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A STUDY OF EFFECTS OF SAMPLE

PREFIARA‘HON AND METHODS OF
EXTRAC'FEQN (EN DETERMENATEON 0F
STARCH EN PLANT MATfiREALS

Thesis for the Dogma of M. S.
MKHIGAN STATE COLLEGE
BMW Ru? fiché‘ssmn

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105912193 1

 
  

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A STUDY 0? EFFECTS OF SAMPLE PRLPARKI‘ION AND
METHODS OF EXTRACTION 0N DETERMINATION
OF STARCH IN PLANT MATERIALS

By
BETTY RUTH JOHNSTON

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirement:
for the degree of

MASTER OF SCIENCE

Department of Chemistry
1953

'“C- .’ - I .‘V'W‘. f‘d

.3 ”eras-“39 9? Y'Tfl‘r’firn 9""qvfztn

I wish to thank Professor C. D. Ball for his
enccuragement and suggestions in connection with the
experimental portion of the present study and pre-
paration or the theaie manuscript. Also, 1 tian to
thank the Bepartment of $011 ecicnce for the privilege

of sampling their experiauutal plate.

Betty Ruth Johnston

TABLE OF CONTENTS

Introduction
Historical
EIperimental

Results and Discussion
Summary

Literature Cited

Page

18
29
31

LIST OF FIGURES AND TABLES

Page Ho.

Figure No. 1. Glucose-Corie Sulfate Relationship 11
Table I. Standardization of Ceric Sulfate 10
Table II. Recovery of Starch from.A1falfa

Dried at 50° C. 14
Table 111. Per Cent Starch in Pure Defatted

Samples 15
Table IV. Effect of Hydrolysis Conditions

on Results of Analysis of Pure

Starch 17
Table V. Per Cent Starch in.Alra1fa Calcu-

lated to Dry‘Weight Baais 19
Table VI. Per Cent Starch in.Reed Canary

Graae Calculated to Dry

Weight Basis 20
Table VII. Per Cent Starch.in Orchard Grass

Calculated to Dry Weight Basie 21

Table VIII. Per Cent Starch in Brome Grass
Calculated to Dry Weight Basis 22

IRTRODUCTIOH

nan: papers on the chemistry or starch.have been
written over the years and are still being published
on various phases of the subject. However, one aspect
which has been comparatively neglected is that of
analytical methods for the quantitative determination
of small amounts of starch in plant materials.

Because of the importance of starch data to studies
or plant metabolism, it seemed desirable to determine
the effectiveness of some of the analytical procedures
which have appeared in the literature. The problem or
such analyses is threefold: l. the preliminary treat-
ment of samples prior to analysis: 2. the complete
extraction of starch; 3. the accurate evaluation of
starch with the exclusion or any other carbohydrates
which may be present. The work to be discussed deals

with these three phases of starch analyses.

VT ”"0"?” 5!!

Some study has been devoted to the effect on
starch content of preliminary treatment or plant
materials prior to analysis. In 1895, Brown and
horris (1) reported observations on the effects of
temperature, loss of actor, and anesthetics on starch
dissolution in tree leaves. Freezing of leaf samples
was found by Spochr and "ilner (9) to prevent deple-
tion of starch. Treatment of fresh plant material with
toluene, chloroform, and other anesthetics was reported
by Spoehr and Hilncr (3) to preserve starch.

or the many agents which have been used to dissolve
starch, concentrated caloiunlchloride solution is one
of the oldest and is still need for that purpose. As
early as 1860, Flueckiger (4) reported that starch
could be dissolved in calcium chloride solution. Pollen-
berg (5, 6, V) devised procedures for the determination
of pure starch and starch in.plant samples using cal-
cium chloride solution as the dispersing agent. Several
studies were made by Lenny (a, 9, 10) comparing calcium
chloride extraction of starch.from plant tissues with
direct acid extraction method or 1Fbunnie(l‘l) and direct
malt diastase hydrolysis procedure used by talton and

Coo (12). Sullivan (13) reported Optimum conditions

for extraction of starch.trcm plent.nettsr with calcium
chloride. core recently, calcium chloride extrection
has been applied to plant asteriels contsining less
than l0 per cent starch by Hoffpsuir (14, 15). A new
method for starch determination in leaf samples use deu
scribed by Chinoy (16) using dilute potassium hydroxide
to extract starch. Riemann, Roberts, and Link (17)
chose en ethanol-nitric acid.nixture to solubilise
starch in wood; tissue prior to extraction of starch
with 20 per cent aqueous ethanol. the procedure de.
scribed by‘Eenes (18) for extracting stereh from plent
materiel such as apple fruit involved the conversion
of starch to e highly soluble form by hosting the tissues
in alcoholic hydrochloric acid. boiling actor was then
used to extract the solubilised starch from.the tissues.
fisssid, ficCrosdy, and floscnfols (19) adapted the method
outlined by Hence (it) to tan determination of starch
in leaves end various other trees of plant reterisls
for which the original procedure one unsatisfactory.
After its isolation. starch has been evaluated
in s number of ways. Fellenbcrg (5, 6, 7) precipitated
starch from solution with iodine and collected the
sterohaiodlne complex on c Gooch crucible. After thor-
oughly washing the precipitate, the crucible was firiud

to constant weight, ignited, and reweirhed. The loss

in weight was considered to be amount of starch present.
Gravimetric evaluation sac used also by flask (ll) who
precipitated starch from solution by addition of ethanol
and collected.the starch by filtration for direct weigh-
ing. Chinoy (16) precipitated starch with.iodine and
weighed the starchniodine complex. Iodine solution was
used also by Sullivan (13) to precipitate starch from
cslcium.chloride solution. However, in this case the
starch was subsequently hydrolysed with hydrochloric
acid and reducing power was taken as a measure of glue
cose, which was used as the basis for calculation of the
amount of starch originally present. Hoffpsuir (14)
published.s modification of the Sullivan (13) procedure.
Walton and Cos (12) incubated plant material malt
B-amylase to hydrolyze starch without preliminary
extraction. Pectin was removed by precipitation with
sleOhol, end the resulting solution was treated with
acid to completely hydrolyse any dextrins and maltose
before determining reducing sugars. Malt B-amylsse

was used by Hanes (18) to hydrolyze starch, whereas
Hassid. McCreedy, and Roaenfels (19) used salivary
amylase in their procedure to convert starch to mal-

tose and dextrins.

EXPERIMENTAL

Plant samples analysed in the present study were
obtained from the experimental plots of the Department
of Soil Science. The plants selected were alfalfa,
Medicago sativa, just before blooming, and second growths
of brome grass, Bromus inermis, reed canary grass,
Phalaris arundinacea, and orchard grass, Dactzlis
glcmerata, hereinafter to be referred by the common
names. None of the plots sampled had been fertilized
or irrigated.

Samples were taken at about two o'clock on after-
noons of clear days with temperatures of 85° F. or
above. ‘All plants in an area of approximately four
square feet were out about two inches above the ground
with shears. As soon as possible after cutting, the
samples were taken to the laboratory where all dead
plant leaves and any extraneous matter were removed.

Each plant material was divided into approximately
four equal parts for curing by four different methods.
The first portion was spread out on a table and allowed
to dry at room temperature until brittle. A second
portion was placed in a drying room at 50° 0. for
twenty-four hours. Another portion was dried for eighteen
hours in an electric oven at 85-90° C. The fourth por-

tion was frosen and then placed in the electric oven

and dried for eighteen hours at 35-90° c. A total of
sixteen samples resulted from four methods of handling
each of the four plant varieties.

After drying, the samples were put through a large
Wiley mill equipped with a coarse sieve prior to final
grinding to 60-mesh with a semi-micro Wiley mill.

Defatted corn starch was prepared by the method of
Schoch (20) for use as a standard in recovery tests.
Lipids were removed from 5 g. of corn starch by Soxhlet
extraction with 95 per cent :5th for 48 hours. Ex-
cess alcohol was removed from the starch by allowing
the paper thimble holding the starch to stand at room
temperature overnight. Final drying was accomplished
by spreading the starch out on a large watch glass and
and heating for 24 hours in an electric oven at 105° C.
The dried starch was placed in a small weighing bottle
and stored over calcififin a desiccator.

A portion of each plant sample was taken for
moisture determination at the same time that samples
were weighed for starch analysis. Moisture content of
the dried plant material was measured by placing 6 g.
of sample in a special aluminum pan and drying at
100° 0. for 24 hours in the Brabender moisture tester.
The per cent moisture was obtained by multiplying the
Brabender scale reading by two since the moisture
tester is calibrated for 10 g. sample weights.

Duplicate amounts of 0.5.1.0 3., depending on starch
content, of each sample of a specific plant variety
were weighed on the same day for extraction by the
three different procedures studied as described in
succeeding paragraphs. For example, six 0.8 g. por-
tions of each of the four alfalfa samples, cured by
different methods, were weighed into apprOpriate ves-
sels, 200 ml. Pyrex centrifuge bottles for ethanol-
hydrochloric acid and.potassium.hydroxide extractions
and 150x25 mm. Pyrex test tubes for calcium chloride
extraction. Thus, all analytical results for a given
plant sample could be calculated to a common dry weight
basis requiring only one moisture determination.

For the solubilization and extraction of the starch,
three procedures were used. The first one studied was
that of Hassid, flcCready, and Posenfels (19). The
second procedure used was based on the method described
by Chinoy (16). The third method followed the sug-
gestions given by Hoffpauir (15).

In the first procedure the samples were refluxed
in a boiling water bath for twenty minutes with 100 m1.
of 95 per cent ethanol. The starch was solubilised
by adding 1 m1. of concentrated hydrochloric acid
through the top of the reflux condenser and boiling for

fifteen minutes longer. After cooling, the sample was

centrifuged to pack the plant residue. The ethanol-
hydrochloric acid was drasn off, and the residue was
washed twice with 25 ml. portions of hot 95 per cent
ethanol. next, the residue was boiled with 100 m1.

of distilled water for five minutes and then placed in
a boiling water'bath for thirty'minutes to dissolve the
solubilised starch. The ndxture eas cooled, centrifuged.
and.the supernatant liqudd.dooanted into a 250‘m1.
volumetric flask. Two 25 ml. portions of hot distilled
water were used to wash the residue, and the washings
were added to the solution in the volumetric flask.
Solubilisation was repeated with.50 ml. of 95 per cent
ethanol and 0.5 ml. of distilled water to dissolve any
remaining starch. The water extract and plant residue
were transferred quantitatively to the volumetric flask.
A rubber policeman was used to scrub the last traces

of starch from the centrifuge bottle. After cooling,
the contents of the flask were diluted to volume and
filtered.

Hydrolysis of the starch in the aqueous extract
was carried.out in the following manner. A 25 ml. ali-
quot was pipetted into a 60 ml. volumetric flask, 2
ml. of 0.2 3 sodium acetate buffer of p3 5.6, 4 ml. of
0.85rh sodium.chloride, and 4 ml. of saliva which had
been diluted 1:1 with water and filtered were added, and

the mixture was kept at 37-40° C. for four hours. After

the hydrolysis period, 1 m1. of saturated.noutrd1 loud
acetate was added to the digest to precipitate water.
soluble proteins und.noaponroou:drutc reducing sub-
stances. The flask was shaken and allowed to stand
about five minutes. fixcess lead was removed by adding
4 ml. of saturated disodium phosphate. the solution
was diluted to volume, mixed, and filtered. A second
25 ml. sliquot taken for s blank was treated in the
same say except that the diluted saliva eddcd.hud been
inactivated.by boiling for five minutes.

Eeducing values 0' the hydrolysed sample and blank
filtrates were determined as follows: Five ml. of the
solution, containing not more than 4 mg. of.ueltose,
and 5 ml. of alkaline potassium forricyanida, containing
8.25 g. of potassium forricyanide and 10.6 g. of sodium
carbonate per liter of aqueous solution, were mire; in
a 150x25 mm. Pyrex toot tube and.hootod in a boiling
water bath for exactly fifteen micutos. After cooling
the mixture by immersing the tube in cold running water
for three minutes, 5 ml. of 53 sulfuric acid were
added and thoroughly mixed with the contents of the tube.
Ten drops of detopaline C indicator were added, and the
solution was titrated to a golden brown color eith
0.0093 R eerie sulfate using a micro burcttc.

The eerie sulfate solution used to titratc the starch

extracts was standardised with glucose solution.

10

Standard glucose solution containing 1 mg. of glucoao

per ml. was prepared by diluting 10 m1. of 1 per cent
glucose solution to 100 m1. From 0.6 to 2.5 m1. of tho
diluted glucose salution were pipetted into large test
tubes and.diluted to 5.m1. with water. Five m1. of alka-
line ferricyanldo solfition was added to each tube {01‘
lowed by heating for fifteen minutes. Aftar cooling, 5
m1. of 5 n sulfuric acid and 10 drape of aetOpallno c
indicatOr were aéaed. ”fie aarnlea mare titrated wlfih
eerie sulfate in the same manner as the plant sample as»
tracts. It was found as shown inflfablo I that 3.820 ml.
of the corio sulfate was equivalent to 1 mg. of glucose.
In terms or maltase, 2.576 ml. or the eerie sulfate was
equivalent to 1.mg. heaaid {21) reported that 3.0 m1.

of exactly 0.01 fl cfirlo sulfata was equivalent to 1 mg.
of glucose. From this relationship, tho normality of
the ceria sulfate used in the present study was calculated

to be 0.0093.

$able I
Standardization 0! Carla Sulphate

 

 

 

1 ’ I W" I I T
0 E1. standard ' Hm. of ' $1. of ceric ' F1. of cario'
' glucose ' gluzaaa ' sulfate ' sulfate per '
' solution ' ' ' mg. glucose '
o ‘ n u o o
1W7 ' 1 m I - ' ' "'l
' 0.0 ' 0.5 ’ 1.600 ' 5.200 ’
' 1.0 . 10° ' 5.255 ' 30295 .
' 1.5 ' 1.5 ' 4.845 * 3.330 '
' 2.0 ' 2.0 ' 0.437 ' 3.214 '
: 2.3 ’ 2.5 ' 8.008 ’ 0.223 '

t t o I

3.1? o :5. 1:30

V 7

11

A linear relationship was found to exist between
glucose oxidised and.rsrrieysnide reduced under the ti-
trstion conditions used.in the present study. By plot-
ting the number of mg. or glucose used to standardise
cerie sulfato against the number of m1. of 0.0093 R
cerie sulfate required to reach titration end-point,

s strsight line scs obtained as shown in Figure I. This
result agreed with the findings or Hoinse snd,Murneek

 

(22).
Figure I
Glucccenccric Sulfate
Rolstionship
205 r
2.0 -.
M8. ,—
Glucose "5
|.0 -
0.5 ’— w
I 1 J I L l I l

 

 

J
I'2 345678
E1. 0.0093 R Ceric Sulfate

To calculate the reducing power in terms of mal-
tose, the not titration value of the plant extract,
obtained by subtracting the ml. of 0.0093 R ccric eulfate

. used to titrate the blank from that required for tho

 

hydrolysed aliquot, was divided by the number of.m1.
of 0.0093 8 eerie sulfate equivalent to 1 mg. 0! mol-
tose: i. 0.. 2.576. Ftnrcn equivolcnt one round by
dividing the smount of maltose by the hydrolysis limit
0.890, ss established by anoid, mcCroady, and Eoeonfels
(19). The shove calculations moy be combined in one
fornnls as follows:

(mi. 0.0093 a mi. 0.0093 3:: )

(eerie sulrsts‘_ coric sulfate)

(for sample for blank 3

(titration titration )x 100 -
_ x 100 ' fl Starch

 

 

.f ,M

as. or plant sample 1.0.890 x 2.576

For the second extraction procedure, that based
on tho method or Chino: (16), an arm‘Opriofe night
of plant eample in a 200:m1. Pyrex centrifuge bottle
fitted with o rotlux.condcnsor was booted with ?0'm1.
of 0.7 per cent potassium hydroxide for forty minutes
in s boiling water both. Aftor tho preliminary heating
period, the sample toe boiled fivo minutoe, cooled,
centrifuged, and the solution was decanted into a 280
m1. volumetric flask. The extraotion one then re-
poatod with 50 m1. of tho 0.7 per cont yotoooium.hyb
droxido. She ontirc contonto of the centrifuge bottle
were transforred to the volunotrio flock, diluted to
volume. and.filtorod.

Two 23 m1. uliquots of the filtrate were taken for

hydrolysis and for blank determination as outlined above

13

except that e B eoetio acid wee added to adjust the
eolution to e pH of 8.6 before addition or the diluted
enlive.

Ehe calcium chloride eolution used for the third
extraction procedure eee preyurod by adding a Lie of
0.8 per cont acetic acid.tor each 100 ml. of slightly
alkaline calcium chloride of 1.3 density as described
by Horfpauir (15). Five ml. of calcium chlorioe was
added to the eemple in e Pyrex test tube measuring 150x25
mm. The tube woe heated.for fifteen minutes in an oil
befib at 120” 0. During the heating period, the one.
peneion wee etirred thoroughly with a glass rod. the
eample one cooled. 80 ml. of water added, and the
tube contente were thoroughlyumlxed. After centrifuging,
the eupernetent liquid was decanted into a 230 m1.
volumetric flack. A second extraction woe carried out,
and.the eample wee tranefcrred quantitatively to the
volumetric flack end diluted to volume with water.

The eolution was filtered and 25 ml. or aliquote
were taken for enclysie. Dilute oodium.hydroxide solu-
tion wee eddod to adjust the eolutione to pfi 6.6. The
eolutione were treated ee before except that no eodium
chloride wee aooed prior to hydrolyeie. In the evelue-
tion of reducing power. the heating period and.manner

of titration were the eeme as described before.

14

However, the amounts of algalino potassium.ferri-
cyanide end of 5 N sulfuric acid were increased from
5 to 10 m1.

Recovery of added starch was determined on duplicate
samplee of alfalfa for each of the three extraction
methods studied. szctly 50 mg. of dry defstted starch
was mixed with 500 mg. of alfalfa which had been dried
at 50° C. otsrch extractione, hydrolysie, and deter—
mination of reducing power were carried out as previously

described. The resulte are given in Table II.

Table 11
Recovery 0! Starch from Alfalfa Dried at 50° 0.

 

 

 

 

50 mg. etsroh'

 

' 1 Ethanol-fial ‘ 0.5 per cent ' fislcium ‘TT
0 ' treatment ' potassium ' chloride '
3 Sample ' followed by ' hydroxide ‘ extraction ’
i ' hot water ' extraction ' of starch '
' ' extraction ' of starch ' ’
' ' ot_stsrch ' i '
1 I I T I
’ 500 mg. of ' 35.39 mg. ' 28.79 mg. ' 38.59 mg. '
. ‘lfau‘ ' 34.96 Fig. ' 27e16 Bible ' 38.63 mg. '
O 9 ! I I
' Average ' 35.18 mg. ' 27.97 mg. ' 38.51 mg. ’
1 I f V '
c v v 1 1 u
' 500 mg. of ' 87.24 mg. ' 71e63 mg. ' 92.90 11180 '
' alfalfa plus ' 86.80 mg. ' 72.86 mg. ' 02.70 mg. '
v c v v
' Average ' 87.02 mg. ' 72.10 mg. ' 92.80 mg. '
I i 9 t I
| T ”— 1 1 t
' Stardh ' 51.84 mg. ' 45.13 mg. ' 54.28 mg. '
' recovery ' 103.68 $ ' 90.26 g ' 108.56 6 '
f I 9 V !

 

1.“;

To test the solubility of pure starch in the extrac-
tion reagents etudied, 100 mg. of starch was weighed into
appropriate vessels and treated separately aith ethanol-
hydrochloric acid followed by hot water extraction, with
0.7 per cent potassium “geronldc; and with calcium
chloride solution. The starch enoaared to dissolve com.
pletely in the hot water and in the potassium hydroxide.
However, samples treated with calcium chloride formed a
gel at the bottom of the tubes which was difficult to
remove completely. On hydrolysis for two hours with
2 ml. of acetate buffer, 2 ml. of 0.25 N sodium chloride
solution, and 2 ml. of diluted saliva an outlined by
Rancid, vcflready, and "osenfele (19) and determination
of reducing values, erratic result. were obtained. The
results in terme or per cent starch for duplicate samples,
except in the case of calcium chloride treatment, are

shown in Table III.

Table III

Per Cent Starch in rure befatted éumplel

 

Kthanol-FCI

v 'W WI

 

1 1 “T
' ' 0.7 per cent ' Calcium '
' treatment ' potassium ' chloride '
' followed by ' hydroxide ' extraction '
' hot water ' extraction ’ '
'4_§xtraction ’ ' '
1 c r o
' 102.76 ' 45.18 ' L4.23 '
' 93.40 ' 77.34 ' '
I I I I

 

16

The aolubilizntion by ethanol-hydrochloric acid
followed with but water extraction and by 0.7 per cent
potassium hydroxide were repeated on 50 mg. of defotted
corn starch. Duplicate samples were analyzed in both
cases. In the h drolysic procedure, aliquots of the
starch solutions eere hydrolyzed for two houra with 2 m1.
of acetate buffer, 2 cl. or 0.;5 N sodium chloride soluu
tion, and 2 m1. of diluted saliva as described by Heeeid, -
McCreedy, and Rosenfelc (19). Second aliquots were
hydrolyzed for four hours with 2 m1. of buffer, 4 m1.

f sodium chloride, and 4 ml. of diluted saliva. Better
agreement of values was obtained with the longer hydro-
lysis period and increased amounts of reagents an shovn
in Table IV. OJ the basic of these exocrizental re-
sults, the extracts from the rlant concise studied were
hydrolyzed for four Laure with the larger volumes of
reagents except for sliéht variations in the procedure
for potassium hydroxide and for calciue chloride extrnetc

ac mentioned previously.

17

Table IV

Effect of Hydrolyeis Conditions on
Reculta of Analysis of Pure Starch

 

 

 

OCQCOQCQCQO¢Ouf-“Od

o 1 ~
~ ' Fthnnol~HCl ' 0.7 per cent
Hydrolysis '- treatment ' potassium
Conditions ' followed by hot ' hydroxide
'eeter extraction ' extraction
9 ‘ 0
"AW I # “‘"I
2 hours with 2 ml.’ ' 95.10 % ' 88.?0 5
buffer, 2 kl. ' 91050 g ' 96031 g
sodium chloride, ' '
end 2 ml. dilute ' '
saliva ' ‘
1 ,1
k “* v * * “fi ' *fi
4 hours with 2 ml.‘ 97.75 % ' 95.51 fl
buffer, 4 ml. e' 97.03 4 ' 95.66 g
sodium chloride, ' '
and 4 m1. dilute ' '
celivn " '

¢.o¢0.-ocaoo--nocod

 

313111.733 A?!) DISCUSSION

The percentages of otoroh round by each of the
three solubilising prooodurss described shove and ob-
tained for each of the four plant substances used or.
31m in Tables V, VI. VII, sod VIII.

Methods for cramming samples for analysis wars
«looted from tho literature and represented the pro-
ooduros commonly used to dry snd grind plant mtorisls.
Ono portion or ouch plant material otudiod on air-.-
drisd st room tmporoturs to simulate as nearly on
possible the drying or boy craps in the field. Drying
at 50° 0. in a com room equipped oith n ventilating
ton snd at 85-90" c. in an even more used to determine
the effect or temperature on starch content. A part of
tho fresh plant materials sos from before final drying
at ass-90° C. to determine the effect or low temperature
on starch dissolution in the plant materials and also in
no effort to rupture the cell soils and thus make starch
extrsotion more complete. occupies dried at room tempers-
tors snd st 50° 6. had a green or yellow-green color
whores. samples dried at 825-90" C. chewed little or
no browning with the exception of bromo groan which
turned dork brm. Samples which were frozen turned
dork brain on drying in the oven.

The dried samples were ground to (SO-mean as

19

Table V

Per Cent Starch in Alfalfa
Calculated to Dry weight Basis

(Each value given represents a separate sanple)

 

1

-C...‘OOCCGQQCC‘O‘CQ.OOOQO-.C.

 

 

 

 

w T 1 7' v Q
' Ethan01~VCl ' 0.7 per cont' Calcium
Sample ' treatment ' potassium. ' chloride
' followed by ' hydroxide ' extraction
' hot water ' extraction '
' extraction ' '
a 9 r
w v I “W WW I A
Air-dried ' 1.121 ' 1.167 ’ 1.027
at room ' 1007‘ . 1.074 ' 0.935
temperature ' 1.110 ' 1.135 ' 1.192
' 1.110 ' 1.000 ' 1.117
I C O
T V f T r T fir? V"
warm room. ' 6.941 ' R.F!3 ' 7.786
at 50° C. ' 7.127 ' 6.988 ' 8.053
' 7.b11 ' 6.071 ’ 7.781
' 7e‘24 ' 5.118 ' 7.963
O O O
' A * i T“ ‘ I * * I
Oven-dried ' 6.706 ‘ 5.429 ' 6.569
at B5°~90° c. ' 6.567 ' 5.159 ' 7.664
' 6.72: ' 4.434 ' 6.72
' 6.589 ' 4.292 ‘ 7.000
O f I
___u l l l w
Frozen and ‘ 7.02d ‘ 5.709 ' 8.097
oven-dried ' 7.675 ' 5.148 ' 7.424
at 850-900 0. ' 7.24. ' 4.:nc ' 7.770
0 7.454 ' 4.445 ' 7.965
t o v

 

..--.--.-.“.‘.‘..-.-‘--.--“-‘

“table 71’

Per Cent utarch in Reed Canary Greee
Calculated to try icight Basil

(Lech value given represents a separate eemple.)

 

 

 

 

 

‘.C-.---‘.‘--.‘.--‘.----O‘CCCQA

“‘ W‘ I * v 1 ~ ** c

' hthnnoluHCI ' 0.7 per cent Calcium ’

figmple v treatment ' potassium ' chloride '

' £0110wcd by ' hydroxide ' extraction '

' hot water ~ ' extraction ' '

' extinctién ' ’ '

I I C I

1 1 ““ 1 ‘7 u

Air-dried ' 0.?fiR ! 0.463 9 0.347 c
at room ' 0.330 ' 0.371 ' 0.440 '
temperature ' 0.255 ’ 0.343 ' 0.595 '
' 0.730 ' 0.255 ' 0.278 '

c I o v

7 I V T I

Larm roan ' 0.322 ' .324 ' 0.524 '
at 50° C. ' 0.370 ’ 00231 . 0.070 .
' 0.594 ' 0.022 ' 0.302 '

: 0.300 ' 0.237 ' 0.370 '

v c t

1,1 1‘ c v— v .
Ovon-drled , 0.985 ' 0.6?2 . 1.745 '
at 8530-900 C.’ 0.036 ’ 0.575 ' 0.885 '
' 0.885 ' 0.096 ' 0.956 '

' 0.898 ’ 0.478 ' 0.862 '

a o o 0

V v I 1. T 1
Frozen and ' 0.468 ' 0.608 ' 0.374 '
oven-dried ' 0.508 ' 0.306 ' 0.258 '
at 350-900 ' 0.53? v 0.515 0 0.422 v
C. ' 0.444 ' 0.501 ’ 0.505 '

c n o v

 

‘7‘}

Table VII

For Cont Starch in Urchard Crass
Calculated to Dry Height basic

(Each value given represents a separate sample.)

 

 

 

 

 

C‘..--.-.‘.-.Q‘CCOO-“---‘-‘..‘

1 1 T w 1

. rthfln01-nc1 c 0,? nor oent' Calcium ‘

Sample ' troatmcnt ' potnceiwm ' ohloridc‘
‘ followed by ' hydroxide 'extraction'

' hot water ' extraction ' '

' extraction ' ' '

O O I i

I l W 1 !

Al. r-dl’i Qd . 0 e 303 ' Co 2342 . 00 573 .
fit r0011} ' OeOEG . 0.0250 ' 0.257 '
temperature ' ;.37 ' 0.534 ' 0.348 '
' .32 ' 0.276 ' 0.422 '

I I Q 7

.1 ~ “ “a . v " 3

term room ' Gui-)0“ . 0e£5U . 0e45,, '
at 50° c. ' 0.46? ' .560 - ' 0.554 '

' 0.73" ' 0.479 ' 0.561 '

' 0.586 ' 0.562 ’ 0.b56 '

v ' : c c

, ‘7 1‘ [A ’ 1 '
O'I'Gn-dr:eld . C o 3:58 . a 3: 2 . 0. 447 .
at 850-900 ' 0.255 " 0.208 ' 0.517 '
C.‘ ', 0.505 ' 0.210 ' 0.074 '

' ' 0.5548 ' . 0.230 . 0.468 .

Q 7 0 7

7 A .__ T l “ i “ I
Frozen and ’ 0.329 ' 0.135 ' 0.507 '
oven-dried ' 0.936 ’ 0.501 ' 0.460 '
at 850-00” v -0.395 ' o.':2 ' 0.533 '
C. ' 2.345 ' 0.198 ' 0.488 '

I I l O

 

1

22

Table VILI

Per Cent boaron 1n aroma Grass
Calculated on Dry Weight 0351:

(Each valuo given represents a separate sample.)

 

 

 

 

 

.‘C‘.’-‘CQ---.‘--‘-.-‘-QCC‘C‘Cfi’

l

1* r ._ I I
' Ethanol-£01 ' 0.? par cont ' Calcium
Sample ' treatment ' potassium. ' chloride
' followed by ' hydroxidc ' extraction
' hot water ' extraction '
’ extraction ' '
o s a
1‘ T‘LT; ‘ T "
Air-dried . 0.237 ' 00518 . 00250
at room ' 0.??? ’ 0.227 ' .272
temporaturc ' 0.955 ' 0.206 ' 0.275
' .230 ' 0.193 ' 0.299
O Q v
a T 1 4 v v
warm room ' 0.365 ' 0.205 ' 0.434
at 50° 0. ' 0.519 ' 0.030 ' 0.300
' 0.033 ' 0.304 ' 0.346
' 0.370 ' 0.208 ' 0.416
O O I
t i T,
Oven-dried ' 0.301 ' 0.162 ' 0.346
at 850-900 ' 0.277 ' 0.182 ' 0.524
' 0.200 . 0.300 ' QRQQ
' 0.293 ' 0.200 ' 0.568
O I O
o 7 1
Frozen and ' 0.375 ' 0.141 ' 0.445
OVfin‘drifld . 0032' ' 0.164 . 00468
at 850-900 ' 0.540 ' 0.140 . 0.418
' 0.372 ' 0.253 ' 0.372
I I !

 

.“-‘--’-------.“------Q.-‘-QA

25

recommended by Hassid, McCready, and Rosenfels (19).
Chinoy (16) ground samples to looemesh whereas Hoffpauir
(15) suggested grinding to finer than BO-mesh. Malhotra
(23) determined the effect of fineness of material on
chemical analyses and found that the point of diminishing
return occurred at 60-mesh. In grinding the samples for
the present study, some of the woody structural portions
of the plant materials remained on the screen of the mill,
but in general, the plant samples were fairly fine and
powderelike.

In the evaluation of reducing power of solutions from
calcium.chloride extraction, a white precipitate formed
on addition of alkaline potassium ferricyanide which dis-
solved readily in 5 N sulfuric acid. The effect of this
precipitation on the reaction of the reducing components
with the potassium ferricyanide was not investigated.
However, to insure an excess of alkali and potassium
ferricyanide, the amount of alkaline potassium ferricyanide
added was increased from.5 to 10.ml. Subsequently, 10
ml. of 5 N sulfuric acid was added to maintain the same
relative acid concentration in the final titration.

In the present study, salivary amylase was used as
the hydrolytic agent for starch in the extracts obtained
with three different reagents. Salivary amylase is
active in the presence of fairly high salt concentra-

tions. Sodium, potassium, and chloride ions activate

the hydrolytic action whereas Kneon, Sandstedt, and
iollenbeck (24) report that calcium ions exert a stabi-
lizing effect on 0¢-amylaees.

There is soms_disagrcement among various authors
concerning the final products of salivary amylase hydro-
1y31. of starch. Swanson (25) resorted slight maltase
activity after incubating corn starch with salivary
amylase for more than twenty-four hours. Bourne, Hayworth,
Haney, and Pest (26) found no increase in reducing power
when salivary amylase was incubated with maltose, indicat-
ing the absence of saltaee. In studying the end products
resulting from.the action of salivary otoamylase on potato
amylase, Roberts and whelan (27) found a mixture of mal-
tose and maltotriose in the molecular preportion of
2.39:1. hassid, EcCready, and Fosenfels (19) calculated
the reducing values found in terms of maltose assuming
that no glucose was formed. Also, the last named authors
found that less than 1 per cent of dextrins remained
after hydrolysis of starch mien salivary amylase.

The limiting hydrolysis value of 0.89 for salivary
amylase was found by Hassid, KcCready, and Posenfels (19)
to be constant for starches from different sources and of
varying concentrations. Also, the hydrolysis limit ap-
peared to be constant for salivary amylase from different

individuals. Gherefore, it was reasonably safe to assume

:5

that the limit held for the present analyses.

Results from ssnples cried in different ways, Tables
V through VIII, show some variations which seem to be
dependent on the type of plant material studied. Alfalfa
which was frozen and then dried contained more starch than
samples prepared by the other curing methods. the oven-
dried sample of reed canary grass had a higher content
of starch whereas the sample of orchard grass cured at
50° 0. contained a greater amount of starch. in the case
of brome grass, the different drying procedures had little
effect on the starch present. All of the plant varieties
had the least amount of starch in the air-dried samples.
The analytical results for the frozen samples might be
explained on the basis of findings of npoehr and Lilner
(3) that some plant materials show an increase in rate
of starch dissolution with a decrease in temperature.
Treatments which destroy protonlesm, such as freezing, do
not necessarily destroy enzyme activity. However, in
general, no starch dissolution occurs perhaps because of
formation or deposition of some sort of protective
materiel around the starch granules on destruction of
protOplssm. Spoehr and Nilner (5) also found that loss
of water from leaves, as in tilting, causes an increase
of starch dissolution which may account for the low starch
content of the air-dried samples. Since amylase activity

seems to follow the reaction rate-temperature rule, i. e.,

.50

a 10 degree increase doubles the rate of reaction, at
temperatures which do not destroy the enzyme, the rate
of starch dissolution should‘be greater in plant samples
dried at 50° O. and at 85-900 C. than at room temperature.
However, if amylase were capable of hydrolysing starch
for s much shorter period of time at elevated tempera-
tures than at room temperature as a result of enzyme
destruction or formation of s protective materiel around
the starch granules, then one might expect 0 higher
starch content in samples dried in.the warm room and dry-
ing even than in those which were sir-dried.

ln general, starch values of samples extracted with
calcium.chloride were higher and.thcse from 0.7 per cent
potassium hydroxide were lower then those from ethanol-
hydrochlcric acid treatment followed by‘hot water ex-
trsction. Tbs blank titrations from ethanol-hydrochloric
acid treatment followed by hot water extraction were
less than 1 ml. of 0.0093 H ceric sulfate indicating the
presence of a small amount of non-starch reducing sub-
stsncs. Blank titrations from 0.7 per cent potassium
hydroxide and from calciun.chloride extractions were
rather high, ranging from 1 ml. to 6 ml. of 0.0093 R eerie
sulfate. Hemicelluloss and poetic substances were pro-
bably extracted with dilute potassium hydroxide. Also,
any sugars present would be extracted and might reset

with the potassium hydroxide. The calcium chloride

27

solution used was acidic and may have reacted with hemi-
celluloss and pectic substances to release reducing
materials. Also, sugars in the plant materials would be
extracted. In both potaeaiun hydroxide and calcium
chloride extraction, a considerable amount of the chloro-
phyll in the plant samples was extracted as evidenced

by green solutions. Addition of lead acetate after en-
zymatic hydrolysis would remove much or the reducing sub-
stances present. flowever, any sugars and other subatances
not precipitated by lead would be determined in the blank
titratione. The magnitude of the various blank titrations
tram potassium hydroxide and calcium chloride extractions
may reflect the amount of sugars present in the plant
samples.

Starch values from ethanol-hydrochloric acid treat-
ment followed by extraction with hot water are more re-
producible than those from the potassium hydroxide and
calciumichlcrids extractions. one rcason.may be the re-
latively high blank titrations in the latter two methods,
particularly in the case of reed canary grace, orchard
grass, and bromo grass which contained less than 1 per
cent of starch and required lees than 0.2 ml. of 0.0093
R oeric sulfate net titration.

In View of findings presented, the procedure, other
than sir-drying, need in curing plant samples for starch

analysis would depend upon the type of plants studied.

The extraction procedure of hasaid, PcCready, and Rosen-
fels (19) is more satisfactory than extraction with
either potassium hydroxide or calcium.chloride because

of the small amount of reducing substances present in

the extract. Also, more reproducible atarch values and
better recovery of added starch acre obtained by ethanol-
hydrochloric acid treatment and water extraction than by

other extraction procedures.

1.

2.

3.

4.

5.

6U: {35/}. RY

samples of alfalfa, reed canary grass, orchard
press, and brows rrase were taken on warm, clear
afternoons from unfertilized and unirrigated
experimental plots of the Department of Soil
Science.

fihe plant samples were each divided into four
portions for drying at room temperature, at

50° 0., at 35-90" c.. and freezing followed by
drying at 85-30” 0.

After grinding the dried samples to 60-mesh,
starch was extracted by three different procedures
involving: (1) solubilisation with ethanol-
hydroohlorio acid followed by hot eater extrac-
tion; (2) 0.7 per cent potassium.hydroxide ex-
traction; and (3) calcium.chloride extraction.
Starch in the extracts was hydrolysed with sali-
vary amylase and reducing power was determined
by treating the hydrolysed extract with alkaline
potassium ferricyanide followed by titration

of ferrocyanide formed with standard ceric sul-
fate solution.

Analytical results indicate that the choice of
curing procedure to preserve the greatest amount
of starch is dependent upon the particular plant

studied.

6.

Beet reproducibility and agreement of starch
values for a specific plant sample were obtained
by the treatment of plant material with ethanol-
hydrochloric acid followed by extraction of
solubilized starch with hot water.

(1)

(2)

(3)

(4)
(5)

(6)
(7)
(8)

(9)

(10)
(11)

(12)

(15)

(14)
(15)
(16)
(17)

(18)

LITLEATURE CITED
Brown, H. T., and.Morris, G. H., J. Chem. 800., gg,
604 (1893).

Bpoehr, H. A., and Milner, H. w., J. Biol. Chem.,
111, 679 (1935).

Spoehr, H. A. and Milner, H. W., Proc. Am. Phil.
300., 53;, :57 (1959).

Flueckiger, F. A., Z. pherm.,'§, 185 (1860).

Fellenberg, T. u. von, mitt. Lebensm. Hyg.,‘z, 369
(1916).

Ibido’ g, 55 (1917).
me” 33, 51 (1929).

Denny, F. E., J. Assoc. Offic. Agr. Chem., 1, 175
(1922)

Donn , F. E., Contrib. Boyce Thompson Inst., g,
129 1934).

Ibid. , 381 (1934) .

Rack, 0. 8., J. Assoc. urtic. Abr. Chem.,‘19, 108
(1927).

Walton, G. P., and Cos, V. R., J. Agr. Research,.§§,
995 (1925).

Sullivan, Jo Fe, A8300. Office Agr. Chem., E. 621
(1935). '

Hoffpauir, c. I... Ibid., 323, 291 (1949).
1mm, 3:3, 910 (1950).
Chinoy, J. J., Analyst, §§, 876 (1958).

Niemenn, 0., Roberts, R. H., and Link, K. P., J.
Biol. Chem., 110, 727 (1955).

Hana”, Co 8., 3106118111. JO, Q, 168 (1936).

 

 

(19)

($30)
(21)

:‘d
(‘3

flaccid, b. 3., KcCreedy, H. n., and Eocenrele, R. 5.,
Ind. 1.1.18. 01131510, 3.1.1151. 31.3., .33, 1‘82 (194.3)-

33110011. T. J" J. Am. 02191.1. 306., 12' 11.1.5 (194.2).

flaccid, E. Ind. Eng. Chem., Aral. d.,'2, 088

(1937).

'I
:14. ’

Heinze, P. 8., and nurneex, A. n., hissouri Agricul-
tural zxpcrimcnt Station, research hulletin 314, 19,
(1940).

yelhotrs, R. 0., Ind. E w. Chom., Anal. Ld.,Ԥ,
398 (1950).

Knees, 3., Scndstcflt, R. V. and Pollenbcck, C. R..
Cereal Chem.,|§§, ’39 (1943 .

Swanson, fl. A., J. Biol. Chem.,llzg, but (1948).

Bourno, L. J.,
J. 13:23:21. 000.,

fisyworth, H., Kacey, 3., and Feet, 8.,
Part 11, 984 (1948).

fiabflrta. 3’. :0 ”I.

873‘ “Elfilflng W'we uTe. v.10C1‘Ume JO.
3:, 1v1 (1951).

ABSTRACT

A STUDY OF EFFECTS OF SAMPLE PREPARATION AND
MEEHODS OF EXTRACTION 0N DETERMINATION
OF STARCH IN PLANT MATERIALS

By
Betty Ruth.Johnston

The present study was made to determine the effective-
ness of some or the analytical procedures for the deter-
minstion or small amounts of starch in plant materials
with.speeis1 attention given to preliminary sample pre-
paration, completeness of starch extraction, and.evalus~
tion of starch with the exclusion of other carbohydrates.

Samples of alfalfa, Medicggo sativa, Just before
blooming and second grosths of brcme grass, Bromus inermis,
reed canary grass, ghalaris arundinacea, and orchard
grass, Dactzlis lomerats, were taken on warm, clear
afternoons tron.unfertilised and unirrigated experimental
plots of the Department of Soil Science. The fresh plant
samples were each divided into four portions for drying
at room.tmmpersture, at 50° 0., at 85-90° 0., and trees-
ing followed by drying at eo-ao° c. After grinding the
dried samples to 60~mesh, starch was extracted by three
different procedures involving: (1) solubilisaticn.sith
ethanol-hydrochloric acid followed by hot water extrac-
tion as described by Hassid, McCready, and Rosenfels;
(2) 0.7 per cent potassiun.hydroxide extraction as used

Betty Ruth Johnston

by Chinoy; and (3) calcium chloride extraction as out-
lined by Hoffpauir. Starch in the extracts was hydrolysed
with salivary amylase. Reducing power was determined

by treating the hydrolyzed extracts with alkaline potassiun
ferricyanide followed by titration of ferrccyanide formed
with standard ceric sulfate solution.

Analytical results indicate that the choice of curing
procedure to preserve the greatest amount of starch is
dependent upon the particular plant studied. Best re-
producibility and agreement of starch values for a speci-
fic plant sample were obtained by the treatment with
ethanol-hydrochloric acid followed by extraction of
solubilised starch with hot water as described by Hassid,
Hccready, and Rosenfels (1).

Literature Cited

(1) Hassid, W. 2., McGready, R. Eh, and Rosenfels, R. E.,
Inde Enge Chane. Angle Ede, 2...?" 1‘2 (1940).

(2) Chincy, J. 3., Analyst, _6_§, 8'76 (1958).

(5) Hoffpauir, C. L., J. Assoc. Offic. Agr. Chem, g,
810 (1950).

HICHIGQN STQTE UNIV. LIBRRRIES

 

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