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A QUANTHATWE STUDY OF

PHYTIC ACID m FIELD BEANS _

(PHASEOLUS VULGARIS L.)

- Thesis for the Degree of M. 3 

MICHIGAN STATE UNIVERSITY
CLAIRE VEHOHH THOMAS
.   '1974 * - ~

 

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ABSTRACT

A QUANTITATIVE STUDY or
Ernie ACID IN FIELD BEANS
( BLASEOLUS VULGARIS L.)
By

Claire Verona Thomas

thtic acid is a phosphorus storage compound present in legume
seeds, cereal grains, and tubers. If present in an animal's diet in
high enough concentration it can cause mineral deficiencies by chelating
the metal ions.

This study examined the level of phytate present in many different

varieties of P. vulgaris, using a hydrochloric acid extraction procedure,

 

followed by prajgitation of the phytate with ferric chloride.

The phytic acid content of commercially canned beans was found to
be considerably lower than the raw bean of comparable type. Apparently
the high pressure and temperature of the canning process can degrade the
phytate. A cooking test was performed to determine whether boiling
could also degrade the phytate. Some decrease in phytate was observed,
but not of the magnitude observed in the canned product.

Since phytate can bind metal ions, and has been specifically observed
to cause zinc deficiency in animals, the possibility that it might be
involved with zinc deficiency in plants was examined. The amount of

phytate in the roots of several varieties of P. vulgaris was examined.

 

Some of the varieties tested were tolerant of low zinc, some were sensi-

tive to it. The amount of phytate present was effectively zero, implying

Claire Verona Thomas

that it is probably not the factor involved in zinc deficiency in beans.

Material was examined from plots with normal or with low zinc
supplies in the soil. No significant differences were found in phytate
content at the two locations.

Material from two experiments was assayed to examine the influence
of environment upon phytate content, and also to observe varietal levels
of phytic acid. The first experiment showed significant variety and
environment effects, but insignificant interaction. The second showed
significant effects for varieties, environments, and interaction.

The correlation between % protein and % phytate was determined to

be only 0.352h.

A QUANTITATIVE STUDY OF
FHYTIC ACID IN FIELD BEANS

(Russows VULGARIS L.)

 

By

Claire Verona Thomas

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Science

l97h

~’ “ AcmowLBDcrmrrrs

I wish to extend my sincere appreciation and fondest regards to my
adviser Dr. M. w. Adams, without whose help and guidance I would never
have been able to complete this work. Also Dr. Fred Elliott has given
me much invaluable advice and direction: I would also like to extend
my appreciation to Dr. Fakdoni for helping me establish an extraction
procedure; to Dr. Knezek and Dr. Ellis for the use of their laboratory
facilities as well as their advice; and to Dr. R. J. Evans for the use
of his and Dr. Adam's data about protein quantities of some of the

material that I examined.

ii

1‘

.u-I

 

 

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . .

I'dATERIjXLS AIVTD )fLETHODS o o o o o o

RESIHJTS O O O O O O O O O O O O O O 0

CONCLUSIONS . . . . . . . . . . . .

BIBLIUK‘IPLA‘oB-{Y O O O O 0 O O O O O O

Ihytate Extraction Procedure . . . . . . .
Cooking Trials . . . . . . . . . . . . .
Effect of Normal vs Low Zinc in the Field
On thtate Concentration in the Seed . . . . . .
Ihytate in the Root. . . . . . . . . . . . . . . . . .
Phytic Acid Content of Assorted Lines . . . . . .
Ihytate Content of Lines Selected on the Basis of
Protein Content . . . . . . . . . . . . . . . .
Environmental and Varietal Effects Upon Phytate Content

Cooking Trials . . . . . . . . . . . . .
High vs Low Zinc . . . . . . . . . . . . . .
Phytate in the Root . . . . . . . . . . . . . . . . .
thtic Acid Content of Assorted Lines . . . . . . . .
Phytate Content of Protein Selections . . . . . . .
Environmental and Varietal Effects Upon Ihytate Content

iii

10
ll

12
12

13
lh

1h
lb.
18
l8
18
18

27
29

Table

10.

LIST OF TABLES

Changes in Ihytate Concentration During Boiling
of Sanilac Navy Beans. . . . . .

High and Low Zinc . . . . . .

Analysis of Variance of % Phytate in Five Lines
Under High and Low Zinc . . . . .

Ihytate Content of Assorted Lines . . .

Protein and Phytate Content of Selected Lines

Analysis of Variance of Material From the
Partial Growth Analysis . .

Comparison of Ihytate Content of E. vulgaris Varieties
From the Partial Growth Analysis.

Analysis of Variance of §.'vulgaris Varieties.
(Silvera, 197A) . . . . . . . . .

Comparison of Phytate Content of I} vulgaris Varieties.

(Silvera, 197M) . . . .

Average % Phytate for Each Variety at Each Location.
(Silvera, 197A) . . . . . . .

iv

Page

l6

l7

l7
19

20

2h

2h

25

25

26

LIST OF FIGURES

Page

Changes in phytate concentration of beans and broth
during first cooking test . . . . . . . . . . . . . . . . . 1h

Changes in phytate concentration of beans and broth
during second cooking test . . . . . . . . . . . . . . . . 15

Relationship between % protein and % phytate .

INTRODUCTION

Leguminous seeds contain the highest average percent protein of

virtually any vegetable product. Bean seeds (Fhaseolus vulgaris L.)

 

contain approximately 2h% protein, compared to 7-8% for rice, 8-lO% for
wheat, and appr. 10% for corn. We know that the sulphur-containing

amino acids act as nutritionally limiting factors in utilization of this
protein. However, even after supplementation with these amino acids, the
results obtained with legumes in animal feeding tests are still not as
good as expected. Autoclaving and supplementation together give a
protein capable of a high rate of utilization. (Kakade and Evans, 1965).
What is in the raw seed that limits the ability of the animal to extract
from it the nutritional value we believe ought to be there?

There has been considerable research done with certain inhibitory
substances. As described by Liener (1973), trypsin inhibitors are
proteins that interfere with the activity of trypsin in some animals.
However, their importance in human nutrition may not be very great since
at least some of the ones that have been studied do not affect human
trypsin. Hemagglutinins are glycoproteins that in some legumes, including
soybeans and Phaseolus beans, cause at least part of the growth depress-

ion observed after consumption of raw beans. However, these are usually

destroyed by

(1‘

dequate heat treatment, so probably do not have any signif-
icant effect in a cooked product. There is a possibility that some of the
same factors that are believed to contribute to flatus may also be

involved in depressing the nutritional value. The problem of flatus has

1

PO

not been very well explained, but it is probably due in part to certain
oligosaccharides that humans cannot digest, and quite likely also
influenced by the rather high percentages of indigestible starches and

proteins that pass through to the lower gut where the microflora do

(D
J

digest them and release gas in th .rocess. At this point they may also

ro-

release toxic degradation products that act as growth depressants.

There is at least one other anti-metabolic factor present in seeds
of E. vulnaris L. and also in cereal grains that could contribute to the
diminished nutritive value of legumes. This is phytic acid. I have
undertaken to compare the level of phytate in different bean varieties
grown at different locations, to determine whether there may be potential
for genetic modification of the amount present in commercially produced
3. vulgaris L. varieties. I also examined the degree of stability under

ordinary cooking methods.

LITERATURE REV IEW

Phytic Acid

myo-inositol 1,2,3,h,5,6-Hexakis
(dihydrogen phosphate)

 

Because of the presence of the hydroxyl groups on phytic acid, it
possesses the capacity to chelate metal ions. The quantification tech-
nique that I used made use of the fact that ferric phytate is insoluble
in 0.6% hydrochloric acid. One mole of phytate theoretically can
chelate h moles of the trivalent ferric ion, Fe+++. Wheeler and Ferrel
(1971) showed that in actual tests the ratio does indeed turn out to be
h Fe: 6 P.

In forming the ferric-phytate precipitate‘by treatment of phytic
acid with FeCl3, it is most logical to assume that the flocculent prec-
ipitate is formed by linkage between different phytate molecules through
the Fe+++. This overcomes the steric hindrances involved in trying to
chelate h Fe+++ ions with one phytate molecule. De Lange 33.31. (1961)

claimed that extraction with trichloroacetic acid gave a molar ratio in

the precipitate of 3 Fe: 6 P. However other workers do not confirm their

results. (Oberleas, 1969; Makower, 1970; de Lange gt_§l., 1961).

One mole of phytate should be able to bind six moles of a divalent

ion. Phytate does indeed complex such metals; according to Maddaiah

gt 31. (l96h) the stability of the phytate-metal complexes are in the
order: Zn++> Cu++) Co++)»Mn++)>Ca++. However VOhra gt_§1, (1965) reported
that when sodium phytate is titrated at pH 7.h against metal ions, the
order is: Cu++> Zn++> Co++) Im++> Fe+++> Ca++. This order is changed
somewhat under different pH conditions.

Phytate is very commonly found as its calcium-magnesium salt,
phytin, which is quite insoluble. However, O'Dell gt El. (1972) claim
that the high phytin concentration often reported for corn is impossible
because most corn phytate is present in the germ, and the germ has a very
low Ca++ concentration.

The binding of zinc by phytate is enhanced even further, both lfl.!i££2
and 13 yiyg, by the inclusion of calcium. (Oberleas gt_gl., 1966; Saio
st. §°a 1967).

Phytate in the seed is found in association with protein, and
initially it was thought that the entire complex was essential for the
binding of metal ions. However, it has been demonstrated that the same
effect can occur with a diet based on free amino acids. Therefore,
phytate does not need to be bound to protein in order to chelate metal
ions. (Likuski and Forbes, 196u).

Because of its ability to chelate metal ions phytate has the poten-
tial to interfere with mineral metabolism of the organism that consumes
it. Especially if the animal were already at a borderline position with
regard to the supply of some metal in its diet, the presence of sufficient
phytate could induce deficiency.

There are two basic kinds of phytases known, which sever the phos-

phate from the inositol. Man and most other animals do not contain

either. Phytase Type One is found during germination in the seeds that
contain the phytate, and in other parts of plantsat other times. This
obviously serves to liberate the phosphate to meet the plant's require-
ments for it during germination. Yeast also possesses Type One phytase,
therefore, during the leavening process, the phytate present in wheat
flour is destroyed. For most cereals and legumes the cooking processes
don't employ yeast and so the phytate is not broken down in that way.
The heat of cooking, whether wet or dry, by itself is generally insuf-
ficient to break down the phytate. Phytase Type Two is found in animals
and has been demonstrated in albino rats, guinea pigs, rabbits, pullets,
chicks and ruminants. (ggtt, Bg1,, 1967, "Effect of Phytate...").

Oberleas gt gt. (1966) studied pH effects upon solubility of phytic
acid-metal complexes. They concluded that zinc salts should be dissoc-
iated at the pH of gastric juice, and be least soluble at the pH of the
small intestine. Because of this, low concentrations of phytate should
not limit zinc availability. "In order for phytate to make zinc unavail-
able, the phytate must be present in sufficient quantity to overcome the
effect of dissociation and dilution in the stomach and to assure formation
of an insoluble compound in the intestine before absorption occurs."
(Oberleas, gt gt., 1966, p. 59) Practically all zinc absorption takes
place in the duodenum so as long as the phytate manages to reassociate
with the metal ion before it gets very far into the duodenum, the sol-
ubilization in the gastric juice will not significantly increase absorp-
tion.

In the presence of a high proportion of calcium, as in most diets,
a zinc-calcium-phytate complex is formed that is considerably more

insoluble than the zinc-phytate one. Virtually 100% of the zinc may

be removed from solution at a ratio of 100 Ca: 1 Zn. (Oberleas, gtht.,
1966). Therefore, a smaller concentration of phytate could be present
and reassociation could still occur, so there is good reason to suspect
that if phytate is present in significant amounts, it may interfere with

zinc metabolism.

Dahmer gt_g_, (1966) have claimed that histidine, even with phytate
and calcium in the diet, can eliminate the symptoms of parakeratosis in
swine.

Phytate in some protein can be at least partially destroyed by
autoclaving, according to some workers. In EEEE: Egg, (1967) it is
claimed that autoclaving soybean protein hydrolyzed the phytic acid and
increased zinc availability. O'Dell (1969) also stated that in his
laboratory, ”Autoclaving an isolated soybean protein for four hours at
115° C destroyed most of the phytate, ... , but under the same conditions
approximately 80% of the phytate in sesame meal remained intact."

Phytic acid in seeds serves as a storage form of phosphorus, inos-
itol, and possibly certain metals required for germination. It is found
in significant concentrations in cereal grains, leguminous seeds, and
potatoes. Very little is found in other parts of the plant, including
bean pods. (Makower, 1969). Phytate formation occurs most rapidly in
the later stages of seed developement. Makower reported a content of
approximately 1% phytate in Pinto beans at maturity.

The distribution of phytate in the seed of corn has been studied
by O'Dell (1969). He reported that practically all of the phytate is

found in the germ. O'Dell gt, 1. (1972) dissected kernels of rice,

wheat, high lysine corn and ordinary corn into germ, endosperm and peri-

carp. They found in general that over 80% of the total phosphorus was

present as phytic acid. In corn about 90% of the phytate was in the
germ, but in rice and wheat, although the concentration in the germ was
high, most of the phytate was in the outer layers. In the same study
they examined mineral distributions and concluded that "A major propor-
tion of the nutritionally important mineral elements is lost to consumers
by milling processes that involve degermination of corn or wheat, or
removal of the pericarp and aleurone of rice and wheat. 0n the other
hand these processes (also) remove a large proportion of the phytate...".

Phytic acid represents quite a significant percentage of the total
phosphorus in seeds. Oke (196A) reported 20 to 60% for pulses, hO - 70%
for cereals, and 8 - 67% for tubers.

As already explained, phytic acid has the capacity to reduce the
availability of essential metals to the organism. It has been observed
to contribute to calcium, iron, magnesium (O'Dell £3,2l-a 1972), and
zinc deficiency.

In countries of the Middle East, and especially in Iran, a rather
high frequency of mineral deficiency diseases occurs. Rickets in
children and osteomalacia in women, iron deficiency anemia, and zinc
deficiency all exist in this area. (Reinhold, 1971; Reinhold gt_gt.,
1973; O'Dell, 1969).

Through the action of phytate, zinc is the first element to become
limiting. Unleavened whole meal bread, eaten by villagers in rural
areas of Iran, has been conclusively shown to lead to zinc deficiency in
humans. (Reinhold gt gt,, 1973; Reinhold, 1971). Zinc deficiency delays
the onset of puberty, retards growth, can lead to anorexia, seborrhea,
loss of hair, testicular atrophy, and parakeratosis. (Caldwell gt al.,

D?70). It came to the attention of officials in this area and in Egypt

when they attempted to draft young men for military service. An amazingly
high percentage of these 18 to 20 year old men resembled 12 to 13 year
old boys, both in terms of size and in developement of sexual organs
and secondary sex characteristics. Presence of calcium in the diet, as
cited earlier, decreases availability of zinc. People not only consume
phytate-rich diets, but the calcium content of human diets tends to be
high in the Middle East, and geophagia is not uncommon, also increasing
calcium intake. Because of the enhancing effect that calcium has upon
binding of zinc by phytic acid, these conditions can only increase the
incidence of zinc deficiency.

The induction of zinc deficiency upon inclusion of phytate in the
diet has been demonstrated several times. O'Dell and Savage (1960)
showed it for chicks. O'Dell gt gt. (196k); Oberleas gt gt. (1965); and
Likuske and Forbes (1965) all demonstrated that calcium aggravates zinc
deficiency in experimental animals. This of course would tend to dis-
courage the use of Ca supplementation in phytate-rich flours for the
purpose of diminishing the rachitogenic effect. Parakeratosis, "A
thickening or hyperkeratinization of the epithelial cells of the skin

and esophagus” (anon., Hutr. Rev., 1967) in swine, has been attributed

 

 

to zinc deficiency. Ten ppm zinc was sufficient to maintain good growth
and healthy skin in the abscence of phytate, but when soybean meal was
fed to the swine, zinc content had to be increased to 80 ppm. In
studies with rats, 1? ppm zinc was sufficient when casein or egg albumin
was fed as protein source, but with soy protein, 18 ppm were required.

In studies with rats and chicks, O'Dell gthl, (1972) established
the following biological availability values for zinc with different

protein sources. With chicks: high lysine Corn, 65%; control corn, 63%;

9
rice, 62%; wheat, 59%; igh lysine corn germ, 56%; control corn germ, 5h%;
sesame meal, 59%; soy bean meal, 67%; egg yolk, 79%; fish meal, 75%;
oysters, 95%; non-fat milk, 82%. The rat assay gave: high lysine corn,
55%; control corn, 57%; wheat, 38%; rice, 39%; egg yolk, 76%; non-fat
milk, 79%; fish meal, Phfi. This showed that rats could not utilize the
zinc as well as chicks, and that zinc in general was more available
from animal protein than from plant sources.

Zinc deficiency in rats has been shown to lead to behavioral modif-
ications of a detrimental nature. (Caldwell gt gt., 1970). The authors
suggest that diets employing only plant protein sources may lead to
maladaptive behavior such as lethargy and reduced learning ability.

Phytate also leads to calcium deficiency in humans. (Reinhold
g a_l_., 1973; Reinhold, 1971). During ww II in Great Britain, 95%
extraction "National Flour" was used, leading to increased incidence of
'rickets. Even after yeast fermentation, the content of phytate is higher
in high extraction flours than in low extraction flours. It is probably
sufficiently reduced by the yeast action that it no longer need be
considered a potential problem. However, in areas where unleavened
breads make up a significant portion of the diet, eg. the Middle East,
India, and Pakistan, with wheat breads, and Central and South America
where corn tortillas are very often consumed, the potential for disturbed

mineral nutrition must be recognized.

MATERIALS AND METHODS

Ehytate Extraction Procedure

Whole beans were dried four days in a forced air oven at 60°C, then
ground for two minutes in a water-cooled micro-mill (Chem. Rubber Co.)
and stored in air-tight bottles. All work was done in glass containers,
and all water used in samples was glass distilled. One gram of bean
powder was extracted in 100 ml of Solution A (1.2% HCl, 10% NagSOh) for
two hours in a water bath shaker cooled to approximately ho C. The
cooling and low concentration of bean powder gave a sufficiently non-
viscous solution that it was possible to filter it. The samples were
then filtered through Whatman #1 filter paper, and ten m1 aliquots taken
of each. To the ten ml samples were added 10 ml of water and 15 ml of
Solution B (2 gm. FeC13-6H20 / 500 ml 0.6% HCl). These were then placed
in a boiling water bath for 15 minutes or longer if necessary for prec-
ipitate to fully form. The tubes were then cooled in a cool water bath,
and centrifuged 15 min at highest speed of an International Clinical
(table-top) Centrifuge. Putting water around the test tube in the bucket
helped to keep the glass tubes from breaking. They were washed two times
with Solution C (0.6% HCl, 5% NagSOh) by resuspending in 5 m1 of Solution
C and then centrifuging as before. The pellets obtained were each
dissolved in 3 m1 of concentrated HESOg and added to some water in a
100 m1 volumetric flask, then brought to volume with more water. They
were then stored in glass bottles until iron determinations were made
' with a P-rkin-Elmer model 303 Atomic Absorption Spectrophotometer. A

10

11

ratio of h Fe: 6 P was used to calculate the amount of phytic acid

present.

Cooking Trials

 

Commercially canned beans were assayed for phytic acid content.
Gazlord brand Pork and Navy beans, Great Northern, (idney and Pinto beans
were removed from the can and dried in a forced air oven at 50° C to
constant weight. Liquid was not drained off. They were then ground and
tested for phytate.

Because of the low values observed in the commercially canned beans,
I decided to test whether any changes occur in phytate concentration
during ordinary home cooking procedures. After forced-air drying, 50 gm
of Sanilac beans were put into each of four glass beakers. These were
soaked over-night and cooked the next day by placing the flasks in a
boiling water bath, so all would be evenly heated. During cooking,
samples of beans were removed from the beakers at various time intervals,
up to two hours, then freeze-dried. At the end of two hours the concen-
tration of phytate in the solids dissolved in the cooking water was also
determined. Weights of samples were not taken, so it was not possible to
determine whether the total concentration of phytate changed. Therefore
I repeated the analysis, this time also determining sample weights, so
the final phytate concentration could be.compared to the original
phytate concentration. In both cooking tests, one of the beakers had

the soak water decanted and replaced with fresh before boiling.

12

Effect of Normal vs Low Zinc in the Field
0n Ihytgte Concentration in the Seed

A limited amount of material was available from plots with high
phosphorus fertilization, which creates a state of zinc deficiency in the
plant, and from plots with normal levels of phosphorus plus supplemental
zinc. Ihytate concentration in the seed was determined to see whether

this particular environmental parameter had any effect on it.

thtate in the Root

Since phytate binds metal ions, and especially zinc, the possibility
existed that perhaps higher then normal phytate levels in the roots of
certain varieties of P. vulgaris could account for their greater sus-
ceptibility to low levels of zinc in the soil. An attempt was made to
determine the amount of phytate in the roots of Sanilac, which is sus-
ceptible to low zinc; Saginaw, which is resistant; Black Turtle Soup,

resistant; and Idaho 59, susceptible, grown both with high and low zinc.

Phytic Acid Content of Assorted Lines
A set of varieties mostly from California and Idaho were assayed
for phytate content. These varieties were obtained from different

sources, so they were not necessarily grown in the same place at the same

time.

Ihytate Content of Lines Selected on the Basis of Protein Content

A group of lines selected for high or low protein content were
tested for percent phytate. These varieties were not all grown in the
same place before they were assayed for protein content. However,
after that determination was made they were all grown together at the

Saginaw Valley Bean and Beet Research farm in 1972. The seed harvested

13
in 1972 was then assayed for percent phytate.

Environmental and Varietal Effects Upon Phytate Content

To obtain further informatiOn about varietal X location effects on
phytate, seeds from two other experiments were analyzed. The first
experiment consisted of 25 entries grown at two locations in Mich. in
1973, the Saginaw Valley Bean and Beet Research Farm, and the Montcalm
County Research Farm near Entrican, and identified elsewhere (Adams,
personal communication) as the Partial Growth Analysis nurseries.

I assayed 12 lines from each of these locations, using two rep-
lications of each line at each location, and two samples for phytate
analysis from each replication. There was not enough material available
from all four field replications to do this analysis, and the analysis
of phytate content is sufficiently time consuming that I did not con;
sider it desirable to use all four replications of each line anyway,
so I simply used any two of the four field replications. Therefore there
is no reason to think thfl:line one at rep one is more like line two at
rep one than it is like line two in rep two. The analysis of variance
was conducted accordingly.

For the second experiment, seed was obtained from a yield component
study conducted by Dr. Gaspar Silvera at three locations (E. Lansing,
Mich.; La Molina, Peru; and Cali, Colombia) during the summer of 1973.
All nine varieties and all three replications of all lines, except one
replication of the variety Liborino at Paru were available from the
material grown in E. Lansing and in PEru. These were analyzed as shown
in Tables 8 and 9. From Colombia only one replication was available of

six of the varieties, as seen in Table 10.

RESULTS

Cooking Trials

Percent phytate determined in commercially canned beans was: Navy
beans, 0.12; Great Northern, 0.20; Kidney, 0.26; Pinto, 0.16. These
figures are considerable lower than raw beans of comparable type. This
could mean that the high temperature and pressure of commercial canning
procedures destroyed some of the phytate or that part of it was destroyed
by yeast action during the drying operation. The latter is very unlikely
decause drying time was not long, and the beans of course were sterile.
when originally removed from the can.

With the first set of the samples I cooked, the concentration of
phytate in the seeds went down, and in the water went up. (Fig. l)
The decrease in phytate concentration was not so obvious the second time,
but there was a decrease in concentration of phytate in the beans, and
an increase in the broth by the end of the two hour period. (Fig. 2)
As can be seen in Table 1, there was a decrease in total phytate as well,
but it was not consistent. The decanted overnight soak water showed no

detectable phytate in either test.

High vs Low Zinc

As can be seen from Table 2, there was no significant treatment or

line effect for this set of data.

19

1h

% Phytate

 

 

 

 

di

35 a3 90

120
Minutes of Cooking Time

Figure 1. Changes in phytate concentration of beans and broth during
first cooking test. (#1) Overnight soak water was decanted before
cooking and replaced with fresh water. The decanted water had no detect-
able phytate. (#2) Beans were cooked in the soak water.

*-

These points are for concentration of phytate in the solids
dissolved in cooking water at the end of two hours cooking time.

15

% Phytate

 

0.5 i

 

 

3o 60 90 120
Minutes of Cooking Time

Figure 2. Changes of phytate concentrations of beans and broth during
second cooking test. (#1) Overnight soak water decanted and replaced
with fresh water before cooking, samples of beans taken at intervals.
(#2) Soak water not decanted, samples taken only at end of cooking per-
iod. (#3 & #h) Soak water not decanted, samples taken at intervals.

These points are for concentration of phytate in the solids
dissolved in cooking water at the end of two hours cooking time.

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17

Table 2. High and Low Zinc.

 

 

 

 

%thytate

iontcalm Saginaw

Variety Normal Zinc Low Zinc
Dark Red Kidney 023 1.6M 1.86
ierithew ‘ 1.73 1.2h
Red Kote l.h9 1.27
Cal. Dark Red Kidney 1.h6 1.h9
Commercial Yellow Eye 0.67 ' 1.31

 

Table 3. Analysis of Variance of % Phytate in Five Lines
Under High and Low Zinc.

 

 

 

Source of Degrees of
Variability Freedom Sum Squares Mean Squares F Value
Total 9 0 9550
Environment 1 0.0029 0.0029 0.0323 ns
Lines A 0.5930 o.lh83 1.651h ns
Error A 0.3591 0.0898

 

l8

Phytate in the Root

 

No detectable precipitate resulted, therefore the concentration
of phytate in the roots must have been close to zero. It therefore would
have been much too low to lend support to an hypothesis of differential

zinc binding in the roots of the several varieties.

Phytic Acid Content of Assorted Lines.

The phytate content ranged from 0.23 % to 1.65 % of the dry weight.
The mean was 1.29 % 1 0.3122. Einto (Univ. of Idaho # 111) and the Red
Mexican variety Big Bend were both more than one standard deviation above
the mean, Regina Nano and White Ventura # 65 (P. lunatus) were more than

one standard deviation below. (See Table h)

Phytate Content of Protein Selections

The percentage phytate in the seed of the selected protein lines
ranged from 2.39 % to 1.10 %. The mean was 1.53 % I 0.31. Six lines
were more than one standard deviation above the mean, and six were more
than one below.

A regression of % protein on % phytate was calculated. (Figure 3)
The regression was significant, but the correlation coefficient was
very low, only 0.352h, so it still should be possible to find varieties

with high protein and low phytate.

Environmental and Varietal Effects Upon Ihytate Content

For the material from the Partial Growth Analysis, the analysis of
variance showed significant line and location main effects, but insig-
nificant line X location interaction. Average % phytate ranged from

1.h6 % to 2.10 %.

For the second experiment the analysis of variance showed that there

19

Table A. thtate Content of Assorted Lines

 

 

Variety % thtate

 

Pinto: Univ. of Idaho # 111

Red Mexican: Big Bend

Black Eye # 5

Great Northern: Univ. of Idaho # llhO
Great Northern: Univ. of Idaho # 59
Small White # 59

Great Northern: Univ. of Idaho # 61
California Red Kidney: Lot lth
Dark Red Kidney

Great Northern: Nebraska # l
Cranberry-type: Regina Rampicante
Small Flat White: Univ. of Idaho # 1
Great Northern: Univ. of Idaho # 31
Red Mexican: Univ. of Idaho # 36
Vicia faba

Pinto: Univ. of Idaho # 11h
Green-seeded Snap Bean: European A
Red Mexican: Univ. of Idaho # 3h
Green-seeded Snap Bean: European B
Cranberry-type: Regina Nano

‘2. lunatus: White Ventura #‘65

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Table 5.

20

Protein and Phytate Content of Selected Lines

 

 

 

Variety Name or P.I. # % Phytate % Protein*
175277 (2) 2.3920 28.8
229815 2.28h6 28.h
13669h 2.1608 21.9
226913 2.1339 28.0
136691 (l) 1.9533 31.5
17586h 1.9370 27.0
205211 1.8326 27.9
175818 1.8066 23.0
3025h2 1.8060 28.5
282693 1.7929 2 -.7
3025u2 1.7379 28.9
177508 1.7313 27.2
136691 (2) 1.7169 27.6
1u6792 1.7lhh 26.3
173030 (30) 1.6313 27.1
Black Turtle Soup 1.6255 20.8
1758h9 1.5919 28.3
173030 (38) 1.5670 26.2
ll3367 (9) 1.5h97 29.8
176703 1.5k71 30.7
l661h5 (1) 1.5152 22.6
173030 (3A) 1.5128 30.0
1758u2 1.5097 19.8
175815 (2) 1.5055 19.5
169768 ' 1.5029 23.9
207hul 1.u798 26.2
150H06 1.hh28 27.u
150u01 l.h0hl 27.6
113367 (3) 1.3786 ----
Sal. 208-N 1.36u9 21.2
278676 1.3h77 27.1
173763 (h) 1.3385 2h.0
17526h 1.3253 28.1
136692 1.3038 21.h
16U27h (2A) 1.3038 21.8
173763 (3) 1.3005 20.3
l6h27h (23) 1.2788 25.h
173039 1) 1.27lu 25.8
1&2899 1.2630 28.
Mercado de PUntarenas 1.2606 19.6

21

Table 5 (c0nt’d.).

 

 

 

Variety Name or P.I. # % Ihytate % Protein*
166151 1.2581 23.6
205361 1.2360 2h.3
175838 (h) 1.2091 19.5
16h3lu 1.186h 23.h
16h306 (h) 1.1372 26.9
1698h6 1.1331 22.2
175838 1.1259 19.3
1697h6 1.1000 32.h

 

* Values for % protein were determined by Dr. R. J. Evans, Michigan
State University. (Personal communication)

% Crude
Protein

2h 0

22 f

21 .

20 n

 

22

r = 0.352%

 

Figure 3.

I I I I ‘ U U

1.2 1.1+ 1.6 1.8 2.0 2.2 2.1+
‘70 Pnytate

Relationship Between % Protein and % Phytate

23
were significant line and location main effects, and line X location
interaction. The reps/env X lines effect was also significant, implying
that the environmental effect can be significant even over a short
range. The Colombian material also shows location effect, although it
cannot be analyzed statistically with the rest. But the effect is obvious
because all varieties have much higher concentrations of phytate at
Colombia than at either of the other locations. The average percent
phytate for the nine lines ranged from 1.00 % to 1.6h %, for the average

of E. Lansing and Peru, and 1.62 % to 2.3h % at Colombia.

Table 6.

Analysis of

2h

Variance of Material from the Partial Growth Analysis.

 

 

Source of Degrees of Sum of Mean
Variation Freedom Squares Squares F Value
Total 95 (l-e-r-s-l) 9.7118
Lines in Env. 23 (e.1-1) 6.3383
Environments 1 (e 1) 1.1389 1.1389 11.5273**
Lines 11 (1 1) b.1122 0.3738 3.783u*
Lines x Env. 11 (e 1)(1-1) 1.0872 0.0988 1.9960 ns
Replications 2h (e-1)(r-l) 1.1886 0.0895 1.0879 ns
Sample Error A8 (eol°r)(s~1) 2.18h9 0.0h55

 

Table 7.

Comparison of Phytate Content of P. vulgaris Varieties
From the Partial Growth Analysis.

 

 

Variety

Average % Phytate

 

Michelite
Jules
Seafarer
Oregon 58
Sanilac
Swedish Brown
Tui

Big Bend
Pinto 11h
Charlevoix
Manitou
Mich-i-cran

2.099 a*
2.05h ab

 

Varieties with the same letter are not significantly different
from one another at the 5% level of significance.

25

Table 8.

Analysis of Variance of 2; vulgaris varieties. (Silvera, 197M)

 

 

 

Source of Degrees of Sum of Mean
Variation Freedom Squares Squares F Value
Total 107 (e.1-r-s-1) 9.0282
~ Environments 1 (e—l) 0.0238 0.0238 10.8182*
Reps/Env h e(r-l) 0.0086 0.0022
Lines 8 (1-1) h.6hh1 0.5805 11.h0h7***
Env x Lines 8 (e-1)(1-1) 1.6588 0.2078 h.07h7**
Reps/Env x Lines 32 e(r-1)(l-1) 1.627u 0.0509 2.5838
Sampling error Sh e-r-1(s-l) 1.0655 0.0197

 

Table 9.
Varieties.

Comparison of Ihytate Content of it vulgaris
(Silvera, 197M)

 

 

 

Variety Average % Phytate
Cocacho 1.6h07 a*
Canario 1.5588 a
Liborino 1.5882 a
Seafarer 1.39h0 b
Sangretoro 1.3133 bc
Compuesto Negro 1.2217 cd
Coleccion 1-63 1.1h56 de
Rinon Oscuro 1.1h28 de
27- R 1.0026 e

 

*-

one another at the 5% level of significance.

Varieties with the same letter are not significantly different from

- Table 10. Average %

Phytate for Each Variety at Each

f‘

d

6

 

 

 

 

Location. (Silvera, 1973).
Location
*

Variety Peru E. Lansing Colombia
Coleccion 1-63 1.30h5 0.9867 1.7h05
27- R 1.0618 0.9h33 1.7838
Rinon Oscuro 1.0652 1.2203 2.3367
Canario 1.5867 1.5228
Seafarer 1.6138 1.17h2
Liborino 1.8032 1.6932 2.1683
Compuesto Negro 1.3197 1.1237 1.6157
Cocacho 1.5738 1.7075
Sangretoro 1.1683 1.h582 2.331h

 

Average for only one replication.

CONCLUSIONS

Some changes should be made in the extraction procedure. The major
improvement would be to use a colorimetric quantification procedure,
because the results from the Atomic Absorption Spectrophotometer are too
erratic. Also a better filtration method would speed up the process,
because that one operation sometimes took as long as an hour. I tried
centrifugation, but that did not give a sufficiently clear extract.~ Also,
there was detectable protein in the extract, and this could tend to give
deceptively high values. Although the HCl extraction has been used
many times, perhaps a TCA extraction would be better. Makower, 1969,
compared TCA to HCl using Pinto beans, and reported better results with
HCl; but her higher values of total Fe and Fe/ P ratios could have been
due to protein extraction in the HCl procedure. I am no longer convinced
that it is really the best possible way to do it.

High temperature and pressure during cooking does seem to have a
reducing effect upon phytate content. However, just boiling does not
produce as great an effect. Since a verysignificant proportion of the
legumes consumed in the "developing" countries are not prepared by
high pressure heating, the potential to reduce phytate content through
cooking is not very great in those areas. Since it is in those same
areas where diets high in plant seed protein are most common, that is
also where control most needs to be exercised.

The amount of phytate in the root is very low, so it probably cannot

account for the differential zinc susceptibility of certain varieties of

27

28
E.'vulaaris. There was no more detectable phytate in them than in non-
susceptible varieties.

Phytate content was found to range from a low of 0.23% for one sample,
white Ventura 65, to a high of 2.39 7. for P. I. 1! 175277 (2). The over-
all average was approximately 1.5 %. Without a doubt one could select
lines that have lower amounts of phytate than most of the commercially
produced varieties which usually had average or above average phytate
content.

It is gratifying to find a very low correlation of % protein and %
phytate, since we would like to increase the protein in the seed to the
very limit of which we are capable, and at the same time to reduce the
phytate level as low as the plant can tolerate and still maintain its
vigor. Because of the low correlation of only 0.3528, it should not be
too hard to maintain high protein while selecting for low phytate,
or vice—versa.

Phytate content is considerably influenced by environment. What
environmental parameters affect it was not examined except for zinc
stress, which for the ones examined was not significant. The possibility
therefore exists to control the level of phytate in the seed by learning
to understand which environmental factors influence it, and controlling

them.

BIBLIOGRAPHY

 

V‘ 10.

11.

BIBLIOGRAPHY

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Anonymous. 1967. Effect of Phytate on Iron Absorption. Nutr. Rev.
25: 218-222.

Caldwell, D. F., D. Oberleas, J. J. Clancy, and A. S. Prasad. 1970.
Behavioral Impairment in Adult Rats Following Acute Zinc Deficiency.
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de Lange, D. J., C. IE Joubert and S. F. M. du Preez. 1961. The
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Likuski, H..J. A., and R. M. Forbes. 196M. Effect of Phytic Acid
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Makower, Rachel U.. 1969. Changes in Phytic Acid and Acil-soluble
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. 1970. Extraction and Determination of Phytic Acid in Beans
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29

1h.

15.

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21.

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25.

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28.

30

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, Merle E. Muhrer and Boyd L. O'Dell. 1966. Dietary Metal-

Complexing Agents and Zinc Availability in the Rat. J. Nutr.
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, Ana R. de Boland, and Samuel R. Koirtyohann. 1972.
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, J. E. Savage. 1960. Effect of Phytic Acid on Zinc
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31
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