Extraction of Total Available Carbohydrates from
Grass and Legume Tissue " 2
Dale Smith, G. M. Paulsen, and C. A. Raguse
Department of Agronomy, University of Wisconsin, Madison
Carbohydrates are the primiiary source of reserve
energy stored in the vegetative organs of biennial and
perennial forage plants. Reserves of available carbohydrates are essential to survival and to the production
of plant tissues during periods when carbohydrate
utilization exceeds photosynthetic activity.
Total available carbohydrate3 levels and trends in
storage organs are useful in indicating the periods of
storage and usage and are useful in evaluating the
potential of plants for regrowth and production following defoliation. A number of nmethods have been
used to extract total available carbohydrates from
forage plant tissue. However, the method of extraction use(l will influence greatly the values obtained
because forages vary in the type of carbohydrate
stored.
The common perennial and biennial forage
legumes, such as alfalfa, red clover, wlhite clover, and
sweetclover, are characterized by the accumulation of
sucrose and starch (3, 9, 11 ). Extraction of total
available carbohydrates from these species has been
accomiiplished largely by the takadiastase enzyme
method developed by Weinmann (16,17) or with acid
solutions (8, 10). The common perennial forage
g,rasses belong to 2 groups according to the type of
reserve carbohydrate stored. Grasses native to tropical and subtropical latitudes, such as bermudagrass,
bahiagrass, and dallisgrass, are characterized by the
accumulationi of sucrose and starch. Extraction of
total available carbohyclrates has been done as cdescribed above for legumes (17, 18). Grasses native
to temperate latitudes, such as timothy and orchardgrass, accunmulate sucrose and fructosan ( 1, 4, 7, 12,
13). Extraction of total available carbohydrates has
been accomplished by acid hydrolysis (8) or with
water (1, 14, 15) since fructosan is soluble in water.
Starch, however, is largely water-insoluble.
This stucly was initiated to compare enzynme, dilute
acid, and wxater extraction methods for the estimation
of total available carbohydrates in forage plant tissue.
Timothy stem bases and alfalfa roots were used as the
test tissues. Timothy stores the largest proportion of
its reserve carbohydrates as fructosan in the stem
bases (4, 7). Alfalfa stores most of its reserve carbohydrates as starch in the roots (3).
1 Received March 2, 1964.
2 Published with approval of the Director of the Wisconsin Agricultural Experiment Station.
3 Total available carbohydrates in this paper are the
carbohydrates that are readily available as a source of
energy to the plant. Included are the sugars, dextrin,
starch, and fructosan but not structural carbohydrates,
such as hemicellulose and cellulose.
Materials and Methods
Selected samples of timothy (Phlemn pratenisc L.)
stern bases and of alfalfa (Medicago satiza L.) roots
were used. Timothy samples were from a season of
growth undler 2 harvests for hay, June 27 anid August
29 (8). Alfalfa samples were from the early part
of a season under a hay harvest on June 3 (10).
The fresh tissue was dried in a forced-clraft oven
at 1000 for one hour and completed at 700. The
dried tissue was ground to pass through a 40-mesh
sieve, placed in glass bottles, and dried at 70° to constant weight. The bottles were tightly capped, and
stored for analysis.
Enzyme extraction Nas accomplished accordling to
the method of Weinmann (16) using the enzyme
prepara,tion and modifications of Lindahl et al (6).
Essentially the sample was mixedI with water and
refluxedl in a boiling water bath to gelatiniize any
starch.The mixture was then incubated at 380 for 44
hours with a bufferedl takadiatase (Clarase) enzyme
preparation. Proteins were remooved with neutral
lead acetate and the excess lead was removed with
potassium oxalate. A 10 ml aliquot of the filtere(d
solution was hydrolyzed with 0.5 ml of 25 (c. HCl for
30 minutes in a boiling water bath. The solution was
cooled, neutralized with NaOH solution, and diluteJ
to volume with distilledI water.
Acid extractions wx ere made with solutions ranging
from 0.2 N to 0.8 N H.SO4. Results are reported
only for the 0.2 N and 0.8 N extractions. Eaclh sample
was placed in a 200 nml round-bottom flask with 50
ml of acicl, and refluxed for 60 mlinutes in a boiling
water bath. The hot solution wxas filtered througlh
Whatman No. 42 filter paper. Proteins xx ere not
removed. The filtrate was cooled, neutralizecl wvith
NaOH solution, anl (dIluted to volume xvith (listilledl
wvater.
Water extractions were macle with hot aind cold
water. Each sample was placed in a 200 nml roundbottom flask with 50 nml of distilled water. The hotwvater samples were attached to reflux condensers and
refluxed 1 hour in a boiling water bath. The coldwater samples were stoppered and placed on a Burrill
shaker for 1 hour at room temperature. The solutions
xvere filtered through Wlhatman No. 42 filter paper,
and the residlue was washed with 25 ml ot (listilledl
wvater. Proteins were not removedl. One nml of 25 %
HCl was added and the solutions were hydrolyzed for
30 minutes at 750. The solutions were cooled. neutralized with NaOH solution, and (lilutedl to volume
with distilled water.
960
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961
SMITH ET AL.-TOTAL AVAILABLE CARBOHYDRATE EXTRACTION
Carbohydrate content of the above solutions obtained by the various extraction procedures was determined by analyzing for reducing power with the
Shaffer-Somogyi copper-iodometric titration method
outlined by Heinze and Murneek (5) using fructose
standard solutions for the timothy and glucose standards for the alfalfa.The results were expressed as per
cent total available carbohydrates on a dry weight
basis (700).
Results
The enzyme method of extraction for total available carbohydrates was used as the check method.
Starch in this method is extracted and partially hydrolyzed by the takadiastase enzyme preparation.
Soluble sugars and fructosan are extracted during
incubation with the enzyme because of their solubility
in water. Complete hydrolysis of the extracted carbohydrates to reducing sugars is accomplished subsequently w-ith acid, and the reducing power of the
neutralized hydrolysate is determined. Thus, both
starch and fructosan are a part of the total available
carbohydrate content.
The trends of total available carbohydrates in the
15 timothy stem base samples are shown graphically
in figure 1, and in the 10 alfalfa root samples in figure
2. The extraction method with values most nearly
like those of the enzyme method was extraction with
0.2 N H2SO4.
Each method of extraction, except for the water
extractions of alfalfa roots, provided results that
showed the same trend and indicated the same periods
of increase and decrease of carbohydrates. The principal difference among extraction methods was the
level of values obtained. In the case of the water
extractions of the alfalfa roots, the values did
not follow exactly the trend as established by the acid
and enzyme extractions. This was probably because
only free sugars were being extracted and that the
insoluble starch remained in the residue.
Okajima and Smith (7) with these same samples after
extraction of the sugars with 80 % ethanol and of the
fructosan with cold water. The differences in values
between the water extractions and enzyme extractions
of timothy were due probably to insoluble carbohydrates, possibly starch, that were not being removed
with water but were removed under the conditions of
the enzyme method. The values averaged 5.0 %
lower for hot water and 6.9 % lower for cold water
than the values from the enzyme method. Okajima
and Smith (7) obtained similar values (averaging
2.7 %) with these same samples when the residue
following extraction of the sugars (with 80 % ethanol) and of fructosan (with cold water) was treated
with the enzyme method of extraction.
The water extractions of the alfalfa removed only
the free sugars. The values were approximately the
same as those obtained with the 80 % ethanol extraction (unpublished data). The insoluble carbohydrates, including starch, remained in the residue.
Slightly higher values were obtained consistently
with hot water than with cold water extraction as
shown clearly in figures 1 and 2. The average dif-
Discussion
Extraction with 0.2 N HSO4 gave carbohydrate
values that were very similar to those of the enzyme
extraction in both timothy and alfalfa. The acid
values averaged only 0.4 % higher for the timothy
and only 0.5 % higher for the alfalfa than the enzyme
values. Extraction with 0.8 N HSO4 gave values
that were considerably higher than with 0.2 N HSO4.
The stronger acid solution was probably extracting
structural carbohydrates, such as the hemicelluloses.
Much less time was involved during extraction
with 0.2 N H2SO4 than with the enzyme method even
though the carbohydrate values obtained were similar.
Acid extraction required about 8 hours for a complete
analysis compared with 12 hours working time plus
44 hours incubation for the enzyme method.
The water extractions gave lower values than the
enzyme method in both timothy and alfalfa. With
timothy, the water extractions were removing both
the free sugars and fructosan. The values were approximately the same as the total values obtained by
APRIL MAY
JUNE JULY
AUG
SEPT
OCT
FIG. 1. Per cent total available carbohydrates in
timonthy stem bases with various methods of extraction.
ference was 1.9 % for timothy and 0.8 % for alfalfa.
The difference may be due to the greater solubility of
a relatively insoluble carbohydrate, possibly gelatinized starch, in hot water.
The determination of total available carbohydrates
by analyzing for water-soluble carbohydrates (free
sugars and fructosan) will give results similar to
those obtained from the enzyme method only when
starch is absent in the tissue or when starch is present
only in very small amounts. The problem is that
fractionations have been made on some legumes and
on only a few grasses so that it is not known to what
extent they store starch and/or fructosan. If starch
is stored in any appreciable amounts, the results of
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Copyright © 1964 American Society of Plant Biologists. All rights reserved.
962
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PLANT PHYSIOLOGY
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FIG. 2. Per cent total available carbohydrates in alfalfa roots with various methods of extraction.
the enzyme extraction method and of water extraction
will not be comparable. Dodd and Hopkins (2) recently reported as much as 11 % starch and 19 %
fructosan in the roots, rhizomes, and crowns of blue
grama grass (Boutelouta gracilis) in the fall growth.
The best method of extraction in (letermining total
available carbohydrates would appear to be the enzyme
or 0.2 N H11S04 extraction methods because the conditions of both methodls will inclu(le the sugars, starch,
and fructosan in the final carbohydrate values.
7.
8.
9.
10.
Summary
A comparison was made of enzymiie, dilute acid,
and water extraction methods for the estimation of
total available carbohydrates in timothy stem bases
and alfalfa roots.
Extraction with takadiastase enzyme an(l 0.2 N
H.,S04 nmethods gave carbohy(drate values that were
very similar, and the conditi.ons of both methods are
such as to include the sugars, starch, and fructosan
in the final carbohydrate values. Extraction with a
stronger acid solution gave higher values probably
because structural carbohydrates were being hydrolyzed. A1uch less total analytical time was involved
in the acidI extractions than wvith the enzyme; something on the order of 8 hours compared with 56 hours.
Carbohydrate values were slightly higher with hot
water than with cold water extraction.
The use of water extraction to estimate total available carbohydrates gave values of the same physiological significance as the enzyme method only when
starch was absent or present in very small amounts.
11.
12.
13.
14.
15.
16.
17.
Literature Cited
1. BAKER, H. K. AND E. A. GARWOOD. 1961. Studies
on the root development of herbage plants. V.
Seasonal changes in fructosan and soluble sugar
18.
contents of cocksfoot herbage, stubble and roots
under 2 cutting treatments. J. Brit. Grassland
Soc. 16: 263-67.
DODD, J. D. AND HOPKINS, H. H. 1958. Yield and
carbohydrate content of blue grama grass as affected by clipping. Trans. Kansas Acad. Sci. 61:
280-87.
GRABER, L. F., N. T. NELSON, W. A. LUEKEL, AND
W. B. ALBERT. 1927. Organic food reserves in
relation to the growth of alfalfa and other perennial
herbaceous plants. Wisconsin Agr. Exp. Sta.
Res. Bull. 80.
HARPER, R. H. AND T. G. PHILLIPS. 1943. The
composition of timothy. II. Storage organs. New
Hampshire Agr. Exp. Sta. Tech. Bull. 81.
HEINZE, P. H. AND A. E. MURNEEK. 1940. Comparative accuracy and efficiency in determinaticn of
carbohydrates in plant material. Missouri Agr.
Exp. Sta. Res. Bull. 314.
LINDAHL, I., R. E. DAVIS, AND W. 0. SHEPHERD.
1949. The application of the available carbohydrate metho(d to the study of carbohydrate reserves of switch cane (Arunidinaria tecta). Plant
Physiol. 24: 285-94.
OKAJI'MA, H. AND D. SoJITH. 1964. Available
carbohydrate fractions in the stem bases and seed
of timothy, smooth bromegrass, and several other
northern grasses. Crop. Sci. 4: 317-320.
REYNOLDS, J. H. AND D. SMITH. 1962. Trend of
carbohydrate reserves in alfalfa, smooth bromegrass, and timothy grown under various cutting
schedules. Crop Sci. 2: 333-36.
RUELKE, 0. C. AND D. SMITH. 1956. Overwintering treinds of cold resistance and carbohydrates in
medium red, ladino, and common white clover.
Plant. Physiol. 31: 364-68.
SMITH, D. 1962. Carbohydrate root reserves in
alfalfa, red clover, and birdsfoot trefoil under several management schedules. Crop Sci. 2: 75-8.
SMITH, D. AND L. F. GRABER. 1948. The influence
of top growth removal on the root and vegetative
development of biennial sweetclover. J. Am. Soc.
Agron. 40: 818-31.
SPRAGUE, V. G. AND J. T. SULLIVAN. 1950. Reserve carbohydrates in orchard grass clipped periodically. Plant Physiol. 24: 92-102.
SPRAGUE, V. G. AND J. T. SULLIVAN. 1952. Reserve carbohydrates in orchard grass cut for hay.
Plant Physiol. 28: 304-13.
\VAITE, R. AND J. BOYD. 1953. The water-soluble
carbohydrates of grasses. I. Changes occurring
during the normal life cycle. J. Sci. Food Agr. 4:
197-204.
WAITE, R. AND J. BOYD. 1953. The water-soluble
carbohydrates of grasses. II. Grasses cut at grazing height several times during the growing season.
J. Sci. Food Agr. 4: 257-61.
WEINMANN, H. 1947. Determination of total
available carbohydrates in plants. Plant Physiol.
22: 279-90.
WEINMANN, H. 1961. Total available carbohydrates in grasses and legumes. Herbage Abstr.
31: 255-61.
WEINMANN, H. AND L. REINHOLD. 1946. Reserve
carbohydrates in South African grasses. J. S.
African Botany 12: 57-73.
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