Agriculture Reference
In-Depth Information
Chromatographic techniques have also been used for soybean cultivar identification.
Buelher et al. [61] developed a chromatographic method in the reversed-phase mode enabling
the separation of soybean proteins in 55 min and Oomah et al. [62] used size-exclusion (with
a separation time of 25 min) and reversed-phase (with a separation time of 40 min)
chromatography for the same purpose. Both groups found promising the use
of HPLC for
cultivar identification despite differentiation among closely related soybean cultivars was
difficult. Cole and Cousin [63] demonstrated the suitability of size-exclusion chromatography
for profiling soybean proteins using separation times of 40 min. They also applied the
proposed methodology to the analysis
of two different soybean varieties observing profiles
with the same number of peaks but with different peak size. More recently, Riblett et al. [6]
and Mujoo et al. [33] have applied reversed-phase high performance liquid chromatography
(RP-HPLC) for the separation of total proteins and the 11S and 7S fractions in different
soybean varieties. Despite it was possible to observe differences among soybean cultivars
studied, the times used for the completion of the separation were so high (40 and 90 min,
respectively) that both methodologies can be considered of limited application for routine or
field studies. Figure 2 shows the profiles obtained for glycinin and β-conglycinin in four
different soybean cultivars revealing
variations in the composition of each fraction.
Our research group has a great expertise in the use of chromatographic techniques in the
reversed-phase and ion-exchange modes using conventional, perfusion, and monolithic
stationary phases and capillary electrophoresis for the analysis of soybean proteins [64-68].
Some of these methodologies have been applied to the differentiation between soybean and
other related legumes (mungbean, azuki bean, etc.) commonly commercialized as soybeans.
Moreover, the differentiation between transgenic and non-transgenic soybeans has also been
dealt with success [68]. We have focused the present work on the characterization of soybean
cultivars. For that purpose, protein profiles obtained by HPLC with rapid perfusion stationary
phases and multivariate classification techniques are proposed. Perfusion stationary phases
enable the drastic reduction of analysis times due to their characteristic structure that alleviate
the low diffusivity of proteins [69]. Moreover, due to the short time needed for the
completion of every analysis a very comprehensive study consisting of the analysis of 91
different soybean cultivars could be performed.
P
ERFUSION
RP-HPLC
A
PPLIED TO THE
R
APID
C
HARACTERIZATION
OF
S
OYBEAN
C
ULTIVARS
B
ASED ON
P
ROTEIN
M
ARKERS
Materials and Methods
1. Chemicals and Samples
HPLC grade acetonitrile (ACN) (Merck, Darmstadt, Germany), HPLC grade water
(Milli-Q system, Millipore, Bradford, MA, USA), and trifluoroacetic acid (TFA) (Sigma, St
Louis, MO, USA) were used for the preparation of mobile phases and soybean solutions.
A total of 91 non-transgenic soybean varieties with different origins and corresponding to
the germplasm collection of the CRF (Centro de Recursos Fitogenéticos del Instituto
Nacional de Investigaciones Agrarias, Madrid, Spain) were analyzed. Most soybean varieties
were from USA: 41 were commercial varieties (Ap-18, Gnome, Elf, Cumberland, Amcor,
Search WWH ::
Custom Search