JFS C: Food Chemistry and Toxicology

Effects of Solvent, Temperature, Time, Solvent-

to-Sample Ratio, Sample Size, and Defatting on
the Extraction of Soluble Sugars in Soybean
ENZO GIANNOC
NZO CAR
IANNOCCARO, YA-JANE WANG, AND PENGYIN CHEN
CARO

C: Food Chemistry & Toxicology

ABSTRA
ABSTRACT CT
CT:: E xtr
Extr action solv
xtraction ent, temper
solvent, atur
temperatur e, time
ature time,, and solv ent-to-sample rratio
solvent-to-sample atio wer
wer e studied to identify the opti-
ere
mal conditions for extr acting sugars fr
extracting om nondefatted so
from ybean. Water and 10%, 50%, and 80% (v/v) ethanol w
soybean. er
wer
eree
used as the solvents. Extraction temperatures of 25 °C, 50 °C, and 80 °C, for 15, 30, and 60 min and solvent-to-
sample ratios of 5:1, 10:1, and 15:1 (v/w) were combined with different solvents. The sugar composition in the
soybean extracts was analyzed by high-performance anion-exchange chromatography with pulsed amperometric
detection. The sugar appropriate to be used as the standard in the phenol-sulfuric acid method in sugar quan-
tification, and effects of sample size and defatting on sugar extraction were also evaluated. The optimum extrac-
tion protocol concluded from this study includes the use of water as the solvent at a solvent-to-sample ratio of 5:1
at 25 °C or 50 °C for 15 min. Sucrose was shown to be the best sugar as the standard to quantify the total soluble
sugars in soybean. Extractions from sample size of 1 g and 0.1 g yielded similar soluble sugars. A greater amount
of sugars was extracted with the defatted soybean meal than with the nondefatted sample.
K eywor ds: so
eywords: ybean, sugars
soybean, sugars,, extraction solv
extraction ent, extr
solvent, action temper
extraction atur
temperatur e, phenol-sulfur
ature ic acid method, HP
phenol-sulfuric AEC-
HPAEC-
PAD

Introduction (1996) observed a significant interaction between solvent and tem-

C arbohydrate is the 2nd most predominant component in soy-

bean seeds, next to protein, representing approximately 1/3 of
the seed composition (Aspinall 1988). Insoluble carbohydrates in
soybean are mainly composed of cellulose, hemicellulose, pectin,
and trace amounts of starch (Liu 1997). Soluble sugars consist of
sucrose, raffinose, stachyose, glucose, fructose, and trace amounts
of arabinose, rhamnose, fucose, ribose, xylose, and mannose (Eld-
ridge and others 1979). Sucrose is responsible for enhancing the
taste of soy food products (Taira 1990; Geater and Fehr 2000),
whereas consumption of the indigestible raffinose and stachyose
is associated with flatulence and abdominal discomfort (Rackis
1975). Research is being conducted to improve the nutritional qual-
ity of soybean by increasing its sucrose level and reducing the both-
ersome saccharides. Therefore, rapid yet reliable methods to ex-
tract and quantify soluble sugars are needed.
Many procedures for extraction and quantification of soluble
sugars in soybean have been reported; nevertheless, discrepan-
cies exist among reported optimal extraction conditions, and defat-
ted soybean meal was used in most studies. Early studies showed
that extractions with water at 25 °C and 100 °C produced compara-
ble results to those using 80% ethanol (Kawamura 1967; Delente
and Ladenburg 1972; Kennedy and others 1985). Shukla (1987) and
Knudsen and Li (1991) found that water and 50% ethanol were
better solvents than 80% ethanol in extracting sugars from soybean,
which was also observed by Cegla and Bell (1976).
Temperature and time have been shown to affect sugar extraction
from plant materials (Shi and others 2002). Johansen and others
MS 20050419 Submitted 7/12/05, Revised 8/22/05, Accepted 10/3/05. Authors
Giannoccaro and Wang are with Dept. of Food Science, Univ. of Arkansas,
Fayetteville, AR 72704. Author Chen is with Dept. of Crop, Soil and Environ-
mental Sciences, Univ. of Arkansas, Fayetteville, Ark. Direct inquiries to
author Wang (E-mail: yjwang@uark.edu).
perature when extracting sugars from toasted soybean meal. Rela-
tively higher amounts of sugar were obtained with increasing tem-
perature when water or 50% and 80% ethanol were used as solvents.
Kim and others (2003) concluded that temperatures below 50 °C had
a positive influence in aqueous extractions, but temperatures above
50 °C decreased the extractability of sugar from defatted soybean.
The phenol-sulfuric acid method by Dubois and others (1956) is
a reliable and simple method and has been widely used for total
sugar determination in soybean. This procedure detects not only
soluble sugars but also oligomeric/polymeric sugars because the high
sulfuric acid concentration can hydrolyze oligomeric/polymeric sug-
ars into monomers. However, divergences were noted on the prepa-
ration of the standard curve for quantification of the sugar concen-
tration. Sucrose and glucose have been used for establishing the
standard curve in the phenol-sulfuric acid method for determination
of soybean sugars (Openshaw and Hadley 1978; Kennedy and oth-
ers 1985; Cober and others 1997; Geater and Fehr 2000; Geater and
others 2000, 2001; Gulewicz and others 2000). It is known that the
color response of the phenol-sulfuric method varies with different
sugars due to their differences in molecular structure (Saha and
Brewer 1994). The quantification of the total sugars present in a par-
ticular sample will, therefore, be affected by the type of sugar used
in preparing the standard curve (BeMiller and Low 1998). However,
little attention has been given to the selection of sugar for the stan-
dard curve to accurately quantify total sugars in soybean.
High-performance anion exchange chromatography coupled with
pulsed-amperometric detection (HPAEC-PAD) has become increas-
ingly popular and has been extensively used for sugar analysis (Rock-
lin and Pohl 1983; Townsend and others 1988; Mohamed and Duarte
1995; Frias and others 1999; Cataldi and others 1999, 2000). PAD is
highly selective and sensitive because only reactive compounds will
give response and at very low concentrations; for example, nano-

© 2006 Institute of Food Technologists Vol. 71, Nr. 1, 2006—JOURNAL OF FOOD SCIENCE C59
Further reproduction without permission is prohibited Published on Web 1/13/2006
 Extraction of sugars in soybean . . .
grams can still be precisely quantified, which is very important for
soybean breeder when only small sample sizes are available.
The objective of this study was to optimize the extraction condi-
tions for soluble sugars in nondefatted soybean meal by evaluat-
ing the effects of solvent, temperature, extraction time, solvent-to-
sample ratio, and sample size. This study also intended to identify
the most appropriate sugar for preparing the standard curve in the
phenol-sulfuric acid method to best estimate the soluble sugars in
soybean.
Materials and Methods
Materials
C: Food Chemistry & Toxicology
Seeds of 5 soybean lines, Hutcheson, Camp, SS-516, MFL-552,
and V99-5089, grown in Fayetteville, Ark., in 2002 with known
amounts of soluble sugars were provided by the Dept. of Crop, Soil
and Environmental Sciences at the Univ. of Arkansas, Fayetteville,
Ark. Fifteen grams of each soybean line was ground in a mill
(Knifetec 1095, Foss, Hoganas, Sweden) for 20 s, and the ground
flour was screened through a 150-␮m sieve (W.S Tyler, Mentor, Ohio,
U.S.A.) and used for sugar extraction without defatting. Glucose,
galactose, sucrose, raffinose, stachyose, and melibiose were ob-
tained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All other
chemicals were ACS grade.
Seed chemical composition
Moisture and ash contents were determined according to Ap-
proved Methods 44-31 and 08-16, respectively (AACC 1997). Protein
content was determined by the combustion method (Campbell
1992), and the conversion factor of 6.25 was used to convert total
nitrogen to protein. Calcium was analyzed by using inductively cou-
pled plasma emission spectroscopy (ICP-ES) (Model Ciros; Spectro
Analytical Instruments, Inc., Marborough, Mass., U.S.A.) following
the methods of Donohue and Aho (1992). Crude lipid content was
measured according to AACC Approved Method 30-20 (AACC 1997)
with the following modifications. Soybean meal (5 g) was extracted
with 70 mL petroleum ether by boiling at 135 °C for 20 min and rins-
ing for 30 min in a Soxtec system (Avanti 2055; Foss North America,
Eden Prairie, Minn., U.S.A.). The percentage of crude lipids in the
original sample was calculated by dividing the amount of lipids ex-
tracted by the dry weight of the original sample. Total fiber content
in all samples was calculated by the difference from the amount of
ash, protein, lipids, and soluble sugars previously obtained. All chem-
ical composition analyses were performed in triplicate, and results
were expressed as percentage on a dry-weight basis.
Standard curve and sugar quantification
Six standard curves for the phenol-sulfuric acid method (Dubois
and others 1956) were prepared with glucose, galactose, fructose,
sucrose, raffinose, and stachyose at concentrations from 10 to 115
␮g/mL for each sugar. The total sugars of each soybean line was
extracted following the optimal conditions reported by Kim and
others (2003). Duplicate samples of 1 g of soybean meal were
stirred into 5 mL of water at 50 °C for 60 min. The extracts were cen-
trifuged and the supernatants were filtered through a 0.2-␮m mem-
brane (HT Tuffryn Nylon). One milliliter of each filtrate was mixed
with 1 mL of 5% (w/v) phenol solution and then quickly mixed with
5 mL of concentrated sulfuric acid, vortexed, and cooled at room
temperature for 30 min. The mixtures were then vortexed again,
and absorbance at 490 nm was measured using a spectrophotom-
eter (model DU-520; Beckman, Fullerton, Calif., U.S.A.). The
amount of total soluble sugars was expressed as a percentage of the
sample on a dry-weight basis.
C60 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 1, 2006
Purification, separation, and quantification
of soluble sugars by HP
by AEC-P
HPAEC-P
AEC-PADAD
Soluble sugars were extracted from ground Hutcheson soybean
meal by following Kim and others (2003), except that the sample
was spiked with melibiose as the internal standard, which was cho-
sen to check recovery and to ensure accurate quantification of sug-
ars (Black and Bagley 1978; Li and others 1985). After the extraction,
the tube was centrifuged at 20000 × g for 10 min, and 2 mL of each
supernatant were pipetted into another centrifuge tube.
The extract was further purified by following the method of Black
and Glover (1980) with some modifications. Three milliliters of ac-
etonitrile was slowly added into the centrifuge tube with constant
shaking to precipitate the residual protein, and the tube was left at
room temperature for 30 min. The tube was then centrifuged at
1500 × g for 10 min, and 1 mL of the clear supernatant was pipetted
into a 1.7-mL microcentrifuge tube and brought to complete dry-
ness using a heat block at 80 °C for 60 min. The residue was redis-
solved in 1 mL 90 mM NaOH, and the solution was quantitatively
transferred to a 100-mL volumetric flask and brought to volume
with 90 mM NaOH. Ten milliliters of the diluted solution was fil-
tered through a 0.2-␮m membrane (HT Tuffryn Nylon) followed by
a cartridge (OnGuard II RP, Dionex, Sunnyvale, Calif., U.S.A.) to
remove possible residual lipids, surfactants, hydrocarbons, and
high-molecular-weight carboxylic acids (Wicks and others 1991;
Kadnar 1998) before injection into the HPAEC-PAD.
The HPAEC-PAD system (Dionex DX500) consisted of a GP-50
gradient pump, ED40 electrochemical detector, a CarboPac PA-10
pellicular anion-exchange resin column (250 × 4-mm inner dia)
preceded by a CarboPac PA-10 guard column (50 × 4-mm inner dia)
and an AminoTrap column (30 × 3-mm inner dia) (Dionex, Sunny-
vale, Calif., U.S.A.). Samples were injected via an AS40 automated
sampler with a 25-␮L sample loop, and sugars were eluted with 90
mM NaOH at a flow rate of 1 mL/min. The mobile phase, 90 mM
NaOH, was prepared by diluting carbonate-free 50% (w/w) NaOH
solution in distilled water, which was previously filtered with a 0.45-
␮m membrane and degassed with a sonicator (Zenith Inc, T800-2H,
Norwood, N.J., U.S.A.) for 30 min. Sugar standards were used to
identify the sugars based on their retention times.
Extraction procedure
Two types of solvents, deionized (DI) water and ethanol, were
investigated, and only 1 soybean line, Hutcheson, was used in all
extraction experiments. For aqueous extraction, samples of 1-g
ground soybean meal were mixed with DI water at ratios of 5:1,
10:1, and 15:1 (v/w) and extraction temperatures of 25 °C, 50 °C, and
80 °C for 15, 30, and 60 min. Only 1 combination of factors was used
with 100 °C as the extraction temperature: a solvent-to-sample ratio
of 10:1 at 100 °C for 15 min.
For ethanolic extraction, dilutions of 10%, 50%, and 80% (v/v)
ethanol in water were studied at solvent-to-sample ratios of 5:1
and 10:1 and temperatures of 25 °C, 50 °C, and 80 °C for 15 min.
Only 1 combination of factors was used with 100 °C as the extraction
temperature: a solvent-to-sample ratio of 10:1 using 80% ethanol
as the solvent at 100 °C for 15 min. Triplicates of 1-g samples were
placed in 50-mL centrifuge tubes with the selected extractant.
Tubes were capped, placed horizontally, completely immersed in a
water bath, and shaken at 200 rpm. Both aqueous and ethanolic
extractions were repeated until no sugars were detected in the su-
pernatant after centrifugation at 20000 × g for 10 min using the
phenol-sulfuric acid method. Only 1 extraction was needed with
water and 10% ethanol as the solvents (no sugar detected in 2nd
extraction), whereas 50% or 80% ethanol required 2 additional ex-
tractions (no sugar detected in 4th extraction). The combined su-
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Extraction of sugars in soybean . . .
pernatants from 50% and 80% ethanol extractions were centrifuged
at 20000 × g for 10 min to separate lipids from the extract, which was
present at the bottom of the tube and carefully removed using a
disposable pipette. The ethanolic supernatant was quantitatively
transferred to a 100-mL beaker and brought to dryness using a hot
plate at 60 °C for 60 min. The residue was then re-dissolved in 100
mL of DI water and filtered through a 0.2-␮m membrane (HT Tuf-
fryn Nylon) for further purification. All extractions were repeated 3
times on different days to check reproducibility.
Sample size and defatting effects on extraction
To study the effect of sample size, 0.1 g and 1 g of Hutcheson
soybean meal were compared using water as the solvent and a sol-
vent-to-sample ratio of 5:1 at 50 °C for 15 min. For the defatting
study, 2 g Hutcheson soybean meal was defatted according to AACC
method 30-26 (AACC 1997) except that hexane was used instead of
petroleum ether as the solvent. The lipid content was calculated
based on the extracted lipid after solvent was completely removed
in the vented hood. Both the defatted and nondefatted soybean
meals were extracted concurrently with water at a solvent-to-sam-
ple ratio of 5:1 at 50 °C for 15 min and their soluble sugars were
determined. The percentage of total sugars in the defatted sample
was calculated based on the initial nondefatted meal weight (db).
Statistical analysis
Two statistical analyses were conducted to investigate the opti-
mal combination of solvent, temperature, extraction time, and sol-
vent-to-sample ratio to extract soluble sugars from soybean seeds
and the interactions of the extraction factors. Student t test was
used to detect significant differences among the extraction condi-
tions in terms of the amount of soluble sugars extracted. A General
Linear Model (GLM) procedure was used to investigate significant
interactions among the extraction factors. In the factorial study, not
all the interactions were possible to be analyzed due to the incom-
pleteness of the experimental design used. The Tukey-Kramer
HSD test was used to detect significant differences among total
sugars extracted from the 2 sample sizes and among soybean meal
samples quantified using standard curves prepared with different
sugars. All statistical analyses were carried out using version 9.0.1
of SAS statistical software (SAS Inst. 2003).
Results and Discussion
Chemical composition
The chemical compositions of the 5 soybean lines studied are
summarized in Table 1. All lines showed a similar total sugar con-
tent of around 12% with exception of V99-5089, which had the low-
est sugar but the highest protein content. Krober and Cartter (1962)
found that an increase in protein content was associated with a
decrease in total soluble sugars for soybean. SS-516 had the high-
est amount of sugars but the lowest lipids and calcium contents.
Hutcheson was high in sugars and lipids, but low in protein and
fiber. The positive correlation between total soluble sugars and lip-
ids, and negative correlation between lipids and protein in soybean
had previously been demonstrated by Hymowitz and others (1972).
The amount of total sugars present in the 5 soybean lines analyzed
was within the range (6.2% to 16.6%) previously reported by Hy-
mowitz and Collins (1974) from their study of 195 soybean lines.
Standard curve for sugar quantification
Significant differences in total sugars were observed when extract
from the same soybean line was analyzed using standard curves
prepared with different sugars (Table 2). Fructose gave the highest
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amount of total sugars, followed by raffinose, sucrose, galactose/
stachyose, and glucose in descending order for all soybean lines. The
results clearly demonstrated that quantification of sugars using the
phenol-sulfuric acid method strongly depends on the sugar used for
the standard curve. To verify that only low-molecular-weight soluble
sugars were present in the extracts and to determine the sugar con-
centration, HPSEC-PAD was used to identify the sugar composition
and to quantify the sugar content in the extract of Hucheson soybean
line. The HPAEC-PAD chromatogram of Hutcheson extract (Figure 1)
shows that only glucose (0.15%), fructose (0.08%), sucrose (5.64%),
raffinose (1.09%), and stachyose (5.97%) were present in the aque-
ous extract. Oligosaccharides with degree of polymerization higher
than 4 were not present or their contents were too low to be detected
C: Food Chemistry & Toxicology
by the HPAEC-PAD system. The total amount of soluble sugars in
Hutcheson was 12.93% based on HPAEC-PAD results, which was
comparable to the results obtained with the colorimetric method
when sucrose was used as the standard.
Although glucose has been conventionally used as the standard
to determine the amount of total sugars in soybean (Geater and
Fehr 2000; Gulewicz and others 2000; Geater and others 2001), the
present results indicate that sucrose standard gave the closest es-
timation for the true value, whereas glucose tended to underesti-
mate the total sugar content. Because different sugars would re-
spond differently to the phenol-sulfuric acid reaction (Saha and
Brewer 1994; BeMiller and Low 1998), the final color from the phe-
nol-sulfuric acid method was assumed to be a combination of the
colors that resulted from the reaction of different sugars. Sucrose as
the standard provided the best estimation probably because it
represents the majority of the soluble sugars present in soybean.
Therefore, sucrose is proposed to be more suitable than any other
sugars for preparing the standard curve in quantifying total solu-
ble sugars in soybean.
Aqueous extraction
The amounts of total sugars in Hutcheson soybean line ranged
from 11.48% to 12.54% when using water as the solvent (Table 3).
Extraction with water at 25 °C and 50 °C generally resulted in higher
amounts of soluble sugars than extractions at 80 °C, regardless of
extraction time and solvent-to-sample ratio. When extraction was
conducted at 80 °C, lower sugar amounts were generally obtained
with solvent-to-sample ratios of 10:1 and 15:1 than with 5:1. The
extraction performed at 100 °C for 15 min with a solvent-to-sample
Figure 1—Separation of soluble sugars in the extract ob-
tained from Hutcheson using high-performance anion ex-
change chromatography coupled with pulsed-
amperometric detection (HPAEC-PAD). Glucose (1), fruc-
tose (2), melibiose (3), sucrose (4), raffinose (5), and stachy-
ose (6).
Vol. 71, Nr. 1, 2006—JOURNAL OF FOOD SCIENCE C61
 Extraction of sugars in soybean . . .

Table 1—Seed chemical composition of 5 soybean lines

Percentage (dry-weight basis)
Line Moisture Total sugars Protein Lipids Fiber Ash Calcium
Hutcheson 7.38ab ± 0.00 12.84ab ± 0.44 41.83d ± 0.18 23. 60a ± 0.09 16.73c 5.00c ± 0.11 0.21c ±0.00
Camp 7.16c ± 0.10 11.57c ± 0.15 41.35e ± 0.61 20.05d ± 0.17 21.56a 5.47b ± 0.09 0.34b ± 0.01
SS-516 7.18c ± 0.04 13.23a ± 0.73 43.30b ± 0.60 18.18e ± 0.08 19.85b 5.45b ± 0.21 0.18d ± 0.00
MFL-552 7.23bc ± 0.08 11.38cd ± 0.15 42.60c ± 0.21 22.83b ± 0.15 18.25c 4.95d ± 0.14 0.20c ± 0.00
V99-5089 7.41a ± 0.05 6.31e ± 0.29 48.22a ± 0.40 21.97c ± 0.07 17.74d 5.75a ± 0.01 0.38a ± 0.01
a Values are means of duplicate measurements. Means in a column with different letters are significantly different (P < 0.05).

Table 2—Quantification of total soluble sugars in Hutcheson, Camp, SS-516, MFL-552, and V99-5089 soybean lines
using standard curves obtained with different sugars as the standarda
C: Food Chemistry & Toxicology

Total sugars (%, db)

Sugar Hutcheson Camp SS-516 MFL-552 V99-5089
Glucose 7.61 ± 0.38e 6.51 ± 0.13e 7.95 ± 0.63de 6.34 ± 0.13e 1.94 ± 0.25e
Galactose 9.29 ± 0.35de 8.28 ± 0.12d 9.59 ± 0.58d 8.13 ± 0.12d 4.14 ± 0.23d
Fructose 31.01 ± 0.67a 29.02 ± 0.22a 31.56 ± 1.12a 28.75 ± 0.22a 21.02 ± 0.45a
Sucrose 12.84 ± 0.44c 11.57 ± 0.15c 13.23 ± 0.73c 11.38 ± 0.15c 6.31 ± 0.29c
Raffinose 17.77 ± 0.39b 16.60 ± 0.13b 18.09 ± 0.66b 16.45 ± 0.13b 11.91 ± 0.26b
Stachyose 9.71 ± 0.42d 8.49 ± 0.14d 10.09 ± 0.14d 8.30 ± 0.14d 3.42 ± 0.28d
a Values are means of duplicate measurements. Means in a column with different letters are significantly different (P < 0.05).

Table 3—Total soluble sugars in Hutcheson soybean seeds under different extraction conditions using water as the
extractanta
Extraction temperature (°C)
25 50 80
Solvent-to-sample
ratio (v:w) 5:1 10:1 15:1 5:1 10:1 15:1 5:1 10:1 15:1
Extraction time
(min)
15 12.12a-f 12.46a 12.17a-e 12.54a 12.35a-c 12.31a-e 12.35a-c 11.80d-i 11.71d-j
± 0.36 ± 0.34 ± 0.31 ± 0.79 ± 0.20 ± 0.09 ± 0.39 ± 0.06 ±0.14
30 12.13a-h 12.07a-i 12.26a-e 12.29a-e 12.49ab 12.40a-c 12.20a-f 11.95a-i 11.53f-j
± 0.33 ± 0.34 ± 0.09 ± 0.23 ± 0.26 ± 0.20 ± 0.21 ± 0.06 ± 0.07
60 12.06a-i 12.06a-h 12.33a-e 12.18a-g 12.39a-d 12.14a-h 11.98a-i 11.69e-j 11.48h-j
± 0.49 ± 0.54 ± 0.32 ± 0.32 ± 0.18 ± 0.19 ± 0.24 ± 0.40 ± 0.100
a Values are means of duplicate measurements. Means in a column with different letters are significantly different (P < 0.05). Means in this table are compared
with means in Table 4.

ratio of 10:1 gave rise to 11.50 ± 0.14 (%, db) of soluble sugars, which
was also significantly lower than results obtained at 25 °C and 50 °C
(P < 0.05). The low amount of sugar extracted from soybean meal
with water at 80 °C could be attributed to the rapid denaturation of
soybean soluble proteins under high temperatures, which thereaf-
ter entrapped soluble sugars and impaired their extractions (Kim
and others 2003).
Kim and others (2003) found significant differences among dif-
ferent solvent-to-sample ratios and concluded that a solvent-to-
sample ratio of 5:1 was the optimal ratio to effectively extract sug-
ars from defatted soybean meal. The quantification of soluble
sugars can be impaired by the dilution effect of using too much
solvent or by reducing the extractability using inadequate amount
of solvent. Nevertheless, all 3 solvent-to-sample ratios produced
similar results in this study. This discrepancy could be ascribed to
the defatted soybean meal used in their study in comparison with
nondefatted soybean meal used in our study.
Ethanolic extraction
No significant differences were observed among extractions
using 10% and 50% ethanol (P > 0.05); however, extractions with
80% ethanol resulted in significantly lower amounts of total sugars
extracted (Table 4). It was also observed that increasing ethanolic
strength and temperature improved sugar extraction when using
80% ethanol as the extractant but had no effect on sugar extraction
with 10% or 50% ethanol. Extraction with 80% ethanol at 100 °C for
15 min produced 11.78 ± 0.18 (%, db) soluble sugars, which was not
significantly different from those extracted with 10% and 50% eth-
anol (P > 0.05). An increase in ethanol strength in the range of 50%
to 90% has been previously shown to significantly reduce the
amount of raffinose and stachyose extracted from plant materials
(Cegla and Bell 1976; Knudsen and Li 1991). The deficiency of 80%
ethanol in extracting soluble sugars could be related to the de-
crease in efficiency of ethanol to extract sugars of increasing molec-
ular weight (Shallenberger and Birch 1975). Therefore, 80% ethanol
was not as effective as water in extracting raffinose and stachyose.
Johansen and others (1996) concluded that 80% ethanol was effec-
tive only at high temperatures in extracting sugars from toasted
soybean meal. The process of toasting soybean might help the ex-
traction of soluble sugars with 80% ethanol. However, Kennedy and
others (1985) found that water and 80% ethanol had the same ef-
ficiency to extract soluble sugars from defatted soybean meal.
The lower extraction efficiency of 80% ethanol in the present study
could also be the result of a simultaneous extraction of lipids and

C62 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 1, 2006 URLs and E-mail addresses are active links at www.ift.org
Extraction of sugars in soybean . . .

Table 4—Total soluble sugars in Hutcheson soybean seeds under different extraction conditions using different ethanolic
solvents as the extractants a
Ethanol (%, v/v)
10 50 80
Solvent-to-sample
ratio (v:w) 5:1 10:1 5:1 10:1 5:1 10:1
Temperature (°C)
25 12.25a-e* ± 0.11 12.32a-e ± 0.18 11.79c-j ± 0.08 11.83b-j ± 0.13 9.71m ± 0.20 10.79kl ± 0.21
50 12.19a-f ± 0.07 12.41a-c ± 0.12 11.89a-i ± 0.13 12.15a-h ± 0.30 10.75l ± 0.22 11.33jk ± 0.38
80 12.02a-i ± 0.13 12.11a-h ± 0.28 11.95a-i ± 0.17 12.11a-h ± 0.28 10.82l ± 0.81 11.49ij ± 0.35

a Values are means of duplicate measurements. Means in a column with different letters are significantly different (P < 0.05). Means in this table are compared

C: Food Chemistry & Toxicology

with means in Table 3.

sugars because nondefatted soybean meal was used, thus decreas- Table 5—Analysis of variance (ANOVA) of main effects and
ing its extraction capacity to interact with the sugars in the matrix. interactions among solvent, temperature, extraction time
(time), and solvent-to-sample ratio (ratio) on soluble sugar
Additionally, because water has a higher dielectric constant than
extraction
ethanol, it is assumed that higher temperatures would be required
for ethanol to more effectively extract the sugars from soybean. Effect Factor F value Pr > f
Main Solvent 239.6 <0.0001
Interactions of extraction conditions Temperature 1.8 0.1865
Solvent, temperature, extraction time, and solvent-to-sample Time 9.1 0.0043
Ratio 0 0.9484
ratio may exert different effects on the extraction of sugars from Interaction Solvent * temperature 68.4 <0.0001
soybean. A GLM procedure was conducted to elucidate the contri- Solvent * ratio 28.6 <0.0001
bution of each main effect and their possible interactions while Temperature * time 0.2 0.6558
extracting sugars from soybean. The results showed that solvent (F Temperature * ratio 7.8 0.0077
Time * ratio 0.7 0.4236
value = 239.6, P < 0.001) and time (F value = 9.1, P < 0.0043) signif-
icantly affected the extraction of sugars from nondefatted soybean
meal (Table 5). Interactions of solvent and temperature (F value =
68.4, P < 0.0001), solvent and solvent-to-sample ratio (F value =
28.6, P < 0.0001), and temperature and solvent-to-sample ratio (F and accessible to solvents after defatting, thus facilitating solvents
value = 7.8, P < 0.0077) also played significant roles in soybean sug- to extract the sugars in the soybean and resulting in improved sugar
ar extractions. extraction. In addition, the higher amount of sugars extracted from
It was observed that higher temperatures were more effective defatted soybean meal could also be attributed to the lack of inter-
for 80% ethanolic extractions, but less effective for water extractions action of lipids and sugars during extraction.
(Table 3 and 4). The solvent and temperature interaction had pre-
viously been observed by Johansen and others (1996); however, Conclusions
they reported that more sugars were extracted with 80% ethanol
only at boiling temperature. The solvent and solvent-to-sample
ratio interaction was also more evident for 80% ethanol extractions,
T he optimum conditions to extract soluble sugars from nonde-
fatted soybean based on the results from this study included
using water as the solvent and a solvent-to-sample ratio of 5:1 at
where more sugars were extracted at a higher solvent-to-sample 25 °C or 50 °C for 15 min. These combinations provided simple, fast,
ratio. For temperature and solvent-to-sample ratio interaction, the and efficient extractions of the greatest amounts of soluble sugars
highest solvent-to-sample ratio (15:1) extracted slightly less of the and there was no difference in total extracted sugars between sam-
sugars than the lower ratios under aqueous extraction at 80 °C. ple sizes of 0.1 g and 1.0 g. Among the 47 extractions conducted in
When using 80% ethanol as the solvent at 80 °C, the amount of ex- this study, the majority of the conditions produced similar results
tracted sugars was significantly affected by temperature and sol- (P > 0.05). Extraction with ethanol, however, presented the disad-
vent-to-sample ratio. vantage of being more laborious than extractions with water be-
cause water required only 1 extraction step, whereas ethanolic ex-
Sample size and defatting effects on extraction tractions required 2 additional steps to completely remove sugars
The total soluble sugars extracted using sample sizes of 0.1 g and from soybean. Additionally, with water being the solvent, minimal
1.0 g were 12.60 and 12.28 (%, db), respectively. The result with 0.1 residual lipids were present; therefore, no additional cleaning step
g was not significantly different (P > 0.05) from that with 1 g. These was needed for further identification and quantification of sugars
results demonstrated that reproducibility could be obtained with by high-performance liquid chromatography (HPLC). However, ex-
a sample size as small as 0.1 g, which could be critical for breeding tractions with nondefatted soybean meals would underestimate
programs where only limited samples are available for the mea- the amount of total sugars compared with extractions with defatted
surement of total sugars in new soybean lines and where a large samples.
number of lines need to be evaluated.
Extraction using defatted Hutcheson soybean meal resulted in Acknowledgments
15.33 ± 0.12 (%, db) of total sugars extracted, which was about 2.5% The authors thank Dr. Ronald W. McNew, professor and coordina-
higher than the sugars extracted from the nondefatted meal tor of the Agricultural Statistics Laboratory at the Univ. of Arkansas,
(12.84%). It is possible that soybean meal became more porous for his assistance with data analysis.

URLs and E-mail addresses are active links at www.ift.org Vol. 71, Nr. 1, 2006—JOURNAL OF FOOD SCIENCE C63
 Extraction of sugars in soybean . . .
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