PROTESN VALUE OF POTATO
fiaE‘éD NAVY BEAN POWDERS:
NUEfRiTiGNAL EVALfiATiON USING
THE MEADOW VOLE
{MICROTUS PENNSYLVAMCUS).

Thesis for the Degree of M. S.
MICHEQAN SIA‘E‘E UNIVERSE?!
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ABSTRACT

PROTEIN VALUE OF POTATO AND NAVY BEAN POWDERS:
NUTRITIONAL EVALUATION USING THE MEADOW
VOLE (MICROTUS PENNSYLVANICUS)

BY

Bertha J. Rios Iriarte

The protein content of navy bean powders pro-
duced from beans cooked in atmosphere and in a retort was
found to average 23.4 g/100 g. The protein contents
analysed by the Kjeldahl and Biuret methods were similar,
indicating that the Biuret method could be used to mea-
sure the protein of potatoes.

Methionine was the low level amino acid. Gluta-
mic and aspartic acids were in higher amounts than their
essential amino acids. The use of Chemical Score was
successful when applied to nutritional studies with mea-
dow voles. Potatoes at 5.28% protein permitted better
growth than beans, indicating that the former had more
balanced proteins. Inclusion of methionine improved the
nutritional value, demonstrating that methionine was the
first limiting amino acid and isoleucine the second

limiting one. The absorption of nitrogen was also

Bertha J. Rios Iriarte

increased when voles received the supplemented pro-
teins.

The nutrive value of navy beans cooked in a re-
tort was less than that of beans cooked in the atmosphere
and may be attributed to intensity of heat applied. The
use of meadow voles (Microtus pennsylvanicus) in nutri-
tional studies deserves attention because of their sen-
sitivity in responding to low level protein diets. Their
low cost, availability and rapid growth could be important

factors in promoting their use in developing countries.

PROTEIN VALUE OF POTATO AND NAVY BEAN POWDERS:
NUTRITIONAL EVALUATION USING THE MEADOW

VOLE (MICROTUS PENNSYLVANICUS)

BY

Bertha J. Rios Iriarte

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science

1969

ACKNOWLEDGMENT S

The author expresses her sincere appreciation to
her major professor, Dr. Clifford L. Bedford for his
guidance and constructive criticism throughout her gradu-
ate work and for his valuable help in the preparation of
this manuscript.

The author also extends her acknowledgment and
thanks to the members of her committee, Dr. Norman R.
Thompson who provided the potatoes and who made arrange-
ments for the use of voles, and to Dr. Laurence G. Harmon
for his helpful suggestions in preparing this manuscript.

The author wishes to express her sincere grati-
tude to the Agency for International Development for its
financial assistance.

Special thanks are also given to Miss Ursula Koch
for her technical assistance and cooperation in determin-
ing the amino acids, to Dr. Fred C. Elliott for allowing
me to use his voles Colony, to John S. Shenk for his aid
with the feeding studies in Crop Science, to Lynn Pope
and Ben Counter who processed the potatoes and navy beans,
and to those friends who made helpful suggestions in the

preparation of the manuscript.

ii

Lastly, the author is especially grateful and
thankful to her parents, her brother and his family for

their helpful encouragement during her graduate studies.

iii

TABLE OF CONTENTS

LIST OF TABLES O O O I O O O I O I O O O O O O O 0

LIST OF FIGURES . . . . . . . . . . . . . . . .

INTRODUCTION 0 O O O O I O O O O O O O O O O O O O

LITEMTURE REVIEW 0 O O O O O O O O O O O O O I O

1.

FACTORS DETERMINING PROTEIN MALNUTRITION ON
PRESCHOOL CHILDREN 0 O O I O O O O O O O O O

A. Protein deficiency . . . . . . . . . . .
B. Protein calorie malnutrition . . . . . .
C. Socio-economic factors . . . . . . . . .

D. Interrelation between energy intake and
nitrogen O O O O O O O O O O O O O O O O

MEANS OF PROVIDING MORE AVAILABLE PROTEIN .
A. Vegetable mixtures . . . . . . . . . . .
B. Mixture of food and protein concentrates
C. Amino acid supplementation . . . . . . .
D. Microorganisms in food production . . .
AMINO ACID SUPPLEMENTATION . . . . . . . . .

A. Classification of amino acids and re-
quirements . . . . . . . . . . . . . . .

B. Concept of protein's first limiting

amino acid . . . . . . . . . . . . . . .
C. Amino acid balance and imbalance . . . .
PROTEIN EVALUATION . . . . . . . . . . . . .
A. Biological methods . . . . . . . . . . .
B. Protein Efficiency Ratio . . . . . . . .

C. Dietary nitrogen utilization . . . . . .

D. The Meadow Vole (Microtus pennsylvanicus)

in bioassay technique . . . . . . . . .

iv

Page
vi

vii

\lmU1U1

10
10
11
13
16
19

19

20
21
23
23
23
25

26

5. POTATOES AND BEANS IN NUTRITION . .
A. Potatoes as food . . . . . . .

B. Beans as food . . . . . . . . .
METHODS AND MATERIALS . . . . . . . . . .

Sample designation . . . . . . . .
Determination of proteins . . . . .
Analysis of proteins . . . . . . .
Amino acid analysis . . . . . . . .
Protein evaluation by animal assays
Composition of diets . . . . . . .

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

Protein content of beans and potato
Chemical score . . . . . . . . . .
Bioassay using voles . . . . . . .

SUMMARY AND CONCLUSIONS . . . . . . . . .
LITERATURE CITED . . . . . . . . . . . .

APPENDIX 0 O O O O O O O O O O O O O O O

Page

28
28
34

40

40
40
41
42
44
45

50

50
53
54

66
69

82

LIST OF TABLES

TABLE Page
I Moisture content of samples g/100 g . . . . . . 46
II Chemical Score of navy beans and potatoes

as related to the amino acids of whole egg . . 49

III Protein content of bean powder and potato
flakes O O O O O O O O O O O O O O O O O O O O O 51

IV Vole growth with navy bean and potato powders.

Non—supplemented and supplemented diets . . . . 55
V Absorbability of nitrogen, non—supplemented

diet O O O O O O O O O O O O O O O O O O O O O 62
VI Absorbability of nitrogen, supplemented diet . 63
VII Diet comPOSition O O O O O O O O O O O O O O O 83
VIII Protein content of 10 strains of potatoes . . . 85
IX Amino acid composition of processed navy

beans and potatoes expressed as 9/16 g N. . . . 86
X Nutritional value of the non-supplemented

proteins . . . . . . . . . . . . . . . . . . . 87
XI Nutritional value of the supplemented pro-

teins O O O O O O O O O O O O O O O O O O O O O 88

XII Comparison of the means of non-supplemented
and supplemented diets. . . . . . . . . . . . . 89

XIII Summary of the comparisons between the means
of non-supplemented and supplemented diets. . . 90

vi

LIST OF FIGURES

Figure

1.

2.

Growth response in voles when supplied potatoes

and bean proteins . . . . . . . . . . .

Nutritional quality measured as protein
efficiency ratio . . . . . . . . . . .

Protein determination: Biuret method.
Standard curve. . . . . . . . . . . . .

A diet feeder for weanling meadow voles

vii

Page
. 58
. 59
. 82

91

INTRODUCTION

One of the most serious problems found today in
developing countries is the shortage of high quality pro-
tein foods. Protein malnutrition, especially among young
children, has become a major concern (Senecal, 1958).
This problem is particularly complex because the various
regions of the world differ not only in topographic and
climatic conditions but in social patterns and economic
resources.

In some areas of developing countries the expense
of protein of animal origin limits its consumption to a
minority of people while the great majority get their
protein food from plant origin.

When the body receives a deficient protein, the
health is injured and particularly in growing children
this insufficiency has deleterious effects (Allison et.
al., 1960). Mortality rates rise considerably when re-
sistance to infections is decreased (Scrimshaw, 1966).
For those who survive, physical growth is impaired (Jack-
son, 1966) and mental ability lessened (Cravioto et al.,
1967). The mind may be irreversibly damaged depending on

the duration of the deficient food intake (Coursin, 1965).

Tizard (1968) emphasized that prolonged and severe
malnutrition in infancy may cause changes in the chemical
composition of the brain, as well as decreased stature
and lack of stamina.

In young experimental animals, learning, memory
and behavior are affected when malnutrition is severe
(Nutrition Foundation, 1967-68). These authors reported
that the brain acquires about 80 per cent of its adult
weight by the age of three years, when the body has
reached 20 per cent of its adult weight. Malnutrition
in children was shown by psychological and behavioral
changes, characterized by apathy (Pearson, 1967).

In adults the inadequacy of food intake creates
an incapacity to work efficiently, which also can be ag-
gravated by psychological changes rendering the mind
apathetic to work. These affections can vary in inten-
sity, depending upon the physiological state of the or-
ganism, which, in turn, is related to the amount of time
of the individual's undernutrition or malnutrition.

Keys et a1. (1950), reported that prolonged undernu-
trition deteriorates not only general behavior but in-
telligence and personality. Brozek (1955) pointed out
that food intake and behavior are complex and interdepend-
ent systems.

A world wide survey has demonstrated the magnitude

of the problem of hunger and malnutrition. Half of the

world's population is suffering from undernutrition or
malnutrition and probably both (FAO, 1963). Undernutri-
tion refers to an inadequate intake of food and calories,
whereas malnutrition refers to an inadequacy in the quality
of food intake, notably in protein, vitamins and minerals.

If one considers the scarcity of good protein
sources in some economically depressed areas of the world
and the larger number of preschool children receiving
subnormal levels of nutrients (70% according to Gyorgy,
1966), the campaign for averting the deleterious effect
of protein deficiency is welcome (United Nations, 1968).
Numerous investigators are dedicated to this important
task of preventing and controlling malnutrition through
the beneficial system of food supplementation. This sup-
plementation increases the quantity and quality of avail-
able protein for metabolic purposes, covering in this way
the shortage of animal protein.

Today there are several ways of enhancing the pro-
tein value of a food. A practical method is to mix two
or three different vegetable foods in such a way that
they will produce a higher quality protein supplying the
required dietary protein (Bressani et al., 1961).

Protein concentrates are also excellent sources
of protein. According to their utilization they may be

arranged as follows: fish flour, soy products, peanut

flour, sesame flour, cotton seed flour, and coconut pro-
tein (Hundley, 1968).

The quality of the protein can be improved by
the addition of the deficient amino acids. This method
is being used today with success not only in animal feed-
ing but also as a means of combating the protein malnu-
trition problem for human beings (Gomez et al., 1958 and
Hansen et al., 1956).

According to Flodin (1953) proper supplementation
of staple foods with amino acids doubles the value of the
dietary protein.

The purpose of the present work is to become ac-
quainted with principles involved in improving the quality
of the protein in order to apply these principles to ag-
ricultural products native to Bolivia, with the objective
of combating the protein malnutrition problems, especially
in children at preschool and school age.

In this study the nutritive quality of drum dried
potato flakes and navy bean powders was evaluated by
determining protein content and amino acid composition
and in feeding trials using nonsupplemented and supple-
mented proteins. Both chemical and bioassay procedures

were used.

LITERATURE REVIEW

1. FACTORS DETERMINING PROTEIN MALNUTRITION IN PRESCHOOL
CHILDREN.

A. PROTEIN DEFICIENCY.

Several scientists from different parts of the
world working on the control of children's health agreed
that protein malnutrition is mostly manifested under the
age of five years in underdeveloped countries.

The high rates of morbidity and mortality in in—
fants in the immediate postweaning period are principally
due to the inadequate intake of protein. Breast feeding
is dominant and the infants do not receive supplementa-
tion during lactation, which may be extended to twelve
months (Scrimshaw et al., 1961). A marasmic condition
could develop when the infant is nourished from a de-
ficient mother, although a combination of parasitic and
nutritional deficiency diseases may obscure the diagnosis
(Behar et al., 1958). The infant's impaired clinical
picture is mostly due to the shortage of dietary protein
because family's food selection for post weaning is basi-
cally of farinaceous origin, thus eliminating the required
protein so essential for growth and maintenance (Hansen
and Howe, 1964). Bengoa et a1. (1959) reported that
malnutrition is more dominant in the postweaning period
and the high mortality rate of children between the age

of one and four years is indicative of the true dimension

of the problem of malnutrition. Over half of the popu-
lation in developing countries dies before the age of
fifteen and in many areas 50% or more of the infants die
before reaching the age of five (Schaefer, 1963). The
infant mortality rate in developing countries could reach
ten to fifteen times more than in developed ones and the
infant mortality rate between one and four years may be

as much as fifty to sixty times higher (Williams, 1966).

B. PROTEIN CALORIE MALNUTRITION.

Today it is well known that protein calorie mal-
nutrition is manifested in two ways: 1) Kwashiorkor re—
sulting from the ingestion of low levels of protein with
considerable amount of calorie intake and 2) Marasmus
representing complete malnutrition having both protein
and calorie deficiency (Altschul, 1965). Kwashiorkor
was described by Scrimshaw et a1. (1961), as a disease
characterized by edema, dermatitis of the hyperkeratosis
type accompanied by hyperpigmentation and desquamation
of the skin. The child is psychologically affected and
anorexia and anemia are symptoms of this disease. The
fact than the hair falls or drops out spontaneously, plus
the absence of uniform hair color was found to be a
specific symptom of kwashiorkor. The diets of children
developing kwashiorkor are deficient not only in protein,

but also in other nutrients, particularly vitamins and

minerals (Behar et al., 1958). Marasmus is character-
ized by marked growth retardation, loss of weight, extreme

muscular wasting and absence of subcutaneous fat.

C. SOCIO ECONOMIC FACTORS.

According to Scrimshaw (1961), Behar (1958), Rao
et a1. (1959), and Autret (1961), the families of child-
ren who manifested kwashiorkor or marasmus frequently
come from less favoured social groups, where ignorance,
food habits, attachment to the past, unsanitary conditions
and apathetic attitudes toward child care are factors
which precipitate the development of these diseases. On
the other hand, the limited availability of protein can
be due to the low fertility of soil, production of ag-
ricultural products that are not good sources of protein
and lack of knowledge about correct selection of food for
fulfilling the children's requirements (Burgess and Dean,
1962).

The availability of native foods creates food
habits which are related to cultural patterns, and these
in turn are influenced by technological, economic and
social factors.

Soil and climatic conditions are factors leading
to undernutrition or malnutrition. In developing countries
the lack of modern technology limits food production. Lack

of proper conditions of storage promotes food spoilage due

to contamination with rodents and insects. Food of high
nutritive value like eggs, meat, fish, milk, fruits and

vegetables need appropriate handling, transport and stor-
age for preservation (Burgess and Dean, 1962). The stock
of foods in the market also depends on the consumer's de—

mand, influenced by socio-economic factors.

D. INTERRELATION BETWEEN ENERGY INTAKE AND NITROGEN
RETENTION

Several investigators have shown that carbohydrate
and fat have a protein sparing effect, with carbohydrate
being the more efficient. Swanson (1959) and Howes and
Spector (1954) pointed out that nitrogen utilization is
influenced by both caloric and protein intake. These
authors have observed that the negative nitrogen balance
of active young men receiving no protein food decreased
when about 700 calories were supplied, and the level of
one thousand calories plus 3 grams of nitrogen increased
the sparing effect similar to the ingestion of high quan-
tities of nitrogen.

When a constant nitrogen intake of 3.82 grams per
day and slightly decreasing carbohydrate diets were given
to dogs, an increase of urinary nitrogen excretion and a
constant balance index were observed. When the carbohy—
drate was severely restricted in the diet, nitrogen ex-

cretion was increased and the nitrogen balance index was

decreased (Rosenthal, 1951). This investigator has ob-
served that responses varied with the physiological state
of the animal.

Albino rats fed 4, 6 and 8 grams daily' of a complete
diet containing either 10% soybean oil meal or methionine
supplemented fibrin had the lowest biological value at 4
grams. Forbes and Yohe (1955) attributed this to the use
of protein for caloric needs.

The importance of consuming a high quality protein
when levels of protein and calories are marginal was studied
by Leverton et a1. (1951) who used two groups of young
women receiving 43 and 63 grams of protein at isocaloric
intakes of 1800 calories. Those receiving lower levels
of protein had a higher nitrogen excretion when 240 grams
of milk were included at the noon meal; no difference in
nitrogen excretion occurred with the second group. When
calories were increased to 2400 with no supplement of
milk protein, the nitrogen excretion of both groups de-
creased with the nitrogen sparing effect being higher for
those with lower protein intake.

Differences in nitrogen retention and in the be-
havior of animals receiving plant and animal proteins have
been observed. Puppies fed wheat gluten retained 0.18 g
of nitrogen per gram of nitrogen consumed, whereas, with
egg protein they showed a retention of 0.5 g per gram

nitrogen intake. Animals receiving gluten were obese

10

and inactive and the others lean and active. However,

the increase of body weight per g of nitrogen intake was

approximately the same for both groups (Allison and Wanne-

macher, 1957). This study showed the importance of the

quality of the protein fulfilling their nitrogen needs.
Gluten protein, being deficient, was not totally

used and was converted to a caloric source, manifested

in the obesity of the animals.

2. MEANS OF PROVIDING MORE AVAILABLE PROTEIN.

A. VEGETABLE MIXTURES.

A great part of the world's population subsists on
vegetable cr0ps, usually of low protein content. The ma—
jority of vegetable proteins are deficient or low in at
least one amino acid. A mixture of foods having different
amino acid deficiencies will produce a protein of better
nutritional value than their components, due to the effect
of supplementation.

In Central America, Incaparina, the low cost mix-
tures containing over 25% protein, were developed for pre-
school children at the Institute of Nutrition of Central
America and Panama (INCAP). Mixture 8 contained 50% lime
treated corn, 35% sesame flour, 3% torula yeast, 1% cal-
cium carbonate and 4500 I.U. of vitamin A acetate. This
formula was found to be equivalent to milk in treating

kwashiorkor (Scrimshaw et al., 1961). Other formulas

11

gave similar results. Number 9 is composed of corn flour
29%, sorghum flour 29%, cotton seed flour 38%, torula
yeast 3%, Ca C03 1% and vitamin A 4500 I.U.

Formula No. 15 is in commercial production; it
contains corn flour 58%, cotton seed flour 19%, soya flour
19%, torula yeast 3%, Ca C03 1%, Vitamin A acetate 4500
I.U. (Behar et al., 1966). In Guatemala, Mexico and
Colombia Incaparina formula 9b is in commercial produc-
tion.: This formula has 29% ground whole maize, 29% ground
whole sorghum, 38% cotton seed flour, 3% torula yeast, 1%
Ca C03, 4500 I.U. Vitamin A and its protein content is
27.5%. Incaparina can be drunk as "atole"; it is made
by cooking 25 g of Incaparina in one glass of water for
25 minutes. Sugar is added to improve taste. It can also
be incorporated into soups, breads and puddings. Incap-
arina is found commercially in polyethylene bags at a

price of three U.S. cents per bag of 75 grams (Shaw, 1964).

B. MIXTURE OF FOODS AND PROTEIN CONCENTRATES

In South Africa Pronutro was develOped as a food
supplement of good quality protein balanced with vitamins
and minerals for preventing kwashiorkor and other forms
of malnutrition and undernutrition. The chemical com-
position at present is as follows: protein 22 g; fat,
approximately 11.5 g; calories, 413; calcium, 460 mg;

phosphorous, 580 mg; iron, 5.5 mg; Vitamin A, 3500 I.U.;

12

thiamine, 1.06 mg; riboflavin 1.5 mg; niacin, 14 mg and
vitamin C 53 mg. The protein source is a mixture of soya
beans, peanuts, yeast, wheat germ, and milk (Odendaal,
1966). Pronutro is found in the market in packages of
one pound being sold at a price of 15 U.S. cents.

Cooked full fat soy products obtained by the ex-
trusion cooking process give a high nutritive value, good
oxidative stability, and mild flavor. Vitamins and the
heat labile lysine are conserved, and the growth inhibit-
ing factors are destroyed. With the cooperation of UNICEF
this product was chemically tested in Taiwan where full
fat soya flour was formulated with sugar and supplementary
vitamins and fed to children from 4 to 12 months of age.
Reports from these studies have indicated that this pro-
duct supports excellent growth, the gains being comparable
to those obtained in babies fed cow's milk (Mustakas et
al., 1966).

Fish protein concentrates are primarily used in
Peru and Chile. In Peru a group of children ranging in
age from 6 to 41.5 months receiving wheat noodles enriched
with 10% fish protein concentrate showed better growth
and weight response than those on a diet of wheat noodles
without supplementation (Graham et al., 1966).

The University of Chile is also engaged in study-
ing the use of fish protein concentrate for human consump-

tion and particularly for children. In 1964 production of

13

fish flour in Chile reached 400,000 tons, and it was ex-
pected that one million tons would be produced in 1965.
With the cooperation of the UNICEF, a plant is working
with the aim of obtaining odorless fish flour. Bread,
spaghetti, and soup are being enriched with fish protein
concentrate, and good results have been obtained in test
groups (Moncksberg, 1966).

New protein foods from inexpensive products like
the oil seeds can be used to repair the dietary deficien-
cies observed in malnourished people. These products are
being produced as protein beverages and textured products

(Altschul, 1967).

C. AMINO ACID SUPPLEMENTATION

Protein problems can be present in two ways; de-
ficiency of total protein when related to consumption and
deficiency of the protein itself, when referred to its
amino acid composition (Jansen et al., 1964).

The value of the protein in the food is mainly
measured by its amino acid content. The deficiency of the
protein can be overcome by addition of small amounts of
the needed amino acid. According to Jansen (1964), in a
corn eating country, tryptophan and lysine will be missing
in the diet. With diets consisting of casava and potato,

the total protein is low, and methionine will be in

14

shortest supply. In rice eating countries there is a
deficiency in lysine and threonine.

Teff (Eragrostis abysinica) is consumed in Ethiopia.
A typical Ethiopian diet was calculated to furnish 65 grams
of protein, of which 45 grams came from teff. This food
is a good source of vitamins and minerals, including iron,
but is deficient in lysine. The protein quality could be
improved by supplementing with the deficient amino acid
(Jansen, 1962).

Children recovering from protein malnutrition in-
creased their nitrogen retention considerably when they
were given an adequately adjusted diet of wheat protein
supplemented with lysine. Bressani et a1. (1960), and
Barnes et a1. (1961), have observed that nitrogen reten-
tion was improved when lysine and potassium were added to
the diet. Better growth response was observed at protein
levels of 1.75 to 3 grams per kilogram of body weight
with 75 to 100 calories and more than 2 milliequivalents
of potassium.

Bressani et a1. (1958) have observed the import-
ance of the physiological state of children. Children
show varying responses to a given level of protein. For
this study a simplified basal diet supplying two grams
of protein per Kg of body weight was given to infants.

The diet was supplemented by adding the level of a given

amino acid listed in the FAO "reference protein." The

15

addition of either tryptophan or lysine did not give as
good a response as when these two amino acids were added
together. Nitrogen retention was considerably increased
when isoleucine was included.

The nutritive value of polished rice protein was
improved by the addition of two essential amino acids,
lysine and threonine. When these amino acids were used
separately, no change in the nutritive value of the pro—
tein was noticed. Albino rats were used in this experi-
ment (Pecora, 1951).

The addition of 0.5% d1 lysine hydrochloride,~
which corresponds to 0.2% l lysine, to a bread diet in-
creased the average weight of weanling rats from 32% to
75%. The beneficial effect was similar when lysine was
added to the bread before baking or when it was incorpor—
ated into the bread diet. Higher prOportions of lysine
e.g. 0.8% or more, gave a growth response similar to the
stock diet (Rosenberg, 1952).

Corn varieties containing low and high levels of
protein showed a difference in protein quality (Sauberlich
et al., 1953). The low protein variety was deficient in
lysine, tryptophan, isoleucine, threonine and valine,
whereas the high protein variety was deficient only in
lysine and tryptOphan.

In diets where cereal grains are the chief staple

food the deficiency in protein is one of quality rather

16

than quantity. Supplementation with lysine, tryptophan
and threonine will improve the quality of the protein to
such a degree that it can be compared with a milk protein
(Howe et al., 1965). Casava, yams and bananas are diet-
ary components of a large number of populations. Because
of their low level of protein, the incorporation of fish
protein concentrates and properly processed oil seed pro-
tein in the diet would be beneficial, and supplementation
with amino acids will bring the protein level of the food

to a satisfactory level (Howe et al., 1965).

D. MICROORGANISMS IN FOOD PRODUCTION

Yeast is a promising food for solving the problem
of malnutrition. The potential for this food is mainly
because it is rich in protein and vitamins. It can also
be produced from inexpensive waste materials such as
molasses, sulphite waste, corn steep liquor and wood
residues.

Yeast is currently in greater use as a food source
for humans and animals than are algae and bacteria which
have also been considered as potential food sources of
protein.

Yeast is added to Incaparina, the vegetable pro-
tein.mixture manufactured in Central America for the treat-
ment of children's protein malnutrition, to provide the

B complex vitamins (Scrimshaw et al., 1961).

17

According to Peppler (1967), yeast production for
1964 was as follows: USA, 28,900 tons of dried yeast
per year; Europe, 52,400 tons; USSR, 52,000 tons; Formosa
about 13,000 tons per year.

Candida utilis contains more than 50% of protein

 

on a dry weight basis and all the vitamins of the B
group. However, its protein is low in methionine and
cystine and requires supplementation. It could be an
important protein and vitamin source for the depressed
areas of the world (Prescott and Dunn, 1959). Frazier
(1967) reported that food yeast may furnish, in varying
amounts, thiamine, riboflavine, biotin, niacin, panto-
thenic acid, pyridoxine, choline, streptogenin, glutaé
thione and probably folic and p-amino benzoic acids. From
the literature it appears that considerable difficulties
arise in promoting the consumption of food yeast on a
large scale basis._ Its incorporation in fairly low con-
centration in certain prepared foods such as biscuits,
seems to be a good method. The important task would be
to find strains which are not disagreeable to consumers.

China, Japan, and South East Asia produce and con-
sume large amounts of fermented products. Molds are used
for the production of exotic and flavorful foods.

Shoyu production is industrially significant in
Japan. Aspergillus oryzae is the microorganism used in

the preparation of shoyu, a salty, dark brown liquid with

18

a characteristic flavor. It is used as a seasoning agent
for poultry, meat, fish and cooked vegetables. Miso also
constitutes a big Japanese industry. It is produced by

mold and yeast fermentation and is rich in glutamic acid.

The fermentative organisms are Aspergillus oryzae and the

 

yeast Saccharomyces rouxii. Miso is also used as a season-

 

ing agent for meats and vegetables.

Sufu or Chinese cheese is a common food in China
and Formosa. Its texture resembles that of cream cheese
and its flavor is suggestive of anchovies. Soybeans are
the substrate and the enzymes are produced by the mold

Actinomucor elegans which is inoculated on the surface

 

of expressed soybean curds called tofu (which are pre—
viously subjected to heat treatment). Once ripened, sufu
cubes are salted in 5 to 10% brine.

Tempeh, another soybean food, is widely consumed
in Indonesia and is produced by inoculating the beans with

Rhizopus oligospurus. Ontjom is the name given to a fer-

 

mented product made by inoculating peanut presscake with
Neurospora sitoPhila.

Ang-Kak or red rice is an exotic colored food pro-
duced commercially in the Phillippines, Indonesia and
China. The fermenting organisms are spores of Monascus

purpureus.

 

The fermentation of these soybean products not only

imparts variety to the diets, but it also improves the

19

digestibility of soybeans, an excellent source of protein
(Nelson and Richardson, 1967).

A 1:1 mixture of wheat and soybeans supported
growth comparable to that obtained when casein was fed.

Rhizopus oligospurus was used as'a fermenting agent. The

 

P.E.R. of the rats fed fermented wheat was higher than
that of those fed the unfermented wheat. The authors
attribute the improvement in the protein to the mold's
proteolytic enzyme action, increasing the availability of

lysine (Wang et al., 1968).

3. AMINO ACID SUPPLEMENTATION
A. CLASSIFICATION OF AMINO ACIDS AND REQUIREMENTS

Rose (1937) showed that excellent growth was ob-
tained in rats when non-essential amino acids were ex-
cluded from the diet. The essential amino acids were
found to be tryptOphan, lysine, histidine, phenylalanine,
leucine, isoluecine, threonine, methionine, valine, and
arginine. Rose and Rice (1939) found that the removal
of any one of these amino acids except arginine from the
diet, produced a nutritive failure in growing rats. They
also stated that amino acids, not essential for the grow-
ing rat, were also dispensable for an adult dOg. Klose
et al. (1938) have shown that arginine, histidine and

tryptophan were necessary for the chick.

20

Final classification of required amino acids for
rats was presented by Rose and Womack (1948). Rose et
al. (1957), working on the amino acids requirements of
man,demonstrated that 8 were essential. Histidine was
not considered critical for maintenance and growth of
an adult male, but it was required by children. Holt and
Snyderman (1967) and Swendseid and Dunn (1958) concluded
that the amino acids required by men were also required
by women.

Rose and Wixon (1955) have shown that l cystine
is capable of replacing 80 to 89 per cent of the methion-
ine needs of an adult man. These authors pointed out the
importance of this finding for the areas of the world
where several foods have methionine as the limiting amino

acid.

B. CONCEPT OF PROTEIN'S FIRST LIMITING AMINO ACID

The concept of the first limiting amino acid was
due to the work of Mitchell and Block (1946). According
to these authors, "The amino acid limiting the nutritional
value for maintenance and growth of the laboratory rat,
for any particular food protein, would be that amino acid
present in the least amount with reference to whole egg
proteins i.e., that amino acid with the greatest percentage

deficit."

21

Mitchell and Block (1946) have expressed the nutri-
tive value of a protein in terms of a Chemical Score; sub-
tracting from 100 the percentage deficit in the limiting
essential amino acid. The greater this percentage deficit,
the lower the nutritive value of the protein. Thus, the
larger the Chemical Score, the better the protein quality.
These data, in a way, predict the value of the protein.

For supplementary effects, the first limiting
amino acid must be added in such amount to balance with
the second limiting amino acid, and these two amino acids
will be in balance when they cover the requirements of

the organism for protein synthesis (Rosenberg, 1959).

C. AMINO ACID BALANCE AND IMBALANCE

To be well utilized for maintenance and growth of
laboratory animals, all amino acids (essential and non-
essential) must be present in the correct amount and at
the proper time (Geiger, 1950). A balanced protein sup-
plies the required amino acids and has a high biological
value (Harper, 1959). Imbalance of amino acids in a
protein is reflected in reduced animal growth. This ef-
fect is reversed by the addition of the most limiting
amino acid (Flodin, 1953). Pellagra, for example, found
where corn is the predominant food, is due to tryptophan
and lysine deficiencies. It can be corrected by supple-

menting the diet with these amino acids (Jansen, 1964)

22

or the vitamin, niacin (Laguna and Carpenter, 1951).
Sauberlich (1959) reported that the growth of weanling
rats was depressed more than 50% when oxidized casein
(methionine and tryptophan destroyed by H202) was used.
Normal growth was restored by the addition of methionine.

When large amounts of gelatin were added to a low
protein diet, the growth rate declined. The depressed
growth was reversed by the addition of tryptophan (Salmon,
1964).

If amino acids are not supplied in the proper
proportions, they can not be used for protein synthesis.
Addition of excessive amounts of amino acids does not
improve the efficiency of use of the dietary protein.

If the imbalance is large, greater amounts of the most
deficient amino acid will be required to restore normal
growth (Natl. Acad. Sci., 1963).

In protein metabolism a relationship between the
amino acids has been observed. Mitchell and Block (1946)
reported that methionine can be converted to cystine, but
the reverse does not occur. Graw and Almquist (1943)
showed the sparing action of dietary cystine on methion-
ine. The requirement for phenylalanine is decreased by
the presence of tyrosine (Block and Bolling, 1944b).

To evaluate the nutritive value of a protein,

balanced patterns of amino acids, either the whole egg

23

protein or the FAO Provisional Pattern, were used as

references (FAO, 1957).

4. PROTEIN EVALUATION

A. BIOLOGICAL METHODS

The quality of a dietary protein is based on the
amount and kind of its amino acids (Allison, 1959). Pro-
tein requirements are in relation to the needs of the
organism for growth, tissue repair, maintenance, and en-
zyme replacement. A diet adequate for maintenance and
repair may be inadequate for growth (Mitchell, 1947).

Many biological methods have been proposed for
evaluating the nutritive value of a protein. Studies have
been conducted in an attempt to set up a method that could
measure growth and maintenance.

Animal assays have shown that there are two ways
to measure the nutritive value of the protein:

1) Methods measuring the growth of the animal

related to gain or loss in body weight.

2) Methods measuring the utilization of the

nitrogen in terms of absorption and retention.

B. PROTEIN EFFICIENCY RATIO (P.E.R.)

One of the most common methods used for evaluating
the protein quality is the P.E.R., defined as grams of

weight gain per gram of protein consumed. Growing rats

24

are commonly used as the test animals. P.E.R. is‘a simple
method, its efficiency depends on age, the sex of the
rats, time of experimentation, and protein concentration.

Chapman et a1. (1959) have observed that the
younger the rat, the higher were the P.E.R. values. Fe-
male rats gave higher P.E.R. values at lower concentrations
of protein than male rats (Morrison and Campbell, 1960).
As the time of the assay was extended,.P.E.R. values de-
creased in both sexes. Barnes et a1. (1946) showed that
a 10% protein level gave a maximum ratio. Morrison and
Campbell (1960) reported that casein was more efficient
at 7% than at 10% or 15%. These authors also proposed
the incorporation of a control group to eliminate dif-
ferences in P.E.R. values due to the use of different
strains.

The addition of 20% water to a purified diet con-
taining 9% protein significantly increased the P.E.R.
values. The same effects were observed at levels of 6%
and 12% (Keane, 1962).

In view of the usefulness of this technique, Derse
(1960) proposed a standardization of the procedures used
in determining the P.E.R., to allow comparison of results
obtained.

From the literature one can see that P.E.R. values

have been shown to correlate with the quality of the

25

protein. The more balanced the protein the better the

growth response of the animal and vice versa.

C. DIETARY NITROGEN UTILIZATION

In order to obtain a satisfactory and simplified
method for evaluating dietary protein quality, nitrogen
balance methods were subjected to a series of modifica-
tions. Mitchell (1923-4) estimated the biological value

. = Retained nitrogen
of the proteins by the formula B.V. AbsorBed nitrogenX100.

 

Retained nitrogen is equal to absorbed nitrogen minus
urinary nitrogen and absorbed nitrogen is equal to food
nitrogen intake minus fecal nitrogen. Bender and Miller
(1953a) determined the protein value by comparing the
gain in body nitrogen of rats after 10 days on a protein
diet with those on a non-protein diet.

Later (1953b) they measured the water content of
the body instead of determining the nitrogen content. In
1955 they established the Net Protein Utilization (N.P.U.)
as a ratio of retained nitrogen/food nitrogen X 100. Re-
tained nitrogen equals the body nitrogen of the test
group less body nitrogen plus nitrogen consumed by the
non-protein group. Food nitrogen is equal to the nitrogen
consumed by test group. Bender (1956) obtained a close
correlation between the Net Protein Utilization and P.E.R.

values.

26

Bender and Doell (1957) proposed the Net Protein
Ratio, which is defined as the sum of the weight loss of
the non-protein group and weight gain of the test group
divided by the weight of protein consumed by the test
group. Morrison et al. (1963) showed good correlation
between Net Protein Ratio and P.E.R.

True digestibility is defined as the fraction of
dietary nitrogen absorbed into the blood stream (Allison,
1955). Absorbed nitrogen is calculated by the following
equation: A = I - (F-Fm) I = nitrogen intake, F = fecal
nitrogen and Fm = endogenous nitrogen. True digestibil-
ity = A/I. If correction for fecal losses is not made
the value is designated as Apparent Digestibility = I -
F/I. Waterlow and Verity (1960) gave the following
equation: Apparent nitrogen absorption = N intake - fecal
N/N intake X 100.

D. THE MEADOW VOLE (MICROTUS PENNSYLVANICUS) IN
BIOASSAY TECHNIQUE

Mice of the genus Microtus are the most abundant
mammals around the world. They live in temperate and
boreal zones, usually in farming fields. The species

M.pennsylvanicus is one of the most wide spread among

 

the several species. In the United States they can be
found in the Northeastern parts of the country. These
animals are easily captured by means of mouse traps.

When housed in metal containers and fed foods with high

27

water content, they breed and exhibit normal behavior.

Males of M.4pennsylvanicus are sexually mature when they

 

are five weeks old. The females have shown to be most
prolific among mammals with a gestation period of three
weeks.

The voles at birth are pink, hairless, and weigh
1.6 to 3 grams. Litters are composed of five to six
animals. Growth is rapid from one to two days after
birth. While the young mice are still nursing they sup-
plement their milk food with vegetables, living on this
diet until they are weaned. They are weaned before they
reach the age of twelve days when they weigh approximately
twelve grams. Once weaned, they grow rapidly, gaining
about one gram per day (Hamilton, 1941).

Whitmoyer (1956) has observed certain character-

istics of the growth rates of M._pennsylvanicus:

 

a) No statistical differences were observed in
the instantaneous growth rates of males and females in a
one month period.

b) During the first three weeks no distinct
growth periods were observed.

c) While animals within litters were consistent
in their rate of growth, the average rates for the litters
have shown fluctuations during the first three weeks.
Also, significant differences were observed between litters

for short periods.

28

d) Heredity, parental history, size of litter,
degree of disturbance, and month of birth are factors
affecting growth rates.

Meadow voles are used as experimental animals
mainly in studies with forage plants, which are charac-
terized by their low protein and high content of fiber.

Studies done by Elliott (1963) have demonstrated

that the meadow voles (M. pennsylvanicus) can be used as

 

a bioassay organism. Specific growth responses were ob-
tained when the meadow voles were placed on experimental
diets for six days (Elliott, 1963). Schillinger and

Elliott (1966) calculated the specific growth (Gsp) from

the equation:

Ge equals average percent weight gain on the experimental
diet. Gc equals average percent weight gain on control

diet.

5. POTATOES AND BEANS IN NUTRITION

A. POTATOES AS FOOD

Potatoes belong to the family of Solanaceae. They
were first cultivated near lake Titicaca on the Peru-
Bolivian border. This zone of the Andes is 9000 to 13500
feet above sea level (Cox, 1967). Interesting varieties

that are difficult to recognize as potatoes exist in these

29

regions. Some are golden yellow, others are purple, blue,
spotted or striped. The shape varies from round or oblong
to cylindrical and the skin may be smooth (Talburt, 1967).

Potato production is spread over the world.
Potatoes constitute one of the largest cr0ps and are a
valuable protein source for the human diet. An advantage
of this food is its long storage time.

The potato was dried at least 200 years ago. The
natives of the mountainous Andes allowed them to freeze
at night, squeezed the free liquid from the thawed potato
with their bare feet and then sun dried the potatoes.

This operation was repeated until the potatoes were suf-
ficiently dry to keep (Talburt, 1967). This dried pro-
duct is still sold in the markets under the name of "chufio
negro." Another type of dried potato, "chufio blanco," is
also produced. It is dried under straw to prevent blacken-
ing.

At the present, the potato processing industry is
expanding in the U.S.A. In 1940 less than 2% of the total
potato crOp was processed, in 1955, slightly over 15%, in
1962, 25% (Feustel et al., 1964). In 1966 the figure
reached over 41% (Smith and Davis, 1968). Although the
amount of potato chips produced has increased steadily
per year, frozen products are increasing most rapidly.

Frozen products represented over 41% of the processed

volume, dehydrated potatoes (potato granules, shreds and

30

flakes) over 20% and the rest was processed as canned
products, such as soups, stews, hash, etc. (Smith and
Davis, 1968).

Although there are more than 350 varieties of
potatoes, at present only a few are being commercially
grown. About 50 varieties are being cultivated in the
USA, and each variety has its own characteristic yield,
shape, size, resistance to disease and degree of adapta-
tion to climatic conditions. The United States Depart-
ment of Agriculture, as well as various State Experimental
Stations, are conducting breeding programs which have pro-
duced new varieties with greater resistance to disease
and proper conditions for processing (Feustel et al.,
1964).

The consistency of the finished product is an im-
portant factor in determining the quality of processed
.potato products. A potato variety with mealy texture
also possesses a high solids content. Usually the higher
the specific gravity or dry matter content, the more
suitable the potato is for processing (Smith, 1968).
Potato varieties such as Kennebeck, Russet Burbank, Chero-
kee, Haigh, Irish Cobbler, Katahdin, Norgold Russet and
Superior are considered the best ones for processing
(Thompson, 1967).

The white potato makes important contributions

to human nutrition. As an energy source it ranks second

31

only to cereals among the products of the vegetable king-
dom used for human food. The literature on the chemistry
of the potato is extensive and from the data it is diffi-
cult to obtain a clear picture of the raw potato composi-
tion. The proximate composition of potato flakes per 100
grams of edible portion is: water, 5.2 g; protein, 7.2 g;
fat, 0.6 g; carbohydrate, 84 9; fiber, 1.6 g; ash, 3 g;
niacin, 5.4 mg; thiamine, 0.23 mg; and riboflavin 0.06 mg
(USDA, 1963). Other vitamins present in potatoes are
folic acid, panthotenic acid, and pyridoxine; which are
present in relatively higher amounts than in other vege-
tables (USDA, 1957). Mineral content is: calcium, 35 mg;
phOSphorus, 173 mg; iron, 1.7 mg (USDA, 1963). Potatoes
are one of the richest sources of potassium. The fact
that potatoes are a good source of potassium may result
in the efficient metabolism of protein. Barnes et a1.
(1961) have shown that adequate amounts of potassium were
required for the good utilization of protein.

It is well known that the cooking method influ-
ences the vitamin content of potatoes. After processing
by different household methods (baking, cooking in a
saucepan and frying) the content of ascorbic acid, dehydro
ascorbic acid, niacin and thiamine was measured. Thiamine
was 85% retained when potatoes were boiled unpared, with
no reduction of the other vitamins. Seventy to 80% re—

tention of ascorbic acid and somewhat higher retention for

32

the other vitamins was observed when potatoes were pared.
Pared, quartered potatoes cooked in a saucepan retained
about 80% ascorbic acid with no destruction of the other
nutrients. Potatoes baked in their skins kept 70% of

(the thiamine and the other vitamines were not affected.
Frying of the raw product gave a retention of 60% ascor-
bic acid, thiamine being almost completely destroyed (Hews-
ton et al., 1948).

The length of storage was shown to have an effect
on the ascorbic acid content. In a period of seven months,
the ascorbic acid content of raw, fresh mashed and recon-
stituted dehydrated potatoes decreased from 29.3; 18.8;
and 8 mg/lOO grams respectively to 10.6; 6.8; and 2.8
mg/lOO grams (Bring et al., 1963). During the process
of potato flaking, 71 to 73% of the natural and added as-
corbic acid was retained. Flakes containing 5% moisture
and antioxidant retained 70 to 76% of the natural and
added vitamin C. When stored under nitrogen at 75° F for
28 weeks, no vitamin destruction was observed (Cording
et al., 1961). The destruction that occurred during cook-
ing was greater for pyridoxine than for niacin; 4.8 and
8.8% losses of pyridoxine for boiled and baked potatoes
in relation to 1.5 and 4.2% for niacin (Page and Hunning,
1963).

Potatoes have substantially more of all essential

amino acids except histidine than whole wheat. The

33

non-protein nitrogen of boiled potatoes showed appreciable
amounts of lysine and arginine and poor levels of the re-
mainder of the essential amino acids (Hughes, 1958).

In the table of amino acids published by Hopper
(1958) white potato contains more tryptophan, threonine
and methionine than the other vegetable proteins.

From studies done with rats, Mitchell (1924b) re-
ported that at a concentration of 5% protein, the biologi-
cal value of potato was lower than that of casein, corn,
oat, rice or yeast. However, when the level was raised
to 10% protein, the biological value of potato protein
was the highest. Mitchell pointed out that the nitrogen
balance method could have failed to measure adequately
the yield of the potato protein, since it contained sig-
nificant amount of non-protein nitrogen.

The biological value of potato protein (50% boiled
potato, 50% commercial flakes) was compared with the bio-
logical value of protein of ground whole barley. Two
levels of nitrogen were used: 1.5% with no casein and
2.4% with 6% casein. The results showed lower values for
potatoes at both protein levels. The authors stated that
the heat from processing could have damaged the protein
(Hutchinson et al., 1943). Rats fed potato protein gave
higher results than those fed wheat, but its apparent

digestibility was lower (Chick and Cutting, 1943).

34

In a theoretical comparative study, Hegsted (1957)
demonstrated that the protein of potato can easily fur-
nish the required protein for a child with an 800 calorie
intake, as related to corn grits and white bread which
cannot be used as the sole protein source.

Flodin (1953) stated that a combination of potatoes
and cereals, especially corn, makes desirable food mix-
tures because of the level of lysine and tryptophan in
potatoes.

Non-protein nitrogen in potatoes increases more
than protein nitrogen when nitrogen fertilization is in-
creased. However, the nutritive value of protein is not
impaired due to the greater gain in protein nitrogen.

During storage the nutritive value of the tubers
is enhanced due to a decrease in both total solids and
non-protein nitrogen. Protein nitrogen decreases in

sprouting tubers (Pol and Labib, 1963).

B. BEANS AS FOOD

Legumes are generally of high protein content
but their nutritional value has to be improved by ade-
quate heat treatments and by the addition of one or two
amino acids. Legumes also contain high amounts of carbo-
hydrate material and low amounts of fat except for soy—

beans and peanuts.

35

Beans are considered among those foods which can
help to solve the big gap of protein deficiency by in-
creasing the protein supply in those parts of the world
where animal protein is scarce or expensive.

In some places beans are a staple food. Individ-
uals in certain communities in Rwanda and Uganda consume
as much as 400 to 500 grams of beans per day. Beans are
said to be the "poor's meat" (Aykroyd and Doughty, 1964).

Nutritional surveys in the developing countries
have shown that consumption of beans ranges from 10 to
50 grams daily. This food fits well in the budget of
the economically unfavoured people. Beans also have
the great advantage of a long storage period, without
the necessity of refrigeration.

Most of the studies have been made on the bean

varieties of the species Phaseolus vulgaris, since they

 

are most.commonly consumed. Kakade and Evans (1964)
reported the chemical composition of navy beans (Sanilac)
expressed in grams per 100 g of beans to be: Moisture,
8.96; ash, 3.71; crude fiber 4.18; carbohydrate, 58.07;
protein 24.0; fat, 1.02; calcium, 0.15; phosphorus, 0.46
and iron 0.007. The same authors in 1965 reported the
amino acid composition of five varieties of navy beans.
Seaway contained more of the essential amino acids and
more glutamic and aspartic acids than the other varieties,

but methionine was lower in Seaway.

36

Variety and location are factors that influence
the nutritive value of a food. Tandon et a1. (1957)
analyzed 25 varieties of kidney beans, produced in two
different areas in Guatemala, for contents and levels
of nitrogen, methionine, lysine, tryptophan, niacin,
thiamine and riboflavin. The protein content ranged
from 20.1 to 27.9%, with an average of 24.1%. The re-
maining values are listed below:

Methionine from 0.17 to 0.33% average 0.25%

Lysine from 1.69 to 2.41% average 1.98%

Tryptophan from 0.14 to 0.22% average 0.17%

Niacin from 1.68 to 2.95% average 2.22%

Riboflavin from 0.16 to 0.23% average 0.18%
Osborne and Clapp (1907) reported that phaseolin, the
principal protein of navy beans, contained all the essen-
tial amino acids. A starch free product made from white
beans did not support the growth of rats. Lafayette et
a1. (1912) attributed this to the presence of hemicellu-
lose and cellulose in the product. McCollum and Simonds
(1917) found that the protein of navy beans did not pro-
mote rat growth, despite the fact that the vitamin and
mineral content of the diet was adequate. The mortality
rate of the rats was also high. A diet of 70% or 45%
bean meal, supplemented with 9% casein, supported growth.
The 45% level of bean meal was shown to promote greater

growth than the 70% level. McCollum and Simmonds

37

theorized that the gas liberated produced distention of
the digestive tract or that some unknown chemical com-
plex which is harmful to the young rat is present in the
bean meal. The results obtained by Lafayette et a1.
and McCollum and Simmonds were confirmed by Johns and
Fink (1920), who thought that the failure of the raw
protein of navy bean to support growth was due to the
low cystine in the diet. Addition of this amino acid
to the diet resulted in maintenance of weight, but growth
responses were not satisfactory. Johns and Fink (1920)
also observed that heating the navy beans increased the
efficiency of protein utilization and the protein qual-
ity was further enhanced when supplemented with cystine.
Everson and Meckert (1944) compared the nutri-
tive value of raw and heated sources of protein, includ-
ing navy, kidney, and pinto beans. They reported that

the proteins from P. vulgaris were improved the most by

 

the cooking.

Feeding raw beans to rats at a concentration of
15% protein, caused death sooner than feeding a diet of
raw beans at a 10% protein level.

It has long been known that legumes contain sub-
stances toxic to the organism that can be destroyed by
heat. These were found to be trypsin inhibitors and

hemmagglutinins. The trypsin inhibitor blocks the

38

enzyme trypsin present in the digestive tract, and hem-
magglutinins agglutinate the red blood cells (Liener,
1962).

Bowman (1944) noticed that one of the fractions
of navy beans containing trypsin inhibitor retarded the
in vitro digestion of milk casein. Jaffe (1949) reported
that soaking beans prior to autoclaving improved their
nutritive quality. The addition of methionine markedly
improved the biological value of kidney bean protein and
made it comparable to that of casein. In 1950 Jaffe
demonstrated that heated beans had greater nutritive
value and these observations were confirmed by Honavar
et a1. (1962). He also reported that fractions contain-
ing hemmagglutinin factors, free from trypsin inhibitors,
were isolated from black beans and fed to rats in diets
containing 10% casein. The growth was strongly affected
and 0.5% of hemmagglutinin was reported to be a lethal
dose.

Kakade and Evans (1963) found that properly heated
navy bean protein was almost completely digested by tryp-
sin; however, beans autoclaved for prolonged periods were
poorly digested by trypsin. In 1964, they reported that
a diet of raw navy beans supplemented with methionine,
vitamin 812 and antibiotics barely promoted rat growth.
Autoclaved navy beans supplemented with methionine sup-

ported growth to the same extent as a casein diet.

39

The fact that autoclaving destroys the trypsin
inhibitor and hemmagglutinin in navy beans was confirmed
by Kakade and Evans (1965). One hundred percent mortal-
ity occurred when rats were fed navy beans and a very
high mortality was observed when autoclaved beans with
2% and 3% trypsin were fed. The same investigators (1966)
found that soaking and germinating the beans did not
eliminate the toxic factors. They believe that soaking
prior to autoclaving was not necessary since heat alone
destroyed the toxic factors.

Bressani and Valiente (1962) reported that maxi-
mum growth in rats was obtained by feeding a combination
of rice and black beans, with rice supplying 60% of the
protein. Evans and Bandemer (1967) found that a mixture
of navy beans and Gary oats, each supplying 5% protein,
was superior to either source alone, at a level of 10%
protein. They also reported that methionine supplementa-
tion of legume diets improved growth rate.

Powrie and Lamberts (1964) reported that the ap-
parent digestibility of navy beans canned in water was
decreased from 69 to 63% when processing times at 121°C
were increased from 20 to 70 minutes. Inclusion of glu-
cose decreased the digestibility to 47%. Weight gains,

P.E.R. and biological values were also reduced.

4O

METHODS AND MATERIALS

Potato flakes and drum dried navy bean powders
prepared in the Department of Food Science laboratory

were used.

Sample designation. Potatoes: Numerically

 

designated clones 709, 58, 1111-2, 711-3, 711—8, 706-32,

706-34, Russet Burbank and the Solanum tuberosum-S.

 

stoloniferum species hybrids 321-65, 322-6 were used.

 

Potato flakes were processed as described by
Pope (1969). The procedure is given in the appendix.
After processing the potato flakes were powdered in a
micromill (Chemical Rubber Co.).

Beans: Navy beans, Phaseolus vulgaris (Var.

 

Sanilac), which had been atmosphere or retort cooked,
then drum dried, were used. The powders were prepared
as described by Counter (1969) and the procedure is
given in the appendix.

Determination ofgproteins. The protein content
of both the navy bean powders and potato flakes was de-
termined by the Kjeldahl method as described by A.O.A.C.
(1965). The protein content of the potatoes was also
determined using the Biuret procedure (Layne, 1955).
The potato protein extract was prepared as follows: 40
ml centrifuge tubes containing 20 m1 of deionized water
were placed in a 0° F freezer until the freezing point

was almost reached. Two grams of powdered potato flakes

41

were very slowly added to the tube while it was being
constantly agitated with a test tube mixer (Deluxe mixer-
Scientific products) to avoid the formation of lumps.
When the total sample was mixed, the walls of the tubes
were washed down with five ml of cold water and shaking
was continued for another three minutes. The tubes were
again placed in the freezer, cooled almost to the freez-
ing point and then centrifuged for 30 minutes at 2800
r.p.m. in an International model U centrifuge. The clear
supernatant liquid was decanted into labeled test tubes.

Analysis of protein. One ml of the extract solu-

 

tion was transferred to small centrifuge tubes (1/2 x 4
inches). Four ml of Biuret reagent was added and the
tubes were inverted five times to mix thoroughly. Tubes
were allowed to stand 30 minutes at room temperature

(70° F) for color development and then were centrifuged
for 10 minutes at 2600 r.p.m. in a clinical centrifuge,
model C.L. The clear supernatant liquid was transferred
to a Bausch and Lomb Spectronic 20 Cuvette (1/2 x 4 inches)
and absorbance determined at 545 mu. The instrument was
set at 0 absorbance using 1 ml H20 plus 4 ml Biuret re-
agent (E. and M. Chemicals, Brinckmann Instruments, West-
bury, New York). The amount of protein present was deter-
mined from a standard curve or formula (Fig. 3). The

curve was obtained as follows: representative aliquots

(0.2, 0.4, 0.6, 0.8 and 1 ml) of a Standard E.M. protein

42

serum containing 6 g protein/100 ml were transferred to
the cuvettes and made up to 1 ml with distilled water.
Four m1 of Biuret reagent were added and the color devel-
opment and absorbance measurement were the same as de-
scribed above.

A straight line relationship was obtained (Fig.
3, appendix), which gave a factor of 15.69 (concentration
of protein mg per ml/absorbance). The protein concentra—

tion of the samples was determined by the formula:

 

Sample absorbance X factor X 1001 X l2 X 100
weight of sample 1000

l -- sample diluted to 25 x 4

2 —- conversion of mg to grams.

Amino acid analysis. Amino acid determinations
were made using a Beckmann amino acid analyzer, model
120 C.

The samples were prepared for amino acid determin-
ation as follows:

1. 10 mg bean powder or 30 mg potato powder was
weighed into 10 ml ampoules, 6 m1 6 N HCl was carefully
added and the contents thoroughly mixed.

2. The ampoules were placed in a dry ice ethanol
bath and evacuated with a vacuum pump until they froze.

3. They were removed, allowed to slowly melt

and examined for the presence of gas.

43

4. If free of gas the ampoules were refrozen in
the dry ice-ethanol bath under vacuum and sealed with a
gas fl ane .

5. The sealed ampoules were transferred to an
oven at 110°C and held for 22 hours to hydrolyze the
proteins.

6. The ampoules were then opened and 1 ml of
norleucine solution containing 2.5 micromoles was added.
The addition of a known amount of norleucine permits
correction for losses in transfer.

7. The hydrolyzate was transferred to a 25 ml
pear shaped flask and evaporated to dryness on a rotatory
evaporator.

8. The residue was dissolved with 0.5 ml of
deionized water and redried.

9. The residue was dissolved in dilutor buffer
(pH 2.2) as described in the manual for the Beckmann
amino acid analyzer, model 120 C, transferred to a 5 ml
volumetric flask and made to volume with buffer.

10. From this sample an aliquot of 0.2 ml for
basic amino acids and 0.3 ml for acidic and neutral amino
acids was transferred to the amino acid analyzer for
analysis.

Since the amino acid analysis has been made on

acid hydrolyzates, tryptophan was not determined.

44

Protein evaluation by animal assays. Two bio-

 

assay techniques were used, namely growth methods, ex-
pressed as Protein Efficiency Ratio (P.E.R.) and gain

in body weight. The absorption of nitrogen was measured
by apparent absorbability, which does not consider en-
dogenous nitrogen. Litters of weanling voles, Microtus

pennsylvanicus, were used. These were obtained from

 

the Department of Crop and Soil Sciences Colony at Michi-
gan State University. The voles were weaned at two weeks
and only those attaining a weight of 11 grams were used.
The voles were assigned randomly to individually dispos-
able plastic laboratory cages equipped with a vole feeder
(Shenk and Elliott, 1969) as described in the appendix.
The voles were fed semi-synthetic diets (starter) for

two days before they were given the experimental diet.
Five voles were used for each diet. The experimental
diet was fed for six days. The voles were weighed every
two days, four weighings in all. Both the water and

food were supplied ad libitum. The food consumption
values were determined by the loss in weight of the en-
tire feeder. The feces were collected at the end of the
experiment from the sheets of filter paper placed under-
neath the wire mesh, dried at 105° F for 22 to 24 hours,
weighed and ground for total nitrogen determination using

the microkjeldahl technique (A.O.A.C., 1965).

4S

Composition of diets. The composition of the con—

 

trol, bean powder and potato powder diets is given in
Table VII (appendix). "Vitamin free casein" was used as
the protein source in the control diet. The diets were
mixed with the amount of water necessary to obtain a
dough like consistency, then molded into wafers of ap-
proximately 1/2 x 2 x 6 inches that would fit into the
vole feeder (Shenk and Elliott, 1969). They were dried
at 105° F for 48 hours, wrapped in aluminum foil and
stored at 0° F until needed. They were thawed at room
temperature for 16 to 18 hours before weighing and feed-
ing. The moisture content of the wafered diets at the
time of feeding was determined using the vacuum oven
method (A.O.A.C., 1965) and values obtained are given

in Table I.

In the first experiment, two processed samples
of navy beans and six samples of potatoes were used.
These included atmosphere cooked navy bean powder and
powders prepared from Russet Burbank potato, potato 58,
potato 321-65, potato 322-6, potato 711-3, and potato
709. These powders were incorporated into the diets so
as to provide 5.28% protein.

In the second experiment, atmosphere cooked navy
bean powder and potato powders 58, 321-65, 322-6, 711-3,
were used. These diets also had 5.28% protein and were

supplemented with methionine. Methionine was added to

46

Table l.-—Moisture content of samples.

 

 

As Experiment Experiment
processed I II
9/100 9

Control casein 6.00* 16.25 15.31
Atm. cooked bean 5.32 19.85 16.85
Retot cooked bean 6.88 18.45 -----
Potato R. Burbank 8.98 21.19 -----
Potato 58 6.52 12.51 32.42
Potato 321-65 6.10 20.45 28.98
Potato 322-6 6.42 21.78 35.92
Potato 711-3 4.82 17.48 27.30
Potato 709 6.38 23.56 -----

 

*Data obtained from Nutritional Biochemical

Corporation.

each 100 grams of atmosphere cooked bean diet at a level

of 0.119 and 0.066g of methionine was added to each 100

grams of potato powder diet. Sodium propionate at 0.3%

level was added to these diets as a fungicide.

The Chemical Score (C. S.) method (Mitchell and

Block (1946) was used to determine the amount of methion-

ine required where:

47

1. The amino acid content of the sample was ex—
pressed as the per cent of the same amino acid in whole
egg protein.

2. 100 - percent found = per cent deficit.

3. 100 - per cent deficit = Chemical Score.

For example, in atmosphere cooked navy bean pow-
der, the methionine content of 0.57g/100 g protein was
14 per cent of that of whole egg protein. Therefore,
the Chemical Score from the above formula is 14.

To determine the amount of methionine to add as
a diet supplement, it is necessary to determine the
Chemical Score of the second limiting amino acid; in both
navy bean powder and potato flakes, this amino acid was
isoleucine (Table II). The Chemical Score for isoleucine
in bean powder was 66. The methionine required was cal-

culated as follows:

C.S. isoleucine x g/100 g protein methionine
C.S. methionine

 

= 66 $40'57 = 2.68 g/100 g protein

 

The diet used contained 5.28 g protein/100 9. Since there
was 0.57 g methionine present, 2.68 - 0.57 = 2.11 g meth-
ionine/100 g protein will be required and the amount of

methionine to add to the diet will be:

2.11 x 5.28/100 g diet.

48

For the potato flake diets, the methionine re-
quired to supplement the diets was determined using the
average Chemical Score of the six varieties. Values
for methionine and isoleucine were 27 and 57.3, respec-

tively.

49

 

 

 

 

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oo OO OO OO so OR OO oO N.O oaHoooH
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moo maog3 mo
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50

RESULTS AND DISCUSSION

PROTEIN CONTENT OF BEAN AND POTATO POWDERS

The total protein content of the bean and potato
powders is given in Table III. The protein content of
the atmosphere and retort cooked bean powders did not
differ significantly and averaged 23.4 g per 100 g. The
protein content of the potato powders determined by the
Kjeldahl procedure ranged from 5.9 to 9.1 g per 100 g, and
by the Biuret method from 5.5 to 9.3 g per 100 g. The
differences in protein content obtained by the two methods
were not significant and indicated that the Biuret method
could be used as a more rapid procedure for determining
the protein content. The ten values obtained for each
sample of potato were tabulated and the degree of rela-
tionship among them was estimated by statistical para-
meters (standard deviation and standard error) and these
values are reported in the appendix (Table VIII).

The extraction of the potato flake powder with
ice cold water and centrifugation of the extract after re-
action with the Biuret reagent eliminated the cloudiness
caused by the presence of starch in the solution.

The amino acid composition of the two samples of
navy beans and the six varieties of potatoes was calculated
following instructions given in section 8 of the manual

for the Beckman amino acid analyzer, model 120 C. The

51

Table III.-—Protein content of bean powder and potato

 

 

 

flakes.
KJELDAHL BIURET
SAMPLE METHODl METHOD2
g/lOOg3 g/lOOg4
NAVY BEANS
Atmosphere cooked 23.6 --
Retort cooked 23.2 -—
POTATOES
321—65 5.9 5.5
Russet Burbank 6.9 6.7
322-6 6.7 6.8
58 7.7 7.9
709 7.6 8.0
711-3 8.7 8.2
711-8 7.9 8.3
706-34 7.9 8.4
706-32 8.3 8.7
1111-2 9.1 9.3
1Mean value of two determinations.
2Mean value of ten determinations.
3Protein(N x 6.25).
4

Total protein.

52

calculated results are reported in g/ng nitrogen (appen-
dix Table IX).

No meaningful differences were found in the amino
acid content of the bean powders, indicating that the
amino acids were not altered by the method of cooking.

The various strains of potatoes analysed vary
considerably in amino acid content. The lysine, threon-
ine, and tyrosine content of strains 321-65 and 322-6 was
higher than that of whole egg. Histidine, except for
strain 321—65, was at Optimum levels and leucine, phenyla-
lanine, arginine, isoleucine, and methionine levels were
all lower than those of whole egg.

If the potato strains are arranged according to
decreases in the number of essential amino acids (plus
tyrosine, a non-essential) that were below the level found
in whole egg, the following order is obtained: 58 > 709 >
Russet Burbank > 711—3 > 321-65 > 322-6. None of the
amino acids of strain 58 were as high as those in whole
egg.

Comparison of the data obtained in this study with
the amino acid composition pattern for potatoes published
by HOpper (1958) shows several differences. Lysine,
threonine, methionine, leucine and phenylalanine were
lower in this study than reported by Hopper and isoleu-
cine was present in higher proportion. Levels of the rest

of the essential amino acids were similar. The amino acids

53

methionine, isoleucine, valine and tyrosine were present
in lower amounts in bean powder than in whole egg. Amino
acid values obtained, except for methionine, were higher
than those previously reported by Kakade and Evans (1965)
for the Sanilac variety.

Tandon et a1. (1957) have shown that soil conditions
influence the nutritive value of food. This could explain
the differences between the results obtained in this study
and those reported in the literature.

Aspartic and glutamic acids were present in con-
siderably higher quantities than the essential amino acids
in both navy beans and potatoes. They ranged from 12.2
to 26.4 and 13.4 to 23.0 g/100 g N in beans and potatoes
respectively. These two dispensable amino acids were
markedly greater in potato 58.

Chemical Score. The chemical scores of the dif-

 

ferent amino acids are given in Table III. In both bean
and potato powders the lowest chemical score was found
for methionine, being 14 for beans and 19 to 31 for
potatoes. In the bean powder, in addition to methionine,
isoleucine, valine, and tyrosine also had low scores.

The chemical score of potatoes can be arranged in an as-
cending order as follows: 321-65 < 322-6 < R. Burbank <
58 < 709 < 711-3. This shows that potato 711-3 has the
highest chemical score.for the amino acids. If the

Mitchell and Block (1946) chemical score is used as a

54

means of predicting the efficiency of the protein utili-
zation for growth it may be said that potato powders
could be better protein sources than bean powders.

Bio-assay using meadow voles. The food consump-
tion of voles was reported on a dry matter basis. The
results of both experiments are expressed as gain in
body weight, and Protein Efficiency Ratio (P.E.R.). The
Protein Efficiency Ratio is calculated as the ratio of
weight gain/protein intake.

The digestive efficiency of the proteins was
calculated as the ratio of nitrogen utilized to the
nitrogen intake and calculated by the following formula:

N. Intake - Fecal N

N. Intake X 100

 

Apparent absorbability =

In the first experiment with the non-supplemented
protein diet, the voles all gained weight. The weight
gain ranged from 0.59 to 1.39, with potato 58 giving the
highest weight gain, and was greater than that of casein
diet (4.77% protein). These results (Table IV, Fig. l,
appendix Table X) indicated that with a protein concen-
tration of 5.28% the essential amino acid levels were
high enough and sufficiently balanced to support vole
growth. The presence of high levels of glutamic and as-
partic acids may have contributed to the increased effic-

iency of protein utilization. However, the P.E.R. values

55

 

 

 

 

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comm
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56

 

 

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pommsm nuouom .maud

 

.muowc Umucofioammsmlcoc wm cosmoOMHsmflm Honoumflumum

.ooooHuoousu.>H oHooa

57

(Table IV, Fig. 2) of the vegetable protein diets with
5.28% protein content were all lower than that of the case-
in diet (4.77% protein).

Food intakes were higher when potatoes 58 and 322-6
were fed, and this decreased their P.E.R. values.

Lowest weight gains and P.E.R. values were ob-
tained with retort cooked bean powder, R. Burbank and
potato 709.

Although the chemical scores of all essential amino
acids of potato 58 were below 100, the higher weight gain
indicated that they were in better balance than in the
other samples. The lower weight gain with R. Burbank
could be attributed to the low intake of food. The aver-
age food consumed was 10.09 g in 6 days which was one of
the lowest among the nine groups (Table IV).

The lower weight gain with diet 709 may have been
due in part to an imbalance of the essential amino acids.
The lower weight gain obtained with retort cooked bean
powder may have been due to the method of cooking.

In the second experiment, the vole weight gain on
the supplemented diets with the exception of those on the
potato 58 diet, were greater than those obtained on the
non-supplemented diets. The greatest vole weight gains
were obtained with diets using atmosphere cooked beans and
potatoes 322-6 and 711-3. The range of the weight gain

was from 1.5 to 2.6 g, as compared to 1.6 g for the casein

58

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fl

 

 

 

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on-supplemented Protein
WI Supplemented Protein
Protein 4.77%
r——1

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60

diet (Table IV, Fig. l). The P.E.R. values were also
greater with the supplemented diets; they ranged from
1.27 to 2.66 as compared to 1.68 for casein (Table IV,
Fig. 2). Potato 322-6 and 58 diets were consumed by the
voles in greater amounts than other potato or bean diets.

No significant differences were found in the
weight gains on the different supplemented diets (Table
XI, appendix). Since feeding trials demonstrated nutri-
tional efficiency, the six comparable diets of the two
experiments were statistically analysed using the ortho-
gonal contrast method (Mendenhall, 1968) (Table XII and
XIII, appendix). The results showed that the weight
gains on diets of the supplemented, atmosphere cooked
navy beans and potatoes 321-65, 322-6 and 711-3 were
significantly greater than those of the non-supplemented
diets.

Digestibility is considered to be a good index
in evaluating the nutritive value of proteins. In the
unsupplemented diets the apparent absorbability of the
potato proteins ranged from 55.34 to 63.82% (711-3 < R.
Burbank < 709 < 321-65 < 322-6 < 58) and that of casein
was 64.52%. Atmosphere cooked navy bean powder had
62.50% absorbability compared to 57.28% for retort cooked
bean powder (Table V). The greatest vole weight gains

were observed when diets of potatoes 58 and 322-6,

61

atmosphere cooked navy beans and potato 321-65 were fed,
which correlates with the values found for nitrogen ab-
sorbability.

In the methionine supplemented diets the apparent
absorbability of the protein was increased and ranged
from 69.5 to 88.6%. Apparent absorbability increased in
potatoes as follows: 58 < 321-65 < 711-3 < 322-6. Only
potato 58 (69.5%) and atmOSphere cooked bean powder
(66.3%) were lower than the control casein diet (77.5%).
The greatest increase in absorbability occurred for the
potato 711-3 and 322-6 diets (Table VI).

The results of these studies are in agreement
with the statement of Flodin (1953) that the nutritive
value of proteins can be doubled if proper amounts of
the deficient amino acids are added to the protein. The
addition of methionine to atmosphere cooked navy beans,
potatoes 322-6 and 711-3 markedly enhanced their nutritive
value.

Keane (1962) reported that the addition of 20%
water to the diet improved the P.E.R. values at protein
levels of 6% and 12%. Since sufficient water was added
to both diets in this study, the improvement in the non-
supplemented diet can not be attributed to added water.

Both navy bean powders were similar in their
amino acid composition. However, the results from feed-

ing experiments have shown that the protein of retort

62

 

 

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63

 

 

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ucmnmmmd Hooch u: . a cmmonuwz c m

 

.Acflmuonm @mucmEoHQQva cmmouuflc mo muflaflnmnu0m3¢|:.H> magma

64

cooked navy beans was impaired. The growth failure as
compared to the value for atmosphere cooked beans could

be attributed to the influence of heat while processing.
Everson and Mackert (1944), Patton (1948), Jaffe (1950),
Honavar (1962) and Kakade and Evans (1965) have shown

that heat is beneficial in the destruction of anti-
nutritional factors but over-heating of the food can be
deleterious. Destruction of amino acids was attributed

to non-enzymatic browning reactions, in which free amino
acids interact with reducing sugars. Amino acids having a
functional nitrogen which is not attached in a polypeptide
linkage are especially prone to destruction by non-enzy-
matic browning (Patton, 1948).

Since beans and potatoes were subjected to heat
treatment while processing (Counter, 1969 and Hope, 1969),
the intensity of heat applied may have reduced the nutri-
tive value of the proteins as, for example, in the case
of retort cooked beans. Kakade and Evans (1963) observed
that prolonged autoclaving of navy beans decreased the
amount of lysine and methionine.

Rats fed autoclaved navy beans supplemented with
methionine responded as well as those fed on 10% milk
protein (Kakade and Evans, 1964). This study using voles

(Microtus pennsylvanicus) as bioassay organisms has shown

 

that supplementation of processed navy beans with methion-

ine doubled the nutritive value, as compared to the

65

non-supplemented beans. This fact shows the sensitivity
of the animal's response to the quality of the protein
fed. Furthermore, it seems that meadow voles are sensi-
tive to small differences in the concentration of the
proteins in the diets, since different results were ob-
tained with casein at 4.77 and 5.28%. Growth and nitro-
gen absorption were improved at 5.28%.

This study has shown that the protein quality of
potato and navy bean powders was improved when methion-
ine was added to the diets. Protein quality was assessed
by growth methods (weight gain and P.E.R.) and by appar-

ent nitrogen absorption.

66

SUMMARY AND CONCLUSIONS

The protein content of navy bean powders produced
from beans cooked in the atmosphere and in a retort was
found to average 23.4 9/1009. The protein content of
powders prepared from a number of potato strains ranged
from 5.5 to 9.39/1009 as determined by the Biuret method
and from 5.9 to 9.1 by the Kjeldahl method. The protein
contents determined by the Kjeldahl and Biuret procedures
were not significantly different, indicating that the
Biuret method could be used for the determination of
potato protein.

The amino acid analyses showed considerable varia-
tion in the levels of amino acids in the various potato
strains. However, in every case, methionine was the
low level amino acid. This was also true for the bean
powder. Glutamic and aspartic acids were present in
greater amounts than the essential amino acids in both
bean and potato powders. Potato 58 contained more of
these two dispensable amino acids than the other strains.

Chemical scores were successfully used in the
studies with Meadow voles, whose amino acid requirements
are not known.

In the non-supplemented plant protein diet con-
taining 5.28% protein, the amino acid content of potato
was better than that of beans and supported growth more

efficiently than beans. The weight gains on potato diets

67

ranged from 0.50 g (R. Burbank) to 1.30 g (potato 58).
The vole weight gain on the casein (4.77%) and atmosphere
cooked bean diets averaged 1.10 g and for retort cooked
beans, 0.70 9. Milk protein showed a higher P.E.R. (1.51)
than vegetable proteins, which ranged from 0.76 (R. Bur-
bank) to 1.25 (atmosphere cooked beans).

Potatoes and beans at a 5.28% level of protein
gave better weight gains when fortified with methionine,
indicating that methionine was the first limiting amino
acid in the atmosphere cooked navy beans and in potato
clones 322-6, 711-3 and 321-65. With potato 58, methion-
ine supplementation did not significantly improve the
growth rate, indicating that methionine was near the
optimum level and additional methionine may have disturbed
its nutritional balance.

Chemical analyses of the atmosphere and retort
cooked beans showed no meaningful differences in their
proteins. However, feeding trials showed that the nutri-
tive value of retort cooked beans was impaired, indicat-
ing the retort or high cooking temperatures decreased the
nutritional value. The low nutritive value observed with
the Russet Burbank potato could be attributed to the
small amount of food consumed on this diet. With potato
709, an imbalanced protein could have caused the low

growth response.

68

Digestibility studies have demonstrated that the
addition of methionine increased the nitrogen absorption
with all diets.

The use of Meadow voles in nutritional studies
merits attention due to some advantages of voles over
other experimental animals. Voles are: a) inexpensive,
b) easily obtained, c) grow rapidly and d) show a sensi-
tive response to different diets at low levels of pro-
tein. These could be factors of primary importance in

biological research in developing countries.

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APPENDIX

 

82

    

 

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84

VITAMIN DIET FORTIFICATION MIXTURE IN DEXTROSE

 

(Grams/100 lbs. diet)

Vitamin A concentrate (200.000 units per gram)
Vitamin D concentrate (400.000 units per gram)
Alpha Tocopherol

Arcorbic Acid

Inositol

Choline chloride

Menadione (K)

P -aminobenzoic acid

Niacin

Riboflavin

Pyridoxine Hydrochloride

Thiamine Hydrochloride

Calcium pantothenate

mgms/100 lbs.

Biotin
Folic acid
Vitamin B12

COMPOSITION OF SALT "W"

 

Calcium carbonate

Copper sulfate (5H20)
Ferric phosphate

Manganous sulfate (anhyd)
Magnesum sulfate (anhyd)
Potassium aluminum sulfate
Potassium chloride
Potassium dihydrogen phosphate
Potassium Iodide

Sodium chloride

Sodium Fluoride

Tricalcium phosphate

.5
..rg
w.

\l
wl-‘I-‘l-‘abU'lemUlUlOob

OOOOU‘IONOOOONUI
U1

diet

90
1.35

21.000%
0.039%
1.470%
0.020%
9.000%
0.009%

12.000%

31.000%
0.005%

10.500%
0.057%

14.900%

85

 

 

 

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86

 

 

 

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87

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88

 

 

 

 

 

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89

 

 

 

 

 

 

 

 

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90

 

 

 

 

 

 

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91

 

Diet. voter

Fig. 2. Side view
of the vole feeder.

 

Vole feeder #7 is shown in fhe pfosfic nesf.
The design features are seen in feeder #2. The die!
cufler used to make the wafer is in fhe foreground.

Folding (tend

 

 

010th hinge

Fig. 4.--A diet feeder for weanling meadow voles (Microtus
pennsylvanicus.) John S. Shenk and Fred C.
Elliot (1969)

92

PROCESSING PROCEDURES FOR POTATO FLAKES AND

NAVY BEAN POWDERS.

POTATO FLAKE:

l.

2.

A 14 to 84 pound sample was taken randomly
from storage for processing.

The condition of the tubers was noted, they
were washed, preheated at 170°F for two
minutes in water.

Potatoes were sliced into 3/8 inch slices,
drained, weighed and returned to the $02
solution.

Slices were washed with running water for
1-2 minutes and precooked for 20 minutes
in 165°F water.

The slices were cooled to 70°F - 80°F in a
cold water bath, taken out and allowed to
stand for 20 minutes before transferring
them to a retort for steaming at 212°F,
for 30 minutes.

Ricing was done using a Kitchen Aid model
K-Sa mixer with the coarse rotatory grater
attachment. Additives or diluting water were
added at this point. Samples were removed
for solids analysis before additives were
incorporated.

 

The mash was dried on an Overton Machine
Company model P-36 double drum dryer, 12

Ihch diameter by 19 1/8 inch length. One
drum was used as an applicator roll, the
other as a drying roll. Simulation of one to
five applicator rolls is possible by a doctor
blade control. Conditions normally used were
8 rpm, 4 layers, uncooled applicator roll

and 85-90 psi steam pressure.

 

 

The sheets were placed in a polyethylene bag.
At the end of processing the sheets were re-
duced to flakes using a rotary slicer with 3/8
inch spacing between blades.

93

BEANS. METHODS OF COOKING.
Two types of cooking process were used:

1. Atmosphere cooked: Beans were soaked and
cooked in water at 212°F for 90 minutes.

2. Retort cooked: Beans were soaked in water
at 210°F for 45 minutes. Transferred to a wire basket

and retorted in steam at 220°F for 30 minutes.

They were dried in a drum drier (described above)
under controlled conditions of 85 psi, 23 1/3 rpm and 4
layers. After drying the sheets were powdered with a
Fithatrick comminuting mill using an 0.125 inch screen.
At this stage they were placed into No. 2 cans (307 x 409)

and sealed hermetically for storing.

MICHIGAN STAT NIVE

 

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