thus the first peak must represent a
portion of the protein present and a shift from components
c, d, and e to a (first component) has occurred as a result
of irradiation.

The electrophoretic patterns for the untreated control
flour (Plate I, Fig. 1) show that there are at least six
separate protein components present. The mean distribution
in percent of the total protein.among the various components
of the control flour and of the irradiated flours are given
in Table 10. (See Appendix, Table #2 for distribution of
components for each sample analyzed).

The proteins were identified by means of their mobility.
The mobility of each component was determined from.patterns
of untreated flour. For subsequent patterns of the irradiated
flours, the mobilities were calculated, and the protein was iden-
tified with the component of the control flour having the same
mobility. Table 11 gives the mean mobilities of the six estab-
lished components of the untreated control and irradiated
flours. (Mobilities calculated for each sample analyzed may
be found in the Appendix, Table #3). It is recognized that
some discrepancy exists in the assignment of mobilities.

This discrepancy can be accounted for in part by recognizing

Table 10.

66

Mean distribution of the protein among the various
electrophoretic components of untreated and irradi-
ated flour (ascending patterns).

 

 

 

 

 

Irradiation ICOmponents
dosages - rep a b c d e f
None - control 19.6 3.6 10.9 8.2 51.8 7.0
300,000 19.0 2.6 10.8 8.3 52.3 6.9
1,000,000 22.0 2.9 8.5 9.6 50.2 6.8
3,000,000. 2h.6 2.9 7.6 11.1 M7.9 5.7
10,000,000 35.3 3.u - - 57.5 3.8
Table 11. Mean electrophoretic mobilities of the protein

components of untreated and irradiated flour
(ascending'patterns).

ElectrophorgtIc mgiIlIty,
sec'

 

 

 

 

 

Irradiation cmflfivOIt' x 0'7
dosage - rep Components
‘b c d e I

None - control 1.96 2.76 3.28 hehE 9.05
300,000 1.89 2.65 3.25 n.21 8.59
1,000,000 1.99 2.78 3.39 n.38 8.99
3,000,000 1.96 2.80 3.33 8.81 8.79
10,000,000 1.65 - - 3. h 8.67

 

that obviously in the case of high levels of irradiation,
certain components have disappeared or shifted. In the
absence of some components, the mobilities of other components
remaining may be altered.

The patterns for the 300,000-rep flour (Plate I, Fig. 2)
were essentially the same as those for the untreated control
flour. The first noticeable change in the patterns occurred
at 1,000,000 rep (Plate I, Fig. 3) where although the patterns

looked quite similar to those of the control, the relative

 

De

67

concentrations of the two largest components were beginning
to change. The mean relative concentration of component a
had increased from.l9.6 to 22.0 percent while component 6

had decreased from.51.8 to 50.2 percent in mean relative con-
centration. At 3,000,000 rep (Plate I, Fig. A), component d
was hardly distinguishable and component c was rounded off at
the top. There was an even greater change in mean relative
concentration at 3,000,000 rep than was true at 1,000,000
rep, with the mean relative concentration of component a
increased to 2h.6 percent and that of component e reduced to
h7.9 percent. At 10,000,000 rep (Plate I, Fig. 5), radical
changes had occurred--components b, c, and d were no longer
distinguishable as separate peaks and a,much.larger mean
percentage (33.7) of the total protein had remained stationary
as component a.

The electrophoretic patterns show no evidence of extra
components appearing as radiation dosage was increased, but
components occurring in the normal patterns have disappeared.
This observation does not eliminate the possibility of protein
hydrolysis. The hydrolysis products may not have remained in
suspension as the samples were being prepared. This observa-
tion simply points to the fact that no hydrolyzed.products
accumulated in sufficient quantities to be detected by the
electrophoresis technique unless they were of low mobility
and therefore remained stationary. It would seem that irradi-

ation of the flour with 3,000,000 rep or less has not altered

the proteins sufficiently to cause a significant change in
the components which have not disappeared (Table 10) A
complete disappearance of some components without formation
of new components would result in a lower value for total
nitrogen. The total nitrogen, however, remained approxi-
mately the same (see Table 9) with increasing dosages of
irradiation so it appears that the loss of components is

a shift from one component to another, rather than a cam-
plete disappearance of them.from the flour.

8 After completion of the electrophoretic determinations
on the whole flour extracts of non-irradiated and irradiated
flours, an electrophoretic pattern also was obtained for a
sample of gliadin (soluble in dilute alcohol) extracted from
the non-irradiated flour (Plate II, Fig. l). A definite
similarity was observed. The main differences were the ab-
sence of components b and f for the gliadin sample. As
mentioned earlier, component f was smaller in area in the
pattern for the crude gluten extract (Plate II, Fig. 1) also
indicating that with both the crude gluten and the gliadin,
this small fast-moving component must have been removed in
the extensive washing procedure. The absence of component
b, in addition to component f, indicates the presence of
just four separate fractions in the gliadin sample--compared

to six in the whole flour extract.

69

SUI-MARY AND C ONC LU S I ONS

Nine lots of wheat flour were exposed to cathode ray
ionizing radiation. A tenth lot served as a control.
Biscuits were made using control flour and flour irradiated
at four dosage levels (20,000, 50,000, 100,000, and 500,000
rep). Crude gluten yields were measured for the control
flour and flour irradiated at eight dosage levels (50,000,
100,000, 250,000, 300,000, 500,000, 1,000,000, 3,000,000,
and 10,000,000 rep). Electrophoretic patterns were ob-
tained for acetic acid extracts of protein from the control
flour and flour treated at four dosage levels (300,000,
1,000,000, 3,000,000, and 10,000,000 rep) in a citric acid-
disodium phosphate buffer, pH 2.2, ionic strength.0.02h,
field strength 6.80 volts/centimeter, temperature 2.100.,
time 90 minutes.

Triangle taste tests showed that 50,000 rep (recommended
dosage for deinfestation) was the dosage level at which
irradiation treatment of flour could be detected in biscuits.
Taste panel scores for color, odor, and baking characteristics
of biscuits made with flour treated with 100,000 rep or less,
however, were not significantly different from those for the
controls. A sterilizing dosage of 500,000 rep brought about
marked changes in odor and eclorr of biscuits.

Dosages of 1,000,000 rep or less resulted in increases
in weight of crude gluten extracted from irradiated flour but
did not affect the volumes of baked gluten balls. The

70

increased weight, therefore, must have been due to greater
water retention rather than to protein. The very small
crude gluten yields at dosages of 3,000,000 rep or more in-
dicated definite changes were occurring in the wheat flour
protein at these levels.

Electrophoretic patterns for the control flour indicated
that there were at least six separate protein components
present, including one which was fast-moving and high in
relative concentration and another which was slowamoving
and medium high in relative concentration. Irradiation of
the flour with 300,000 rep produced no changes in the electro-
phoretic patterns obtained, but at 1,000,000 rep or more, the
relative concentrations of the two largest components were
changed--the slow-moving component becoming larger and the
faster-moving component smaller with each increase in dosage.
Also with increased radiation dosages, the peaks of the
smaller components became less distinct until only two main
peaks were distinguishable at 10,000,000 rep. Irradiation
apparently brought about a shift of several of the components
to the slow-moving component rather than a complete disappear;
ance of them from the flour.

The results of this study confirm the idea that changes
in palatability and physical properties of irradiated flour
are apparent at the low dosage levels required for deinfesta-
tion and sterilization, but that alterations in flour protein
are difficult to demonstrate chemically except at very high

dosage levels.

I.

71

CKNOWLEDGMENTS

The writer is deeply grateful to Dr. B. Elaine
Rutherford for advice during the experimental phases
of this study and for guidance during the preparation
of the manuscript. She also wishes to thank Dr. R. a.
Grimes, Assistant Professor of Agricultural Chemistry,
for his valuable help in the use of the electrophoretic
equipment; Mr. N. E. Miant, Professor of Agricultural
Engineering, for irradiating the flour; Miss Mary Morr,
Assistant Professor of Foods and Nutrition, for her
counsel in the palatability phase of the study; and
members of the faculty and graduate students in the
Department of Foods and Nutrition who served on the

taste panels.

72

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Cereal 801. Today. 2:130-131 (1957).

Milner, Max, and Yen,‘Yin-Chac. Treatment of wheat with
ionizing radiations. III. The effect on breadmaking
and related properties. Food Technol. 10:528-531

(1956).

Morgan, B. H. Proc. Symposium.Radiation Sterilization of
Foods. Quartermaster Food and Container Institute,
Chicago, June 8-10 (1955).

Nicholas, R. C., Meiske, Donna P., Jones, Mary E., Wiant,
D. E., Pflug, I. J., and Jones, Evelyn M. Radiation-
induced changes in bread flavor. Food Technol. 12:52-
SA (1958). .

Nicholas, R. G., and Wiant, D. E. Radiation of important
grain-infesting pests: order of death.curves and
survival values for the various metamorphic forms.

FOOd Techn01. 13:58‘62 (1959).

01cctt, H. S., Sapirstein, L. A., and Blish, M. J.
Stability of wheat gluten dispersions toward reducing
agents in the presence and absence of a gluten pro-
teinase. Cereal Chem. 20:87-97 (l9h3).

Osborne, T. B. The proteins of the wheat kernel. Carnegie
Inst. Wash. Publ. Bk (1907). '

Poling, C. E.,‘Warner, W. D., Humburg, F. R., Reber, E. F.
Urbain, W. M., and.Rice, E. E. Growth, reproduction,
survival and histopathology of rats fed beef irradiated
with electrons. Food Research 20:193-21h (1955).

Pomerantz, Reuben. Atoms strike the cereal industry. Cereal
Sci. Today. 1:23-29 (1956).

f'

 

77

Proctor, B. E., and Bhatia, D. 8. Effects of high-voltage
cathode rays on aqueous solutions of trypt0phane,
osine, phenylalanine and cystine. Biochem. J. 51:

t

Proctor, B. E., Coleman, M. T., and Goldblith, S. A.
Effect of high energy cathode rays on the catalase

activity of Micrococcus gogenes var. aureus. J.
B80t6r1010 6 3 " c

Proctor, B. E., Lockhart, E. E., Goldblith, S. A., Grundy,
A. V., Tripp, G. E., Karel, M., and Brogle, R. C. The
use of ionizing radiations in the eradication of insects
in packaged military rations. Food Technol. 8:536-5110

(195M .

Richardson, L. R., and Brock, Ruth. The nutritional value
of a synthetic diet sterilized by gamma rays, as
measured by reproduction and life span of rats. J.

Nutrition 65:353-360 (1958).

Robinson, R. F. Some fundamentals of radiation sterilization.
Food Technol. 8:191-19LL (1951+).

Roessler, E.B., Baker, G. A., and Amerine, M. A. One-tailed
and two-tailed tests in organoleptic comparisons. Food
Research 21:117-121 (1956).

Roessler, E. B., Warren, J., and Guyman, J. F. Si ficance
in triangular taste tests. Food Research 13: 03-505

(19118) .

Runner, G. A. Effect of Roentgen rays on the tobacco, or
cigarette, beetle and the results of experiments with
a new form of Roentgen tube . J. Agr. Res. 6(11):383-

388 (1916).

Sandstedt, R. M., and Blish, M. J. A new characterization
of the gluten proteins. Cereal Chem. 10:359-366 (1933)-

Schwert, c. w., Putnam, F. w., and Briggs, D. R. An electro-
phoretic study of gliadin. Arch. Biochem. 11:371-387
1

911A).

Setlow, Richard. Radiation studies of proteins and enzymes.
Ann. N. v. Acad. Sci. 59:h7l-h83 (1955).

Stockelbach, Lois s., and Bailey, 6. H. The amino acid dis-
tributicn in proteins resulting from the thermal frac-
tionation of wheat gluten. Cereal Chem. 15:801-811

(1938).

78

Sullivan, Betty. Proteins in flour. Review of the physical
characteristics of gluten and reactive groups involved
in giange in oxidation. J. Agr. Food Chem. 2:1231-123h
(19 ).

ifaboada, Oscar. Some effects of radiant energy on the beetles,
Tribolium confusum. Duv, Sitophilus anarius (L.):
and Ascant occe es obtectus (Say7} npu l shed M. S.
thesis, Michigan State University (1953).

 

(reply, L. J., and Kline, B. E. Wholesomeness and possible
carcinogenicity of radiated foods. Federation Proc.
15:927-929 (1956).

VJhite, G3 D. Weight loss in stored wheat caused by insect
feeding. J. Econ. Ent. u6(u):609-610 (1953).

AP PEND IX

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Date
I

FORM III

ELECTROPHORESIS EXPERIMENT 8?-

 

SAMPLE
Type

Operator

Plate No.

 

No.

 

Description

 

 

BUFFER

Composition

pH

 

CON DUCTIVITY lmhos)
Buffer

Diluted with Butter

 

 

Conc. in Buffer

 

 

Solution

 

CELLS (cross-sectional a
Ascending

ELECTROPHORESIS

Initial boundary Ascending

Scanning boundory

Time
Initial

 

rea l

: Descending

 

Average

 

 

 

“ Light_—_ Dark
Milliamps.

 

 

 

 

 

 

 

 

Final

 

 

Average

Potent. Grad.

 

 

Descending

 

" Light __ Dark

 

Gear ratio

 

Plate type

 

CON DUCTANCE

 

v/ cm. Migration

Aperture ..____ Filter

 

 

Sample

R-selector Ratio Slidevire
Switch Reading

R-cell

 

 

 

 

 

 

 

 

 

 

 

 

 

 

83

Table 12. Odor scores for biscuits made with irradiated flour.

 

 

Judgo COntroI 20,000 I'SSOSOOO 100,000 #5001900

6
6
3
2
S
5
S
i
u,

H
H

III

Fl
«<
UIOWR ‘JHTUI kp¥rv1 (bULF'

u:
0
(a
UIOWfl. brown. on\n ov<4r

U)

C

U

0
r—nm
O C
H-P"
0‘

 

Table 13. Color of the crust scores for biscuits made with
irradiated flour.

 

 

 

Lev6I of’irradiafion - rag
Judge Confrol 20.000 ¥_50,UOO ‘IO0,000 500.000
I

B. S.
R. A.
S. S.
II
B. S.
R. A.
S. S.

boU'LO‘» 4 ON

III
B. S.
R. A.
S. S.

.pqrvI croun. U104?

IV
B. S.
R. A.
S. S.

\p-JO\ \anUI 4?~nn_ Urpwb
F-F‘Ch Falox «if-40‘ mom.

U’IOO‘ FUII

u20~i
r:
. 3‘ $?U10~ \anF‘ uuoxp' #TUmF'

U1
<3
0
\o
G\
(3
\o
C)
HUI
.0
#?C>
[.1

 

Table 1h. Color of the crumb scores for biscuits made with
irradiated flour.

 

   

I n 0 on - rep
Judge Control 20,000 50,000 #109,000 #;SO0.000
j:

 

II

rrnxp‘ $7bah'

III

ChUun. ofir~1 VIOWR

IV

{:21

O

U)

0
VIOWF OWFO‘ (fiKPQ OW>~J
(fiOVQ OWFO\ OflT~J UTOWP
\nou
om: mrm mrm mom
43m:— rm:

U1

0
u)
(DUI
'CDUT

C)

S.D.

 

Table 15. Flavor scores for biscuits made with irradiated
flour.

eve o rra a on - rep
Judgp Cohtrdi' 20,000 _;§Q1OOO 1001000 500,000
I .

 

B. s.

R. A. g .5. g l r
s. s. 6 S S 3
II
L. s. 6 6 5 1+ S
R. A. S 6 6 5 3
s.& u S 6 6 5

B Q S S III 1+ ’4-

PZX: S E
S. s. 1+ 1% ll % 6
Iv
:2- i- i S g; a E
s: s: S I: u 3 5
Mean 5.0 5.0 has he? 309
S.D. 0.95 0.73 0.80 0.89 1.50

 

85

Table 16. Tenderness scores for biscuits made with irradi—
ated flour.

fevel o? irradiat on - rep .-

Judge ContrOI 20,000 50,000 100,000 500,000

 

 

 

I
B. s. S S h 6 3
R. A. 2 u 6 6
s. s. 6 S S 6
II
B. s. 6 S 6 S 6
R. A. 3 S 6 6 3
s. S. 5 S 6 6 S
III
B. s. 6 6 5 h -
R. A. 5 3 3 u 3
s. s. 3 u u 7 6
IV
B. s. 7 6 - 5 u
R. A. S u S S u
s. s. 5 S S S 5
Mean u.8 u.8 .8 5.3 u.6
S.D. 1.h9 1.15 1.15 0.89 1.2h

 

Table 17. Lightness scores for biscuits made with irradiated
flour.

feveI of irradiatIon - rep =='

Judge “control’ 20.000 50,000‘ 100,000 500.000
I
5

 

 

B. S.
R, A.
S. S.
II
B. S.
R. A.
S. S.

UJNN ONULP‘

III
S.
A.
S.

\J‘l-F'U‘L U'lO\U'L U'LUIUI

IV

0\U10\ 4:21:01 U‘toxm ~F‘U‘LU1
\nv10~ 0An0\ UVJHT \nvtp'

o\\n\n MOP-l? UIU'l-F'

U'LUII
-P\9U1 \ani

O.
$rc>
n)
(301
00
ova
.4
3g?

 

86

Table 18. Texture scores for biscuits made with irradiated
flour.

 

 

feveI oI‘irradiation - rep
Judgp control 0720.900 50.000 1001900 500L000
I

B. s. 7 5 6 h 5
R. A. S S h 3 g
S. S. 6 6 7 6
II
B. s. S 6 6 5 6
R. A. 5 6 S 2 2
s. s. 6 5 S 6 S
III
B- .. s 5 2 2 -
R. A.
s. s. 6 g, S 6 g
6 IV S 5
B. S. .-
R. A. g h 6 S g
s. s. S u u 5
Mean 503 ,+09 sol-l- Ll-08 LlOLl-
S.D. 1.07 0.79 0.90 1.27 1.08

 

Table 1?. Moisture scores for biscuits made with irradiated
flour.

eve 0 rice 8 0n - reg
Judge on r0 0, 1 _, 00’

’I
B. S.
R. A.
S. S.
II
B. S.
R. A.
S. S.

Own-J oonI

III

rrvun. 047U1 \RUVQ

IV

¥Towr \mw70~ Uun$r 04:.3
\mw70\ \nnfln. 04TU1 <bv10~

VUn$r \nvur' omao\ UIOWb

\nun» OAR!

2 6.9 5.2 5.0 u.7
O 0.8h 009s ' 1056

 

Table

20. General eating quality scores for biscuits made

with irradiated flour.

87

 

 

IControl

5
LI
6

UIOWR \»#7U1 \nUII

20.000
5

O\«F“O\ F—F’U't FU‘Li

50.000
I'

II

III

IV

UUHUI

UH:¢? \nUII

4mm

P
4100}000

S
S
S
5
LI
6
g
S
S
E-

88

 

 

 

 

 

Table 21. Compressibility of the dough made with irradiated
flour and tartrate baking powder (1/10 mm.).
TrIpIIcate==_ LevEI‘ST'irradiationAt‘reE:
samples ContESI‘ 20.000 150.000 100.000 500.000
I
1 96.0 97.0 111.0 105.0 90.0
2 100.0 87.0 105.5 106.0 106.0
3 92.0 89.0 96.0 91.5 86.5
II
1 105.0 108.0 106.0 85.5 73.0
2 107.5 96.0 105.5 9%.5 106.5
3 103.0 99.0 105.0 10 .0 92.0
III
1 92.5 102.0 98.5 100.0 91.5
2 102.5 110.0 106.5 90.0 86.5
3 105.5 103.5 101.5 . 99.0 82.0
' Iv
1 88.0 95.0 95.5 99.5 83.5
2 96.0 106.0 98.5 96.5 96.5
3 99. 90.0 99. 101.0 99.5
v
1 106.5 103.5 80.0 93.0 91.0
2 96.5 92.0 87.5 95.5 93.
3 108.0 100.5 89.0 106.5 100.0
VI
1 95.0 86.5 91.5 90. 83.5
2 89.5 85.0 86.0 96.0 85.5
3 90. 88.5 87.5 92.5 81.0
M881]. 9805 96014» 9609 96.8 9002
S.D. 6.55 7.96 8.5 6.01 8.60

 

 

 

 

 

Table 22. Elasticity of the dough made with irradiated
flour and tartrate baking powder (l/lO mm.)
friplivcate ALeveI Elf—Irradiatfon -"rep
samples ControT 20,000 150,000 7.00.000 300:000
I
1 19.0 20.0 19.5 19.0 16.5
2 23.0 25.0 23.5 18.5 21.5
3 27.0 26.0 15.5 22.0 20.5
II
1 17 .5 15 .5 15 .5 15 .5 10 .5
2 16.0 12.0 13.5 21.5 20.0
3 21.5 12.0 8. 18.5 20.5
III
1 21.0 20.0 17.5 15.0 16.0
2 16.0 17.0 16.0 15.0 15.0
3 17.5 16.5 18.0 17.5 18.
Iv
1 1295 2105 2%.; 22.0 180
2 25.0 16.0 1 .0 15.0 19.5
3 20.5 16.0 21.0 17.5 16.5
v
1 26.0 20.0 12.5 20.0 13.0
2 20.0 10.5 17.5 9.0 11.5
3 20.0 15.5 16.0 .17.0 16.0
VI
1 19.5 25.0 18.5 16.5 6.0
2 11.0 20.5 16.0 18.5 09.0
3 900 1900 114-03 1700 8.5
Mean 18.8 18.2 16.7 1705 114.09
SOD. “-085 “-050 3093 3011 Ll-aéll.

 

9O

 

 

 

 

Table 23. Volume of the biscuits made with irradiated flour
and tartrate baking powder (l/lO mm.).
5"Biscuit ___ *Level of Irradiation - rep
unit Control 20.000 :50.000 100,000 8500.000
I
1 250 250 255 250 255
II
1 275 275 275 285 280
III
1 285 285 280 280 255
IV
1 300 300 290 280 275
V
l 280 255 280 280 265
VI
1 285 285 315 275 275
Mean 279 275 282 275 268
S.D. 16.6 19.2 19.7 12.6 10.3

 

91

Table 2h. Analysis of variance for odor scores of biscuits.

Source of Begrees OI Sum.oI fiean I

 

variation freedom. squares squares values
TOtal 59 71093

Treatments 8 11.93 2.982 2.68%
JUngS 2 0.91 Owl—[.55 Ooh-l
Error 53 59.09 1.115

 

*Significant at the .05 level.

Table 25. Analysis of variance for color of crust scores of

 

 

 

biscuits.
SBurce 0?; ‘Degrees of? Sum.of’ ‘Méan :IIFT —I=
variation freedom. squares squares values
TOtal 59 80.85
Treatments h h.10 1.025 1.01
Judges 2 22.80 11.h00 ll.20*%
Error 53 53.95 1.018

 

eeSIgnificant at the .OITIevel.

Table 26. Analysis of variance for color of crumb scores of

 

 

 

biscuits.
Source 03'; Degrees of Sum 03“ Mean F
yariation freedom. squares squares values _
Total 59 100.98
Treatments h 6h.23 16.058 2h.29%*
Judges 2 1.73 0.865 1.31
Error 53 35.02 0.661

 

aeSignificant at the .01 level.

92

Table 27. Analysis of variance for flavor scores of biscuits.

 

 

 

Source 0? Degrees of Sum.of ‘Mban ‘JL IT
variation freedom. squares squares values
Total 59 66.18

Treatments 6 9.60 2.600 2.35
JUdgeS 2 20h3 10215 1019
Error 53 58.15 1.022

 

Table 28. Analysis of variance for tenderness scores of

 

 

 

biscuits.
Source or* iDegrees 0?: *Sum of' Mean ‘F
variation freedom. squares gquares values
Total 59 72093
Treatments 8 3.76 0.980 0.86
Judges 2 11.83 5.715 5.25*%
EI'I‘OI‘ 53 57 071l- 10089

 

*eSignificant at the .01 level.

Table 29. Analysis of variance for lightness scores of

 

 

 

 

 

 

biscuits.
Source of Degrees of Sum of Imean 7F ““
yariation freedom. squares squares values
Total 59 50.33
Treatments 8 5.16 1.290 1.61

JUdgGS 2 2.6 0315 loéu
Error 53 82.5 0.803

 

93

Table 30. Analysis of variance for texture scores of bis-

 

 

 

cuits.
Source of fiegrees of Sum.o? MeanIZT F
variation freedom. squares squares values_
TOtal 59 66098
Treatments 8 7.90 1.975 1.98
Judges 2 6.13 3.065 ‘ 3.07
Error 53 52.95 0.999

 

Table 31. Analysis of variance for moisture scores of bis-

 

 

cuits.
Source of fiegrees of Sum.oI fiean fi
variation freedom. squares squares value§q_
Total 59 72098
Treatments 8 ' 2.06 0.515 0.50
Judges 2 16.23 8.115 7.86%%
Error 53 58.69 1.032

 

”SSIghIIicant at the .01 level.

Table 32. Analysis of variance for general eating quality
scores of biscuits.

 

 

 

Source 0? Degrees of Sum of #Yean F
variation freedgm squares squares values
Total 59 168.61

Treatments 8 .00 1 .000 0 . 38
Judges .23 2.615 0.89

2
Error 53 155.38 2.932

 

98

(Table 33. Analysis of variance for compressibility of the
dough scores of biscuits.

 

 

 

 

Source of Degrees of* Sum.of Iflean 1E
variation freedom. squares squares values
Total 89 5, 652.08

Treatments 8 781.21 185.30 3.18%
Triplicates 2 7.37 33.68 0.58
Error 83 8,883.50 58.36

 

rSigniIicant at the .05 level.

Table 38. Analysis of variance for elasticity of the dough
scores of biscuits.

 

 

 

Source of Degrees of Sum of Mean F
variation freedom. squures squares values
Total 89 1,703.89

Treatments 8 166.51 81.63 2.27
Triplicates 2 12.28 6.12 0.33
Error 83 1,528.78 18.37

 

Table 35. Analysis of variance for volume scores of bis-

   

 

cuits.
‘oarao‘o '"" -ogreee o ""‘”"um.o" ' ‘ ean'“
variation freedom squares squares values
Total 29 7,30%
Treatments 8 75 1895 0.728

Error 25 6,586 2615

 

95

{Pablo 36. Yields of crude gluten extracted from.non-irradi-
ated and irradiated all-purpose flour.

 

 

Weight - gm.

 

 

 

IRepli- IrradiatIOn dose 9 - rep

cations Control 50.000 9100.000 2 0.000 500.900
1 10.0 10.3 9.9 9.9 11.8

2 9.0 10.5 11.7 11.2 11.3

3 8.9 10.5 9.8 10.6~ 11.2

5 9.0 10.g 10.8 10.8 11.2
9.8 9. 10.0 10.0 10.1

6 9.7 9.5 9.6 10.1 10.3
Mean 9.8 10.1 10.2 10.8 10.9
Sol). 0050 00,4.0 0078 OQLLB 0055

 

Irradiation dggage - rep
Control _300.000 1.000.000 3.000.000 10.000.000

 

1 9.8 10.8 10.3 0.9 0.0

2 10.3 11.0 10.8 1.0 0.0

a 9.8 10.6 10.7 0.3 0.0
90% 10.1 10.5 0.2 0.0

5 9. 11.7 10.8 0.6 0.0

6 9-8 10.2 10.3 0.8 0.0
M6311 9.6 1007 10.5 0.6 000
S.D. 0.37 0.61 . 0.16 0.32 0.00

 

Table 37. Volumes of baked gluten balls made with non-
irradiated and irradiated all-purpose flour.

 

 

‘vqume - cc.

 

 

 

Repli- Irradiation dosage - rep
cations Control' 50.000 4100.000 250.000 4500.000
1 1 103 91 59 82
2 “l8 98 86 88 80
3 109 91 87 82
8 82 78 88 78 80
5 83 8 88 88 88
6 62 77 107 102 86
Mean 83.7 9007 9102 8307 83.0
S.D. 16.9 6.1 8.3 18.3 3.3

 

Irradiation dosage - rep
controlfi.300.000 71.000.000 3.000.0007‘10.OOQ.000

l 95 83 121 7 O

2 92 82 82 10 0

3 88 109 100 3 0

l-l- 93 77 89 3 O

5 8O 78 100 2 0

6 91 68 82 3 0
lean 89.2 8298 9507 ’40? 0.0
S.D. 5.8 1309 114-08 301

96

 

{9

/’

 

97

Table 38. Analysis of variance for yields of crude gluten
extracted from.non-irradiated and irradiated
all-purpose flour.

 

 

Source 0? Degrees of Sum.of Mean F‘
variation freedom squares squares values
Total 58 519.03

Treatments

508.28 63.53 268.71**
Error 85 10.79 0.28

 

**Significant at the .01 level.

Table 39. Analysis of variance for volumes of baked gluten
balls made with non-irradiated and irradiated
all-purpose flour.

 

Source of Degrees of Sum.of Mean F
variation freedom. squares squares values
Total 53 83.770.83

Treatments 8 37,561.66 8,695.21 38.03**
Error 85 6,209.17 137.98

 

**Significant at the .01 level.

98

Table 80. Protein concentrations of acetic acid dispersions
of flour after centrifugation and dialysis for
72 hours against citric acid-disodium phosphate
buffer, pH 2.2.

 

 

Protein concentrations 6? solutions
Irradiation Triplicate samples

 

 

dosagq- rep_ 1 2 l‘lean 5.131.
a: T 3% a

None-control 0.89 0.73 0.79 0.80 0.081

300,000 0.80 0.82 0.87 0.83 0.036

1,000,000 0.82 0.79 0.88 0.82 0.025

3,000,000 0.79 0.78 0.82 0.78 0.080

10,000,000 0.81 0.78 0.83 0.79 0.087

 

Table 81. Analysis of variance for protein concentrations
of acetic acid dispersions of flour.

 

Source OI fiegrees 0? Sum of fiean 5
variation freedom squares squares values
Total 18 0.0288

Treatments 8 0.0081 0.00102 .815

Error 10 0.0287 0.00287

 

/‘

 

99

Table 82. Distribution of protein among the various electro-
phoretic components of untreated and irradiated
flour (ascending patterns).

 

 

Relative concentration - %:

 

 

 

Irradiation _‘ Components

dosage - rep a ‘b c d' e .f
COntrOI l '1609 208 10.8 909 51.3 803
2 22.0 2.3 11.1 6.0 52.7 6.0
3 1908 208 10.8 8.7 Blah 6.6
300,000 1 23.1 3.0 8.9 9.5 89.8 6.1
2 21.0 2.5 11.8 6.7 52.6 5.6
3 13.0 2.2 12.2 8.8 5’.9 8.9
1,000,000 1 23.1 2.3 7.6 10.8 50.8 5.3
2 21.7 3.8 9.5 8.6 89.5 7.2
‘ 3 21.2 3.0 8.5 9.3 50.2 7.8
3,000,000 1 28.9 2.6 8.3 9.8 85.8 8.8
2 20.' 3.5 8.2 12.2 88.6 7.1
3 28. 2.6 6.8 11.3 89.7 5.3
10,000, 000 l 3802 308 "' " 58.0 LL01
2 30.7 3.8 - "' 61.8" 3.8
3 36.9 2.7 - - 56.8 3.6

 

Table 83. Electrophoretic mobilities of the protein com-
ponents of triplicate samples of untreated and
irradiated flour (ascending patterns).

“ EIegfirophoretic

 

lOO

 

 

mgbilit?

 

 

 

 

cmd'volt‘l sec.’ -x O
Irradiation Components

dosage - rep ’b c d‘ E, ‘7?
Control 1 2019 2086 3014-7 LL.52 9.20
2 1.87 2.66 3.23 8.31 8.62
3 1.82 2.77 3.13 8.52 9.32
300,000 1 1.97 2.68 3.28 8.16 8.60
2 1.78 2.58 3.15 3.97 8.36
3 1.93 2.72 3.37 8.51 8.81
1,000,000 1 2.08 2.96 3.56 8.52 9.81
2 2.01 2.72 3.30 8.30 8.75
3 1.88 2.67 3.32 8.33 8.81
3,000,000 1 1.97 2.95 3.88 8.36 8.86
2 1.99 2.70 3.27 8.27 8.75
3 1.93 2.75 3.27 8.30 8.76
10,000,000 1 1.65 - - 3.81 8.77
2 1.63 " " 3090 8.52
3 1.68 "' - 3.80 8.72

 

lOl

11‘

 

EXPLANATION OF PLATE III

Triplicate electrophoretic patterns of wheat
protein extracted directly from untreated all-purpose
flour. Ascending boundary on left, descending on
right. Citric acid-disodium phosphate buffer pH 2.2,
ionic strength 0.028; field strength 6.80 volts/centi-

meter; temperature 2.loC.; time 90 minutes.

 

 

PLATE III

 

 

.8
1L
1

103

 

EXPLANATION OF PLATE IV

Triplicate electrophoretic patterns of wheat
protein extracted directly from flour treated with
300,000 rep ionizing radiation. Ascending boundary
on left, descending on right. Citric acid-disodium
phosphate buffer pH 2.2; ionic strength 0.028; field
strength 6.80 volts/centimeter; temperature 2.100.;

time 90 minutes.

108

PLATE IV

 

Fig. 1

 

Fig. 2

 

F

   

Fig. 3

105

 

EXPLANATION OF PLATE V

Triplicate electrophoretic patterns of wheat
protein extracted directly from flour treated with
1,000,000 rep ionizing radiation. Ascending bound-
ary on left, descending on right. Citric acid-
disodium phosphate buffer pH 2.2, ionic strength
0.028; field strength 6.80 volts/centimeter; tempera-
ture 2.100.; time 90 minutes.

106

PLATE V

 

Fig. 1

 

F180 2

 

F

   

107

EXPLANATION OF PLATE VI

Triplicate electrophoretic patterns of wheat
protein extracted directly from flour treated with
3,000,000 rep ionizing radiation. Ascending bound-
ary on left, descending on right. Citric acid-
disodium.phosphate buffer pH 2.2, ionic strength
0.028; field strength 6.80 volts/centimeter; tempera-

ture 2.10C.; time 90 minutes.

 

 

 

108

PLATE VI

 

109

 

EXPLANATION OF PLATE VII

Triplicate electrophoretic patterns of wheat
protein extracted directly from flour treated with
10,000,000 rep ionizing radiation. Ascending bound-
ary on left, descending on right. Citric acid-di-
sodium phosphate buffer pH 2.2, ionic strength 0.028;
field strength 6.80 volts/centimeter; temperature
2.1%.; time 90 minutes.

 

PLATE VII

 

Fig. 1

 

Fig. 2

F'F r

 

HI Fig. 3 +"1

111

VITA

Elsie Beth Alsup
candidate for the degree of
Doctor of PhiloSOphy

Final Examination, October, 1959

Thesis: The Effects of High Voltage Cathode Ray Ionizing
Radiation on Some of the Physical and Chemical
Properties of Wheat Flour Protein

Outline of Studies

Major subject: Foods and Nutrition
Minor subject: Physiology

Biographical Items
Born, June 25, 1925, Kopperl, Texas
High School, Albuquerque High School, New Mexico
Undergraduate Studies, University of New Mexico, 1982-85

Graduate Studies, Ohio State University, 1988-1989,
Michigan State University 1953-1958,
1956-1958

Experience: Home Economics Teacher, Belen (New Mexico)
High School, 1985-1987, Raton (New Mexico)
Jr. High School, 1987-1988; Graduate
Research Assistant, Ohio State University,
1988-1989, Assistant Professor of Foods and
Nutrition Research, South Dakota State
College, 1989-1953, 1958-1956; Graduate
Research Assistant, Michigan State University,
1956-1958; Associate Professor of Foods and
Nutrition, Kansas State University, 1959-

Member of Kappa Omicron Phi, Pi Lambda Theta, and
Sigma Delta Epsilon

 

 

3888 “SE gm

 

"IMMINWIK

93 030

II

llHIHIIINH

1A2 0645

31