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ISSfllAflC iJ? OF S^AitOH
m
11 P lA lf. 1
Robert [email protected] Powell
A Dissertation Submitted to the
Graduate Paeulty in Partial [email protected] of
©ne Requirements for the Degree of
ooctor of .pmosofiir
Major Stibjeets Plant Physiology
Approved;
W .«g©
Iowa State dollege
1950
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UMI Number: DP11869
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Page
INTRODUCTION.. .* ** 1
REVIEW OF LIT! ^ * Ai t j . * * . . 3
Grinding of flant Materials. *3
Extraction of starch, * 4
Determination of S t a r c h * 10
MATERIALS- AM) METHODS, 1*7
Plant M a t e r i a l s 1*7
Enzymes* 19
Equipment... • * * , . 19
Analytical V etho&s 20
EXPERIMENTAL .V. BETS*, 22
Enzyme Studies 22
Effect of concentration of saliva. 22
Effect of time of incubation with saliva**.. . 27
Effect of tatea-diastase concentration*•»»...* 32
Clearing and D e l e a d i n g , 55
■ Clearing of the plant extract* ....... 35
Deleading with dibasic potassium phosphate* * * 38
Acid Hydrolys is * 42
Gel&tlnizatlon. • 43
Effect of Grinding,..A,*,,,....,...,.*,*,#****..,, 45
A Method of Starch D e t e r m i n a t i o n * 54
Analysis of various plant materials*,**.**,., 58
DISCUSSION. ..... 58
SUMMARY. ... * 84
LIT i.TXJRE CIT. d............ ........................... . .......... 65b
ACKNOWLEDGMENTS , * * .. *..... 38
r ^ r o i
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IHTRODUCTIGH
Determination of staroh has been of Interest to biological
chemists* plant physiologists and food technologists for over
a century* Reports on the determination of stanch are found
in' the literature as far back as 1831* Plants are composed of
a great many different types of polysaccharide© which might
Interfere with the determination of starch* Th& number and
type of those compounds vary tremendously from on© species of
plant to the next* The food technologist is usually working
on a particular species* and* therefor©* may use a method that
is applicable to that type of plant without consideration of
its adaptability to other plants* Th© biochemist and plant
physiologist* however* are interested in a large number of
different species* It is necessary for these research workers
to have available a method that may b© applied to plants in
general* The work presented-in this paper was done with this
objective in mind*
frobably the most prevalent method of starch determination
used by plant physiologists Is that of Loomis and Shull (17)*
This method* starting with dry material from which sugars have
been removed, consists of an extraction with 10 percent
alcohol to remove dextrine* Water is added to the residue
and heated to gelatinize the starch* which Is then digested
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with saliva and extracted with water. Neutral lead [email protected]
is added to remove gums, and the solution filtered and
deleaded* An aliquot of the clear solution is hydrolyzed
with hydrochloric acid In a concentration equal to 1,0 ml
concentrated acid for each 20 ml of solution (1 4 20),
Dextrine may be determined by application of the same pro
cedure to the 10 percent alcohol extract, eliminating the
gelatlniaation and ©mymatic hydrolysis. The resulting
glucose Is determined and the starch calculated by using
a conversion factor of 0,90, The work presented her© will
be a study of the various phases of this method*
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3
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hbview op lit
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■One of the oldest methods fop the estimation of starch
Is that of Rermbstaedt {13) which appeared in 1631# This
method was essentially the- [email protected] isolation and grav
imetric determination of starch* Present day methods consist
of some means of freeing starch or its hydrolytic products
from the remainder of the plant tissue and the determination
of the starch or sugar content of the extract. It has also
been stated that preliminary treatment of the plant material
Is of great Importance*
Grinding of Plant Material
The fineness of grinding necessary to extract starch
quantitatively from plant tissue has been the subject of
considerable disagreement among various authors,, probably due
to, various methods of extraction and differences in plant
tissue* Sullivan (26), working with apple twig terminals
and a calcium chloride extraction, found that the material
must be able to pass through an 80 mesh screen, and recom
mended that it pass through a 100 meshscreen* Bass Id,
MeCready and Rosenfels {12), using hot, dilute, alcoholic
hydrochloric acid as a preliminary extractant, claimed that
grinding to pass through a 60 to 60 mesh screen was sufficient*
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Griffiths and Potter (10) state that difficulty In extraction
of starch say be partiy due to Inadequate grinding* They did
not* however* give any experimental data*
Ho data have appeared in the literature concerning the
effect of prolonged grinding on starch* Schoch (24)* however*
stated that prolonged dry grinding of starch in a pebble mill
eventually yielded a product that stained red with iodine*
He believed that hydrolytic and oxidative changes may occur,
as well ms dextrin!aation due to heat ’-generated■in the grind
ing process* He further stated that mechanical rupture of,
the bonds in a molecule such as starch Is entirely possible*
and that a similar phenomenon was found with proteins.
Extraction of Starch
Starch Is found In plants .In a great many, different
types of tissues* It Is always formed inside a plastld in
a living cell* These are usually parenchyma cells and
therefor© thin walled* However* these cells may be surrounded
by woody tissue* as in the xylem of the root and stem* Th©
starch must be removed fro® th© colls and freed from any
woody material surrounding those cells*. .Several means of
extraction have been proposed,
Probably one of the oldest means of extracting starch
takes advantage of th© hydrolytic activity of eneymes*
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This, in many eases* is coupled with a subsequent acid
hydrolysis# With, these techniques* it is necessary to
determine the hydroltyic products formed and calculate the
starch* Dayla and Daish (5) showed that* under proper
conditions* digestion of starch with taka-dlastase yields
glucose and maltose only* They then determined the glucose
and maltose polarlmetrieally and by copper reduction* The
quantity of each was calculated by simultaneous equations
and the amount of starch estimated* Several papers then
appeared with conflicting results on the use of taka-dlaatasa
for the determination of starch* Horton (14) suggested that
the hydrolysis to glucose and maltose is not complete and
that some dextrin remains* Collins (4) reported glucose
values of 98*5 to 101*1 percent by hydrolysis of atarch
when 50 ml of 0*5 percent starch solution was incubated at
40°C for 56 hours with 1 ml of 10 percent taka*dlastase and
5 ml of acetate buffer (pH 5*0)« Widdowson (28) could not
find a constant ratio of glucose to maltose with enzymatic
hydrolysis* These discrepancies were explained by Denny (6)*
He used dlalyzed taka«*diastase and showed that the pro
portion of glucose to maltose to dextrin depended upon the
concentration of enzyme used, and the conditions of the
incubation* In the same paper he also showed that the
postulation of the destruction of glucose by -acid hydrolysis
was invalid* He showed that the previous data did not merit
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