DOCTORAL DISSERTATION SERIES

mi m mir/m m m m of
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AUTHOR.

S m i4 iM M H

UNIVERSITY

DEGREE

MCM' SfATE C0U<

u

DATE

. PUBLICATION NO..
III

/Hi

Mi

a_i

|TM UNIVERSITY MICROFILMS

THE NUTRITIONAL EVALUATION OF CERTAIN NUT PROTEINS

By
Sushela Lingaiah

A THESIS
Submitted to the School of Graduate Studies of Michigan State
College of Agriculture and Applied Science in partial
fulfillment of the requirements for the degree
of
DOCTOR OF PHILOSOPHY

Department of Foods and Nutrition
Year

1952

▲CraOWLEDGEBMENTS

The author wishes to express her appreciation to Dr, Margaret
A, Qhlson for her expert guidance, advice and encouragement
throughout this study; to Miss Ruth Ingalls for patient instruction
in microbiological assay methods; and to Miss Mary Mills for willing
assistance in the laboratory.
Acknowledgement is also made to Dr, Dena Cederquist, Dr* Wilma
Brewer, Mrs, Annanell Jubb, and Miss Louise Kelley for helpful
suggestions given during the progress of this study*

TABLE COP CONTENTS

?2±iag.
INTRODUCTION..................................................

1

REVIEW OF LITERATURE ..........................................

3

Evaluation of the Proteins of Almonds, Cashews, Pecans
and English Walnuts

..............

3

D i g e s t i b i l i t y ....................

3

Studies of growth

• • ...................................

4

Biological value

.......................................

7

Other Nutrients Found in N u t s .............................

9

Evaluation of Rice Proteins

.............................

11

Studies of g r o w t h .......................................

11

Biological value

........................... ............

Amino acid c o n t e n t .......................................

14

The Vitamins Pound in Rice • . • ..........................

16

T h i a m i n ..........

16

Riboflavin, niacin and other B vitamins

...........

20

Pat soluble vitamins, A, E andD . . . ....................

24

Loss of B Vitamins in Washing andCooking R i c e ..............

24

Supplements to Indian Rice Diets . • • • • • • •

27

........

•

EXPERIMENTAL P R O C E D U R E .......................................
Proximate Analysis of Nuts and Rice

30

.................

30

Moisture determination .....................................

30

Ether e x t r a c t .............................................

^

Pag®
Crude p r o t e i n ............................................

31

Ash d e t e r m i n a t i o n ........................................

31

Carbohydrate determination . . . . . . . .

31

............. .

Cross e n e r g y ....................... ; ..............
Microbiological Assay of Ten EssentialAmino Acids in Nuts •
Preparation of hydrolysates

foraminoacid assays

• . • •

................................

Organisms used

31
32
32
34

Basal m e d i a ..............................................

34

Assay m e t h o d ..............

34

Growth Experiment on B a t s ..................................

37

Nitrogen Balance E x p e r i m e n t ...............................

42

RESULTS AND DISCUSSION ........................................

44

Chemical A n a l y s i s ...........................................

44

Essential Amino Acid Composition ofNuts

............

45

Efficiency of Rice and Nut Protein Mixtures for the
Growth of B a t s ............................................

50

Nitrogen Intake, Excretion, and Retention by Rats on
Test Diets

..........................................

58

Nutritive Value of Rice DietsSupplemented with Nuts . . . .

63

SUMMARY AND C O N C L U S I O N S ......................................

67

REFERENCES CITED ..............................................

58

A P P E N D I X .......................................................

75

LIST OF TABLES
Page
In Body of Thesis
1.

Thiamin content of brown, white, and parboiled rice • • •

19

2.

Biboflavin content

21

3.

Iliacin content of brown, white, and parboiled rice

4.

The average content of pyridoxine, pantothenic acid

of brown, white, and parboiled rice

and blotin in brown and white rice

...

22

...............

23

5.

Methionine content

of fat extracted and whole nuts

6.

The composition of

the rice control diet

7*

The proximate composition of nuts and rice

8.

Essential amino acid content of brown rice and white
rice and whole egg

9*

..........

38

...........

45

47

• • • • • • . . .

•• • • • • • • • • • • • •

52

Statistical comparison between protein efficiency on
57

Nitrogen intake, excretion, and retention per day per
animal on the proteins of the experimental diets

13.

48

Growth in weight in relation to protein intake of

the various diets • • > • • . . . • • • • • • • • • • •
12.

32

Partition of nitrogen from cashew, pecan and walnut

rats on experimental diets
11•

...

.................

globulins reported in the literature
10*

•

•

• •

59

Estimated true digestibilities and biological values
of the proteins of experimental diets • • • • • • » . •

61

fiass
In Appendix
i

Essential amino acids in four nuts and recoveries
of added amino acids

ii
iii

.......................

The composition of rice diets supplemented with nuts
The composition

of whole

egg control d i e t .........

• •

85
89
92

LIST OP FIGURES

1*

Titration Values Obtained from Streptococcus faecalis R for
Known Concentrations of DL-Methionine*

2*

Titration Values Obtained from Streptococcus faecalis R for
Known Concentrations of DL-Tryptophane*

3*

Titration Values Obtained from Streptococcus faecalis R for
Known Concentrations of DL-Threonine*

4*

Titration Values Obtained from Strept ococcus faecalis R for
Known Concentrations of L-^Lrginlne hydrochloride*

5*

Titration Values Obtained from Streptococcus faecalis R for
Known Concentrations of L-Histidine hydrochloride*

6.

Titration Values Obtained from Lactobacillus arabinosus P—60
for Known Concentrations of DIr-Valine*

7.

Titration Values Obtained from Lactobacillus arabinosus 17—6
for Known Concentrations of DL-Isoleucine*

8.

Titration Values Obtained from Lactobacillus arabinosus 17-5
for Known Concentrations of DL—Phenylalanine•

9.

Titration Values Obtained from Lactobacillus arabinosus 17-5
for Known Concentrations of L-Leucine.

10*

Titration Values Obtained from Leuconostoc mesenteroldes P—60
for Known Concentrations of L-Lysine hydrochloride*

IHTB03XJCTION
Almonds, cashews, English walnuts and pecans have a high content
of protein and fat.

These concentrated foods have been found to be

readily digestible when Incorporated in a mixed diet (Jaffa, 1903),
Certain nuts have been studied and have been found to have a
variable biological value.

Mitchell and Beadles (1937), using the

rat as the eaqperimeutal animal, recorded a biological value of 73 for
cashews, which was only slightly less than that of beef protein (76),
while pecans, almonds and English walnuts had biological values
ranging from 50 to 60.

The biological values were determined after

expressing some of the oil from the nuts in order to incorporate
14 per cent of protein in the different diets.
Very few studies have been made on the nutritive value of
proteins of whole nuts.

Also an examination of Block and Bolling's

(1951) compilation of the amino acids in common foods includes no data
on the amino acid composition of any nuts other th an peanuts.

Buts

have been widely advised as a means of increasing the nutritive value
of a vegetarian diet.

Precise knowledge of the composition of this

food group is needed.

The supplemental value of nuts to a dietary

pattern characteristic of a vegetarian population merits investigation.
Bice forms the staple food of a large section of the Indian
people.

Sample surveys of food intake of the rice eating sections

-2-

in different parts of India revealed that the pattern of consumption
of the rice diet was very similar in all parts of the country
(Aykroyd, 1940).

In addition to rice, very small amounts of pulses,

vegetables, fruit, milk and meat were included in the diets of the
low income groups*

In a series of experiments by Aykroyd and

collaborators (1937-1940), it was demonstrated that milk formed a
most effective supplement to this diet*

Because of the difficulties

of producing milk at a low cost in India, and because of the non­
acceptability of proteins of animal origin by sections of the Indian
people, the investigators tried a number of vegetable proteins to
improve the basic rice diet*

It was found that soya beans, pulses,

peanut milk in varying amounts did not form a satisfactory supplement
to the diet*

Ho work has been performed on the supplementary value

of almonds, pecans, cashews and Sngllsh walnuts to the rice diet
although nuts are grown in India and eaten when available*
It was the purpose of this study to investigate the essential
amino acids and total protein content of certain nuts and to study
the supplementary value of varying amounts of these nuts to a basal
diet, the pattern of which was similar in all essentials to the rice
diet of low income groups in India*

REVIEW QT LITERATURE

Evaluation of the Proteins of Almonds,
Cashews, Pecans and English Walnuts

Digestibility.
Jaffa (1901, 1903) found the digestibility of nut proteins to
be satisfactory.

Diet studies were conducted on California

fruitarians and xxutari&ns who had lived for many years on diets
consisting mainly of nuts and fruits.

A few university students,

who assumed the nutarian diet during the experimental period also
were studied.

A careful analysis of the proximate composition of

several fruits and nuts was made.

Among the nuts studied were

almond, pecan, English walnut, Brazil nut, peanut and coconut.
balance period lasted usually for four days.

A

On an exclusively

fruitarian diet, which consisted of two types of fruits as sources
of carbohydrates and one nut as source of protein and fat, the
coefficient of digestibility varied from 70 to 82 per cent.

On a

mixed diet containing 13 to 29 gm. of protein from nuts per day
per person, Jaffa reported a protein utilization of 90 per cent.
Cajori (1918) supported Jaffa's conclusions regarding the
utilization of nut proteins, when fed in mixed diets, where almond,
pecan, English walnut, lichi or the Brazil nut formed the sole source
of protein.

Generally the nut pastes (pulverized whole nuts) were

"better utilized by men than when whole nuts formed part of the diet*
Cooking caused no marked change in the digestibility of the nuts*

In

the case of almonds* the thoroughly roasted nuts showed no advantage
over the boiled or the raw product*

The utilization of the digestible

carbohydrates determined as soluble

reducing sugars after hydrolysis

with two per

cent hydrochloric acid appeared to be excellent in diets

containing the various nuts*
Mitchell and Beadles (1937) recorded the digestibility of the
cashew nut, almond and English walnut as 96, 94, and 84 per cent
respectively*

Studies of growth*
Cajori (1920) tested the value of the proteins of the almond,
the English walnut, filbert, pecan and pine nut by the rat growth
method*

The shelled nuts were passed through a meat grinder and

subjected topressure in a hand press.
considerable

The process

removed

quantities of oil from the nut and the nitrogen

determination of the residual press cake indicated that the protein
content had been sufficiently concentrated to incorporate the press
cake in the diet at the desired level of 18 per cent of protein*
The experiment was conducted from 10 to 18 weeks and in each group
there were 10 to 32 rats*

Growth curves were drawn and compared with

the rat growth experiment reported by Osborne and Mendel (1915) on
a diet containing 18 per cent of casein.

Growth was comparable to

-5-

that on the casein diet In the case of almond, English walnut, filbert
and pine nut diets, but the growth rate on the pecan diet was poor*
Zn this investigation nut diets furnished an opportunity to test
ths efficiency of the dietary protein for milk production*

The mother

rats were able to nurse young successfully on diets in which almond,
English walnut, pine nut, and the filbert were the sole source of
protein*
In 1921 Cajori removed the skins of pecans which contain tannin,
and the nuts were dried and pressed through a meat grinder and
subjected to pressure

to remove enough oil to obtain a press cake

of sufficiently high protein content to provide a diet with 18 per cent
nut protein*
pecans*

Five young rats grew normally on the diet of treated

Thus the presence of tannin in pecan skin was shown to be

the limiting factor for the growth of the rat*
Mignon (1923) demonstrated the adequacy of the walnut globulin
as the sole source of dietary protein for white mice, and mixed walnut
protein for normal development of both rats and mice*

Preliminary

experiments indicated that the tannin in the skin of the nut was
injurious to the animals.

The skin was removed by hand, using hot

water as the lye method employed by Cajori (1921) to remove pecan
skin was found unsatisfactory.

The blanched nuts were ground in a

meat grinder, transferred to a muslin bag,and as much oil as possible
was pressed out by hand using the ordinary type of hand press.
resulting cake was fat extracted.
45 per cent protein*

The

The fat extracted material contained

-6—

Before experimenting with the walnut globulin as the sole source
of protein, two rats and three mice were fed on a diet in which walnut
press cake was the sole source of protein, incorporated to form 18 per
cent protein of a diet adequate in all other respects.

The animals

consumed the ration well and even when nut protein formed only 12 per
cent of the diet, rat growth was entirely satisfactory.

On a diet

containing walnut globulin alone in an amount providing 18 per cent
of protein to the diet, five mice grew normally.
Lane (1931, 1936) reported that almond milk, a vegetable milk
prepared to resemble human milk in composition and consisting mostly
of protein from almonds, gave satisfactory results in infant feeding.
She reared a pair of twins, children of a young woman, whose diet for
many years was mainly vegetarian, on almond milk.

The twins were

started on this milk when they were three months old.

During the

entire nursing period, there was no sour stomach or regurgitation
as is so frequent with cow's milk formulae.

The strength and endurance

of the children seemed to be above that of the average child.
teeth erupted at the normal time and were healthy.

The

When the children

were eight months old, a pediatrician evaluated them as being normally
healthy, rickets-free children.

When two years old, they weighed

29 and 30 pounds, the height of the boy was 33 5/8 inches and the
girl 33 l/2 inches.
was satisfactory.

Their health and development in all its phases
Growth was followed until five years later, when

almonds still furnished a good portion of the diet which was entirely

vegetarian*

Cfcrowth was normal.

Lane reported that case studies of

adults and children receiving almond milk appeared to prove that the
human organism can use a generous daily quantity of almonds to
advantage for indefinite periods*

Biological value*
Mitchell, Burroughs and Beadle (1936) in their extensive research
on the significance and the accuracy of the biological values of
protein computed from nitrogen metabolism data, have made an
interesting comparison of beef proteins and pecan proteins*

They

used rats as the experimental animals*
Preliminary digestion experiments indicated a marked difference
in the digestibility of the two proteins*

Beef prctein was completely

digested and whole pecan protein was digested to the extent of 70 per
cent only*

Hence the experimental rations were made up to contain

the same percentage of digestible protein, which formed 9*4 per cent
of the diet*

The investigators found the biological value averaged

75 for beef protein, and 60 for pecan protein*
The above values indicated that 1*33 gm* of digestible nitrogen
from beef covered the body *8 requirement for one gm* of nitrogen,
while for the same purpose 1*67 gn* of nitrogen from the pecan were
required*

Hence, one gm. of absorbed beef nitrogen was the nutritive

equivalent of 1.26 gm. of absorbed pecan protein*
When the authors used the paired feeding method for studying
growth of rats, ei^it pairs of rats weighing from 50 to 58 gm. were

-8-

investigated and the nutritive equivalence of nitrogen of beef round
to that of pecan protein was as one is to 1*24, almost the same
result as was obtained by the nitrogen balance method*
Mitchell and Beadle (1937) found by using the nitrogen balance
technique with rats that the cashews had a biological value of 73
which was higher than that estimated for the almond, filbert, or
Xnglish walnut*

The nuts were analyzed in the routine way*

The

experimental rations contained approximately 9*2 per cent of protein,
2 2 per cent of fat, five per cent of Wessen salt mixture as modified

by Osborne and Mendel, one per cent of sodium chloride, and 10 per
cent of sucrose*

The fat was provided by the test sample, butter fat

and a lard-cod liver oil mixture in the ratio of four to one to make
22 per cent of total fat*

Vitamins B and 0 were provided by one per

cent of dried yeast and Harris vitamin powder given in separate small
dishes at the rate of five mg* per gm* of food*

Cashew nut, almond

and Xnglish walnuts were found to have biological values of 73, 51,
5 6 respectively*
Ujsaghy (1940) found that in healthy infants fed almond milk,
the absorption of nitrogen was satisfactory, but the biological value
of almond milk while equal to that of cow's milk was slightly inferior
to that of human milk*
Campous (1946) reported that the proteins of cashew nuts had
high biological value*

Roasted cashew nuts forming 40 per cent of

the diet produced good growth in rats and corrected the deficiency

da* to a poor protoin derived from dried peas*

The biological value

of cashew protein was found to be 77*2 per cent •

Other Nutrients Found in Nuts

Levine (1932) found the pecan nut to be a good source of
vitamin A*

Zt was estimated that the pecan nut sample used in rat

feeding experiments had a vitamin A potency of approximately 3.6
units per gram of nut, one unit of A according to the definition of
Sherman and Smith (1931) being the amount which when fed daily to a
standard test animal would suffice to support an average gain in body
weight of three grams per week during the test period of four to
eight weeks*
De Caro and VranceBchini (1939) assayed the vitamin A content of
several nuts by biological tests on rats depleted of vitamin A*

Th*

vitamin A value expressed in I*U* per gm. of material was, for fresh
walnut 1*2, dried walnut 1*3, peanut 3.6, hazel nut 4.4, and almond 5*8*
The vitamin Bi content was estimated by the same authors*

The

minimum daily dose of test material, required to secure the survival
for 60 days of young rats deprived of vitamin

was 2*82 micrograms

per ©a* of walnuts and 2 *8 , 1*2 and 2*0 micrograms per gram of hazel­
nuts, almonds and pecans respectively*
tyke, Melville and Sarsan (1942) found unripe walnuts to be a rich
source of vitamin C containing 410-1800 mg* per cent.
nuts retained only a trace of this element.

The dry mature

Most methods of preparing

-10-

sweet confections from ths unripe nut destroyed all the ascorbic acid
bat if the unripe xxat was heated in water and pickled in vinegar or
made into Jam most of the vitamin C was retained.
Sverson.et al.

(1948) found the riboflavin in almonds was not

as readily available as the riboflavin of ice cream.

light women

subjects consumed a basal diet supplying approximately 2.4 mg.
riboflavin per day.

Their urinary excretion of riboflavin was

surprisingly constant.

When the weighed amount of basal diet was

supplemented with one mg. of pure riboflavin, an average of 42 per
cent of the test dose was recovered in the urine.

When a supplement

of one mg« of riboflavin was supplied in the form of ice cream, the
urinary excretion of riboflavin was comparable to that produced by
the pure vitamin.

This was taken as indicating that riboflavin of

ice cream was as available as that of the pure vitamin.

When either

frosen peas or almonds furnished one mg. of riboflavin as a supplement,
much less riboflavin was excreted in the urine.

It was thought that

these differences might be due to variations in the absorption of the
vitamin.
Jentsch and Morgan (1949) assayed the riboflavin, thiamin
niacin content of four varieties of walnuts.

and

Thiamin was assayed

by the thtochrome method and by rat growth assay.

The values obtained

from the latter method were 20 to 30 per cent larger than those
obtained by the thiochrame procedure.

The three varieties had an

average of 37 mg. per cent thiamin by the thiochrome method.

The

11

riboflavin content of the walnut samples determined by the micro­
biological method was found to be 2 0 per cent less than the values
obtained by the fluorometric method which gave an average of 0.148 mg.
per cent riboflavin.
Niacin was determined microbiologically and gave an average
value of 0.71 mg. per cent niacin*
Peterson, Skinner and Strong (1943) reported the percentage of
major mineral elements in almonds to be as follows:

calcium, 0*228;

magnesium, 0*275; potassium, 0.756; sodium 0*024; phosphorus, 0*465;
chlorine, 0.037; sulphur, 0*164.
Almonds, cashews, pecans and English walnuts wculd a p p e a r to be
valuable sources of vegetable protein.

The evidence is that the

protein is readily digestible and supports satisfactory growth and
nitrogen retention.

Pew studies have been made with whole nuts*

Some form of press cake or protein isolated from nuts has been used
in most of the studies recorded*
Evaluation of Rice Proteins

Studies of growth.
gjlc (1940) compared the growth promoting value of proteins of
whole rice and polished rice at 5*5 per cent protein in the diet,
using the paired method of feeding rats.
130 days.

The experiment lasted for

In all the nine pairs of rats fed whole rice, the animals

utilized their food better than those fed polished rice.

The average

-12-

gain in weight per gm. of protein intake wae 1*8 gm. for the animals
fed the whole rice ration and 1* 6 6 gm. for those fed the polished
rice ration*

The difference between the two weight gains was

statistically significant*
The growth promoting value of rice bran and rice polish also
were studied at an intake of eight per cent protein*
lasted for 112 days*

The experiment

In all nine pairs of the rats, less nitrogen

from rice polish was needed to promote the same gain in weight*

The

average gain in weight per gm. of protein consumed was 1*79 gm* for
the animals fed the rice polish ration and 1*48 gm* for those fed the
rice bran ration*

The difference of 0*31 gm* was statistically

significant*
In further paired feeding experiments, Kik (1940) showed that
additions of 25 mg. of cystine, methionine or lysine daily supplemented
the proteins of whole rice and polished rice to a slight extent*

The

addition of tryptophane did not show any beneficial effect*
Sure (1946) found the growth promoting efficiency of rice protein
wae greater than that of wheat*

Thirty rats were given a diet

adequate in all respects containing 5*8 per cent of protein from
polished rice.

The average gain in weight per gm. of protein was

1*86 gm* while on the same plane of protein nutrition the average

gain per gram of protein of whole wheat flour was only 0*72 gra.
Substitution of one, three and five per cent of polished rice
with equivalent amounts of strain G yeast increased the growth rates
of rats by 41 to 69 per cent (Sure, 1947).

-13-

Jones, Caldwell and Widness (1948) compared the growth promoting
value of the proteins of whole rice, polished rice, yellow corn,
hard and soft wheat, pearled “barley, rye and rolled oats.
experiments were performed on rats for six weeks*
fed ad libitum*

The

The animals were

At an intake of 7*5 per cent protein, brown rice gave

the best growth in rats*

However, at an intake of 4*5 per cent protein,

brown rice produced less growth than polished rice*

Oats and rye

produced slightly greater growth than polished rice.

Biological value *
Bice protein has a satisfactory biological value compared to the
proteins of other cereals*

Employing the nitrogen balance method

on rats, Mitchell (1934) o b s e r w d that whole rice proteins had an average
biological value of 86.1 per cent, on a diet supplying five per cent
of protein*

At the same protein intake, the biological values of

corn and oat proteins were found to be inferior to rice protein, the
value for c o m being 72*0 and for oats 78*6 per cent.
Baaru and Basak (1947) investigated the biological value of the
Airmn and Aus varieties of Bengal rice and found both varieties had a
biological value of 80.0 per cent.

Parboiling did not affect the

biological value of either variety.
Kik (1939) reported the biological value of whole rice to be
73 per cent on a diet supplying five per cent of protein.

Thia value

was lower than that observed by Mitchell (1924) and Basu and Basak (1937)

-14-

for whole rice*

On diets containing five per cent of protein,

recorded the biological value for polished rice as 6 6 *6 , rice bran
as 84*9 and rice polish as 82*9 per cent*

At a protein intake of

eight per cent rice bran and rice polish had lower biological values*
Shis was thought to be due to the fact that the economy with which
amino acids were used decreased as the supply increased*
Sure and House (1948) demonstrated that the biological values
of milled and whole rice proteins were superior to those of other
cereals*

At an intake of five per cent of protein, the biological

value of milled rice was 75*1, wheat 60*0, rye 63*1 and corn 32*

The

findings for whole grains were rice 80*0, wheat 76*1, rye 73*2, c o m
78*8 and rolled oats 75*6 per cent*

Amino acid content«
(1941) determined the cystine, tryptophane, lysine, arginine
and histidine content of whole rice, polished rice, rice bran and
rice polish by chemical methods.

The amino acid content compared

favorably with that for c o m and wheat*

In comparison with casein,

the rice proteins contained less tryptophane, lysine and histidine*
Five varieties of rice, Arkansas 155, Shoemed, Acadia, Zenith
and Fortune, were assayed for the above five amino acids*

Considerable

variation was noted in the amino acid content of these varieties*
Ultchell and Block (1946) calculated the percentage deviations
of the content of essential amino acids of white rice protein from

-15-

the concentration of the corresponding amino acids found in whole egg
protein, both standardized to a nitrogen content of 16 per cent,
lysine was the amino acid most deficient in white rice, and probably
limited the nutritional value of the rice for maintenance and growth
of the laboratory rat.
lyman and Silken (1949) found that unpolished rice had 6.39 per
cent crude protein.

The percentage of 10 essential amino acids in

the sample was as follows:

arginine, 0.54; histidine, 0.14;

lsoleucine, 0.28; leucine, 0.51; lysine, 0.28; methionine, 0*14;
phenylalanine, 0.31; threonine, 0.22; tryptophane, 0.10; valine, 0.40*
The amino acid composition of polished rice was comparable to that
of wheat.
Pecora and Hundley (1951) observed that the addition of two amino
acids, lysine and threonine, produced a growth response in white rats
three times greater than that obtained with an unsupplemented diet*
Neither lysine, nor threonine, nor any other essential amino acid added
to rice individually produced better growth than rice alone*
▲ threefold excess of arginine or methionine when added to rice
completely supplemented with all other essential amino acids produced
a significant growth depression*

-16-

The Vitamins Found in Bice
Thiamin.
lijkman (1897) reported that an illness of fowls similar to
beriberi in roan was produced by feeding fowls with polished rice.
This illness was prevented or cured by an extract of rice polishings.
The active principle was isolated as vitamin B^ and later characterized
as thiamin (Sherman, 1945).

Since that time, the antineuritic vitamin

content of rice has been of special interest to nutritionists.
Fraser and Stanton (1910) investigated the relationship between
the degree of milling to which different kinds of rice had been
subjected and the capacity to protect human beings from beriberi and
birds from polyneuritis.

A. convenient way to estimate the vitamin B^

content was to measure the amount of gezm and pericarp present.

Most

of the phosphate present in rice was to be found in the embryo and the
integuments.

The phosphate content estimated as P2®5* depended on the

degree of milling of rice and was suggested as a useful index of the
vitamin B^ value.

The ingestion of unmilled rice or parboiled rice,

which had a phosphate content of 0.4 per cent was never associated
with beriberi in humans.

The Investigators found that polished rice

contained less than 0.4 per cent ^2^5

rice was likely to

cause beriberi.
McCarrison and ITorris (1934) observed that various samples of
parboiled rice even when milled to a high degree

protected pigeons

from polyneuritis when fed to the birds as the sole source of
vitamin B^.

17-

Aykroyd (1932) estimated the vitamin

potency of a number of

rice samples, raw as well as parboiled, by the rat growth method*
Groups of rats were given a basal diet deficient In vitamin B^*

The

rats Increased In weight for two to three weeks, after which growth
ceased*

When the weight was stationary for about one week, the test

material was fed either In the form of separate dally additions to
the basal diet, or the basal diet was modified to include a definite
proportion of the substance under examination*

The Increase in weight

was observed over a further test period of four weeks*

The vitamin B^

value of different rice samples was estimated and compared by observing
the average weekly gain in weight corresponding to the various daily
doses of rice in the basal diet.

The vitamin B^ content of unmilled

samples of raw and parboiled rice was equal, but when each was milled
to an equal degree, the parboiled sample retained the vitamin, while
the raw sample did not.
The estimation of the P 2 O5 content of rice seemed to serve as an
index of the vitamin B^ value of raw rice.

This investigation

supported the suggestion of Fraser and Stanton (1910) that raw rice,
containing less than 0.4 per cent of P 2 O5 was apt to be low in
vitamin B^ and gave rise to beriberi.

The phosphate content of

parboiled rice, however, was no guide to the vitamin B^ value, since
percentages as low as 0.28 might be present in the samples which
contained an abundance of the vitamin*

-

18-

The investigator was of the opinion that daring the parboiling
process, when paddy was soaked in water for 24 to 36 hours, subjected
to steam at atmospheric pressure for 15 to 20 minutes and then dried
and hulled, the antineuritic vitamin was transferred to the inside of
the grain and thus not lost to it.
The quantitative estimations of the thiamin content of brown,
white and parboiled rice as reported by various investigators have
been presented in Table 1.

Callieau, Kidder and Morgan (1945) and

Miller (1945) determined thiamin in rice by biological assay.

JLU

the other investigators assayed thiamin by the thlochrome method.
The distribution of thiamin in the rice was examined by Hinton
(1947) by dissecting the various layers and examining them separately.
He found 44 to 50 per cent of the total thiamin in the scutellum,
34 to 35 per cent in the pericarp aleurone layers and in the covering
of the gexro, seven to nine per cent in the endosperm and nine to twelve
per cent in the remaining parts of the embryo*

He noted that the

parboiling process led to the redistribution of thiamin.
was enriched at the expense of the embryo.

The endospera

There was a marked retention

of scatellum in parboiled rice as compared with raw milled rice, due
largely to the gelatinization of the endosperm immediately below the
germ.

This acted as a cementing tissue so that the germ was not so

readily knocked out of the grain during subsequent milling.
Simpson (1951) studied the distribution of thiamin and riboflavin
in rice by the simple and ingenious photographic method first developed

TABLE 1
Thiamin content of brown, white and parboiled rice

_
Investigator

Type of rice________
Brown

White

Parboiled

og/gn

ug/gm

Aykroyd, et al. (1940)

2.90

1*00

2.40

Swaminathan (1942)

—

1*00

2*10

Kik (1923)

3.00

0.60

1.66

Williams, Knox and Pieger(1943)

4.20

0.80

—

Calliaeu, Kidder and Morgan(1945)

3.80

0.90

2.20

Miller (1945)

2.10-2.98

—

Kik and Williams (1945)

3.55

0.84

1.35-1.74
2.50*

Jones, et al. (1946)

3.71

—

2.00—2.30

Simpson (1951)

3.84

0.60

* Converted rice

2.03

-20—

1)7 Somers, Coolldge and. Haworth (1945) for locating these vitamins In

wheat kernels*

Appropriate filter combinations to record specifically

the fluorescence excited by thiochrome and riboflavin, when illuminated
by ultra-violet radiation were used in this investigation*

In all cases,

the maximum amount of thiamin occurred in the scutellum and in the cells
of the endosperm adjacent to the embryo*

The remainder of the embryo

other than the scutellum did not appear to be rich in thiamin, but
riboflavin seemed more evenly distributed throughout the tissues of the
embryo than in the case of thiamin*
This work brought visual confirmation of the fact that a
considerable portion of the thiamin of the scutellum was absorbed into
the endosperm during the parboiling process and would remain in the
endosperm, even after the parboiled rice was highly milled*

Riboflavin, niacin and other B vitamins*
The riboflavin content of brown, white and parboiled rice as
determined by investigators by fluorometric analysis has been
presented in Table 2*
The niacin content reported by two groups of investigators has
been given in Table 3*
Williams, Knox and Fieger (1943, 1944) determined the pantothenic
acid, pyridoxine and biotin content of three typical Louisiana
varieties of rice.

Pantothenic acid and biotin were assayed

microbiologically and pyridoxine by Scudi's (1942) colorimetric method*
The average content of these vitamins is presented in Table 4*

TABLE 2
Riboflavin content of brown, white and parboiled rice

Type of rice
Investigator

Brown

White

ug/gm

ug/gn

vg/m

Kik and Landingham (1943)

0*53

0*36

0.38

Williams, and Rieger (1944)

0*61

0.24

—

Kik and Williams (1945)

0.60

0.27

0.37
0.25*

* Converted rice

Parboiled

SABLE 3
Niacin content in brown, white and parboiled rice

Type of rice
Investigator

Brown

White

ug/gm

ug/gm

Williams, Knox and Feiger
(1943)

47.2

12.7

Kik and Williams (1945)

53.08

19.62

Parboiled
We®

Microbiological
46.4
32.5*

* Converted rice

Method

Calorimetric
method of
Melnick (1942)

TABLE 4
The average content of pyridoxine, pantothenic acid and biotin
in brown and white rice*

Type of Rice

fyridoxlne
us/S®

Pantothenic

Blotla

ug/gn

ug/gm

Brown

17.00

10.30

0.12

White

6.40

4.50

0.04

* Williams, Knox and Fieger (1943, 1944).

24-

Kat soluble vitamins. A, E and D.
Todd et al.

(1937) obtained a complex mixture of oily

crystalline esters by acetylatlon of a purified concentrate from
the unsaponifiable fraction from the rice germ oil*

The crystalline

esters on separation and hydrolysis yielded three approximately
homogenous crystalline isomeric alcohols, which were biologically
inactive.

The purified oils remaining after the removal of alcohols

from rice germ oil had high vitamin 1! activity*
Kik (1942) reported that rice and its by products were deficient
in vitamins A and D*

P 0lished rice, whole rice, rice polish and rice

bran were Investigated using young albino rats*
were fed the foodstuffs alone*

Groups of six animals

Addition of one drop of viosterol

daily was followed by improvement in growth.

After a maintenance curve

was reached, a farther growth response was obtained after administration
of four drops of cod liver oil daily, which furnished vitamin A and
additional amounts of D as well*

The results showed that rice needed

fortification with vitamins A and D to produce good growth.

Loss of B Vitamins in Vashing and Cooking Rice

Aykroyd et al.

(1940) reported the average losses of thiamin

and niacin during washing and cooking of raw milled rice were from
40 to 60 per cent.

Swaminathmm

(1941, 1942) estimated that raw rice

lost 60 per cent of its thiamin and niacin content and parboiled rice
eight per cent of the thiamin and 12 per cent of niacin during washing.

-2 5 -

A n additional 25 per cent of the thiamin in both raw and parboiled
rice was dissolved into the cooking water.

Cooked parboiled rice

even when the cooking water was discarded, contained 1 .4 ug. per
gram of thiamin.
Miller (1945) determined the loss of thiamin as a result of
washing and cooking brown rice and partially polished rice.

Unwashed

brown rice was cooked for 50 minutes and brown rice was washed five
times and cooked in a small amount of water in exactly the same
manner as the unwashed sample.

Unwashed partially polished rice was

cooked in a small amount of water for 40 minutes.

A sample of the

same rice was washed five times and cooked in the same way as the
unwashed partially polished rice.

Th« different types of cooked

rice were fed to rats as the sole source of thiamin.

As a result

of washing and cooking rice, there was an apparent loss of 12 per
cent of thiamin.

Statistical analysis of the difference between the

mean gains of the rats fed washed and unwashed brown rice showed
unimportant differences.

The differences between the mean gains of

the groups of rats fed washed and unwashed partially polished rice
appeared to be significant and the apparent loss of thiamin was
about 20 per cent.

No measure of statistical significance was given

by these authors.
wv

and Williams (1945) performed washing experiments on brown,

white and converted rice and reported the cooking losses of riboflavin,
niacin and thiamin during these processes.

One cupful of rice was

26-

washed twice in a capful of water•

Brown rice so washed lost

20*1 per cent of its thiamin, 7.7 per cent of riboflavin and
23.0 per cent of niacin.

White rice lost 43.0 per cent of thiamin,

25.9 per cent of riboflavin and 23.0 per cent of niacin.

Converted

rice lost 6*5 per cent of thiamin, 10.5 per cent riboflavin and
16*0 per cent niacin*
during washing*

Converted rice had a lesser loss of vitamins

This confirmed an observation made by Swamlnathaa

(1943).
The investigators cooked rice samples by the doable boiler and
the open kettle method.

One cup of rice and one and one half cups

of boiling water were placed in the top of the double boiler and
cooked until all the water was absorbed in the cooked rice.

In open

vessel cooking, one half cap of rice was added to eight cups of
boiling water, the rice was cooked, transferred to a colander and
drained.

The vitamin determination on cooked rice showed that all

types of rice lost an average of 4.3 per cent of thiamin in the
doable boiler method compared to 46.9 per cent when c ooked in the
open vessel.
The average losses of riboflavin and niacin using the doable
boiler type of cooking were 6.7 per cent and 3.4 per cent respectively
versus 43 per cent for riboflavin and 44.8 per cent for niacin when
the open vessel type of cooking was used.

The investigators expressed

their opinion that all vitamins present in rice before cooking could
be saved when the method of cooking used the minimum amount of
cooking water and the rice was not rinsed after cooking.

-27

Brown rice contains appreciable quantities of the B vitamins,
especially thiamin and niacin*

Milling rice reduces its vitamin

content while parboiling conserves it*

Xxcessive washing and careless

cooking was detrimental to the retention of B vitamins in cooked rice*

Supplements to Indian Rice Diets

S^nce McCarrlson's (1936) animal experiments revealed the
nutritional Inadequacy of Indian rice diets, several investigations
have been carried on to improve the rice diets of low Income groups
by appropriate supplements.

Aykroyd and Krishnan (1937, 1939, 1940)

fed rats with rice diets which closely resembled the diets actually
consumed by poor rice eaters in various parts of India*

On these

diets young rats showed very poor growth, an average increase in
weight of three to four gm* per week*

The addition of milk greatly

Improved the nutritive value of the diet for rats*

The general

condition of the animals improved and the weight gains doubled or
trebled on the milk supplemented diet*
Yeast also was an effective supplement to the rice diet*

Meat

and eggs had some supplementary value but less than that of milk*
Soya beans and pulses in varying quantities did not form an effective
supplement.

The most striking observation of the investigators was

that most of the supplemental value of milk could be produced by a
calcium salt.

The addition of calcium lactate or calcium phosphate

to the basal diet improved the growth rate considerably*

Parallel

-28

experiments vith undernourished children corroborated these results.
▲ daily intake of 0*5 to one &n. of calcium lactate or 28 to 48 gnu
of skinned milk powder produced an acceleration of growth in mal­
nourished children.

The investigators were of the opinion that the

rice diets were not so much deficient in protein, as rice proteins
were of high biological value but tended to be very deficient in
calcium and vitamin A concentration,
Banganath and Ban (1938) confirmed Aykroyd and Erishnan* s (1937)
results concerning the improvement of a rice diet by the addition of
calcium salts.

They added two per cent calcium carbonate to the rice

diet characteristic of the Madras area and observed the apparent
improvement of the biological value of the protein of this diet,
Deshikachar (1948) reported milk prepared from ground nuts had
no supplemental value to the rice diet.

This was surmised to be mainly

due to the fact that ground nut milk was deficient in calcium.
Moorjani, et al.

(1950) investigated the supplemental effect

of ground nut milk fortified with calcium and blended with germinated
soya bean to the rice diet.

The growth rate of rate was doubled on

this supplement.
The nutritive value of the Indian rice diets was improved
considerably by the addition of milk or calcium salts to the diets.
This improvement was thought to be mainly due to the increase of the
ratio of calcium to phosphorus in the diet.

The ratio of calcium to

phosphorus in rice was approximately as one is to ten, which was

-29-

thought to 1)9 unfavorable for maximum availability and utilization
of phosphorus*

Supplements in the form of milk or as calcium salts

appeared to favor phosphorus assimilation and thus increased the
nutritive value of rice diets*

EXPERIMENTAL PROCEDURE

Proximate Analysis of Pate and Rice

The proximate analysis of nuts and rice was made as a knowledge
of the major food constituents was necessary in order to prepare the
test diets for biological assay.

Moisture datormination.
Moisture was determined by drying the foods at 70° C. with the
aid of a Brabender moisture tester.

The nuts were coarsely powdered

in a Waring blender and rice was ground with a pestle and mortar.
Triplicate samples of 10 gm. were weighed into the pans of the
instrument.

Percentage moisture was read directly at intervals of

one half an hour.

When the difference between two readings was within

0.05 per cent, the final reading was taken as the percentage moisture
of the sample.

Ether extract.
The moisture free samples were fat extracted in a Soxhlet
extractor using ethyl ether.

Pat extracting thimbles were first

extracted for one half an hour and after all trace of ether had
evaporated, were placed in a desiccator.

Triplicate samples of

9.5 to 10 ©n. of moisture free samples were weighed into the dry

-

31—

fat extracted thimbles and fat extracted until the samples reached
constant weight•

Crude 'frroteln*
Hltrogen was assayed on duplicate samples of one gm. of the fat
extracted materials by the Kjeldahl method.

The quantity of nitrogen

was maltiplied by the factor 6.25 to express the crude protein
concentration of the samples.

A s h determination.
Ash was determined on powdered whole zmt and rice samples.
Duplicate samples of 0.5 to one ©n. were weighed Into ashing dishes
and placed in a muffle furnace.

The temperature of the furnace was

raised gradually to 525° C. in order to prevent rapid burning of the
materials.

The samples were left in the furnace at 525° C. until a

white ash of constant weight was obtained.

Carbohydrate determination.
Carbohydrate content was obtained by difference.

The percentage

of water, ether extract, crude protein and ash were added and the
difference of this sum from 1 0 0 , was taken as the carbohydrate content
of the samples.

Carbohydrate by difference included available and

non—available carbohydrates and any organic acids present.

Cross energy.
The gross energy value of nuts and rice was determined by the
aid of a bomb calorimeter.

-

32-

llicroblological Assay of Ten Essential Amino Acids
In Almonds, Cashews, Pecans and English Walnuts

It was planned to study the essential amino acid content of the
nuts as this would give an indication of the nutritional value of nut
protein.

However, it was necessary first to determine the effect of

the high percentage of fat in the nuts on the growth of the micro­
organism.

Therefore, a preliminary experiment was carried out in

which the methionine content of fat extracted and non-fat extracted
nuts was determined.
presented in Table 5.

The results of this experiment have been
Slightly higher values were consistently found

in fat extracted samples.

Therefore, the fat extracted nuts were

used for the analysis of amino a d d s *

Preparation of hydrolysates for amino acid assays.
Acid hydrolysates for the assay of arginine, histidine,
methionine, threonine, lysine, leucine, isoleucine, valine and
phenylalanine were made by adding 100 ml. of 2N hydrochloric acid
to 1.5 gm. of each fat extracted sample of nuts in an Erlenmeyer
3.
flask. The recovery of added amino acids was studied by treating a
sample of defatted cashew nuts as follows.

A hydrolysate was prepared

by adding to the above mixture of nut and acid, two ml. of DL—
phenylalAnine, two ml. L-histidine hydrochloride solutions and four ml.
each of solutions of DL-Valine, L-Leucine, DL-isoleucine, DL-threonine,
DL-raethionine, L-arginine hydrochloride and L-lysine hydrochloride.

TABLE 5
Methionine content of fat extracted and whole nate

____________
Method

Walnut

Type of nut___________
JLlmona
Cashew
Fecaa

mg/gn

og/gm

og/gm

mg/e#

Fat extracted samples

1.719

1.900

3.176

1.536

Whole nut sample

1.679

1.632

2.716

1.453

-

33-

The solution of amino acids used each contained 10 to 40 mg. of the
material or its hydrochloride.

The flasks were plugged with glass

wool and autoclaved for five hours at fifteen pounds pressure
(Schweigert ,et al*

1946)*

The hydrolysates were cooled to room

temperature, filtered, made to volume with distilled water and stored
in screw-top bottles in the refrigerator*
Alkaline hydrolysis was employed for the assay of tryptophane,
using cysteine as a stabilizing agent (Euiken, Lyman and Hale, 1947)*
Two hundred mg* portions of L-Cysteine were weighed into five alkaline
resistant Irlenmeyer flasks.
hydroxide were added.

Into each flask, 32 ml* of 4N sodium

The flasks were stoppered with cotton and

autoclaved at 15 pounds pressure for one hour*

While the solutions

were still hot, another 200 mg* portion of L-Cysteine and 0*4 gm*
of the sample were added to each flask*

Duplicate samples of almonds

were prepared and to one sample of almond 2*5 ml* of tryptophane
solution containing 12 to 15 mg. of DL-tryptophane were added for the
purpose of determining the percentage recovery of the amino acid*
The mixtures in the five flasks were autoclaved for fourteen hours
at fifteen pounds pressure.

Bach sample was transferred to a 100 ml.

volumetric flask, while still hot, using warm rinse water.

When

cool the samples were diluted to volume and stored in screw top bottles
in the refrigerator*

-

34-

Organisms used.
Leueonostoc mesenteroldes F-60 was used for lysine assay*
Lactobacillus arabinoarus 17-5 was utilized in the assay of
isoloucine, leucine, phenylalanine and valine*
Streutococcus faecalis R was used for the assay of arginine,
histidine, methionine, threonine and tryptophane*

Basal mediam*
For the assay for all amino acids except methionine, the mediu .
of Schweigert, et al* reported in 1949 was employed*

For the

determination of methionine, a raedii,, was used which was a combination
of that of Schweigert, et al* (1949) and Iyman, et al* (1946), in
which the amino acids were replaced by hydrogen peroxide treated
peptone, cystine, tyrosine and tryptophane.

Kills (1951) reported

the composition of the median for methionine as employed in this
laboratory*
Assay method*
The method was essentially that of Sauberllch and Bauman (1946)
with slight modifications as routinely followed in this laboratory
(Ingalls, et al. 1950)*
The organisms were maintained on stab cultures of yeast extracti

dextrose—agar•
48 hours.

Transfers were made weekly said incubated at 37° C. for

Inoculums for the assay tubes were made by transferring

cells of the organism from stab cultures to tubes containing five ml*

35-

of the complete basal media plus five ml. of water*

This liquid

culture was incubated for 24 hours at S7°C* and then stored in the
refrigerator*

Inoculum, which was over five days old was discarded*

At the time of inoculation, the inoculum was centrifuged and the
supernatant liquid discarded*
of 0*9 sterile saline solution*

The cells were suspended in 20 ml*
One drop of this suspension was used

to inoculate each arsay tube*
Assays were carried out in pyrex culture tubes and in a total
volume of two ml*

Gtraded amounts of the amino acid to be assayed

were added to a series of tubes in order to establish a standard
curve*

The pH of the standard solutions of amino acids from which

the dilutions were obtained was adjusted to suit the needs of the
assay organism*

For streptococcus faecalls R the pH was adjusted

to 8 * 4 to 8*6 with thymol blue as indicator and for I^c tobacillus
^7-S and Leueonostoc mesenteroldes P-60 the pH was adjusted
to 6.6 to 6*8 with brom thymol blue as indicator*
Twenty-four tubes were used to establish the standard curve;
triplicate tubes were employed for the first six dilutions and
duplicate tubes for the three highest dilutions*

Water was added

to each tube to bring the volume to one ml*
Aliquots of the sample hydrolysates were taken and further diluted
to a suitable volume after adjusting to the desired pH*

IXxplicate

tubes at dilutions of 0 *3 , 0*6 and one ml* respectively of the sample
were prepared.
in each tube*

Water was added to bring the total volume to one ml*

-36

The basal mediu to be used was mixed omitting the amino acid to
be assayed.

The pH of the medium was suitably adjusted before diluting

the solution to 100 ml* volume.

One ml* of the medir*. was added to all

tubes to bring the total volume to two ml*
The racks of tubes were covered with heavy towelling and auto­
claved for eight minutes at 20 pounds pressure.

The cooled tubes were

Inoculated with one drop of inoculum, suspended in sterile saline
solution and incubated in a water bath for 72 hours at 37° C*
At the end of the incubation period, the growth response of the
test organisms was measured by titrating the lactic acid produced
against 0*05 N sodium hydroxide*^

Brom thymol blue was used as

indicator for tests using Lactobacillus arabinosus 17-5 and
Leueonostoc mesenteroldes P-60 as the test organisms, and thymol
blue for those using Streptococcus faecal is R.

During titration,

air was blown into the tubes to provide complete mixing.
Standard curves were drawn by plotting the milliters of 0*05 N
sodium hydroxide used in titration against the micrograms of standard
amino acid.

The amount of amino acid in the samples was obtained by

interpolation from this standard curve*

Typical standard curves

obtained for the ten essential amino acids have been shown in
Figures 1 to 10 in the Appendix*

* The 0.05 H sodium hydroxide was standardized against a
standard solution of potassium acid phthelate*

The recoveries of the added amino acids were usually within —
per cent, Table i, Appendix.

10

However in the case of tryptophane the

recoveries were consistently low.

Possibly there was a slight de­

struction of tryptophane during the preparation of the alkaline
hydrolysate, even though cysteine was used as a stabilizer, which
according to Kuiken, Lyman and Hale

(1947) should

destruction of tryptophane under these conditions.

completely prevent
Recoveries in

the case of threonine were found to be consistently hi$i.

The reason

for this is not clear.
Growth Experiment on Rats

A growth study was conducted for a four week period on young
female albino rats, weighing 55 to 75 gm. to test the growth promoting
value of nut supplements to a rice diet.

The rice diet, which was

used as a negative control diet was similar to Aykroyd*s (1940)
"Poor rice eater*s diet," in that it contained little else than rice.
Calcium hydroxide was added because of the evidence that calcium
greatly increased the growth promoting value of the rice diet and
because uany Indians obtain significant amounts of added calcium from
the practice of chewing betel nuts.

Irradiated ergosterol was added

because the rice consuming section of Indian people live in a climate
full of sunshine and do not lack vitamin D.

Vegetable fat was added

to the diet, as Indian people do not commonly use animal fat in their
diets.

The composition of this ’iet is given in Table 6.

TABLE 6
The composition of the rice control diet

Food^

Weight

Calories*

gm*

100.0

Bice
Ca( OH)-

380.2

Protein

Pat

gm.

gm.

7

1

1.0

Ergosterol
Vegetable fat
Total

13.9

131.4

114.8

511.6

13.9
7.0

14.0

• Gross energy value*
1 About 6 gm. of greens were fed to animals every alternate day

daring the experimental period*

-

39-

Two xrat supplemented rice diets were prepared from each of the
four experimental nuts hy displacing five per cent and 25 per cent
of rice protein in the rice control diet hy proteins of the Intact
nuts*

The object of this procedure was to find out if the supplementation

of rice protein with nut proteins improved the growth promoting value
of the rice protein alone*

A 11 of the eight nut supplemented diets

were isocalorlc with the energy value of the rice control diet and,
like it, contained 511*6 calories, seven gm. of protein, 14 gm* of
vegetable fat, one gm. of calcium hydroxide and 1600 I.U. of ergosterol*

A positive control diet was prepared from fat extracted dried defatted
whole egg to match the rice control*

The protein in this diet was

derived solely from the dried whole egg*

The composition of the

various nut supplemented rice diets and the whole egg control diet
have been given in Tables il and lii in the Appendix*
In the preparation of the rice diets, converted rice was washed,
cooked and dried*

Two pound lots were washed with water once and

cooked in an open pan for 15 minutes in a large amount of water*

The

cooking water was discarded, the cooked rice was dried and ground to
a fine powder.
Pecans and English walnuts were prepared with the skins.

Almonds

were blanched and the coats of the cashew nuts were peeled as the nuts
were purchased for the experiment*

-

40-

Incorporation of the nuts into the diets presented a problem
as it was difficult to grind the nuts into small pieces without loss
of oil*

Therefore, all the dry ingredients of each diet were weighed

into a large pan, mixed and ground in a Taring blender and passed
through a sieve and then the requisite amount of vegetable oil was
added to each diet and mixed thoroughly.

Two ml* of ether solution

of vlosterol containing 1600 I.TJ. of vitamin D were sprinkled on each
diet*

After all the ether had evaporated, the diet was sieved three

times and mixed well*
Groups of five rats were fed each experimental diet.
in each group were of approximately the same weight*

The rats

The animals

were placed singly in cages with large mesh screen floors and given
diets and distilled water, ad libitum*
of each animal was kept.

A record of the food intake

The animals were weighed on alternate days*

Every other day each animal was fed abort six gm. of green cabbage
which supplied a source of vitamin A*
At the end of a week, the animals in all the groups showed very
poor growth*

The consumption of food was* low and the physical condition

unsatisfactory*
noticeable.

Tood scattering by some animals in each diet was

The egg control animals were in a slightly better condition

than the other animals*

During the first part of the second week of the

experiment, 11 animals discharged orange-red colored urine.

On the 10th

day of the experiment, one rice control animal died and on the 12 th day,

-

41-

Oiie a n im a l on rice-cashew diet 1 died.
deteriorated noticeably,

The condition of the animals

k few lost weight.

majority reached a plateau.

The weights of the

The coats of the animals on the rice

control and rice and nut diets became rough*

The coat of one of the

animals on rice-cashew diet 2 was distinctly oily.

Except for the

changes noted above, the animals showed no specific vitamin deficiency*

The diets contained 6.1 to 6.6 per cent protein which should have
been adequate for slow growth in the rat.

There was sufficient calcium

in the diet to supplement the minerals of rice to give satisfactory
growth (Kik, 1942).

The test diet, with the exception of the egg

control diet consisted of converted rice or largely of converted rice.
The rice was cooked as was characteristic in parts of India by the
open kettle method in a large amount of water and the cooking water
was discarded, and this cooking method leadB to large losses of thiamin,
riboflavin and niacin (Kik and Williams, 1945).
It was surmised that poor qppetite, failure of growth and the low
condition of animals might be due to lack of some of the water soluble
vitamins.

Therefore it was decided to supplement the diets with water

soluble vitamins.

k vitamin mixture1, of the composition below was prepared.
Calcium Pantothenate

0.280 gm.

Kiacin

0.100 gm.

Biboflavin

0*050 gm*

1 By courtesy of Mrs. Annanell Jubb.

used in this laboratory.

This mixture was routinely-

-4 2

Thiamin

0.040 gm.

Pyridoxins

0*050 gm.

Inositol

2.000 gm.

Ascorbic Acid

2.000 gm,

p-amino Benzoic Acid

2.000 gm

Choline H

5.000 gm

The vitamins were w e l d e d separately Into a small beaker and
made up to 20 gm. with cornstarch and mixed well*

Two gm. of the
♦

mixture were added to each kilogram of ration*

About six gm* of

spinach were fed to each animal instead of cabbage to increase the
vitamin A content of the food*
By the end of the third week, the animals on the whole improved
in physical condition and showed weight gains and the urine became
normal in appearance in all animals*
was much better*

The muscle tone of the animals

By the end of the fourth week, most of the animals

showed substantial weight gains*
Nitrogen Balance Ibqseriment

During the last three days of the 28 day growth period a balance
period for determining nitrogen retention was done*
collections were made.

Urine was absorbed on filter paper, following

Mitchell's (1924) procedure.

The feces for all the animals in each

group were pooled into a single sample.
acid was used as a preservative.
the urine collection*

Urinary and fecal

Twenty per cent hydrochloric

The same procedure was followed for

A careful record of the food intake of each

-

group of animals was made.

43-

Weights of animals at the beginning and

the close of the balance period were taken.
The urinary and fecal samples were digested on a hot plate until
a brown digest uniform in color and consistency was obtained.

The

brown digests were passed through sieves into volumetric flasks and
made to volume.
bottles.

Thoroughly mixed samples were stored in screw topped

The nitrogen analysis of diet samples and urinary and fecal

samples were made by the Kjeldahl method.
After the balance period, all the animals were sacrificed and
autopsied.

About 55 per cent of the animals were mildly infested with

Taenia taenlaformin cysts.

RESULTS AND DISCUSSION

Chemical Analysis

The results of the chemical analyses of the experimental nuts
and converted rice with the gross energy value of these foods are
presented in Table 7.

The moisture, crude protein, total ash of nuts

and the ether extract values for almond and walnut were within the
range of values reported by Mitchell and Beadles (1937), Peterson,
Skinner and Strong (1943), Winton (1945), Campcus (1946), Watt and
Merrill (i960) for the above nuts.

The ether extract values for

pecans and cashews were two and five per cent lower*
The gross energy value of nuts per 100 gm*, determined with the
aid of a bomb calorimeter, was 705 calories for almonds,659v 806,
and 773 calories for cashews, pecans and English walnuts, respectively*
Mitchell and Beadles (1937) reported the gross energy value of nuts
in calories per 100 ©a* as follows:
pecans 769; and English walnuts 732*

almonds, 676; cashews, 648;
These values were lower than

the values obtained in this study in comparison with the percentage
composition of nuts in the two studies*

Cross energy values were

computed using as average heats of combustion 4*1 calories per gm*
for carbohydrates, 9*45 calories per gm. for fat, and 5*65 calories
per gm* for protein (Sherman, 1946)*

The calculated values, expressed

in calories per 100 gn* for nuts used in this study were:
cashow, 639; pecans 772; and English walnuts 750.

The

almonds, 702

Table 7
The proximate composition of nuts and rice

Type of food

Moisture
per cent

Ether
extract

Crude
proteixf

Total
ash

per cent

per cent

per cent

Carbohydrate
by difference
per cent

Oross energy
value/100 gnu
calories

Nuts
Almond

4.16

53.34

23.49

2.99

16.00

704

Cashew

4.65

42.75

19.35

2.58

30.47

659

Walnut

3.80

63.47

15.85

1.88

15.00

773

Pecan

4.02

60.06

9.87

1.33

15.72

808

0.10

7.04

0.39

81.87

380

Bice, cooked
and dried

10.6

• I x 6.25

1

Acknowledgement is made to Mrs. Dorothy Dunsing for determination of gross energy
values.

difference* between the computed caloric values and the actual
determinations were within five per cent and are believed to be
more consistent with the proximate composition than the data reported
by Mitchell and Beadles (1937).
The proximate analysis of converted rice, cooked by the open
kettle method and dried after discarding the cooking water has not
been reported in the literature.

Essential Amino Acid Composition of Almonds,
Cashews, Pecans and English Walnuts
The amino acid content of four nuts, as obtained in this study
has been given in Table 8.

Kuiken and Iyman’s (1949) analysis of

whole egg and -unpolished rice and amino acid composition of white
rice calculated from compiled data (Block and Bolling, 1951) also are
included in this table for comparison.
There were no studies found in the literature on the essential
amino acids of total proteins of the nuts studied in this investigation.
However Cajori (1921) partitioned the nitrogen present in globulin by
the method of Van Slyke.

Following the same method, Mignon (1923)

partitioned the nitrogen of English walnut globulin, and Damodaran
and Sivaewany (1936) partitioned the nitrogen of cashew nut globulin.
Table 9 summarises the data of their investigations.

These data show

that the extracted proteins of cashews, pecans and walnuts have a high
content of arginine and fair amounts of histidine and tryptophane.

fable 8
Essential amino acids of four nuts, brown rice, white rice and whole egg

Amino acid

Brown
rice1

White
rice2

Whole
egg1

Walnut

Almond

Cashew

Pecan

per cent

per cent

per cent

per cent

per cent

per cent

per cent

Arginine

3*32

4.42

4.92

2.01

0.54

0.54

0.85

Histidine

0.47

0.70

0.70

0.36

0.14

0.11

0.33

Isoleucine

0.70.

1.12

1.07

0.49

0.28

0.38
.

0.80

Leucine

1.08

1.54

1.55

0.65

0.51

0.59

1.23

Lysine

0.45

0.70

0.97

0.30

0.28

0.24

1.03

Methionine

0.17

0.19

0.32

0.15

0.14

0.26

0.45

Phenylalanine

0.71

1.16

1.14

0.48

0.31

0.48

0.66

Threonine

0.67

0.82

0.91

0.37

0.22

0.30

0.64

Tryptophane

0.10

0.11

0.13

0.07

0.10

0.10

0.21

Valine

0.73

1.10

1.23

0.52

0.40

0.47

0.93

1 Iyman and Kuiken1s (1949) analysis*
2 Calculated from compiled data, Block and Bolling (1952)*

Table 9
Partition of nitrogen from cashew, pecan and walnuts globulins reported in the
literature

Source of I

Cashew^

Pecan2

per cent

per cent

Walnut**

per cent

Amide N

11.91

9.8

9.99

Humin I?

1.40

3.6

1.84

21.57

22.9

39.13

Histidine IT

4.78

3.7

5.74

Lysine N

1.54

6.2

10.61

51.7

30.36

0.8

0.88

Arginine N

Mono-amino N

29.5

Non-amino N

10.09

1 Damod&ran and Sivaswamy (1936)*
2 Cajori (1921).
3 Mignon (1936).

-

49-

This r e m i t v&s in agreement with the findings of the present study
on total protein*

However, the lysine content of total proteins of

pecans and walnuts was found to he much lower than that of nut globulins
alone•
She nuts on the whole were richer in lysine and lower in methionine
than white rice*

Zn comparison with whole egg all the nuts were low in

methionine as well as lysine.

Tang and Butts (1949) reported an

analysis of amino acids in filbert globulins* which suggested that the
globulins were deficient in lysine and methionine*

In the case of

lysine this finding confirms the earlier studies*

Biological assay

(Tang and Batts, 1949) showed that lysine and methionine were the
growth limiting amino acids in filbert globulins for rats.

Whether

this was true of the proteins of almonds, cashews, pecans and English
walnuts was not known, as the proteins of the test diets was not
derived solely from the above nuts*
The arginine content of cashews was more than four times greater
than that of whole egg*

Also histidine, isoleucine, leucine,

phenylalanine and threonine occurred in greater concentration in cashews
than in whole egg, while lysine, methionine and tryptophane were found
to be in lower amounts*

The pecans have a lower content of all

essential amino acids compared to whole egg, with the exception of
arginine*

In walnuts and almonds, arginine and histidine occurred in

greater concentration than in whole egg; lysine, tryptophane and
methionine were present in lesser amounts.

Isoleucine, leucine.

50-

pheagrlalanizi*, threonine and valine occurred in greater amounts In
almonds than in whole egg, while these amino acids were found in
comparable amounts in walnut and whole egg*

Efficiency of Bice and But Protein Mixtures for the Growth of Bats

The weight gains of animals in relation to the protein of the
diet are given in Table Id*

The individual differences in growth

were great even though the protein intake was approximately the same*
The animals infested with Taenia taeniaformin seesMd to have grown
as well as the healthy animals, under the conditions of this experi­
ment (Table 10) •

This also was true of a preliminary experiment not

reported here*
The growth promoting efficiency of the protein of whole eggs
was superior to that of rice*

Bice protein promoted slightly better

growth than most of the rice and nut protein mixtures*

The order of

the protein efficiency computed as ©n. gain per gm* protein ingested,
for the different diets was as follows:
whole egg control
rice control
walnut—rice diet 2
cashew—rice diet 1
cashew-rlce diet 2
walxxut-rice diet 1
pecan-rlce diet 1
almond-rice diet 2
almond-rlce diet 1
pecan-rice diet 2

2.19
1.50
1.36
1.13
1.01
0.91
0.77
0*64
0.50
0.28

The differences between the protein efficiency of the rice
control and the egg control diets and rice control and each of the

-51

following dletss

pecan—rice l v pecan-rice 2, almond-rice 1,

almond-rice 2, cashew-rice 1, cashew-rice 2, were statistically
significant*

Th® differences between the rice control and each of

the walnut-rice diets warenot statistically significant (t— test,
Vischer, 1938)*

The "t" values obtained in the statistical analysis

are presented in Table 11*
The average protein efficiency of whole egg on a diet containing
6*8 per cent of protein was found In earlier work on rats in this
laboratory* to be 3*0 gm* gain in weight per gn. of protein ingested*
Buegmer, Poling and Lockhart (1950) and Arnrich, Hunt, Axelrod, and
Morgan (1951) report a protein efficiency of 15 gm* gain per gm* of
nitrogen ingested, or 2.4 gm. per gm. of protein ingested, on diets
containing 9*5 to 10 per cent protein derived from powdered whole egg*
The efficiency of proteins of the rice control diet in promoting
growth was also comparable to studies reported in the literature*
On a diet containing 5*5 per cent protein, the protein being derived
solely from rice, Kile (1940) reported a protein efficiency of 1*80 gm*
Sure (1946) reported the protein efficiency of polished rice at 5*8
per cent protein Intake as 1*86 gm*

The protein efficiency of rice

in this study was found to be 1*50 gm*

However, in making these

comparisons, it was noted that the studies quoted above were on diets
complete with respect to all dietary essentials, while in the present
investigation the diet was that characteristic of a human population but
which had never been demonstrated to give maximum growth in the rat*

^ TTrvnfn'hT i

data, bv courtesy of Miss Louise Xelley*

Table 10
Growth in weight in relation to protein intake of rate
on experimental diets

Type of diet

Eice Control

Animal
number

Total
Weight
gain

Total
food
intake

Protein
in diet

Total
protein
intake

Gain per gram
of protein intake
gm.

gm*

gm.

per cent

gm.

17

171

6.2

10.60

1.60

31

21

190

11.78

1.78

4

14

189

11.72

1.19

51*2

16

183

11.35

1.41

17.0

183

11.36

1.50

I1

29

159

10.49

2.76

21

26

162

10.69

2,43

3s

11

148

9.78

1.12

4

25

160

10.56

2.37

52

22

148

9.77

2.25

22.6

158

10.26

2.19

1
2s

Average
Igg Control

Average

6.6

Table 10 (contd)

Type of diet

Animal
number

Total
Weight
gain
gm.

Pecan Bice Diet 1

per cent

Total
protein
Intake

Gain per gram
of protein intake

m*

gm.

10.40

0.29

3

160

21

16

171

11.12

1.44

3

1

139

9.04

0.11

4

11

169

10.99

1.00

10

153

9.95

1.01

158

10.30

0.77

10.88

-1.84

f

Average

Average

m*

Protein
in diet

l1

61,2

Pecan Rice Diet 2

Total
food
intake

8.2

6.5

1

-2

170

21

10

178

11.39

0.88

31

11

205

13.12

0.84

41

8

183

11.71

0.68

52

10

184

11.78

0.85

184

11.78

0.28

7.4

6.4

fable 10 (contd)

Type

of diet

Walnut Bice Diet 1

Animal
number

Average

Total
food
Intake

protein
in diet

Total
protein
intake

Qain per
of protein

gm.

gm.

per cent

gm.

gn.

I1

1

172

6.6

11.35

0.08

2

4

172

11.35

0.35

3

16

183

12.08

1.32

4

19

172

11.35

1.67

5

12

159

10.49

1.14

10.4

172

11.32

0.91

I1

15

180

10.98

1.37

2

18

191

11.65

1.55

32

19

184

11.22

1.69

41

13

199

12.14

1.07

52

12

175

10.68

1.12

15.4

185

11.33

1.36

Average
Walnut Bice Diet 2

Total
weight
gain

6.1

Table 10 (contd)

Type of diet

Almond Rice Diet 1

Animal
number

Average

Total
food
intake

Protein
in diet

gm.

gm.

per cent

i1

2

170

6.1

31

3

31

Total
protein
intake

Gain per gram
of protein intake

gn.

gm.

10.37

0.19

152

9.27

0.32

13

169

10.31

1.26

41

3

150

9.15

0.33

51

4

171

10.43

0.38

5.0

162

9.91

0.50

11.71

1.20

Average
Almond Rice Diet 2

Total
weight
gain

I1

14

192

3

-1

165

10.07

-0.10

3

0

158

9.64

0.00

41

9

172

10.49

0.86

51

14

186

11.35

1.23

175

10.65

0.64

7.2

6.1

Table 10 (contd)

gm.
Cashew Rice Djet 1

Average

gm*

Protein
in diet
per cent

Total
protein
Intake
gm.

Gain per gram
of protein intake
gm.

6*5

I2
2

18

208

13.52

1.33

3

14

176

11*44

1.22

4

13

184

11*96

1*09

5

9

155

10.27

0.88

13.5

182

11.80

1.13

I1

19

188

11.66

1.63

21’2

10

171

10.60

0.94

31

14

181

11.22

1.25

41

6

163

10.11

0.59

51

8

200

12.40

0.65

11.4

181

11.20

1.01

Average
Cashew Rice Diet 2

Total
food
intake

6.2

* Animals mildly infested with Taenia taenlaformin*
2 Animals with orange-red urine, cause undiagnosed*.

- * 3-

Type of diet

Animal
number

Total
weight
gain

Table 11
Statistical comparison between protein efficiency on the various diets

"t1* values
Comparison of diets

Obtained

Bequired for significance
Probability < ,01

Probability < .05

Bice and egg

2.464

3.499

2.365

Bice and pecan-rice 1

2.920

3.499

2.365

Bice and pecan-rice 2

2.346

3.499

2.365

Bice and walnut-rice 1

1.967

3.499

2.365

Bice and walnut-rice 2

1.167

3.499

2.365

Bice and almond-rice 1

5.250

3.499

2.365

Bice and almond-rice 2

2.965

3.499

2.365

Bice and cashew-rice 1

4.111

3.707

2.447

Bice and cashew-rice 2

2.578

3.499

2.365

-

58-

The growth-promoting value of the protein of the zzat supplemented
rice diets was in no case equal to that of rice*

It was apparent that

the proteins of nuts do not form an effective supplement to the rice
diet as measured by growth per gm* of protein fed*

Nitrogen Intake Excretion and Retention by Bats on Test Diets

The nitrogen intake, excretion and retention by young rats on the
test diets is given in Table 12*

The data show that the fecal losses

of nitrogen of rats on rice and nut supplemented rice diets were much
greater than the fecal nitrogen loss on whole egg protein, indicating
that the vegetable proteins were not as digestible as the whole egg
or that the other constituents of the vegetable food interfered with
the digestion of protein*

Fecal losses were greater on walnut-rice

diets than on the rice diet, while this loss on the other nut supple­
mented rice diets was comparable to that on rice.

True digestibility

of proteins of test diets was not obtained since endogenous excretion
levels were not tested*

The apparent digestibility^- of the whole egg

protein surpassed that of the other diets.

The digestibility of this

protein was found to be 85 per cent which was comparable to A m r l c h ' s
(1950) figure of 89 per cent on the apparent digestibility of whole
egg*

The apparent digestibilities of rice and nut supplement rice

diets ranged from 70 to 78 per cent*

None of the apparent

1 Formulae for computing apparent digestibility: ^

x

Table 12
Nitrogen intake, excretion, and retention per day per animal
on the proteins of the experimental diets

Ave.
initial Food
Diet________ wt.
intake

—

—

N
Fecal
intake
N

Urine
IT

N retainN
N
Apparent
ed/gm.
absorbed retained digestibility absorbed
gm.

*
N retained
N. intake

lm»

gm.

gm.

per cent

0.0545

0.0227

77

0.4165

0.3224

0.0630 0.0095 0.0191

0.0535

0.0344

85

0.6430

0.5460

5.6

0.0577 0.0165 0.0269

0.0412

0.0143

71

0.3471

0.2478

77

6.4

0.0650 0.0154 0.0310

0.0496

0.0186

76

0.3750

0.2862

ilnut rice 1

78

6.3

0.0662 0.0197 0.0322

0.0465

0.0143

70

0.3075

0.2160

ilnut rice 2

80

6.7

0.0664 0.0180 0.0267

0.0484

0.0217

73

0.4483

0.3268

Lmond rice 1

67

5.8

0.0566 0.0146 0.0305

0.0420

0.0115

74

0.2719

0.2032

Lmond rice 2

70

6.3

0.0615 0.0150 0.0305

0.0465

0.0160

76

0.3441

0.2602

isnew rice 1

75

6.2

0.0635 0.0141 0.0286

0.0494

0.0208

78

0.4211

0.3276

ishew rice 2

68

6.4

0.0638 0.0141 0.0323

0.0497

0.0174

78

0.3501

0.2727

gm.

gm.

gm.

gBU

gm.

Lee control

73

7.2

0.0704 0.0159 0.0318

5g

77

6.0

jean rice 1

77

jean rice 2

control

gm.

-

60-

digestibllities of the rice euppleaented diets can be said to be
better than that of rice.
Nitrogen retained per gm. nitrogen intake

again showed that

whole egg protein promoted greater retention of nitrogen than either
rice proteins or proteins of nut supplemented rice diets.

Whole egg

protein showed a nitrogen retention of 0.5460 gm. per gm. nitrogen
intake.
The rice diet and the walnut and rice diet 2 and cashew and rice
diet 1 have the same order of nitrogen retentions per gm. of nitrogen
intake, namely, 0.3224; 0.3268; and 0.3276 gm. in comparison with
0.5460 for the egg diet.

The remaining diets resulted in lower

retention values in all cases.

Trom the point of nitrogen retention

it can be generalized that none of the nut supplemented diets provided
for better protein nutrition than the rice diet alone, since the
retention of nitrogen on these diets ranged from 0.2034 to 0.3276 gm.
Endogenous nitrogen excretion was not determined on this series
of rats.

However, in a recent study in the department endogenous

nitrogen excretion was determined on a series of animals of comparable
size and under comparable conditions of management.

B(y using the

average endogenous nitrogen^- excretion from this study, the true
digestibility and biological values were calculated and these are
presented in Table 13.

The egg control diet was superior to all other

diets in both estimated true digestibility and biological value (Table 13.)

1 Unpublished data by courtesy of Miss Louise Kelley.

Table 13
Estimated true digestibilities and biological values
of the proteins of experimental diets

Diet

IT
Fecal
intake E
gm.

gm.

Meta­
bolic
Fecal N
©a.

Fecal E
-Meta- Urine
bolic E
E
©n.

gm.

Endo­
genous

Urine E
True
-endoH
E
digesti- Biological
value
N genous B absorbed retained bility

gm.

gm.

gm.

©n.

per cent

per cent

,ce control 0.0704 0.0159 0.0089

0.0070 0.0318 0.0126

0.0192

0.0634

0.0442

90

70

g control

0.0630 0.0095

0.0006 0.0191

0.0065

0.0624

0.0559

99

90

ican rice 1 0.0577 0.0165

0.0076 0.0269

0.0143

0.0501

0.0358

87

71

ican rice 2 0.0650 0.0154

0.0065 0.0310

0.0184

0.0585

0.0401

90

69

ilnut ricel 0.0662 0.0197

0.0108 0.0322

0.0196

0.0554

0.0358

84

65

dnut rice 20.0664 0.0180

0.0091 0.267

0.0141

0.0573

0.0432

86

75

Lmond ricel 0.0566 0.0146

0.0057 0.0305

0.0179

0.0609

0.0330

90

65

Lmond rice2 0.0615 0.0150

0.0061 0.0305

0.0179

0.0554

0.0375

90

68

ishew ricel 0.0635 0.0141

0.0052 0.0286

0.0160

0.0583

0.0423

92

74

ishew rice 2 0.0638 0.0141

0.0052 0.0323

0.0197

0.0586

0.0389

91

66

Average metabolic nitrogen per rat per day, lelley (1952)*
Average endogenous nitrogen per rat per day, Eelley (1952).

-62

Kellftr(1952) found the average true digestibility and biological values
to be 96 per cent and 89 per cent respectively on a diet containing
6*8 per cent protein derived from defatted whole egg.

These values

are in accord with the findings of this experiment.
The true digestibility of protein which was found for rice
(90 per cent) was slightly lower than 97 per cent reported by Kik (1943)
for brown rice.

True digestibilities of rice diets supplemented with

nuts ranged from 84 to 92 per cent and were in agreement with Jaffa's
report (1903) and Cajorl's (1921) observation that the nuts in a mixed
diet gave a 90 per cent protein digestibility.
The calculated biological value observed for the rice diet was
70 per cent.

Kik (1943) reported a biological value of 73 per cent for

brown rice, but Mitchell (1924) and Basu and Basak (1937) found 80 to
86 per cent of the proteins of brown rice used by the animal body.
The biological values of the proteins of cashew-rice diet 1 and walnutrice diet 2 and pecan-rice diet 1 ranged from 71 to 75 per cent, the
proteins of other test diets had a lower biological value than rice
alone.

The biological values of protein calculated as outlined above

showed close agreement with protein efficiency.

Whole egg protein had

the highest biological value and the best protein efficiency.

The rice

control diet, walnut-rice 2, cashew-rice 1 had about the same biological
value and about the same protein efficiency.

The pecan-rice diet 1 had

a biological value approximately the same as that of rice alone but the
protein efficiency of this diet was poor.

Almonds had low protein

-

63-

efficiency as well as low 'biological value*

The other nut supplemented

rice diets had lower efficiency and lower "biological values than the
rice diet*

It was evident that the addition of nuts to a rice diet

did not improve the digestibility or the biological value of the
proteins over that of rice under the conditions of this experiment*
Hone of the diets were comparable to dried egg in the efficiency of
the protein supplied*

Hutrltive Value of the Bice Diets Supplemented with Buts

The supplementation of rice diets with nuts did not improve the
nutritive value of the experimental diets over that of the rice control*
Cashews contain more of all the essential amino acids than white
rice*

Almonds, pecans, and English walnuts have a higher concentrate

of amino acids, with exception of methionine, than white rice
(Table 8)*

It was expected that the amino acids in the nuts would

supplement the amino acids of rice, and this would result in good
growth and better utilization o f nitrogen of the nut mixed rice diets*
The results of this experiment did not support this hypothesis; possibly
the decrease of the nutritive value of the nuts and rice mixed diets
may be the result of amino acid imbalances due to addition of nut
protein to rice protein*

Arginine imbalance could be a factor in

decreasing the nutritive value of nut mixed rice diets*

Pecora and

Hundley (1951) reported a three fold excess of arginine in the rat
diet produced by adding arginine to polished rice caused significant

-

growth depression in rats*

64-

Bose (1937) estimated the growth require­

ment of rat for arginine as 0*2 per cent of the diet*
contained 0*54 per cent of arginine*
percentage of arginine (Tattle 8).

Polished rice

The rmts also have a high

Addition of nuts to rice increased

the amount of arginine in the nut-rice mixtures*
diet, the arginine content was 0*47 per cent*

In the rice control

In the various rice

diets supplemented with nuts the arginine content ranged from 0*50
to 0*71 per cent, and the deviation of arginine from the growth
requirement of the rat ranged from * 150 to ♦* 255 per cent in the diets*
This excess of arginine in diets could be harmful to the growing animal*
lorman and Xlvehjem (1951) also have reported that amino acid imbalances
resulted on the addition of excessive amino acids to a casein-gelatin
diet, as measured by the growth depression of weanling rats*
It may be possible that there were interfering factors in the rmts
which decreased the nutritive value of the nut supplemented rice diets,
and prevented proper utilisation of the protein of the diet*

Gajori

(1921) found tannin in the skins of pecans toxic to the rat*

Mignon

(1923) reported the tannin in the skin of English walnuts as harmful
to the rats*

Pecal nitrogens were found to be higher on all the

vegetable diets than on that of the egg control diet, indicating a
lower digestibility for the vegetable diets than that of the egg
control diet.

Urinary nitrogens also were higher for the vegetable

diets than the egg control indicating that the amino acidsabsorbed
were less available to the animal body*

-

65-

Ths rics control diet and the nut supplemented rice diets were
protected by addition of calcium.

This, according to Akyroyd (1940)

and Banganath and Baa (1937) should stimulate the growth efficiency
of the diets.

The diets also contained greens, ergosterol, and the

water soluble vitamins which should have made the diet effective, but
in spite of these additions, the growth was slow on all the experi­
mental diets except the rice control.
for four weeks per animal.

It averaged from five to 15.4 gm.

Three extra animals from the same consign­

ment, one of them infested with Taenia taeniaformin, were kept on the
stock diet routinely used in this laboratory.

The growth of the

animals during the four week period was 67, 75 and 86 gm.

It is of

interest that the infected animal made the greatest growth.

It was

clear that the experimental animals did not attain maximum potentiality
for growth on a diet simulating an Indian rice diet, or on the experi­
mental rice diets supplemented with nuts.

Evidence from this

experiment supports the observations concerning the slow growth of
human beings whether in India or elsewhere in Asia on a rice diet.
The improvement of the rice diet for India and other Asian
countries has been the concern of nutritionists.

Milk, eggs, or meat

have been found to supplement the rice diet very efficiently,(Aykroyd
and Collaborators ,1937-1940).

Owing to the difficulties of the

production of milk and non—acceptability of other animal proteins by
some section of the Indian people, other sources of protein to
supplement the basic Indian rice diet need to be explored.

From

-66

this experiment it was found that nuts do not form an effective
supplement to the rice diet.

A consistent testing program is

necessary for other forms of vegetable protein of high biological
value which would both be acceptable to the Indian population and
which might improve the quality of the rice diet*

Cereal germs and

brewer's yeast are possible products for this purpose, both of which
could be produced economically*

SUMMABT A N D CONCLUSIONS

The nutritive value of four nuts, almonds, cashews, pecans and
walnuts was investigated for proximate composition and percentage of
10 essential acids.

The effectiveness of the supplementation of the

basic rice diet of low income groups in India with nuts also was
studied.
Weanling rats were maintained on the experimental diets for four
weeks.

The protein efficiency was measured as gain in weight per gm.

of protein ingested.
follows:

The efficiency of the different diets was as

defatted whole egg 2.19 g m . , rice 1.50 gm., and from 0.28

to 1.36 gm. for the eight nut supplemented rice diets.
The apparent digestibility of the proteins and nitrogen retention
per gm. of nitrogen intake was investigated for each diet use of the
balance experiment.

!foe apparent digestibility of proteins of test

diets was found to be:

whole egg 85 per cent, rice 77 per cent, end

nut supplemented diets from 70 to 78 per cent.
The nitrogen retained per gram intake was for whole egg 0.5460 g m . ,
rice 0.3224 gm.

Th« range for nut supplemented rice diets was from

0.2032 to 0.3276 gm.
Displacement of five and twenty-five per cent of the protein of
the basic rice diet by the proteins of four whole nuts did not improve
the growth promoting efficiency, the digestibility, or nitrogen retention
of the proteins of mat supplemented rice diets over that of rice alone.
Therefore, it was concluded that the nuts did not form an efficient

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FIGURE

X

5.0

Milliliters of 0.05 N NaQH

4.0

S.O

1.0

0

2

4

6

8

io

14

16

Micrograms of DX.— 1.1«-th.i onine

Titration Values

O'b'fcained. from

Streptococcus f a e c a l Is
Known Concentrations

of

R for

D L — Methionine

18

20

FIGRJKE 2
6,0

5.0

Milliliters

of 0,05 N Na OH

4.0

.0

;:.o

o
0

1

2

3

4

5

6

7

8

Micrograms of DL-Tryptophane

Titration Values Obtained from
Streptocoecus faecalis R for
Known Concentrations of DL— Tryptophane

9

10

i

FIGURE 3

Milliliters

of 0.06 N Ufa. OH

5.0

1.0

0

5

10

15

20

25

30

35

40

Micrograms of DL— Threonine

Titration Values Obtained from
Streptococcus faecalis R for
Known Concentrations of DL—Threonine

45

50

FIGUBE 4
6.0

3.0

Milliliters

of 0.05 N Ha OH

4.0

1.0

0

4

8

12

16

20

24

28

32

Micrograins of L-Arginine hydrochloride

Titration Values Obtained from
Streptococcus faecalis E for
Known Concentrations of L-Arginine hydrochloride

36

40

FIGURE 5

Milliliters

of 0.05 H Na OH

4.0

3.0

2.0

1.0

0
0

1

2

3

4

5

6

7

8

9

Micrograms of L-Histidine hydrochloride

Titration Values Obtained from
Streptococcus faecalIs R for
Known Concentrations of L-Histidine hydrochloride

10

FIOtJRE 6

6.0

Milliliters

of 0.05 N Ka OH

5.0

4.0

0

0

1.0

0
0

5

10

15

20

25

30

35

Micrograms of DL-Valine

Titration Values Obtained from
Lactobacillus arabinogus P—60 for
Known Concentrations of DL-V&line

40

45

50

PIQUES 7
7.0

6.0

Milliliters of 0.05 N Ha OH

5.0

4.0

2.0

1.0

0

10

15

20

25

30

35

40

Microcrams of Dir-Isoleucine

Titration Values Obtained from
Lactobacillus arabinosus 17-5 for
Kn otm Concentrations of DL-isoleucine

45

50

FIGURE 8

Milliliters

of 0.05 N Na OH

4.0

3.0

3.0

1.0

0
0

1

2

3

4

5

6

7

8

9

Micrograms of DL—Phenylalanine

Titration Values Obtained from
Lac t obac i1 lus arabinosus 17— 5 for
Xnown Concentrations of DL— Ihenylalanine

10

FIGUHE 9
7.0

6.0

4.0

Milliliters

of 0.05 U Na OH

5.0

1.0

0

2.5

5

7.5

10

12.5

15

17.5

Micrograaas of L-Leucine

Titration Values Obtained from
Lactobaci1lus arabinosus 17-5 for
Kno rm Co nc entrations of L— leucine

20

22.5

25

FIGURE 10
5.0

Milliliters

of 0.05 N Na OH

4.0

2.0

1.0

0

5

10

15

20

25

30

35

40

45

Micrograms of L-Lysine hydrochloride

Titration Values Obtained from
Leuconostoe roesentoroides P—60 for
Known Concentrations of L-Iysine hydrochloride

50

85-

T a b le

1

Essential amino acids in four nuts and recoveries of added amino acids

Test
Humber

Walnuts

Almonds

Cashews

mg/gm

mg/ gm

mg/gm

Pecans

Recovery

mg/gn

per cent

Threonine
1.

6*633

7.522

8.488

3.651

116.9

2.

6.713

8.357

8.879

3.912

116.1

3.

6*641

8.857

9.852

3.660

109.6

Average

6*662

8.246

9.073

3.741

Valine
1.

7.350

10.962

13.021

5.535

94.3

2.

7.102

11.234

12.126

5.177

95.4

3.

7.467

10.796

11.826

4.793

100.5

7.306

10.997

12.325

5.185

Average

Methionine
1.479

101.6

1.

1.705

1.833

2.797

2*

1.718

1.862

3.026

3*

1.779

1.909

3.909

1.545

103.0

4.

1.673

1.996

2.971

1.569

96.0

Average

1.719

1.900

3.176

1.531

_

.

94.2

—86—

S a b le

Test
lfumber

Walnuta
mg/gn

Almonds
mg/gm

1

(c o n td )

Cashews
mg/gm

Pecans
mg/gn

Becovery
per cent

Tryptophane
1.

1*162

1.217

1.558

0.784

71.5

2.

0.876

0.946

0.835

0.574

73.2

3*

0.944

0.997

1.426

0.620

75.0

4.

1.066

0.925

1.003

0.804

79.3

5.

1.074

1.624

1.805

0.842

81.7

Average

1.034

1.142

1.325

0.723

Histidine
1.

4.311

7.946

7.462

4.006

111.7

2.

4.808

6.659

7.386

4.267

103.1

3.

5.084

6.475

6.675

3.058

4.

4.682

6.847

6.450

3.138

Average

4.721

6.982

6.993

3.617

104.9

Arginine
1.

35.892

46.846

50.000

19.982

98.3

2.

30.424

47.966

55.556

21.906

91.2

3.

33.227

37.801

42.010

18.490

107.5

Average

33.181

44.204

49.189

20.126

87-

V a b le

Test
Humber

1

(c o n td )

Valnats

Almonds

Cashews

mg/gm

ng/m

mg/gm

Pecans

Becoverr
per cent

Isoleucine
1.

7*000

10.939

10.707

6.142

2.

8*253

11.326

9.947

4.298

90.8

3.

7.623

11.232

11.182

4.669

97.9

4.

7.480

11.326

10.873

4.375

97.2

7.589

11.206

10.677

4.871

Average

—

Leucine
1.

11.314

15.138

15.847

6.901

110.0

2.

10.962

16.470

16.168

6.430

100.9

3.

10.209

14.738

14.351

6.103

102.7

Average

10.828

15.449

15.455

6.478

lysine
1.

4.766

6.910

9.344

3.034

92.5

2.

3.955

7.768

10.470

3.221

104.4

3.

4.476

6.376

9.230

2.963

98.0

4.

4.746

6.959

9.572

2.964

99.4

4.486

7.003

9.654

3.046

Average

—8 8 —

T a b le

1 (c o n td )

Test
Kturiber______ Walnmts_____Almonds_____ Cashews_____ Pecans_____ Recovery
mg/gm

mg/gn

mg/©a

mg/ga

per cent

Phenylalanine
1.

6.601

11.326

11.214

4.712

94.7

2.

7.128

11.834

10.090

4.829

86.5

3.

7.537

11.590

13.010

4.931

84.8

Average

7.089

11.583

11.438

4.824

S a b le

11

The composition of rice diets supplemented with nuts
food1"

freight

Calories

gm*

Protein
gm.

Fat
gm.

Almond-Rice Piet 1
75.00

285.15

5.25

0.08

Almonds

7.45

52.49

1.75

3.98

Vegetable fat

9.94

93.93

Cornstarch

19.52

80.03

Total

112.91

511.60

Rice

9.94

7.00

14.00

Almond-Rice Piet 2
Rice
Almonds
Vegetable Fat

95.00

361.19

6.65

0.10

1.49

10.45

0.35

0.80

13.1

Cornstarch

3.94

Total

114.53

13.10

123.8
16.16
511.6

7.00

14.00

Pecan-Rice Piet 1
Rice

75.00

285.15

5.25

0.08

Pecan

17.73

143.33

1.75

12.25

1.67

15.78

Cornstarch

16.42

67.34

Total

111.82

511.60

Vegetable fat

1.60

7.0

14.0

-90-

Table 11 (contd)

Weight

Food

Calories

gm*

Protein

Fat

gm.

0D.

Pecan-Bice Diet 2
95.00

361.19

6.65

0.10

3.55

28.66

0.35

2.45

11.45

108.20

Cornstarch

3.30

13.55

Total

114.30

511.60

Bice
Pecan
Vegetable fat

11.45

7.00

14.00

Walnut-Rice Diet 1
Bice

75.00

285.15

5.25

0.08

Walnut

11.04

85.39

1.75

7.01

6.91

65.30

Cornstarch

18.48

75.76

Total

112.43

511.60

Vegetable fat

6.91

7.00

14.00

Walnut-Bice Diet 2
95.00

361.19

6.65

0.10

2.21

17.08

0.35

1.40

12.50

118.13

Cornstarch

3.71

15.20

Total

114.42

511.60

Bice
Walnut
Vegetable fat

12.50

7.00

14.00

- 91-

Table ±i (contd)

Food

Jfatahfc

Calories

Protein
Cm,

Fat
Cm,

Cashew-Rice Diet 1
Rice

75.00

235.15

5.25

0.03

9.09

59.40

1.75

3.87

Vegetable fat

10.05

94.97

---

Cornstarch

17.53

71.38

---

Total

112.77

511.40

Cashews

10.05

7.QC

14.00

Cashew-Rice Diet 2
95.00

341.19

6*65

0.10

1.81

11.92

0.35

0.78

13.12

123.93

---

Cornstarch

3.54

14.51

---

Total

114.47

511.40

Rice
Cashews
Veget able f at

7.00

13.12

14.00

One jn. of CaCOH)^ and 1600 Intern vii ••w.al units of irradiated
ercostercl vrere added to each test •-.•Vet. Animals on the diet
were fed six gm. of creens ever^ alternate ooy during the
experimental period.

-92-

Table ill
The con^osition of whole egg control diet

Weight
gin.

Food1

Calories

Protein

Fat

gm.

gm.

Dried whole egg

10.00

o0.35

7.00

2.00

Fat

12.00

113.40

---

12.00

Cornstarch

82.40

337.70

---

---

Total

105.40

511.40

7.0C

14.00

^ Aboiit six ■tti. of greens vrere fed to and .sis on the whole egg
control diet during the experimental period# 1600 International
units of irradiated ergosterol were added to the diet#