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A STUDY OF THE BSOLOGICAL VALUE
AND COMFOSEHON OF BREADS FROM
VARIOUS COUNTRIES

Thesis for ”1-9 Degree 0‘ M. S.
MiCHIGAN STATE UNWERSITY
Simin D. Boiourchi

1963

mms

LIBRARY

Michigan State
University

 

 

 

 

ABSTRACT

The purpose of this study was to compare the nutritional
value of bread baked in Iran and Syrian bread with the Ameri-
can white enriched bread; to determine the nutritional con-
tent of these breads; and to evaluate the supplementary ac-
tion of dried milk solids at the 4% level and lentil powder
at the 7.5 and 15% level on Iranian bread.

Two feeding experiments were carried out. Four groups
of 10 male weanling rats were fed diets composed of 90%
Persian bread, Syrian, American white enriched and Alladin
bread. Breads were analyzed for fat, moisture, and nitrogen.
Growth and food intake were measured. At the end of 4 weeks
the animals were sacrificed, and the following analyses were
done on the rats' livers: moisture (by drying to constant
weight); nitrogen (by the Kjeldahl technique); fat (ether
extraction using the Goldfisch apparatus); and liver xanthine
oxidase activity (using the Warburg apparatus).

In the second experiment Persian bread was supplemented
with 4% dry milk solids, 7.5% lentils and 15% lentils. Rats
were sacrificed at the end of 3 weeks and the same chemical
analyses were carried out on the livers of the rats. Growth

and food intake were measured. Hemoglobin levels were also

determined.

Simin D. Bolourchi

These studies showed the growth promoting effect of
Persian bread was the same as that of American white en-
riched bread. Supplementation of the Iranian bread with 4%
dry milk solids or 7.5% or 15% lentils improved its nutri-
tional quality as shown by the increase in weight gain of
the rats.

The amino acid intake of rats and humans fed bread diets
were calculated. These calculations indicate that man's re-
quirement for amino acids may be met by a diet in which bread
serves as the source of 70% of the total calories. These
calculations also show that the bread diet does not provide
adequate amounts of amino acids necessary for the growth of

weanling rats.

A STUDY OF THE BIOLOGICAL VALUE AND COMPOSITION

OF BREADS FROM VARIOUS COUNTRIES

By

Simin D. Bolourchi

A THESI

tn

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

Master of Science

Department of Foods and Nutrition

1963

ACKNOWLEDGMENTS

The author wishes to extend her thanks and gratitude to
Dr. Olaf Mickelsen under whose guidance these studies were
completed. The author also wishes to thank Dr. Dorothy
Arata for her help in initiating these studies.

To all of the people in the Department of Foods and
Nutrition go the author's grateful appreciation for their

assistance and encouragement.

To My Parents

TABLE OF CONTENTS

INTRODUCTIONCOOCOOO0.00.0...O...OOOOOOOOOOOOOOOOOOOOO.
REVIEW OF LITERATUREOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.

1. Wheat and Wheat Flour Proteins.................
2. Composition of Wheat and Wheat Flour...........
3. Amino Acid Loss during Milling and Baking......
A. Milling.0‘00...OOOOOOOOOOOOOO0.00.00.00.00.
B. BakinSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOQ0.0.0!
C. Toasting and Staling......................
. Lysine Requirement.............................
5. Bread and Flour Enrichment Program.............
A. Fortification of Bread with Milk..........
B. Supplementing Bread with Wheat Gluten.and.
Other High Protein Foods..................
C. Supplementation of Wheat Bread and Flour
with Lysine...............................
6. Measures of Nutritional Status.................

GENERAL EXPERIMENTAL PROCEDURE........................
EXPERIMENT I

EXPERIMENTAL PROCEDURE................................

RESULTS...............................................

DISCUSSION............................................
EXPERIMENT II

EXPERIMENTAL PROCEDURE................................

RESULTSOOOOOOOOOIOOOOOOOOOOOOO'OOOOOOOOOOOOOOO0.0.0....

DISCUSSION...0.0:...»0000000000......OOOOOOOOOOOOOOOOOI.

EPImGUEOOOOOOOO-0.0....OOOOOOOOOOOOOOOOOOOOOOOOOOOOO-O.
GENERAL SUMMARY AND CONCLUSIONS.......................
BIBLImMPfIY. O O 0 O O O O O O O O O O O O O O 0 O O O O O O O O 0 O O O O O O O O O O O O O O

23
25
28

32
3.4
38

42
49
52

LIST OF TABLES
page

Table 1. Composition of breads used in the four
experimental diets.......................... 26

Table 2. Mean food intake per week, weight gain,
protein efficiency ratio and gain per gram
Of fOOd eatenOOOo.OOOOOOIOOOOOOOOOOOOOOO00.0J 26

Table 3. Level of statistical significance of
differences between groups.................. 26

Table 4. Liver fat, nitrogen, moisture, and xanthine
OX1daSe activ1ty0000OOOOOOOOOOOOOOOOOOOOOOOO 27

Table 5. Level of statistical significance of
differences between groups.................. 27

Table 6. Diet composition and percent protein in
the dietOOOO0..0.00.00...OOOOOOOOCCOCCOOOOOO 35

Table 7. Mean food intake per week, weight gain,
protein efficiency ratio and gain per gram
Of fOOd eaten-cocooncoo-00000000000000.0000. 35

Table 8. Level of statistical significance of
differences between groups.................. 35

Table 9. Fat, moisture and nitrogen content of the
livePSOOOO...0.000000000IOOOOOOOOOOOOO0.0.0. 36

Table 10. Level of statistical significance of
differences between groups.................. 36

Table 11. Hemoglobin levels of blood of rats.......... 37

Table 12. Level of statistical significance of
differences between groups.................. 37

Table 13. Comparison of the extent to which bread
provides the amino acids required by adult
human beings and growing rats............... 43

INTRODUCTION

An adequate food supply is one problem facing the world
as its population continues to increase at an ever accelera-
ting rate. Protein is the most difficult nutrient to pro-
duce. The less expensive sources of protein including cere-
als are considered to be poor in nutritional quality. In
most areas of the world, bread and cereal make up a large
share of the diet. Protein, B vitamins and minerals are den
ficient in cereals and bread.

More than one half of the world derives over one half
of their calories from cereals, chiefly rice and wheat.
Cereals are the cheapest source of calories, and throughout
the world they are consumed in large proportions by people
of low income levels.

Cereals are stated to supply 72.5 percent of the total
energy of the diets of people in the Middle East. The esti-
mated total protein intake for these people is 69.2 grams
per person per day, of which 83.4 percent is supplied by
cereals, mainly wheat, 8.7 percent by nuts and seeds, and
7.9 percent by animal food. Some signs of protein deficiency
among young children have been reported from certain areas
(Sabry, 1961).

Wheat is the staple food for a large percentage of
Iranians. Approximately three~fourths of the usable land in

l

Iran is given over to the raising of wheat. Most of the
wheat is made into bread in local bakeries from 75 percent
extraction flour, prepared in government owned mills. The
mills in turn buy their supply of wheat from farmers and
land owners.

Different varieties of wheat that are raised in differ-
ent parts of the country are as follows: common wheat, durum
wheat, Polish wheat, Polard wheat, shord wheat, Persian wheat
and a few others.

Several different kinds of bread are made in Iran.

These contain wheat flours, water and salt. The leavening

agent is a yeast starter carried over from the previous bak-

ing. The bread is baked on hot gravel in sheets about 30x12
inches and 28-40 mm thick. Thhs bread provides 70 percent

of the caloric intake of the people of lower economic groups

and farmers. The people in this group comprise more than 80
percent of the population. The other foods in the diets of

these peOple do not completely supplement the bread nutri-
tionally. As a consequence, many of the peOple show mild
symptoms of protein and B vitamin deficiency (Browne et al. 1961).

Since bread is the main dietary constituent for the peo-
‘ple in the lower and average income groups, it seemed worth-
while to attempt to improve the quality of protein in bread.
Any addition of dietary essentials to the wheat flour is
likely to be reflected in improvement of the nutritional

status of a large proportion of the population.

REVIEW OF LITERATURE

Nutritional studies have shown that the proteins of
wheat flour‘, eSpecially gluten, are among the poorer pro-
teins consumed in large amounts. Munaver and Harper (1959)
in a study with rats concluded that "wheat gluten due to its
very low level of lysine is so severely unbalanced that the
lysine it contains is not completely utilized, and lysine re-
quirement of the rat for maximum growth is increased when
this protein is the sole source of lysine in the diet".

Block et a1., (1958) indicate that growth retardation of ani-
mals fed wheat flour and bread as the only source of protein,
is due to the low percentage of complete protein in wheat

flour and bread.

1. Wheat and Wheat Flour Proteins

Protein supplies approximately 13 percent of the total
calories of the wheat flour (Block et a1” 1958). Howard et
al. in 1958 showed that the rate of growth of weanling rats
is directly related to the dietary levels of complete pro-
tein when wheat flour protein supplies the major part of the
diet. Furthermore, addition of proteins which provides non-

supplementary essential amino acids as well as excess

 

I Wheat flour prepared in the United States is usually 72
percent extraction and white flour is made by further re-
fining of wheat flour to different degrees, depending upon
the parts of the country and the type of bread. (The
EXperimental Study of Foods, Ruth M. Griswold, 1962).

3

quantities of dietary dispensable amino acids (all amino
acids except lysine) do not per se influence the rate of
growth.

The studies of Osborne and Mendel on the nutritional
values of protein emphasized the poor biological value of
the protein of wheat flour, especially gluten. Because of
the low levels of lysine in flour protein, animals fed flour
as the sole source of protein can utilize less than half the
protein to form new tissue (Block et a1., 1958).

Work by many investigators indicated that the total pro-
tein content of a food is not a good criterion of its growth-
promoting action when it is fed to rats. To overcome this
difficulty, Howard et a1., (1960) suggested the use of the
term "complete" protein. They defined this as "that fraction
of the total dietary protein which is completely utilized for
anabolic purposes”. The amino acids required for growth of
the young rat Ewe used in calculating the fraction of cereal
protein which was "complete". On this basis the "complete"
protein should contain 5.3 g lysine, 4.6 g valine, 5.0 g
sulfur-containing amino acids (cystine plus methionine), 3.0
g threonine and 1.1 g tryptophan per 16 g nitrogen (Howard
et a1., 1958; 16 g nitrogen 2 100 g protein).

To test the application of this theory, Howard et a1.,
(1960) fed rats wheat flour rations which contained 15%
protein. On this basal ration, only 50% of the total protein

was complete. Addition of increasing amounts of lysine

improved the growth rate and with that, the fraction of the
wheat protein which was ”complete". In agreement with the
report of Bender (1958) however, Howard et a1., (1960) were
unable to get growth rates in rats fed the lysine supple-
mented wheat flour ration which were more than 65 to 70% of
that secured when rats were fed a diet containing bread sup-
plemented with lactalbumen, soybean and lysine.

From the results of the amino acid analysis they made
of a variety of break3(table 13), Block and Mandl (1958)
calculated the percentage of "complete" protein in each of
the three commercial breads they studied. These calculations
were based on their conclusion that "lysine is the primary
amino acid deficiency" in wheat products and that "there is
no closely limiting second amino acid". With such assump-
tions, they calculated that the following values represented
the fraction of the total protein which was "complete":
wheat flour, 40%; white bread, 52%; whole wheat, 60%; Special
formulacracked wheat bread, 70% and lactalbumen-soybean milk
bread, 80%.

If the assumed estimated amino acid requirement is cor-
rect, according to the above figures the second limiting es-
sential amino acid in flour is cystine plus methionine
(Howard et a1., 1960). However, reports by Bender (1958) and
Sure (1957) suggest that threonine is the second limiting

amino acid in wheat protein. There is considerable evidence

that when an amino acid mixture simulating a good animal

protein is used in the test ration, the threonine require-
ment is in the order of 3.0 to 3.8 g per 16 g nitrogen. If
the requirement for threonine is higher when wheat proteins
are used as the source of amino acids the possibility of an
amino acid imbalance causing this increased need should be
considered.

Rosenberg and Rohdenburg (1954) found that rats raised
on a white bread diet supplemented with minerals and vita-
mins including vitamin 812 reached an adult size which was
only 75 to 80 percent as great as that of control rats fed a
mixed ration in which most of the protein was derived from
milk, meat scraps and soybean meals. Flodin (1953) indicates
the strong probability that quantity and especially quality
of protein in the diet exerts a predominant influence on
growth rate and adult size. Racial differences in size are
notapparent in the early months of life. However, marked
retardation of growth develops as the diet of infants changes
from the high quality protein of mother's milk to the low
quality_protein of vegetables. The adult height and weight
of the peOple in the various countries of the world varies
directly with the amount of protein in the diet (Flodin, 1953).

When protein is markedly deficient in the diet, produc-
tion of plasma proteins declines and the weight of circula-
ting blood proteins per kilogram of body weight diminishes.
Prolonged protein deficiency decreases the formation of anti-

bodies and resistance to infections. These defects can be

quickly cured by feeding adequate amounts of high quality
protein.

If a pregnant woman consumes less than 75 grams of pro-
tein daily during the later part of pregnancy, her infant
would tend to be short and light in weight. Mothers produce
more milk on high protein diets than on high fat or high
carbohydrate diets (Flodin, 1953).

The lysine content of wheat flour limits its protein
efficiency ratio (gain in weight per gram of protein eaten)
to about half that of the high quality protein. Rats fed
wheat gluten had low plasma choline esterase levels while
the addition of lysine and methionine elevated the level of
enzyme activity1 (Barrows, 1958). Pearson (1937) has shown
that a lysine deficient diet suppresses not only growth but

also the estrous cycle.

2. Composition of Wheat and Wheat Flour

J. R. Murlin et al.in 1941 in a study of wheat grain
and other cereals showed that the various layers of cereal
grains differ not only in their vitamin and minerals, but
also in their general protein makeup. The fractions which
contain the pericarp and germ and which serve as the dis-
tinguishing ingredients of the dietetically superior whole

grain flour, possess a much higher nutritive value than

 

I The level of choline esterase activity was considered a
means for detection of nutritional status but there are
reports that indicate this is not a significant way for
measuring nutritional level.

fractions which are composed mainly of endosperm.

Wheat germ has been recognized as having a high nutri-
tive value. It not only has a high thiamine and flavin con-
tent, but it also contains 30% protein (Hove et a1., 1943).

McDermotte et a1. (1960) in studies on amino acid com-
position of the protein in wheat determined the lysine con-
tent of six different varieties of flour. They found that
the different varieties of wheat have protein contents rang-
ing from approximately 6.7% to 14%. As the protein content
of the flour increases the percent lysine in the protein
decreases. The same inverse relationship exists between the
content of protein and amount of lysine in the protein of
endosperm obtained by microdissection from wheats of differ-
ent protein content.

Lawrence et a1. (1958) confirmed the preceding when they
found no significant difference in lysine content of differ-
ent varieties of wheat. The overall mean lysine content on a
unit protein basis was 2.89% for wheat of about 13.5% or more
protein. But below this level there was a highly significant
inverse relationship between protein content and lysine in
protein. The lysine content was not affected by the seasons
nor the region in which the wheat was grown.

The availability of lysine in wheat, flour and bread

was found to be 75, 72 and 76 percent, respectively, when

wheat gluten was fed as the protein in the basal diet‘.
With an amino acid basal diet the percent availabilities
were 78, 80, 83 and 80 for wheat, flour, bread and gluten,
respectively (Calhoun et al., 1960).

Kuiken and Lyman (1948) found 92.8% of lysine in the
whole wheat to be available to the rats as determined by
fecal excretion2. Guthneck et al. (1953) reported the lysine
of wheat germ to be 65% available as measured by gains in
body weight of protein depleted adult male rats. Gupta et
al. (1958) reported the lysine of wheat flour to be 70%
available as measured by the growth of young rats.

The animal work suggests that the lysine in wheat bread
is only about 70% available. No work has been found on the
availability of lysine in wheat or wheat products for the
human organism. Some of the lysine may be bound in the
browning reaction which occurs in the crust of the bread and

some might not be absorbed.

3. Amino Acid Loss during Milling and Baking
A. Milling
When converting wheat to flour, the level of the amino

acids in flour decreases. Hepburn et al. (1957) reported

 

TR A diet containing 20% gluten, 70% starch and 10% salts,
corn oil and vitamins plus histidine, methionine, trypto-
Jphan, and threonine.

2 This value may have been too high since there is a possi-
bility that the bacteria in the gastro-intestinal tract
may have metabolized the lysine.

10

that in converting wheat to flour the following amino acids
were significantly reduced: lysine, 24%; arginine, 19% and
aspartic acid, 19%. Hutchinson et al. (1956) indicated that
the lysine content of flour is proportional to the extraction
rate. The lower the percentage of flour secured from the
wheat the lower the lysine content of the flour. Consequent-
ly, whole meal bread has a higher lysine content than white
bread.
B. Baking

Baking bread from flour produced an 11% decrease in
cystine and 19% decrease in lysine (Hepburn et al., 1957).
McDermotte and Pace (1957) reported that there are 15 to 16
percent losses of phenylalanine, tyrosine and lysine in the
bread; most of the loss is in the crust (browning reaction).
Rosenberg and Rohdenburg (1951) showed that considerable
loss of lysine (from 9.5 to 23.8%) occurs during the baking
process.

C. Toasting and Staling

Toasting further reduces the lysine content from 5 to
10 percent and similar losses occur when bread becomes stale
and dry (Rosenberg and Rohdenburg, 1951). Howard et al.
(1958) state that browning products formed in the crust
during baking are primarily responsible for the loss of ly-

sine in the bread.

4. Lysine Requirement
The minimal daily requirement of 1-lysine is the most

ll

variable of all essential amino acids - it appears to have a
great interindividual variation. The average lysine require-
ment of man as found by Clark et a1. (1957) is between 500

to 900 mg per day. Rose et al. (1955) in their study found
that the minimal daily L-lysine requirement was 0.7 gram.
Since some individuals required more than this, Rose et a1.
tentatively proposed 0.8 g as the minimal daily requirement.
They suggest that 1.6 grams of this amino acid is a safe
dietary intake for an adult.

Numerous experiments using both human and animal sub-
jects have demonstrated that the protein of whole wheat flour
and bread in spite of its low digestibility is superior to
that of white flour and white bread (Caflson et al., 1946).

Commercial bread, because of its content of dry milk
solids should be decidedly better in protein value than
wheat flour (Block et al., 1959). Block et a1. rank the dif-
ferent breads they studied in the following order of increas-
ing nutritive value: white bread, whole wheat bread, special
formula, cracked wheat and lactablumen-milk-soyabean flour

enriched white bread.

5. Bread and Flour Enrichment Program

Before the bread and flour enrichment program started
in the United States, the medical literature was filled with
reports of pellagra, beriberi, mild anemia and ariboflavin-
osis. Thiamin was also low in the diets during 1920 and the

following decade. The body does not store this vitamin and

12

it needs to be replenished to prevent the deficiency symp-

toms associated with a low intake.

The enrichment of bread and flour was one factor that
contributed to the decreased death rate for pellagra and
beriberi that occurred in the subsequent years. It also
contributed to the disappearance of ariboflavinosis.

There have been many studies which demonstrate how the
nutritive value of bread, flour and cereal grain can be im-

proved. Milling removes the wheat germ from flour and so re-

duces very drastically the vitamin, mineral and protein con-
tent of the flour. There are a variety of means that have

been proposed for overcoming these deficiencies in flour.

They are:
A. Fortification of Bread with Milk
Non fat dry milk solids which supply among other things

lysine and valine, have been used in white bread rather ex-

tensively by the baking industry.

Most commercial white breads contain 2-6 percent dry
Inllk solids. Bakers add the milk solids to bread to increase

14:8 softness and stability rather than to improve the bio-

JJDSical value of bread protein. The improvement results from

tile fact that the lysine content of milk protein is 7.5 per-

c3°nt and its valine content is 4.5 percent. Commercial bread .

bile been prepared containing as much non fat dry milk solids

as 6 percent of the flour.
Parks et al. (1954) showed that addition of milk improves

13

the amino acid composition of white bread. The milk also
increased the calcium content of the bread. Light et al.
(1943) states that addition of 6% dry skim milk powder to the
bread formula produces a bread which is equal to whole wheat
bread in so far as growth of rats is concerned. Welton et
al. (1959) reported that a high nutritive type of white bread
containing non fat milk solids at a level as high as 25% of
the flour could be made. The addition of this very high
level of non fat dry milk solids increased the protein con-
tent of the bread about 30 percent,-depending on the type of
flour used. The biological value of this bread, as shown by
rat growth tests, was twice as great as that of commercial
bread containing 4 percent non fat dry milk. Thus the nutri-
tional value of the protein (biological value x protein con-
tent) is about 2.5 times that of the usual commercial bread.

Jahnke et al. (1957) reported that even when 12 percent
non fat dry milk solids were incorporated in white bread,
the biological value thereof could be further increased by
the addition of lysine. Animals that were fed the bread con-
tainhm;l2% milk solids showed an improved growth rate when
0.25% lysine was added to the bread. They concluded that
the rate of growth of rats fed the bread is directly related
to the lysine and tryptOphan content of the ration.

Kennedy et al. (1954) showed that acceptable bread could
be obtained from white flour containing as much as 22% milk

solids. The maximum amount of milk solids that could be

14.

added depends to some extent upon the flour. According to
L. V. Rogers (1959) when bread containing a high percentage
of dry skim milk is made, it is generally necessary to in-
crease the fermentation time due to the increased buffering
capacity resulting from the added milk solids.

B. Supplementing Bread with Wheat Gluten and Other High
Protein Foods

A number of studies have been carried out to determine
how the biological value of bread might be improved by adding
a variety of proteins or foods high in protein, other than
milk solids. Hutchinson et al. (1962) showed that wheat
gluten did not improve the lysine deficiency of white flour.
Supplementation of bread with 10~50 percent wheat gluten
caused only a small increase in the growth of the experi-
mental animals. For this reason, gluten is not considered
a good supplement for white flour.

Wheat germ which is high in protein, iron, threonine,
riboflavin and niacin, can be added to bread after the fat
has been removed (Westerman et a1., 1952). The addition of
fat free wheat germ to bread increased the level of the
above mentioned nutrients in the diet. When 4 to 6 percent
of defatted wheat germ was added to unenriched flour, it in-
creased the growth rate of rats. The only objection to the
use of defatted wheat germ is that it is not very palatable.

The protein of soybean flour due to its high lysine and

valine content may also be used to increase the nutritional

quality of wheat flour protein (Carlson et al., 1946).

15

Sure (1952) reported that the proteins in whole rye are
superior in nutritional value to those in whole wheat. This
was true at all levels of flour fed to rats. Prior to this,
Ron and Markuze (1931) obtained results indicating the superi-
ority of rye bread with respect to the biological value of
the protein.

A new type of bread made with rice and wheat flour has
been offered as a substitute for the usual white bread. The
use of rice does not change the organoleptic quality of white
bread. Alejandro—Mosqueda-Suarez (1958) showed that a suit-
able flour could be made by replacing 30 percent of white
flour with rice flour. The protein efficiency of rice-wheat
bread compared with wheat bread was increased by 9.1 percent.
They suggested that the improvement in protein efficiency
probably resulted from the changed amino acid pattern pro-
duced by the rice. This bread requires 25 percent more yeast
and also needs a longer fermentation time.

Algae have also been used for supplementing white flour.
Hundley et al. (1956) reported that chlorella is a better
source of thiamine than purified soy protein. When chlorella
was added to white flour on an isonitrogenous basis, it was
equal to several animal proteins of high biological value.
They also suggested that algae might serve as a source of
protein for the supplementation of various flours and cereals.

Sure (1946, 1947, 1948) in an extensive series of stud-

ies on the supplementation of cereal grains observed that

16

rats fed enriched white flour supplemented with 1-5 percent
nonviable dried yeast exhibited large increases in weight
gain and food utilization as compared to those fed unsupple-
mented enriched white flour. These increases were not en-
tirely accounted for by the lysine content of the yeast. The
diet used by Sure contained adequate amounts of 8 crystalline
B vitamins. Seeley et al. (1950) stated that "Although dried
yeast contains 3.5 percent lysine and is therefore an excel-
lent source of this substance, the amount of lysine and va-
line which it (the yeast at a 3% level) provides at the low
level employed evidently is not enough to produce a large in-
crease in the biological value of the bread protein". The
increase in the weight of the rats fed the yeast-supplemented
bread depended on the protein, vitamins and minerals in the
yeast as well as the lysine.

Parks et a1. (1954) showed that the level of thiamine
was increased in bread made with brewer's yeast.

0. Supplementation of Wheat Bread and Flour with Lysine

Many studies have been carried out to determine the ef-
fect of lysine in improving the protein of white flour and
bread. In 1943, Light and Ferry reported that the protein
of white and whole wheat bread can be further improved by
supplementing the flour with lysine and valine. Rosenberg
and Rohdenburg in 1952 reported that if sufficient lysine
was added to the bread diet to bring its total lysine con-

tent to 0.8% or more, growth similar to that obtained with

17

the stock diet was observed). Sure (1955) found that the
addition of lysine and valine increased the protein effici-
ency ratio of the proteins in wheat flour from 0.68 to 3.11.
Lysine and threonine were found to play a "significant spe-
cific" role in food utilization. In 1954, Sure carried out
rat growth studies with a whole wheat cereal breakfast food
and with whole wheat. Feeding these cereals at an 8 percent
pr0tein level, he observed an increase in protein efficiency
when lysine alone was added. However, he obtained a further
increase by adding valine and threonine to the lysine sup-
plemented cereal.

When lysine supplemented white bread was fed at a 12.5%
protein level no supplementary growth effect was obtained
from the addition of valine and threonine. He also reported
that vitamin 812 in the presence of lysine and threonine in-
creased the protein efficiency of the processed wheat. The
vitamin B12 also increased food intake. The addition of
lysine alone to wheat gluten has been shown to raise its
protein efficiency ratio to a value equal to those for ani-
mal proteins. This was shown to be true for both infants

and adults (Flodin, 1956).
Hutchinson et al. (1956) found that the difference in

 

I Composition of the basal bread diet was: dried bread, 90%;
salt mix no. 351, 3%; crude soybean oil, 4.5%, cod liver
oil, 0.5%; biotin or starch (15 ug/g), 1.0% and vitamin
concentrate, 1%. Bread used in this experiment was com-
mercial loaf bread baked according to the standard for-
mula. It contained 3% non fat milk solids and 2 parts of
yeast per 100 parts of flour.

l8

lysine content was responsible for the increased growth rate
of male weanling rats fed rations containing protein from
whole wheat meal as compared with that of white flour. They
showed that the optimum response in growth rate occurred with
a lysine addition of between 0.2 and 0.3 percent to white
bread. Addition of more than 0.3 percent lysine depresses
growth rate and also food intake of rats. The latter is
probably due to an amino acid imbalance.

Culik et al. (1958) showed that lysine supplementation
of bread improved reproduction and lactation performance of
rats uniformly in each of 7 litters of the same generation
and in successive generations. Rations containing, as the
sole source of protein, flour supplemented with 0.25% L-_
lysine HCl produced normal or nearly normal reproduction and
lactation of rats.

Rosenberg and Rohdenburg (1958) showed that the protein
efficiency ratio of bread protein can be increased from 1.01
to between 1.89 and 2.12 by fortification with lysine. Brown
et a1. (1959) concluded that "for protein efficiency ratios
in the high quality range of 2 or higher it appears necessary
that the bread protein contain at least 200 mg of L-lysine
per gram of nitrogen".1

Clark et al. (1961) reports that supplementation of

 

T' In my experiment the protein efficiency ratio was found
to be 1.34 for American standard white bread with 2%
dried milk solids added (table 3).

l9

flour diets with lysine up to the level of 0.5 percent sig-
nificantly increased the food intake and weight gain. Maxi-
mal weight gains were noted with 0.2 percent of added lysine.
The biological value of flour protein (the percentage of ab-
sorbed protein retained by the experimental animals) was in-
creased in a stepwise fashion by lysine supplementation up

to the 0.25 percent level. Higher levels did not further
increase the biological value of the protein.

The nitrogen efficiency value reached a maximum when
lysine supplementation was at a level of 0.20 or 0.25 per-
cent.

From the above observations, it is well established that
wheat bread and flour are poor sources of complete protein.
If they supply the major part of the diet, growth retarda-
tion in animals results. Various methods of bread and flour
enrichment have been studied and several supplements were
used such as dry milk solids, soybean flour, rye flour, cul-
tured and brewer's yeast, rice, algae, pure amino acids, etc.

At present in Iran it is economically unfeasible to con-
sider the fortification of flour or bread with pure amino
acids. However, skim milk powder is produced by a number of
plants throughout Iran. This product has not found a very
extensive market largely because the peOple are not accus-
tomed to this food. For this reason it should be possible
to mix skim milk powder with the flour prepared in the gov-

ernment mills. It may also be possible to use legumes that

20

are indigenous to Iran for supplementing the protein in the

flour.

6. Measures of Nutritional Status

Liver xanthine oxidase activity is a method used by in-
vestigators to study the levels of protein in the diet. W.
Westerfield et al. (1951, 1956) showed that this enzyme is
very senSitive to levels of protein intake. Low protein
diets cause a loss or decreased activity in liver xanthine
oxidase. Measuring the activity of this enzyme was used in
this experiment to compare the protein content of the 4 types
of bread used in Experiment I.

Choline esterase activity was used by Barrows (1958) for
measuring the quality of wheat protein. McCance et al. (1948)
reported that mean serum choline esterase levels are related
to the protein and food intake, but Saunders et al. (1952)
showed there is no relation between the level of this enzyme
and protein intake. In view of the controversial nature of
this method, it was not used in this experiment.

Another factor that"is very sensitive to the level of
dietary protein is hemoglobin levels. In starvation studies,
Keys et al. (1950) showed that both the level of hemoglobin
in the blood and total circulating hemoglobin reflect the
protein losses more accurately than any other factor. The
adequacy of the protein in the 4 diets in Experiment II was
assessed by the hemoglobin levels in the rats at the end of

the study.

GENERAL EXPERIMENTAL PROCEDURE

This study consisted of two experiments. Each experi-
ment will be discussed separately. The first experiment is
a comparison of the growth produced by rations in which the
primary ingredient was either Persian, Syrian, Alladin or U.
8. white enriched bread (see p. 23 for description of breads).

The second experiment was designed to study the improve-
ment of Persian bread by supplementing the bread with dry
milk solids and/or lentils.

Male weanling rats of the Sprague-Dawley strain, weigh-
ing45-55 grams were used in both experiments. The animals
were divided into groups of ten on the basis of weight. The
mean weight of all groups in the same experiment did not vary
by more than 0.5 gram. The animals were housed individually
in wide mesh, wire bottom elevated cages. Food and water
were given ad libitum. All animals were weighed weekly dur-
ing the experimental period. Food intake was measured and
recorded.

The composition of the basal diet was:

Dried ground bread 90%
Corn oil1 5%
Mineral mixture2 4%
Vitamin mixture 0.25%
Choline chloride 0.15%
Sucrose 0.60%

 

1 Containing 7.5 mg alpha tocopherol per 5 g.
2 Salt mixture W, obtained from Nutritional Biochemicals

Corp.
21

22

The vitamin mixture provided in each 100 g of diet:
0.5 mg thiamine, 0.5 mg riboflavin, 1.0 mg niacin, 0.25 mg
pyridoxine HCl, 2.0 mg calcium pantothenate, 10 mg inositol,
0.02 mg folic acid, 0.002 mg vitamin B12, 0.01 mg biotin,
1.0 mg peaminobenzoic acid, 10 mg vitamin A, 0.18 mg vitamin

D, and 0.38 mg menadione.

EXPERIBENT I

EXPERIMENTAL PROCEDURE

Four groups of 10 rats each were fed rations in which
the dried ground bread was: Group I, American white enriched
bread which was obtained on the open market; Group II,
Syrian-Lebanese bread, baked in Detroit and having a label
which indicated the bread was made of flour, water, salt,
yeast and preservatives; Group III, Persian bread which was
bought, dried and ground in Iran and shipped here; Group IV,
Alladin bread which was ordered from New York. The label on
the latter bread indicated that the same ingredients were
used in this bread as those in the Syrian bread.

All four types of bread, including the dried bread from
Iran, were dried in a vacuum oven at 500C for a period of
5 hours then ground to a fine powder.

The rats were fed these diets for a period of four
weeks. Food intake and body weight were recorded each week.
From these values, protein efficiency ratio (gain in body
weight per gram of protein consumed) and gain per gram of
food consumed were calculated. .Atthe end of the 4th experi-
mental week, 5 rats from each group were sacrificed with a
sharp blow on the head and decapitated. The livers were re-
moved, homogenized with water in a Potter-Elvehjem apparatus
and then dried to constant weight in an oven maintained at
100°C (requiring 12 hours). On the 29th experimental day,

23

24

one rat from each group was sacrificed. One gram of its
liver was immediately homogenized and a fraction of it added
to a Warburg flask for the determination of xanthine oxidase
activity. On each of the next 4 days, another rat from each
group was sacrificed and one gram of its liver used for the
determination of xanthine oxidase activity. This was done
for all of the rats killed during the 5 days after the exper-
iment prOper was terminated. The liver remaining after the
removal of the 1 gram sample was homogenized. The moisture
in the liver was estimated by drying to constant weight at
100°C. After drying, the livers were ground to a fine pow-
der in a small Wiley mill. The fat from one gram of the
dried, ground liver was extracted by ether on a Goldfisch ap-
paratus. Nitrogen was determined by the Kjeldahl technique.
For liver xanthine oxidase activity, the procedure of Axelrod
and Elvehjem (J. Biol. Chem., 1403725, 1941) for the Warburg
apparatus was used.

The breads in the four diets were analyzed for nitrogen
and fat and the protein contents of the breads were calcula-
ted using 5.7 as the conversion factor (Assoc. Off. Agric.

Chem., Official Methods of Analysis, p. 215.).

RESULTS

Table 1 presents the results of the bread analyses.

This table shows that the nitrogen content of Persian bread
is almost equal to that of American standard bread (with 2
percent milk solids). Alladin bread has the highest nitro-
gen content. The level of fat in American bread is 1.36 due
to the addition of 5 percent tri- and diglycerides (mostly as
stearate) while the other 3 breads are much lower in fat
since they have none added.

The growth per week of rats in Groups III and IV were
similar to those of the control group (American white bread,
table 2). Group II gained significantly less weight than the
control animals (P<0.05, table 3). The protein efficiency
ratio of Group I is significantly greater than that for
Group II and Group IV and that of Group III is significantly
greater than Groups IV and II (P<0.0l).

The liver nitrogen of animals in Group I is significant-
ly less than that of Groups III and IV (P<0.01, tables 4 and
5) and the animals in Group II have significantly less nitro-

gen in their livers than Group IV (P<0.05).

25

 

 

 

 

 

 

 

 

 

Table 1. Composition of breads used in the four experimental
diets. All values are expressed as percent of the
dried breads or diets prepared for the rats.

Nitrogen Fat in Protein Protein

Group Bread in Bread Bread Moisture in Bread in Diet

I American 2.17 1.36 1.29 12.37 11.13
II Syrian 2.47 0.13 4.29 14.08 12.67
III Persian 2.00 0.39 9.86 11.40 10.26
IV Alladin 2.78 0.31 4.37 15.85 14.26

Table 2. Mean food intake per week. weight gain, protein

efficiency ratio and gain per gram of food eaten.

Food Protein Gain/g Gain/g

Group Intake Growth Intake Protein Food
g7week g/week

I 6300:296 904510055 7013 103410005 0015i00006
II 66.8:2.8 8.80:0.42 8.46 1.04t0.03 0.13:0.004
III 76.6i3.3 10.40i0.48 7.86 1.32:0.04 O.l4io.004
IV 63.4i2.7 9.93:0.70 9.04 l.08i0.04 0.16t0.005

Table 3. Level of statistical significance of differences
between groups (see table 2).

Groups Food Gain/g Gain/g
Compared ,Intake Growth Protein Food
I vs II none none 1% 5%

I vs III 2% none none none

I vs IV none none 1% none

II vs III 5% 5% 1% none

II vs IV none none none 1%

III vs IV 2% none 1% 1%

 

26

Table 4. Liver fat, nitrogen, moisture and xanthine oxidase
activity. All values are expressed as percent of
the fresh livers.

 

Xanthine Oxidase

 

Group Moisture Fat Nitrogen ul Oo/houngggva?
I 72.510.6 4.3610.3 2.1510.05 175226
II 72.810.8 4.1510.l 2.28i0.09 196138
III 75.0i0.7 3.88:0.1 2.45:0.08 214140
IV 70.Bil.1 4.08:0.1 2.59io.09 196144

 

Table 5. Level of statistical significance of differences
between groups (see table 4).

 

 

Groups Xanthine
Compared Moisture Fat Nitrogen Oxidase
I vs II none none none none
I vs III none none 1% none
I vs IV none none 1% none
II vs III none none none none
II vs IV none none 5% none
III vs IV none none none none

 

27

DISCUSSION

Body weight gain. During the 4 weeks of the study, the
animals in Group III fed the Persian bread diet had an aver-
age weight gain which was significantly greater (P<0.05) than
that for the rats in Group II, fed the Syrian-Lebanese bread
(10.4 g vs 8.8 g). In the other groups the differences in
weight gain were not significant.

Food intake. The amount of food consumed by the rats
was measured by recording the differences in the weights of
the feed cups when empty and when filled with food. The
amount of spilled food was recovered and weighed to obtain
more accurate food intake values.

The average food intake of the animals in Group III was
significantly greater than Groups I, II, and IV; these
groups consumed the same weight of food.

These differences can very well be the result of food
spillage. It was observed that in Group III, the animals
fed the Persian bread diet spilled their feed more than the
rats in the other groups. However, special attention was
given to make sure that this spilled food was accounted for.

Liver analyses. There were no significant differences
in liver moisture and fat among the 4 groups of animals. The
nitrogen concentration in the livers of the rats in Group I
was significantly less than in Groups III and IV (P<0.01) and

28

29

that in Group II was significantly less than in Group IV
(P<0.05). This could be due to the better protein efficiency
of the bread fed to the animals in Groups III and IV. The
nitrogen content of the bread used for the diet of the ani-
mals in Group IV was higher than that fed the controls (table
1).

The primary purpose of this part of the study was to see
whether the biological values of the bread proteins were the
same. 0n the basis of the above results, there are no sig-
nificant differences between the growth promoting effects of
the American commercial white bread, Persian bread and Alladin
bread. But that of Syrian-Lebanese bread is significantly
less than that of the Persian bread. In other words, the
growth promoting effect of Persian bread is equal to American
standard white bread which is made with 2.7 percent yeast,

2% milk solids and vitamin and mineral enriched flour. This
could be due to several factors. First, Iranian bread is
baked in the oven for only 5 minutes or less, at a high tem-
perature. But on the other hand, this bread being baked in

a sheet has more surface area exposed to the heat and con-
sequently the browning reaction is greater. Theoretically,
there should be more lysine lost in this bread than American
bread baked in loaves. According to Rosenberg and Rohdenburg
(1951) and McDermotte and Pace (1957), the amino acids de-
stroyed due to the browning reaction in the crust of bread

amounts to between 9.5 and 23%. Thus, the time and heat

30

involved in baking the bread does not explain the similarity
between the nutritive values of Iranian bread and white Ameri-
can bread. Another factor may be the lower extraction of
flour in Iran. They discard a smaller portion of the wheat
in the milling process and thus more of the protein is con-
served in the flour than is conserved in the American white
flour (the usual extraction of flour in Iran is between 75
and 80%). The type of wheat may be another factor. Accord-
ing to McDermotte et al. (1960) different varieties of wheat
have a protein content of from 6 to 14 percent. However,
Lawrence et al. (1958) indicated that there were no signifi-
cant differences in the lysine content of different varieties
of wheat.

For a variety of reasons, the different varieties of
wheat in Iran are bought and pooled at the mills. Usually
the wheat flour that is used for bread is prepared from a
mixture of different varieties of wheat, depending on yield,
price and season and other factors as well. If the pattern
of amino acids in different varieties of wheat differs, they
may balance each other and as a result the mixture may have
a better protein content.

The length of the fermentation can be another factor.
The dough which is thin (almost like a batter) is left over-
night to rise, the raised dough is not as elastic as the
dough used for American bread. According to Clifton (1957)

31

the number of yeast cells increases with time. The rate of
cellular division remains constant, but the actual number of
bacteria or amount of bacterial protoplasm formed per unit of
time, during this phase increases with time since growth is
proportional to the number of cells present at any given
time. Huffman and coworkers (1941) found that in standard
dough containing 1.67% of yeast fermented at 80°F there was
practically no increase in the number of yeast cells during
the first 2 hours. The greatest increase of about 26% took
place between the second and fourth hour. The rate of in-
crease drOpped to about 9% between the 4th and 6th hour of
fermentation. The long period of fermentation of Iranian
bread may help increase the total amount of yeast cell proto-
plasm thus increasing its protein content.

The factors that may be important in bringing the bio-
logical value of the Iranian bread up to that of American
white bread containing 2% skim milk powder are: 1) short
baking period of Iranian bread; 2) lower extraction rate of
Iranian flour; 3) the variety of wheat used in making Iranian

bread; and 4) the long period of fermentation.

EXPERIMENT I I

EXPERIMENTAL PROCEDURE

Forty male weanling rats were divided into 4 groups of
10 each and fed the following diets for a period of 3 weeks:
Diet I, basal diet which was the same as that listed on page
23 and made with Persian bread; Diet II, the same as Diet I
except that 4% skim milk powder (Carnation instant) replaced
an equal amount of bread; Diet III, same as Diet I except
that 7.5% ground lentils (Lens esculenta) replaced an equal
weight of bread; Diet IV, same as Diet I except that 15%
ground lentils replaced the same amount of bread.

The measurements for food intake and body gain in weight
were taken as previously described. The chemical analyses
were also the same as previously described.

Hemoglobin levels in the blood of these rats were de-
termined in all animals in place of liver xanthine oxidase
activity determinations.

Blood samples were taken from the rats' tails prior to
sacrificing. The blood was transferred into spot plates
which contained crystalline potassium oxalate to prevent
blood clotting. Then 0.02 ml of blood was transferred to
tubes containing Drabkin's solution. Drabkin's solution was
made from 1.0 g NaHCO3, 50 mg KCN and 200 mg K3FE(CN)5 and
brought up to 1 liter volume. The tubes were read in a
Bausch and Lomb colorimeter and percent hemoglobin was read

32

33

from the standard curve prepared using known concentrations
of cyanomethemoglobin standard (Drabkin, 1932).

The nitrogen content of diets was determined and protein
content was calculated using 5.7 as the conversion factor.
The average protein intake of animals was also calculated

(table 7) as previously described.

RESULTS

In table 7 the average group food intake per week is
shown for the 3 week period. Average growth per week, gain
per gram protein eaten and gain per gram food intake were
calculated therefrom.

The food intake, body weight gain, protein efficiency
ratio and grams of body weight gain per gram of food intake
in the rats in the 4 groups were significantly different (P<
0.01, table 8).

The moisture content of the livers of the rats in Group
III was significantly less than that of rats in Groups I, II
and IV (P<0.0l, 0.02 and 0.01, respectively; tables 9 and 10).
The fat content of the livers of the animals in different
groups were not significantly different but the nitrogen con-
tent of the livers of the rats in Group III was significantly
greater than that of animals in the other groups (table 9 and
10).

There was a significant difference (P(0.05) in the hemo-
globin levels of the rats in Groups I and III, Group III
being greater. Hemoglobin levels in the blood of animals in
other groups were not significantly different (tables 11 and

12).

34

Table 6.

Diet composition and

percent protein in the diet.

 

 

 

%N in %Protein

Group Composition Diet in Diet

I Persian 1.91 10.89
(9.86% moisture)

II Persian + 1.98 11.29
4% skim milk

III Persian + 2.01 11.46
7.5% lentils

IV Persian + 2.16 12.31

15% lentils
Table 7. Mean food intake per week, weight gain, protein

efficiency ratio and gain per gram of food eaten.

 

Food

Grou Growth Intake
g7week g/week

I 10.8i0.5
II 18.1iO.4
III 21.310.4
IV 31.6il.O

63.4:2.1
75.1:3.3
86.0il.3
105.613.4

Gain/g
Food

0.17:0.005 6.92
0.24:0.005 8.48
0.25:0.004 9.86

O.30i0.00S 12.99

Protein

Intake Protein

Gain 5

1.57:0.05
2.13:0.04
2.1710.03
2.4310.04

 

 

 

Table 8. Level of statistical significance of differences
between groups (see table 7).

Groups Food Gain/g Gain7g
Compared Growth Intake Food Protein
I vs II 1% .l%’ 1% l
I vs III 1% 1% 1% 1%

I vs IV 1% 1% 1% 1%
II vs III 1% 1% none none
II vs IV 1% 1% 1% 1%
III vs IV 1% 1% 1 1%

 

Table 9.

Fat, moisture and nitrogen content of the livers.
All values expressed as percent of the fresh liver.

 

 

 

 

 

Group Moisture Fat Nitrogen
I 71.56i0.33 3.84i0.15 2.1710.07

II 71.2210.26 3.84:0.13 2.2710.05
III 71.5110.19 3.81:0.14 2.41:0.03
IV 70.4010.15 4.1820.15 2.2310.06

Table 10. Level of statistical significance of differences

between groups (see table 10).

Groups

Compared Moisture Fat Nitrogen

I vs II none none none

I vs III none none 1%

I vs IV 1% none none

II vs III none none 5%

II vs IV 2% none none

III vs IV 1% none 5%

 

36

Table 11.

Hemoglobin levels of blood of rats.
at 18th day of experiment.
3 weeks” duration.

Sample secured

The experiment was of

 

 

 

Group Bread Hemoglobin Level
I Persian ll.5510.l6
11 Persian + 4% milk solids ll.65$0.24
III Persian + 7.5% lentils 12.05:o.15
IV Persian + 15% lentils 11.9510.28
Table 12. Level of statistical significance of differences

between groups (see table 11).

 

I vs
I vs
I vs
II vs
II vs
III vs

Groups Compared

II
III
IV
III
IV

IV

Hemoglobin Level

none
5%
none
none
none

none

 

37

DISCUSSION

The bread used in the rations for all groups was Persian
bread which was used at a level of 90% in the control diet.
For the other groups, the bread was supplemented with 4%
dried milk solids, 7.5% and 15% ground lentil powder, respec-
tively. The amount of bread used in Groups II, III and IV
was 86%, 82.5% and 75%.

Food intake and growth. There was a significant differ-
ence (P<0.01) for all four groups of animals in so far as
body weight gain and food intake were concerned. This indie
cates that the supplements used in this study enhanced the
rate of body weight gain. For the lentils, the higher the
level of supplementation, the better was the food intake and
growth. The addition of 4% dried milk solids increased the
food intake of the animals during the 3 week study. Lentil
powder (7.5% and 15%) further increased the food intake and
growth which were proportional to the percent of supplementa-
tion.

Table 7 shows that protein efficiency ratio of the diet
increased as the level of supplementation increased.

Liver analyses. The moisture content of the livers in
Groulns I, II and III were significantly greater than those
in eraup IV (P<0.01, 0.02 and 0.01, respectively). There was
no signdificant difference between any of the groups in the
percerit fat in the livers.

38

39

Liver nitrogen in Group III was significantly greater
than that in Groups I and II (P<0.0l and 0.05, respectively).
The nitrogen in Group IIIvms significantly greater (P<0.05)
than that in Group IV.

The hemoglobin level in the blood of all animals was
determined and the statistical analyses show that Group III
had significantly greater (P<0.05) than that of the control
group. There were no significant differences between the
level of hemoglobin in the blood of animals in the other
groups.

The level of supplementation of the Iranian bread with
dried skim milk solids was only 2% above that of the American
white bread. In the first experiment it was found that un-
supplemented Iranian bread produced the same growth in rats
as the standard American white bread, containing 2% milk
solids. In the second experiment, rats fed Iranian bread
which was supplemented with 4% dry skim milk solids, gained
almost twice as much weight as the rats fed the standard
American white bread in the first experiment (18.1 g vs 9.45
s).

Supplementation of the bread with lentils at the levels
of 7.5% and 15% respectively in the diets of Groups III and
IV in Experiment II further increased the weight gain in pro-
portion to the level of supplementation. However, the liver
nitrogen in Group IV was significantly less than the liver

nitrogen in Group III. This might be due to the larger size

40

of the livers in Group IV (an average of 5.45 g vs 4.51 g).

The lower moisture content and nitrogen content of the
livers in Group IV could be due to the toxicity of lentils
(legumes) at high levels. anseti et al. (1952) reported
that higher levels of legumes in human dhfls causes lathyrism.
Geiger, Steenbock and Parsons in 1933 noted scoliosis and
hernias in white rats fed a diet containing 50 percent sweet
pea seeds. Those authors found that the water extracts of
peas fed to the rats were toxic while the extracted peas fed
at the 50 percent level were not toxic. Lewis et al. (1948)
noted spinal curvature in rats fed various species of legumes.
When peas are eaten as a smaller proportion of the diet, they
are said to be harmless.

Stockman (1929) believed that the toxic principle was
an alkaloid which he obtained from the peas and used to pro-
duce paralysis in frogs. Acton and ChOpra (1922) reported
paralysis in the hind leg of monkeys, rats, etc., produced
by a pure hydrochloride of an amine obtained from peas.
Young (as reported by Stockman, 1929) suggested that the dis-
ease was due to a vitamin A deficiency but sources of vitamin
A did not protect animals against the disease. Normal growth
was obtained in 20 weeks in rats fed 12.5% and 5% sweet peas
in the diet.

Lentils, being a legume, might cause the same problem as
peas do when used to supplement the diet at high levels. If

so, this would be a limiting factor in trying to supplement

41

the bread with higher levels of lentils. Another factor
would be the taste of the bread. This should be tested and
the acceptability of the lentil supplemented bread should be

proved before any attempt to actually supplement bread with

lentils is begun.

EPILOGUE

This study was started with the purpose of improving
the nutritional quality of the protein of Iranian bread.

Many experiments conducted by various investigators over long
periods of time have shown that bread is a poor source of
protein as evidenced by feeding experiments. These studies
further showed that the quality of the protein in the bread
could be improved by adding different proteins or amino acids
to it. Most of these experiments were done using the rat as
the experimental animal. The results of the 2 feeding experi-
ments with the rat that are reported here are in agreement
with other experiments done before by others. They show that
the Persian bread can be improved nutritionally by supple-
menting it with 4% dried milk solids, or with 7.5 and 15%
lentils.

After the experiments were completed, it seemed desir-
able to find out how much of the amino acid requirement of
man was met by a diet consisting of 70 percent unsupplemented
bread. This would be comparable to the diet of the Iranian
farmers and people in the lower classes. Surprisingly, the
results of our calculations showed that the above mentioned
diet would meet all the amino acid requirements of human
beings. Further research of the literature and calculations
showed that the same bread is definitely deficient in amino
acids as far as the growth of the rat is concerned (table 13).

42

43

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44

This leads to the question: Are the results obtained from
studies with rats directly applicable to man especially in so
far as the adequacy of the amino acid composition of the diet
is concerned?

Assuming the average caloric intake of Iranians to be
2500 calories and Iranian bread to have the same lysine con-
tent as American white enriched bread (containing 2% dry milk
solids), lysine intake from bread alone, of a diet composed
of 70% bread would be 1.6 grams. This was calculated using

the equation:

2500 x .go x .2521 = 1.6
27

According to W. C. Rose (as reported by H. H. Williams,
1947) the minimal daily lysine requirement of man is 0.8 g.
The safety requirement for lysine brings this value to 1.6 g.
The above mentioned Iranian diet seems to meet the safety
lysine requirement of the adult man, even with bread proteins
alone. On the other hand, this study shows that supplement-
ing the bread with 4% dry milk solids and 7.5 and 15% lentils
caused weanling rats to gain 2 and 3 times as much, respec-
tively, as control rate. If the human lysine requirement is
met by the above diet and if lysine is the most limiting

amino acid, the requirement for other amino acids will also

 

1 0.252 g lysine/100 g bread (table 13)
2 276 calories are obtained from each 100 g of bread
(U.S.D.A. Handbook no. 8)

45

be met. The values give in table 13 prove this point.
Mitchell (1958) indicates that the amount of absorbed
nitrogen required for nitrogen equilibrium in human beings
is 4.61 mg/basal calorie and for the rat, 3.32 mg/basal cal.
when wheat flour is fed. However, when the total caloric
requirement is considered for both rat and man, the ratio
for man decreases to almost one-half, while that of the rat
remains almost the same. This is based on the fact that the
total energy requirement for man is about twice his basal
needs. If the individuals do as much physical work as the
Iranian laborers and farmers do, the basal energy required
may have to be multiplied by a factor greater than two.
Under such circumstances, the reduction in the above ratio
for man becomes even greater. For the rat, there is probably
little difference between its basal and total energy require-
ment. The rat confined to the small cage in which these
studies were carried out is restricted in his total activity.
Consequently the energy above basal needs to provide for
spontaneous activity is very small.
A possible explanation for the differences in the re-
sults secured with rat feeding trials and the extent to
which bread appears to meet the amino acid requirement of
man may be the difference in the ratio of calories to amino
acid requirement. The weanling rat receiving the bread diet
containing the Iranian bread consumed 9 grams of diet which

contained 90% bread with 9.86% moisture. Their bread intake

46

was 7.3 grams per day. The rat requires 138 mg lysine per
day, therefore the animals obtained each mg of their lysine
intake from .223 calories. Man requires 800 mg lysine per
day. If he receives 1750 calories from the same bread, each
mg of lysine is obtained from 2.2 calories. This ratio of
caloric and lysine requirements differs by a factor of 10
from that of the rat.

From the amount of amino acid available to rats eating
a diet consisting primarily of bread, it can be shown that
this diet does not meet the amino acid requirement of the
rat (table 13). The rats in Experiment I, Group I, that
were fed the diet consisting of 90% American white enriched
bread, and the rats in Experiment II, Group I that were fed
a diet consisting of 90% Iranian bread had an average food
intake of 63 g and 63.5 g per week, reapectively. Since the
amino acid content of American bread is known, the amino acid
intake of rats on this diet can be calculated. The rats had
an average intake of 9 grams each day of the diet that had
90% bread, or 8.1 grams of bread each day. Using the amino
acid content of bread (table 13) the intake of each individual
amino acid was calculated. The result of this calculation
shows that the amino acid requirement of the rat is not met
by a bread diet.

J. C. Winter (1947) in a feeding experiment with rats
fed the animals freshly prepared human diets to determine

the effect of bread enrichment. She concludes "these adverse

47

factors (sterility of females, failure of survival of young
and many still-births and the young beingeaten by their moth-
ers) rendered statistical comparison of the effects of en-
riched versus unenriched diets impossible. No significant
effect of enrichment could be obtained. Such human diets
appear highly defective for nutrition of rats during the
reproductive cycle".

She concluded therefore, that the human diet under study
was not satisfactory for the rat. There is a possibility of
food spoilage as a result of enzymatic changes that may have
occurred in the fresh moist human food left in the rat cages
for an indefinite period of time. If she had used a dried
sample of human food this problem of changes, resulting from
enzymatic or microbial activity in the food would have been
minimized. Nevertheless, Winters made no attempt to evaluate
the diet she tested in terms of man's nutrient requirements.

In view of the above calculations we can conclude that
the rat is not a satisfactory animal to use for studies of
the extent or degree to which human diets may meet man's re-
quirement for amino acids. The studies of McCance and Wid-
dowsen confirm this statement. They studied children in
German orphanages who drew most of their caloric intake from
bread. They concluded that diets consisting largely of breads
and vegetables provided the children with double the amount

of all essential amino acids (except methionine) for nitrogen

equilibrium.

48

Improvement of Iranian bread may be possible as far as
minerals and vitamins are concerned. Since no calcium or
vitamins are added to the bread, such supplementations are
important since the Iranians are not accustomed and do not
have access to milk and milk products. The possibility of
calcium and vitamin deficiency occurring among the Iranians
is great. Supplementation of bread with calcium and needed
vitamins, especially B vitamins, would be an excellent way
to incorporate these daily essentials into the diets of the

middle and lower economic classes of people in Iran.

GENERAL SUMMARY AND CONCLUSIONS

Bread provides about 70% of the calories for a large
pr0portion of the people in Iran. The bread used by the
Iranian people is made with a flour that is comparable to
the 72% extraction flour used in the United States. Since
studies by many other investigators showed that the nutrie
tional value of the protein in wheat and bread is poor, it
appeared desirable to determine how the Iranian bread might
best be improved. To be adopted by the people of Iran, any
proposed improvement would have to utilize other foods that
are readily available in Iran.

To this end, two feeding studies were carried out with
rats. They were done to compare the nutritional value of
bread baked in Iran with American white enriched bread and a
Syrian-Lebanese bread secured from local grocery stores.

In the second experiment, a study was made of skim milk pow-
der and lentils as supplements for improving the nutritional
value of Iranian bread.

Four groups of 10 male weanling rats were used in each
experiment. In the first experiment, the nutritional values
of breads from Iran, Syrian-Lebanese and American white en-
riched bread were compared. All animals were sacrificed at
the end of 4 weeks. Body weight gain and food intake were
measured and recorded. The livers of the animals were used
for the following analyses: liver xanthine oxidase activity

49

50

was determined on a part of the livers from 5 rats in each
group. The remaining liver from these animals and the livers
from the rest of the animals were dried to a constant weight
(for moisture content) and groundto a fine powder.

In the second experiment, the same number of rats was
used as in Experiment I. This experiment was designed to
evaluate the effectiveness of skim milk and lentils as sup-
plements to the Iranian bread. For these studies, the cone
trol group of rats was fed a ration containing 90% of Iranian
bread plus adequate amounts of minerals and vitamins. Other
groups of rats received similar rations in which a part of
the bread was replaced with 4% skim milk powder, or lentils
at either a 7.5 or 15% level. The changes in body weight and
food consumption were measured for a 3 week period. Chemical
analyses similar to those described for the first eXperiment
were carried out with this group. In addition thereto, hemo-
globin levels were determined on blood samples taken from all
rats prior to sacrifice.

The results of the first experiment showed that Persian
bread, despite having no nutrients other than flour in sig-
nificant amounts, has the same nutritional value as American
white bread containing 2% skim milk powder. This was shown
by the rat growth studies. Syrian bread was inferior as
compared with Iranian and American bread. In the second
experiment, it was shown that the nutritional quality of pro-

tein in Iranian bread can be improved by supplementing it

51

with 4% dried skim milk powder or 7.5 and 15% lentil powder
as far as the growth of rats is concerned. The growth pro-
moting effects of the breads increased with the level of
supplementation when lentils were used.

After the biological studies were completed, the contri-
bution of Iranian bread to the amino acid requirement of
adults was calculated. This calculation was based on the as-
sumption that the amino acid pattern of Iranian bread is simi-
lar to that of American enriched white bread. Furthermore, A
it was assumed that 70% of a 2500 calorie intake would come
from bread. Under such circumstances, it could be shown that
an adult would receive all essential amino acids in adequate
amounts from the bread. The amounts of amino acids provided
by the bread according to these calculations equalled the
"safe" levels suggested by W. C. Rose for adults.

0n the basis of these calculations, it appears desirable
to evaluate the adequacy of bread as a source of amino acids
for human subjects. An extension of this problem should be
directed that way.

This study emphasizes the fact that although rations
may be inadequate for supporting normal growth of rats, such

results may not be applicable to man himself.

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