# Sugar Variation in Soybean Seed Assessed with a Rapid Extraction and Quantification Method

Authors: A. Hou; P. Chen; A. Shi; B. Zhang; Y.-J. Wang
Journal: International Journal of Agronomy
 (2009)

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## Abstract

Sugar content in soybean [Glycine max (L) Merr.] seed is an important quality attribute for soyfood and feed. Rapid extraction and quantification of soluble sugars in soybean seed are essential for large-scale breeding selections. In this
study, using water as extractant combined with a high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) system, variability and repeatability of sugar content were tested in 20 diverse soybean genotypes. Individual sugars were clearly resolved and identified, including sucrose, stachyose, raffinose, glucose, fructose, and verbascose. Analysis of variance (ANOVA) revealed a highly reproducible estimation of sucrose, stachyose, and raffinose. PI 243545 was confirmed as a unique germplasm with the highest sucrose
(105.48 mg/g) and total sugar (148.76 mg/g) content.
Two low-oligosaccharide accessions, PI 200508 and 03CB-14, were confirmed with extremely low concentrations of raffinose and stachyose. PI 417559 was identified as a high glucose and fructose germplasm. The identified germplasm with unique sugar profiles will be valuable in breeding specialty soybeans for improved sugar content. The sugar testing method will facilitate the screening of seed sugar profiles in a large-scale soybean breeding program.


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## Body

## 1. Introduction

Soybean [Glycine
max (L) Merr.] seed is one of the major food
sources for human and
livestock. In addition to its rich protein (40%) and oil (20%) content, soybean
seed also contains approximately 33% carbohydrates, up to 16.6% of which are soluble
sugars [1]. The major sugars present in soybean seed include glucose, fructose,
sucrose, raffinose, and stachyose [2, 3]. Sucrose makes up
41.3–67.5%, raffinose
5.2–15.8%, and
stachyose 12.1–35.2% of the total
soluble sugars in soybean seed [3]. Trace amounts of other sugars have also
been reported such as pinitol, myo-inositol, verbascose, galactose, arabinose,
and mannose [2, 4]. Sugars in soybean seed affect soyfood quality,
digestibility, and nutritional values. Soyfood such as soymilk, tofu, and natto
is considered as healthy diets, and consumption is highly recommended by
nutritionists and medical doctors [5]. In tofu, soymilk, natto, and many
other soyfood products, desirable sugars including glucose, fructose, and
sucrose contribute to the favorable sweet taste and are ready-to-digest, while
raffinose and stachyose are indigestible and cause undesirable flatulence and
diarrhea [6].Soybean
cultivars are bred and grown for different end uses, such as regular beans for
high protein and oil extraction, low-phytate beans for animal feed, low-linolenic
beans for special low-trans-fat oil, large-seeded for tofu and soy milk, and
small-seeded for natto production. Seed sugars vary in different soybean
genotypes [1, 7–9]. The concentration of various sugars is also
significantly affected by processing methods in soybean meals [10]. Significant
association (−0.81) was found between the total sugar amount and the sum of
protein and oil content in soybean seed [11].
Hymowitz et al. [8] reported that
the total sugar and oil content in soybean seed were positively correlated
(0.26), but both were negatively associated with protein (−0.19 and −0.63, resp.).
The soyfood markets prefer soybeans with high-sucrose, low-raffinose and
stachyose content. One efficient way to improve the soybean sugar composition
and marketability is through conventional breeding and genetic enhancement.
Soybean breeding for improved sugar content has received considerable attention
in recent years. Soybean breeding populations have been constructed using low-oligosaccharide
germplasm PI 200508 and evaluated for developing soybean cultivars with reduced
raffinose and stachyose [12]. Quantitative trait loci
(QTLs) have also been mapped with molecular markers for soybean
sucrose and oligosaccharide content [13–15]. The identified molecular
markers will facilitate the breeding process for soybean sugar improvement.
However, soybean cultivars with improved sugar profiles have not been reported.Rapid
sugar extraction and quantification are essential for large-scale soybean
breeding programs because selections need to be accomplished for a large number
of plant populations in a short period of time. A rapid and reliable procedure
is also necessary for genetic screening and gene mapping. There are several
protocols reported and used for sugar extraction in some soybean research
programs, however, those protocols are not standardized and their repeatability
is yet to be validated. Generally, sugar extraction is prepared from defatted
or whole soybean meals using either ethanol or water as the solvent [8, 9, 16].
Effective sugar extraction has been achieved using 50–80% ethanol as
solvent, however, complete extraction usually requires heating and repeated
extraction steps with extraction time varying from 30 minutes to 24 hours [8, 9, 14].  Recently,
Giannoccaro et al. [16]
investigated the effects of solvent type, temperature, extraction time,
solvent-to-sample ratio, and sample size on soybean sugar extraction. In that
particular study, complete sugar recovery was achieved by either three repeated
extractions using 50–80% ethanol or a single extraction using water
as solvent. Sugar recovery by aqueous extraction remained comparable when
extraction temperature varied between 25 and 50°C. Their results further
concluded that aqueous extraction for whole soybean meals with sample size of
0.1 g, solvent-to-sample ratio of 10:1, and extraction time of 15 minutes at 25°C
achieved comparable sugar results to various ethanolic methods. However, only
one cultivar Hutcheson was evaluated in that study, and variations due to different
extractions and quantifications were not examined. The objective of this study
was to compare the variations of sugar content among selected diverse soybean
genotypes and different extractions, following the procedures described by
Giannoccaro et al. [16] in order to find a rapid and reliable procedure suitable
for sugar analysis in large-scale soybean breeding.


## 2. Materials and Methods

### 2.1. Plant Materials and Sugar Extraction

Twenty soybean genotypes (Table1)
were used for this study, and seeds were harvested from the field in 2005 at
the University of Arkansas Agricultural Experiment Station in Fayetteville,
Ark, USA. These 20
genotypes were selected with various unique agronomic traits including six
regular cultivars and germplasms (5002T, Jack, Ozark, PI 504510, PI 323556, and
PI 417559), four small-seeded natto beans (Camp, SS516, V97-3000, and MFS-591),
two large-seeded tofu beans (MFL-159 and MFL-552), one high protein line
(R95-1705), one low phytate line (CX1834-1-2), one high total sugar line (PI
243545), and two low oligosaccharide lines (03CB-14 and PI 200508). Two
accessions of wild species, PI 326581 (G. soja) and PI 153292, (G.
gracilis) were also included for comparisons among species. Fifteen grams
of seed were taken from each genotype, and two duplicate extractions were
prepared from each sample. For each extraction, two repeated quantifications
were conducted for soluble sugar concentrations. Standard sugar melibiose
(Sigma Chemical Co., St. Louis,
Mo, USA)
was added in each sample as internal standard. CB03-14 and Ozark were also used
as checks in each extraction and quantification to monitor the testing
consistency and accuracy.Table 1
List of soybean genotypes used in this study and
detected mean sugar content.
GenotypeSpeciesTypes and usesSugar content(mg/g)(a)Sucrose(b)Raffinose(c)Stachyose(d)Glucose(e)Fructose(f)Total  sugar(g)PI 243545G. maxHigh-total sugar105.4814.2327.850.950.25148.7603CB-14G. maxLow oligosaccharide88.093.764.141.330.9598.275002TG. maxRegular cultivar77.0212.6839.951.550.33131.53JackG. maxRegular cultivar64.3710.5837.370.680.25113.25PI 504510G. maxRegular germplasm63.025.936.70.80.3106.72OzarkG. maxRegular cultivar58.0212.0933.441.250.4105.2SS516G. maxSmall-seeded56.877.144.981.180.25110.13CX1834-1-2G. maxLow phytic acid56.099.940.480.850.23107.55R95-1705G. maxHigh protein55.143.4537.291.871.399.05V97-3000G. maxSmall-seeded53.946.4843.681.480.73106.31MFS-591G. maxSmall-seeded52.475.3849.951.180.2109.18CampG. maxSmall-seeded52.0410.738.680.380.13101.93PI 323556G. maxRegular germplasm43.79.4734.531.330.3789.4MFL-552G. maxLarge-seeded43.5615.539.51.580.25100.39S02-529G. maxRegular germplasm40.1412.7337.031.680.992.48MFL-159G. maxLarge-seeded31.6819.7336.881.630.1390.05PI 200508G. maxLow oligosaccharide30.710.855.150.30.237.21PI 417559G. maxRegular germplasm29.825.9324.516.812.2789.32PI 326581G. sojaWild species22.144.0530.11.831.059.12PI 153292G. gracilisWild species61.017.8541.71.450.23112.24Standard(a) sugar melibiose was added in each
sample as internal standard. External sugar standards
at concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0 μg/600 μL were prepared for creating calibration
curves for each of the major sugars. The concentration of each sugar was
estimated by regression of concentration and absorption of individual standard
sugars; LSD0.05(b)=9.07;
LSD0.05(c)=1.35;
LSD0.05(d)=4.26; LSD0.05(e)=0.46; LSD0.05(f)=0.36; LSD0.05(g)=9.13.Seed sample was ground using a SmartGrind
coffee bean grinder (Applica Consumer Products Inc., Fla,
USA) and screened through a
250μm standard testing sieve (VWR International, USA). Sugar extraction was
conducted following the procedures optimized by
Giannoccaro et al. [16]. Briefly,
duplicates of 0.15 g samples were weighed and placed in 2 mL Eppendorf
microcentrifuge tubes containing 1.5 mL distilled deionized water. The mixture
was vortexed and incubated at room temperature in a horizontal shaker at 200 rpm for 15 minutes. The suspension was then centrifuged at 13000 rpm for 10 minutes,
and 500 μL of the clear supernatant was transferred into a new 1.5 mL
microcentrifuge tube and purified with 700 μL 95% acetonitrile at room temperature
for 30 minutes, and then centrifuged at 13000 rpm for 10 minutes. A volume of
200 μL  of the supernatant was transferred into a 1.5 mL microcentrifuge tube
and dried at 95°C in a Dry
Bath Incubator
(Fisher Scientific, Pittsburgh, Pa, USA) for approximately 45 minutes. The residue
was redissolved in 200 μL 65% acetonitrile and filtered through a 0.2 μm
membrane disc filter (Pall Co., Mich, USA).Seed moisture was determined to
adjust the sugar content to a dry weight basis. Two grams of ground soybean
flour for each genotype were weighed in a predried aluminum weighing dish and
dried in an oven at130°C for 1 hour. The moisture content was calculated as
moisture (%) = 100 × [(weight
before drying)  − (weight after drying)]/(weight before
drying).


### 2.2. High-Performance Liquid Chromatography (HPLC) System

A Dionex DX500 HPAEC-PAD system equipped
with a GS50 pump, an ED40 pulsed amperometric detector, an AS40 automated
sampler with a 25μL injection loop, and a chromeleon chromatography management
system (Dionex, Sunnyvale, CA, USA)
was used for sugar identification and quantification. An analytical CarboPac
PA10 pellicular anion-exchange resin column (250  ×  4 mm)
preceded by a CarboPac PA10 guard column (50 × 4 mm)
and an AminoTrap column (30 × 3 mm)
was used for sugar separation.An aliquot of 24μL purified samples
was diluted with 576 μL distilled water prior to HPLC injection. Sugars were
eluted with 90 mM NaOH at a flow rate of 1.0 mL/min under isocratic conditions.
The mobile phase 90 mM NaOH was prepared by diluting carbonate-free HPLC grade
50% (w/w) stock solution in distilled water, filtered with a 0.45 μm membrane,
and degassed with compressed nitrogen gas for 30 minutes prior to being loaded.
The data acquisition for each sample was 30 minutes, and the analyses of 40
samples were generally completed in 20 hours.


### 2.3. Sugar
Identification, Quantification, and Statistical Analysis

Nine standard sugars, including
pinitol, myo-inositol, galactose, glucose, fructose, sucrose, raffinose,
stachyose, and verbascose, were obtained from Sigma Chemical Co. Sugars in the
extracts were identified by comparing their retention times to those of the
standard sugars. Sugar standards at concentrations of
0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0μg/600 μL were
prepared for creating calibration curves for each of
the major sugars, including glucose, fructose, sucrose, raffinose, and
stachyose. The sugar concentration in soybean seed was expressed as mg per g of
dry seed matter. Analysis of variance (ANOVA) was performed using the general
linear models (Proc GLM) of SAS
9.1 (SAS Institute Inc., Cary,
NC, USA).
Means of individual sugars in different genotypes were compared and separated
by SAS Waller grouping at P =  .05. Correlation analysis among different
sugar compositions was conducted using Excel CORREL function.


## 2.1. Plant Materials and Sugar Extraction

Twenty soybean genotypes (Table1)
were used for this study, and seeds were harvested from the field in 2005 at
the University of Arkansas Agricultural Experiment Station in Fayetteville,
Ark, USA. These 20
genotypes were selected with various unique agronomic traits including six
regular cultivars and germplasms (5002T, Jack, Ozark, PI 504510, PI 323556, and
PI 417559), four small-seeded natto beans (Camp, SS516, V97-3000, and MFS-591),
two large-seeded tofu beans (MFL-159 and MFL-552), one high protein line
(R95-1705), one low phytate line (CX1834-1-2), one high total sugar line (PI
243545), and two low oligosaccharide lines (03CB-14 and PI 200508). Two
accessions of wild species, PI 326581 (G. soja) and PI 153292, (G.
gracilis) were also included for comparisons among species. Fifteen grams
of seed were taken from each genotype, and two duplicate extractions were
prepared from each sample. For each extraction, two repeated quantifications
were conducted for soluble sugar concentrations. Standard sugar melibiose
(Sigma Chemical Co., St. Louis,
Mo, USA)
was added in each sample as internal standard. CB03-14 and Ozark were also used
as checks in each extraction and quantification to monitor the testing
consistency and accuracy.Table 1
List of soybean genotypes used in this study and
detected mean sugar content.
GenotypeSpeciesTypes and usesSugar content(mg/g)(a)Sucrose(b)Raffinose(c)Stachyose(d)Glucose(e)Fructose(f)Total  sugar(g)PI 243545G. maxHigh-total sugar105.4814.2327.850.950.25148.7603CB-14G. maxLow oligosaccharide88.093.764.141.330.9598.275002TG. maxRegular cultivar77.0212.6839.951.550.33131.53JackG. maxRegular cultivar64.3710.5837.370.680.25113.25PI 504510G. maxRegular germplasm63.025.936.70.80.3106.72OzarkG. maxRegular cultivar58.0212.0933.441.250.4105.2SS516G. maxSmall-seeded56.877.144.981.180.25110.13CX1834-1-2G. maxLow phytic acid56.099.940.480.850.23107.55R95-1705G. maxHigh protein55.143.4537.291.871.399.05V97-3000G. maxSmall-seeded53.946.4843.681.480.73106.31MFS-591G. maxSmall-seeded52.475.3849.951.180.2109.18CampG. maxSmall-seeded52.0410.738.680.380.13101.93PI 323556G. maxRegular germplasm43.79.4734.531.330.3789.4MFL-552G. maxLarge-seeded43.5615.539.51.580.25100.39S02-529G. maxRegular germplasm40.1412.7337.031.680.992.48MFL-159G. maxLarge-seeded31.6819.7336.881.630.1390.05PI 200508G. maxLow oligosaccharide30.710.855.150.30.237.21PI 417559G. maxRegular germplasm29.825.9324.516.812.2789.32PI 326581G. sojaWild species22.144.0530.11.831.059.12PI 153292G. gracilisWild species61.017.8541.71.450.23112.24Standard(a) sugar melibiose was added in each
sample as internal standard. External sugar standards
at concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0 μg/600 μL were prepared for creating calibration
curves for each of the major sugars. The concentration of each sugar was
estimated by regression of concentration and absorption of individual standard
sugars; LSD0.05(b)=9.07;
LSD0.05(c)=1.35;
LSD0.05(d)=4.26; LSD0.05(e)=0.46; LSD0.05(f)=0.36; LSD0.05(g)=9.13.Seed sample was ground using a SmartGrind
coffee bean grinder (Applica Consumer Products Inc., Fla,
USA) and screened through a
250μm standard testing sieve (VWR International, USA). Sugar extraction was
conducted following the procedures optimized by
Giannoccaro et al. [16]. Briefly,
duplicates of 0.15 g samples were weighed and placed in 2 mL Eppendorf
microcentrifuge tubes containing 1.5 mL distilled deionized water. The mixture
was vortexed and incubated at room temperature in a horizontal shaker at 200 rpm for 15 minutes. The suspension was then centrifuged at 13000 rpm for 10 minutes,
and 500 μL of the clear supernatant was transferred into a new 1.5 mL
microcentrifuge tube and purified with 700 μL 95% acetonitrile at room temperature
for 30 minutes, and then centrifuged at 13000 rpm for 10 minutes. A volume of
200 μL  of the supernatant was transferred into a 1.5 mL microcentrifuge tube
and dried at 95°C in a Dry
Bath Incubator
(Fisher Scientific, Pittsburgh, Pa, USA) for approximately 45 minutes. The residue
was redissolved in 200 μL 65% acetonitrile and filtered through a 0.2 μm
membrane disc filter (Pall Co., Mich, USA).Seed moisture was determined to
adjust the sugar content to a dry weight basis. Two grams of ground soybean
flour for each genotype were weighed in a predried aluminum weighing dish and
dried in an oven at130°C for 1 hour. The moisture content was calculated as
moisture (%) = 100 × [(weight
before drying)  − (weight after drying)]/(weight before
drying).


## 2.2. High-Performance Liquid Chromatography (HPLC) System

A Dionex DX500 HPAEC-PAD system equipped
with a GS50 pump, an ED40 pulsed amperometric detector, an AS40 automated
sampler with a 25μL injection loop, and a chromeleon chromatography management
system (Dionex, Sunnyvale, CA, USA)
was used for sugar identification and quantification. An analytical CarboPac
PA10 pellicular anion-exchange resin column (250  ×  4 mm)
preceded by a CarboPac PA10 guard column (50 × 4 mm)
and an AminoTrap column (30 × 3 mm)
was used for sugar separation.An aliquot of 24μL purified samples
was diluted with 576 μL distilled water prior to HPLC injection. Sugars were
eluted with 90 mM NaOH at a flow rate of 1.0 mL/min under isocratic conditions.
The mobile phase 90 mM NaOH was prepared by diluting carbonate-free HPLC grade
50% (w/w) stock solution in distilled water, filtered with a 0.45 μm membrane,
and degassed with compressed nitrogen gas for 30 minutes prior to being loaded.
The data acquisition for each sample was 30 minutes, and the analyses of 40
samples were generally completed in 20 hours.


## 2.3. Sugar
Identification, Quantification, and Statistical Analysis

Nine standard sugars, including
pinitol, myo-inositol, galactose, glucose, fructose, sucrose, raffinose,
stachyose, and verbascose, were obtained from Sigma Chemical Co. Sugars in the
extracts were identified by comparing their retention times to those of the
standard sugars. Sugar standards at concentrations of
0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0μg/600 μL were
prepared for creating calibration curves for each of
the major sugars, including glucose, fructose, sucrose, raffinose, and
stachyose. The sugar concentration in soybean seed was expressed as mg per g of
dry seed matter. Analysis of variance (ANOVA) was performed using the general
linear models (Proc GLM) of SAS
9.1 (SAS Institute Inc., Cary,
NC, USA).
Means of individual sugars in different genotypes were compared and separated
by SAS Waller grouping at P =  .05. Correlation analysis among different
sugar compositions was conducted using Excel CORREL function.


## 3. Results and Discussion

### 3.1. HPAEC-PAD Chromatogram

Soybean seed sugars were eluted in
the sequence of pinitol/myo-inositol, glucose, fructose, sucrose, raffinose,
stachyose, and verbascose based on their retention times
(Figure1). Because
pinitol and myo-inositol were eluted at the same retention time (2.3 minutes),
they could not be distinguished from each other by the HPLC system conditions in
this study. Galactose was not detectable in mature dry soybean seed. A group of
small molecules present before 3.8 minutes were not identifiable by the
standard sugars used in this study. The internal standard melibiose was eluted
at 8.0 minutes. Verbascose was identified as a minor sugar with a retention
time of 21.3 minutes, which has not been reported in previous research. Future
quantification of verbascose is important and valuable, especially for
vegetable soybean seed during seed development or storage, when verbascose
concentration is relatively high. Sucrose and stachyose represented the major
sugars in large quantities identified in soybean genotypes
(Table 1). Significant
difference was detected for all the soluble sugars among the 20 soybean genotypes.
The genotypic differences for sucrose, stachyose, and total soluble sugar
appeared larger than those for raffinose, glucose, and fructose. Highly
consistent estimates for major sugars were achieved across different
extractions and replications (Tables 2 & 3).Table 2
Analyses of variance for
five major sugar and total sugar contents in twenty soybean genotypes.
Sugar/source(a)DF(b)Mean squareF valuePr>FSucroseGenotype191188.8025.55<.0001Extraction169.801.50.2284Genotype x extraction1961.691.33.2258StachyoseGenotype19535.9951.04<.0001Extraction10.070.01.9950Genotype x extraction1913.981.33.2227RaffinoseGenotype1987.5282.00<.0001Extraction10.260.24.6265Genotype x extraction191.561.46.1572GlucoseGenotype1937.58303.28<.0001Extraction11.7213.88.0006Genotype x extraction193.5228.39<.0001FructoseGenotype1921.67290.03<.0001Extraction10.537.11.0114Genotype x extraction190.243.33.0009Total sugarGenotype192102.6343.72<.0001Extraction161.631.28.2649Genotype x extraction193204.593.51.0005Two(a) duplicate extractions following
Giannoccaro et al. [16] were prepared from each sample, and each extraction was
analyzed in two replicates; degree(b)  of freedom.Table 3
Means of individual sugars
in 20 soybean genotypes over replications of two duplicate extractions.
SugarAverage sugar content(mg/g)(a)Extraction IExtraction IIReplication 1Replication 2Replication 1Replication 2Glucose0.180.170.130.12Fructose0.120.120.110.05Sucrose4.494.474.824.68Raffinose0.860.910.910.96Stachyose3.413.423.453.45Total sugar9.059.099.359.12Two(a) duplicate
extractions following Giannoccaro et al. [16] were prepared from each sample,
and each extraction was analyzed in two replicates.HPAEC-PAD chromotograms of soybean seed sugars in Ozark (a), 03CB-14 (b), and
PI 417559 (c): glucose (1), fructose (2), melibiose (3), sucrose (4), raffinose
(5), stachyose (6), and verbascose (7). Unidentified peaks are not labeled.
(a)(b)(c)The
use of water as extractant significantly shortened the extraction time when
compared with using 80% ethanol as
extractant [8, 9, 16], therefore,
that would allow for the screening of a large
number of breeding lines and facilitate the development of soybean cultivars
with desirable sugar profiles. Using water as extractant is also low cost
associated with less labor input. The HPAEC-PAD detection method has been
reported as sensitive, fast, and capable of separating individual soluble sugars
in soybean seed [17]. The sensitivity and specificity of the present HPLC system
enabled clear identification and consistent quantification of soybean sugars
without much derivation as often observed in gas chromatography (GC) with flame
ionization detection [8]. In previous reports using  GC system, only a few major
sugars, including sucrose, raffinose, and stachyose, were detected [8, 9].
Identification of minor sugars such as glucose, fructose, and verbascose using
methods in this research makes feasible the studies of these sugars in
specialty soybean genotypes, and their changes during seed development and food
processing. With the combination of rapid extraction and sensitive detection,
following the procedures used in this study, the analyses of 40 samples can be
completed in 24 hours.


### 3.2. Sucrose

Sucrose was eluted at the
retention time of 9.6 minutes (Figure1). The ANOVA results revealed
significant variation for sucrose content among the 20 genotypes tested (P < .0001), whereas no significant  difference was detected between duplicate extractions
(P=.2284) (Table 2). There were no significant interactions between
genotype and extraction on sucrose content (P = .2258). These results
indicate that the extraction and quantification methods and conditions used in
the present study are highly reproducible. Evidently, current methodology is
very accurate in assessing sucrose content in given genotypes and, therefore,
very efficient in ranking soybean genotypes for breeding selection.Sucrose was the predominant soluble
sugar, accounting for an average of 55.16% (33–90%) of the total
sugar (Table1). The average sucrose content was 53.45 mg/g among all G. max accessions screened, with more than three-fold differences between the
extremes, ranging from 29.82 to 105.48 mg/g. The highest sucrose content was
detected in PI 243545 (105.48 mg/g, 79% of total sugar), which was known as a large-seeded
soybean germplasm with high total soluble sugar in our breeding program. The
low-oligosaccharide
breeding line, 03CB-14, also contained high sucrose (88.09 mg/g), accounting
for 90% of its total sugar. However, another low-oligosaccharide line, PI
200508, only had 30.71 mg/g sucrose content. The two large- and four small-seeded
food grade cultivars contained relatively low- (below average) sucrose content
(31.68–56.87 mg/g). The regular commercial cultivars
contained relatively high (above average) sucrose, whereas PI 417559 contained
only 29.85 mg/g sucrose, accounting for 33% of its total sugar. The sucrose
content in high-protein line, R95-1705 (55.14 mg/g), and low-phytate line,
CX1834-1-2 (56.0 mg/g), was moderate. The lowest sucrose content was detected
in the wild species G. soja PI 326581 (22.14 mg/g, 37.4% of total
sugar). In contrast, another wild species, G. gracilis PI 153292, had
moderately high-sucrose content (61.01 mg/g), accounting for 54% of its total
sugar.Hymowitz and  Collins [1] reported
that the sucrose content in 195 diverse soybean genotypes ranged from 30 to 102 mg/g, which is very similar to the sucrose variation observed in this study.
The average sucrose content was 61 mg/g, accounting for 57.5% of total soluble
sugars on average. Hymowitz et al. [8] also showed that the sucrose content
varied with soybean maturity groups (MG), ranging from 41 to 54 mg/g in MG 00
to IV, representing 55.4 to 62.8% of total sugars. More recently, Hartwig et
al. [7] reported a mean sucrose content of 39.6 to 55.2 mg/g in multiple
soybean cultivars evaluated over two years. The average sucrose content and its
proportion to total sugars in 18 G. max accessions screened in this
study were comparable to the values reported previously in other soybean
genotypes [1, 7, 16]. The sucrose content of 105.48 mg/g in PI 243545 was
similar to that of the highest sucrose genotypes identified by Hymowitz and
Collins [1]. In contrast, low-sucrose content was observed previously in the
wild species G. soja (24 mg/g on average) and G. gracilis (36 mg/g on average) [1]. In this study, the G. gracilis PI 153292 (61.01 mg/g) contained much higher sucrose than previously reported [1], whereas G.
soja PI 326581 (22.14 mg/g) was similar to what was reported previously [1].


### 3.3. Stachyose

Stachyose was the second richest
soluble sugar detected in soybean seed with a retention time of 16.8 minutes
(Figure1). The ANOVA indicated a significant variation among genotypes (P < .0001), but not significant between extractions (P = .9950) and
genotype x extraction interactions (P = .2227)
(Table 2). Therefore, the
extraction method and HPLC conditions used in this study produced reliable stachyose
results in differentiating and ranking genotypes for breeding selection.Relatively high stachyose content
was detected in most ofG. max lines tested, excluding 03CB-15 and PI
200508, ranging from 24.5 to 45.75 mg/g. High stachyose content was also
detected in two wild species (41.7 mg/g in G. gracilis and 30.1 mg/g in G.
soja, resp.). The average stachyose content in 18 G. max soybeans
was 31.97 mg/g, and the average percentage of stachyose in total sugar was
33.97%. The average content and percentage of stachyose in total sugar were
similar to those in previous reports [7, 8, 16]. However, the two
low-oligosaccharide lines, 03CB-14 and PI 200508, contained significantly lower
stachyose content (4.14 and 5.15 mg/g, resp.) than other genotypes, accounting
for only 4.2 and 13.8% of their total sugars. The stachyose content observed in
PI 200508 in this study was in good agreement with the previous report, in
which breeding populations derived from PI 200508 contained low-stachyose
content of less than 8.1 mg/g [12]. The high-sucrose and low-stachyose content
in 03CB-14 make it a valuable and unique germplasm for breeding specialty food
grade soybeans (Figure 1(b)). In addition, the high-sucrose line, PI 243545,
and the low-sucrose germplasm, PI 417559, also contain relatively low stachyose
(27.85 mg/g and 24.5 mg/g, resp.) and may be valuable for future genetic
studies or breeding purposes. Stachyose constitutes the most of undesirable
oligosaccharides in soybean seed. Reduction of stachyose improves food quality
for human and other animals by increasing digestibility and metabolizing
energy. The low-stachyose lines identified or confirmed in this study will
benefit future food grade soybean breeding as unique germplasm resources.


### 3.4. Minor Sugars

Low concentration of minor
sugars including glucose, fructose, raffinose, and verbascose was eluted at
6.2, 6.8, 9.6, 15, and 16.8 minutes, respectively
(Figure1). The average
content of raffinose was 8.54 mg/g in 18 G. max soybeans studied
(Table 1), accounting for 8.5% of total sugars. The lowest raffinose content (0.85 mg/g) was detected in PI 200508, confirming it as a low-oligosaccharide
germplasm. Genetic variation for raffinose was significant (P <
.0001) among the 20 genotypes tested, with a range of 0.85–19.73 mg/g. There was no significant variation
for extractions and genotype x extraction interactions, indicating a reliable
detection of this sugar and consistent sorting of the genotypes. Therefore, the
extraction method and HPLC conditions used in this study are adequate for
separating and ranking soybean genotypes in breeding selection.The glucose and fructose contents
were below 2 mg/g in all genotypes except PI 417559, which contained much higher
concentration of glucose (16.8 mg/g) and fructose (12.27 mg/g) than other
genotypes tested. This six- to eight-fold increase in glucose and fructose may
have resulted from a genetic mutation
(Figure1(c)). Therefore, PI 417559 may
serve as a valuable germplasm for genetic studies and breeding implications.
The significant variation due to extractions and genotype x extraction interactions
suggested a poor reproducibility in quantifications of glucose and fructose
under conditions used in this study. This may be due to the fact that the
concentration of these two sugars was very low and it is not feasible to
determine the actual concentrations. However, in breeding efforts to improve
glucose and fructose content, the current approach can be used to screen
genetic materials with high glucose and fructose such as PI 417559. The high
glucose and fructose germplasm will have extra values if incorporated in the
high-sucrose and low-oligosaccharide genetic background.


### 3.5. Total Sugar Content and Correlation Analysis

Significant variation existed in
total soluble sugar content among all soybean genotypes tested (P <
.001, Tables 1 & 2), whereas the variation between extractions was not
significant. The total sugar content among the 20 genotypes ranged from
37.21 (PI 200508) to 148.76 (PI 243545) mg/g, with a mean of 96.9 mg/g.
Different end-use soybean types contained similar levels of total sugars except
PI 243545 being significantly high and PI 200580 and PI 326581 (59.12 mg/g)
extremely low. The low-oligosaccharide line PI 200508 contained the least
amount of total soluble sugar (37.2 mg/g) with 82.5% of which being sucrose.
The total sugar content is primarily composed of sucrose and oligosaccharides
(raffinose and stachyose). The glucose, fructose, or verbascose content in soybean
seed has not been quantified in previous investigations due to the trace amount
of these sugars [1, 8]. Total sugar content in soybean seed ranged from 62 to
166 mg/g in previous reports [1, 8], which was very similar to the results
obtained in this study. The total sugar amount previously reported for G. gracilis ranged from 48 to 99 mg/g [1], which was much lower than that observed in PI
153292 in this study (112.24 mg/g). However, the total sugar content in G.
soja PI 326581 was in the range as reported for G. soja before [1].Correlation analyses revealed that
total sugar content was positively and significantly correlated with sucrose (r=0.7930∗∗), raffinose (r=0.4827∗∗), or stachyose (r=0.4954∗∗),
respectively. Positive and significant correlations also existed between
raffinose and stachyose (r=0.3687∗∗). These correlationships coincided well
with previous results [8]. Additional analyses showed a strong positive
correlation between glucose and fructose (r=0.9796∗∗), but a negative relationship
between glucose and sucrose (r=−0.3125∗∗). A weak and negative correlation was
also found between fructose and sucrose (r=−0.2980∗), stachyose (r=−0.2219∗), raffinose (r=−0.2022), or total sugar (r=−0.1420). This negative
correlationship is well illustrated by PI 417559, which had the highest glucose
and fructose content but low-total sugar (89.32 mg/g) and sucrose (29.82 mg/g).
The increase of glucose and fructose content was apparently at the cost of
other sugars such as sucrose and oligosaccharides. Further investigation is
needed to improve the digestible sugars (glucose, fructose, and sucrose), while
reduce the oligosaccharides (raffinose and stachyose) using the unique
germplasm lines such as 03CB-14 and PI 417559.


## 3.1. HPAEC-PAD Chromatogram

Soybean seed sugars were eluted in
the sequence of pinitol/myo-inositol, glucose, fructose, sucrose, raffinose,
stachyose, and verbascose based on their retention times
(Figure1). Because
pinitol and myo-inositol were eluted at the same retention time (2.3 minutes),
they could not be distinguished from each other by the HPLC system conditions in
this study. Galactose was not detectable in mature dry soybean seed. A group of
small molecules present before 3.8 minutes were not identifiable by the
standard sugars used in this study. The internal standard melibiose was eluted
at 8.0 minutes. Verbascose was identified as a minor sugar with a retention
time of 21.3 minutes, which has not been reported in previous research. Future
quantification of verbascose is important and valuable, especially for
vegetable soybean seed during seed development or storage, when verbascose
concentration is relatively high. Sucrose and stachyose represented the major
sugars in large quantities identified in soybean genotypes
(Table 1). Significant
difference was detected for all the soluble sugars among the 20 soybean genotypes.
The genotypic differences for sucrose, stachyose, and total soluble sugar
appeared larger than those for raffinose, glucose, and fructose. Highly
consistent estimates for major sugars were achieved across different
extractions and replications (Tables 2 & 3).Table 2
Analyses of variance for
five major sugar and total sugar contents in twenty soybean genotypes.
Sugar/source(a)DF(b)Mean squareF valuePr>FSucroseGenotype191188.8025.55<.0001Extraction169.801.50.2284Genotype x extraction1961.691.33.2258StachyoseGenotype19535.9951.04<.0001Extraction10.070.01.9950Genotype x extraction1913.981.33.2227RaffinoseGenotype1987.5282.00<.0001Extraction10.260.24.6265Genotype x extraction191.561.46.1572GlucoseGenotype1937.58303.28<.0001Extraction11.7213.88.0006Genotype x extraction193.5228.39<.0001FructoseGenotype1921.67290.03<.0001Extraction10.537.11.0114Genotype x extraction190.243.33.0009Total sugarGenotype192102.6343.72<.0001Extraction161.631.28.2649Genotype x extraction193204.593.51.0005Two(a) duplicate extractions following
Giannoccaro et al. [16] were prepared from each sample, and each extraction was
analyzed in two replicates; degree(b)  of freedom.Table 3
Means of individual sugars
in 20 soybean genotypes over replications of two duplicate extractions.
SugarAverage sugar content(mg/g)(a)Extraction IExtraction IIReplication 1Replication 2Replication 1Replication 2Glucose0.180.170.130.12Fructose0.120.120.110.05Sucrose4.494.474.824.68Raffinose0.860.910.910.96Stachyose3.413.423.453.45Total sugar9.059.099.359.12Two(a) duplicate
extractions following Giannoccaro et al. [16] were prepared from each sample,
and each extraction was analyzed in two replicates.HPAEC-PAD chromotograms of soybean seed sugars in Ozark (a), 03CB-14 (b), and
PI 417559 (c): glucose (1), fructose (2), melibiose (3), sucrose (4), raffinose
(5), stachyose (6), and verbascose (7). Unidentified peaks are not labeled.
(a)(b)(c)The
use of water as extractant significantly shortened the extraction time when
compared with using 80% ethanol as
extractant [8, 9, 16], therefore,
that would allow for the screening of a large
number of breeding lines and facilitate the development of soybean cultivars
with desirable sugar profiles. Using water as extractant is also low cost
associated with less labor input. The HPAEC-PAD detection method has been
reported as sensitive, fast, and capable of separating individual soluble sugars
in soybean seed [17]. The sensitivity and specificity of the present HPLC system
enabled clear identification and consistent quantification of soybean sugars
without much derivation as often observed in gas chromatography (GC) with flame
ionization detection [8]. In previous reports using  GC system, only a few major
sugars, including sucrose, raffinose, and stachyose, were detected [8, 9].
Identification of minor sugars such as glucose, fructose, and verbascose using
methods in this research makes feasible the studies of these sugars in
specialty soybean genotypes, and their changes during seed development and food
processing. With the combination of rapid extraction and sensitive detection,
following the procedures used in this study, the analyses of 40 samples can be
completed in 24 hours.


## 3.2. Sucrose

Sucrose was eluted at the
retention time of 9.6 minutes (Figure1). The ANOVA results revealed
significant variation for sucrose content among the 20 genotypes tested (P < .0001), whereas no significant  difference was detected between duplicate extractions
(P=.2284) (Table 2). There were no significant interactions between
genotype and extraction on sucrose content (P = .2258). These results
indicate that the extraction and quantification methods and conditions used in
the present study are highly reproducible. Evidently, current methodology is
very accurate in assessing sucrose content in given genotypes and, therefore,
very efficient in ranking soybean genotypes for breeding selection.Sucrose was the predominant soluble
sugar, accounting for an average of 55.16% (33–90%) of the total
sugar (Table1). The average sucrose content was 53.45 mg/g among all G. max accessions screened, with more than three-fold differences between the
extremes, ranging from 29.82 to 105.48 mg/g. The highest sucrose content was
detected in PI 243545 (105.48 mg/g, 79% of total sugar), which was known as a large-seeded
soybean germplasm with high total soluble sugar in our breeding program. The
low-oligosaccharide
breeding line, 03CB-14, also contained high sucrose (88.09 mg/g), accounting
for 90% of its total sugar. However, another low-oligosaccharide line, PI
200508, only had 30.71 mg/g sucrose content. The two large- and four small-seeded
food grade cultivars contained relatively low- (below average) sucrose content
(31.68–56.87 mg/g). The regular commercial cultivars
contained relatively high (above average) sucrose, whereas PI 417559 contained
only 29.85 mg/g sucrose, accounting for 33% of its total sugar. The sucrose
content in high-protein line, R95-1705 (55.14 mg/g), and low-phytate line,
CX1834-1-2 (56.0 mg/g), was moderate. The lowest sucrose content was detected
in the wild species G. soja PI 326581 (22.14 mg/g, 37.4% of total
sugar). In contrast, another wild species, G. gracilis PI 153292, had
moderately high-sucrose content (61.01 mg/g), accounting for 54% of its total
sugar.Hymowitz and  Collins [1] reported
that the sucrose content in 195 diverse soybean genotypes ranged from 30 to 102 mg/g, which is very similar to the sucrose variation observed in this study.
The average sucrose content was 61 mg/g, accounting for 57.5% of total soluble
sugars on average. Hymowitz et al. [8] also showed that the sucrose content
varied with soybean maturity groups (MG), ranging from 41 to 54 mg/g in MG 00
to IV, representing 55.4 to 62.8% of total sugars. More recently, Hartwig et
al. [7] reported a mean sucrose content of 39.6 to 55.2 mg/g in multiple
soybean cultivars evaluated over two years. The average sucrose content and its
proportion to total sugars in 18 G. max accessions screened in this
study were comparable to the values reported previously in other soybean
genotypes [1, 7, 16]. The sucrose content of 105.48 mg/g in PI 243545 was
similar to that of the highest sucrose genotypes identified by Hymowitz and
Collins [1]. In contrast, low-sucrose content was observed previously in the
wild species G. soja (24 mg/g on average) and G. gracilis (36 mg/g on average) [1]. In this study, the G. gracilis PI 153292 (61.01 mg/g) contained much higher sucrose than previously reported [1], whereas G.
soja PI 326581 (22.14 mg/g) was similar to what was reported previously [1].


## 3.3. Stachyose

Stachyose was the second richest
soluble sugar detected in soybean seed with a retention time of 16.8 minutes
(Figure1). The ANOVA indicated a significant variation among genotypes (P < .0001), but not significant between extractions (P = .9950) and
genotype x extraction interactions (P = .2227)
(Table 2). Therefore, the
extraction method and HPLC conditions used in this study produced reliable stachyose
results in differentiating and ranking genotypes for breeding selection.Relatively high stachyose content
was detected in most ofG. max lines tested, excluding 03CB-15 and PI
200508, ranging from 24.5 to 45.75 mg/g. High stachyose content was also
detected in two wild species (41.7 mg/g in G. gracilis and 30.1 mg/g in G.
soja, resp.). The average stachyose content in 18 G. max soybeans
was 31.97 mg/g, and the average percentage of stachyose in total sugar was
33.97%. The average content and percentage of stachyose in total sugar were
similar to those in previous reports [7, 8, 16]. However, the two
low-oligosaccharide lines, 03CB-14 and PI 200508, contained significantly lower
stachyose content (4.14 and 5.15 mg/g, resp.) than other genotypes, accounting
for only 4.2 and 13.8% of their total sugars. The stachyose content observed in
PI 200508 in this study was in good agreement with the previous report, in
which breeding populations derived from PI 200508 contained low-stachyose
content of less than 8.1 mg/g [12]. The high-sucrose and low-stachyose content
in 03CB-14 make it a valuable and unique germplasm for breeding specialty food
grade soybeans (Figure 1(b)). In addition, the high-sucrose line, PI 243545,
and the low-sucrose germplasm, PI 417559, also contain relatively low stachyose
(27.85 mg/g and 24.5 mg/g, resp.) and may be valuable for future genetic
studies or breeding purposes. Stachyose constitutes the most of undesirable
oligosaccharides in soybean seed. Reduction of stachyose improves food quality
for human and other animals by increasing digestibility and metabolizing
energy. The low-stachyose lines identified or confirmed in this study will
benefit future food grade soybean breeding as unique germplasm resources.


## 3.4. Minor Sugars

Low concentration of minor
sugars including glucose, fructose, raffinose, and verbascose was eluted at
6.2, 6.8, 9.6, 15, and 16.8 minutes, respectively
(Figure1). The average
content of raffinose was 8.54 mg/g in 18 G. max soybeans studied
(Table 1), accounting for 8.5% of total sugars. The lowest raffinose content (0.85 mg/g) was detected in PI 200508, confirming it as a low-oligosaccharide
germplasm. Genetic variation for raffinose was significant (P <
.0001) among the 20 genotypes tested, with a range of 0.85–19.73 mg/g. There was no significant variation
for extractions and genotype x extraction interactions, indicating a reliable
detection of this sugar and consistent sorting of the genotypes. Therefore, the
extraction method and HPLC conditions used in this study are adequate for
separating and ranking soybean genotypes in breeding selection.The glucose and fructose contents
were below 2 mg/g in all genotypes except PI 417559, which contained much higher
concentration of glucose (16.8 mg/g) and fructose (12.27 mg/g) than other
genotypes tested. This six- to eight-fold increase in glucose and fructose may
have resulted from a genetic mutation
(Figure1(c)). Therefore, PI 417559 may
serve as a valuable germplasm for genetic studies and breeding implications.
The significant variation due to extractions and genotype x extraction interactions
suggested a poor reproducibility in quantifications of glucose and fructose
under conditions used in this study. This may be due to the fact that the
concentration of these two sugars was very low and it is not feasible to
determine the actual concentrations. However, in breeding efforts to improve
glucose and fructose content, the current approach can be used to screen
genetic materials with high glucose and fructose such as PI 417559. The high
glucose and fructose germplasm will have extra values if incorporated in the
high-sucrose and low-oligosaccharide genetic background.


## 3.5. Total Sugar Content and Correlation Analysis

Significant variation existed in
total soluble sugar content among all soybean genotypes tested (P <
.001, Tables 1 & 2), whereas the variation between extractions was not
significant. The total sugar content among the 20 genotypes ranged from
37.21 (PI 200508) to 148.76 (PI 243545) mg/g, with a mean of 96.9 mg/g.
Different end-use soybean types contained similar levels of total sugars except
PI 243545 being significantly high and PI 200580 and PI 326581 (59.12 mg/g)
extremely low. The low-oligosaccharide line PI 200508 contained the least
amount of total soluble sugar (37.2 mg/g) with 82.5% of which being sucrose.
The total sugar content is primarily composed of sucrose and oligosaccharides
(raffinose and stachyose). The glucose, fructose, or verbascose content in soybean
seed has not been quantified in previous investigations due to the trace amount
of these sugars [1, 8]. Total sugar content in soybean seed ranged from 62 to
166 mg/g in previous reports [1, 8], which was very similar to the results
obtained in this study. The total sugar amount previously reported for G. gracilis ranged from 48 to 99 mg/g [1], which was much lower than that observed in PI
153292 in this study (112.24 mg/g). However, the total sugar content in G.
soja PI 326581 was in the range as reported for G. soja before [1].Correlation analyses revealed that
total sugar content was positively and significantly correlated with sucrose (r=0.7930∗∗), raffinose (r=0.4827∗∗), or stachyose (r=0.4954∗∗),
respectively. Positive and significant correlations also existed between
raffinose and stachyose (r=0.3687∗∗). These correlationships coincided well
with previous results [8]. Additional analyses showed a strong positive
correlation between glucose and fructose (r=0.9796∗∗), but a negative relationship
between glucose and sucrose (r=−0.3125∗∗). A weak and negative correlation was
also found between fructose and sucrose (r=−0.2980∗), stachyose (r=−0.2219∗), raffinose (r=−0.2022), or total sugar (r=−0.1420). This negative
correlationship is well illustrated by PI 417559, which had the highest glucose
and fructose content but low-total sugar (89.32 mg/g) and sucrose (29.82 mg/g).
The increase of glucose and fructose content was apparently at the cost of
other sugars such as sucrose and oligosaccharides. Further investigation is
needed to improve the digestible sugars (glucose, fructose, and sucrose), while
reduce the oligosaccharides (raffinose and stachyose) using the unique
germplasm lines such as 03CB-14 and PI 417559.


## 4. Conclusions

Significant variation was observed for
individual and total sugar contents in 20 diverse soybean genotypes tested in
this study. Reproducible estimates were achieved for sucrose, stachyose, raffinose,
and total sugars with the HPLC system used. Due to the low concentrations of
glucose and fructose in soybean seed, quantification of such sugars was
variable and relatively inaccurate. However, it is possible to screen and
select soybean germplasm such as PI 417559 with high glucose and fructose.
Sucrose was the predominant sugar in soybean seed followed by stachyose. The
highest sucrose (105.48 mg/g) and total sugar (148.76 mg/g) content was found
in PI 243545, whereas the least sucrose and total sugar was found in wild
speciesG. soja PI 326581 (22.14 mg/g) and PI 200508 (37.21 mg/g),
respectively. Two low-oligosaccharide breeding lines, PI 200508 and 03CB-14,
were confirmed with extremely low concentrations of raffinose and stachyose.
The identified germplasm with unique sugar profiles is particularly valuable in
breeding specialty soybeans for food and feed purposes.  Total sugar content was positively correlated
with sucrose, raffinose, and stahcyose content. Positive correlations were also
found between raffinose and stachyose, and between glucose and fructose. Negative
correlations existed between glucose and sucrose, fructose and sucrose, and
fructose and stahcyose. Further investigations are needed to examine the
environmental effects on sugar contents in the unique soybean germplasm
identified in this study.



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*Source: 484571-2008-10-27.xml*