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Structural changes in kafirin extracted form a

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germination
ARTICLE in JOURNAL OF CEREAL SCIENCE MARCH 2012
Impact Factor: 2.09 DOI: 10.1016/j.jcs.2011.10.007

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Journal of Cereal Science xxx (2011) 1e6

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Journal of Cereal Science

journal homepage: www.elsevier.com/locate/jcs

Structural changes in karin extracted from a white type II tannin sorghum during
germination
Dominique M.R. Georget a, *, Abd Elmoneim O. Elkhalifa b, S. Belton Peter a
a
b

School of Chemistry, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK

School of Pharmacy, Ahfad University for Women, Omdurman, Sudan

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 June 2011
Received in revised form
14 October 2011
Accepted 17 October 2011

Karin proteins were extracted from a Sudanese cultivar of sorghum (Feterita). The extracted materials
were characterized by SDS PAGE. Under non-reducing conditions as germination was progressing from
0 to 72 h, there was visually a decrease in b-karins whereas a small effect was observed on a-karins.
The occurrence of interactions between tannins and karin might explain this behavior when compared
to other cultivars. Thermogravimetric analysis (TGA) revealed that the rst change in weight between
room temperature and 100
C was due to the loss of water. The protein extract started to degrade
thereafter with the decomposition rate increasing with germination time. Fourier Transform Infrared
(FT-IR) analysis showed that the secondary structure of karin was found to be mainly governed by
a-helices with some b-sheets. Upon germination, there was a change in protein conformation, mainly an
increase in a-helices. Scanning Electron Microscopy (SEM) showed that after extraction, karin proteins
formed aggregated particles. This study is the rst to show in the case of Feterita cultivar that upon
germination, the physical properties of the karin proteins were signicantly affected and relied on
composition. This could shed some light on the effect of processing such as germination on sorghum
karin proteins originated from Feterita.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Sorghum
Germination
Karin
FT-IR
SDS PAGE
SEM

1. Introduction
Grain sorghum (Sorghum bicolor (L.) Moench) is the staple food
for a large population in Africa, India and the semi-arid parts of the
tropics. Sorghum together with millet is commonly consumed by
the poorer section of the population in many countries and it forms
a major source of proteins and calories in the diet of large segments
of the population of Africa (Belton and Taylor, 2004). It is the most
important cereal crop in Sudan and is generally consumed as fermented at bread (Kisra), thick porridge (Aceda), thin fermented
gruel (Nasha), non-alcoholic beverages (Abreh and Hulu-mur) and
alcoholic beverages (Merissa and Assaliya). A protein quality
problem, which is apparently unique to sorghum, is that the
digestibility of sorghum protein signicantly decreases upon wet
cooked and other processing of sorghum grains (Mertz et al., 1984;
Axtell et al., 1981; Hassan and El Tinay, 1995). In developing
countries where cereals are the staple diet, the quality of proteins
plays an important role from a human consumption perspective.

* Corresponding author. Tel.: 44 01603 593 145; fax: 44 01603 592 003.
E-mail address: d.georget@uea.ac.uk (D.M.R. Georget).

Due to the importance of sorghum, considerable research has

been undertaken (Wang and Fields, 1978; Taylor et al., 1985;
Elkhalifa et al., 2004; Belton and Taylor, 2004) with the objective
of improving the digestibility and nutritional attributes of sorghum
proteins using traditional technologies such as malting and
fermentation. Germination is an inexpensive and simple method to
improve protein quality of sorghum (Oumarou et al., 2005) by
increasing enzyme activity and converting carbohydrates and
proteins into a more digestible form and this can be achieved in
a short period of time. Numerous studies have reported higher
levels of nutrients, such as amino acids, digestible proteins, available carbohydrates, and other compounds, and lower levels of nonnutritive factors in cereal sprouts as compared to ungerminated
seeds (Shem et al., 1990; Elkhalifa and El Tinay, 1994; Elmaki et al.,
1999). Germination is a common practice in sorghum producing
areas. Grains are malted for the production of weaning foods,
opaque beers and other traditional dishes. Germination triggers the
enzymatic activity of sprouting seeds, leading to the breakdown of
proteins, carbohydrates and lipids (Nout and Ngoddy, 1997). This
processing method activates intrinsic amylases, proteases, phytases
and ber-degrading enzymes thereby increasing nutrient accessibility (Taylor et al., 1985). Malted sorghum has traditionally been
used in several countries in Africa, but always after careful removal
of the toxic parts (roots and sprouts).

0733-5210/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2011.10.007

Please cite this article in press as: Georget, D.M.R., et al., Structural changes in karin extracted from a white type II tannin sorghum during
germination, Journal of Cereal Science (2011), doi:10.1016/j.jcs.2011.10.007

D.M.R. Georget et al. / Journal of Cereal Science xxx (2011) 1e6

Karin, the prolamin protein fraction of sorghum, which

constitutes 65e75% of the total protein in the whole grain
(Hamaker et al., 1995), shares similarities with maize storage
proteins in molecular weight, solubility, amino acid composition
and structure (Belton et al., 2006; Shull et al., 1991). It can be
extracted in 70% aqueous ethanol with reducing agent (Gao et al.,
2005). Four classes of karin protein (Belton et al., 2006) have
been identied. The a, b-and g-have been classied based on their
solubility, electrophoretic mobility, amino acid composition and
sequences, molecular masses and immunochemical crossreactions. The fourth group, d-karin, similar to d-zein has been
identied based on sequences of cloned DNA (Izquierdo and
Godwin, 2005). It is important to study the changes that occur in
karin, the main sorghum protein, during germination as this
would enable better understanding of the effect of processing
conditions on the quality attributes of these proteins during the
manufacture of sorghum based food. Therefore, the aim of the
present study was to investigate the effect of the germination
process at three different stages on the chemical properties of
karin proteins from a white type II tannin sorghum cultivar by SDS
PAGE. Additionally the physical characteristics relevant to protein
functionality were studied using FT-IR, TGA and SEM.

the ethanol was removed from the extract by evaporation in a fume

cabinet at ambient temperature until viscous liquid sediment
formed. Chilled distilled water (<10
C) of approximately equal
volume was used to dilute the sediment. The pH was then reduced
to pH 5 with 1 M HCl to liberate all the protein from suspension. The
precipitated protein was then recovered by centrifugation at
2000  g for 5 min at 4
C. The resulting wet protein was frozen
at 20
C and then freeze-dried. The freeze-dried karin preparations were weight milled into a ne powder, and then defatted
three times for 1 h each with hexane at room temperature. The
defatted protein powder was then air-dried in a fume cabinet and
stored in an airtight bottle at 4
C.

2. Materials and methods

Karin powder samples were mounted on aluminum pin stubs

using conductive self-adhesive carbon label. The specimens were
sputter coated with a layer of gold approximately 50 nm thick in
a sputter coater S150B (Edwards, UK). All samples were examined
in a JEOL 5900LV scanning electron microscope (JEOL Ltd, UK) at an
accelerating voltage of 20 kV.

2.1. Materials
In Sudan, for all practical purposes, most of the sorghum malt is
prepared from Feterita, a white type II tannin cultivar. Rural
Sudanese strongly believe in the nutritional superiority of Feterita
over other sorghum cultivars (Dirar, 1993). Sorghum Feterita
cultivar was grown in Gadarif, Sudan, and harvested in the year
2009. The material was carefully cleaned and stored at 4
C.
2.2. Sorghum germination
Sorghum grains were germinated according to the modied
traditional Sudanese method of germination as described by
Elkhalifa (1998). The sorghum grain (500 g) was steeped in distilled
water overnight with two changes of water during the day to
remove dirt and husk. The wet grain was subsequently soaked in
1e2 volumes of 0.2% formaldehyde solution for 40 min to retard
mold growth during germination. The soaked grains were then
washed with distilled water several times and soaked in water for
20 min to remove residual formaldehyde. The wet sorghum was
spread out thinly on a jute bag saturated with distilled water and
covered with another jute bag. Grains were allowed to germinate in
the dark for three days at room temperature (27  2
C). Samples
(100 g) were taken every day. Water was sprayed on the grains
when necessary. The germinated grains were dried to constant
weight at 40
C (to obtain nal moisture content of approximately
5%). The root portions were manually removed. The grains
(germinated and ungerminated) were ground in a hammer mill to
pass through a 0.4 mm (60 BSS mesh) screen and the ours were
stored at 4
C.
2.3. Karin extraction
Karin was extracted from milled sorghum using a modied
Carter and Reck (1970) method with a solution of 70% (w/w)
absolute ethanol in distilled water and 0.5% (w/w) sodium metabisulphite at a ratio of 1:5 (w/w) our to extractant with vigorous
stirring at 70  0.1
C for 1 h. Insoluble matter was removed by
centrifugation at 1000  g for 5 min at ambient temperature. The
clear supernatant was decanted into large glass Petri dishes, and

2.4. SDS PAGE

The protein prole of germinated sorghum our was analyzed
by SDS PAGE under reducing and non-reducing conditions using
12 cm long, 1 mm thick gels on Hoeffer/Pharmacia Biotech vertical
electrophoresis system (SE600) (Uppsala, Sweden) as described by
Duodu et al. (2002).
2.5. Scanning electron microscopy (SEM)

2.6. Thermogravimetric analysis (TGA)

The change in the weight of karin powder extracted from
sorghum seeds germinated at 0, 24, 48 and 72 h when heated was
determined using a thermogravimetic analyzer TA Instruments
HI-Res TGA 2950 (TA Instruments Ltd, Crawley, UK) at a heating rate
of 10
C/min to 350
C. This allowed the water loss to be calculated
when detected. Experiments were carried out in triplicate. Any
measurements presented are the means and the standard error of
the mean (sem).
2.7. Fourier Transform Infrared spectroscopy (FT-IR)
FT-IR spectra were recorded on a Bruker FT-IR spectrometer
(Bruker Optics Limited, Coventry, UK, model IFS 66/S) (Elkhalifa
et al., 2009). Powder samples were tested without any prior
moisture conditioning straight from the airtight jar. Further analysis will reveal that the moisture content was similar irrespective of
the germination stage. Specimens were placed on a singlereection diamond ATR (attenuated total reectance) accessory
(SPECAC, Orpington, U.K.) and pressed down to ensure a good
contact with the ATR crystal. The spectra (200 spectra at 4 cm1
resolution for each sample) were averaged. Three replicates of each
sample were taken using the empty ATR crystal as a reference.
Spectra analysis was performed using Omnic v6.1A software
(Thermo Nicolet Cooperation, Madison, WI) as described earlier
(Elkhalifa et al., 2009; Wellner et al., 1996). Each spectrum was
baseline corrected following the method of Wellner et al. (1996) by
ensuring that the spectrum was zeroed at 1800 cm1 where the
baseline was relatively at. Fourier self-deconvolution (FSD) was
also carried out with an enhancement factor of 1.3 and bandwidth
of 30. Positions of the absorbance peaks located in the amide I and
amide II regions were determined using the second derivative
(Herald and Smith, 1992). Values of the mean and the standard
error of the mean were calculated.

Please cite this article in press as: Georget, D.M.R., et al., Structural changes in karin extracted from a white type II tannin sorghum during
germination, Journal of Cereal Science (2011), doi:10.1016/j.jcs.2011.10.007

D.M.R. Georget et al. / Journal of Cereal Science xxx (2011) 1e6

2.8. Statistical analysis

The data were analyzed statistically using ANOVA with the
software package SPSS. Using a Bonferroni test, signicant difference was considered at the level of p < 0.05.
3. Results and discussion
3.1. SDS PAGE
The extracted karin from ungerminated and germinated
sorghum was evaluated by SDS PAGE (Fig. 1 a and b) to determine
the general characteristics of the protein extracted. Results
obtained showed the presence of g-, a1-, a2-, and b-karin, with
a1-karin (Mr 24e25 kDa) predominating. The band patterns
were in agreement with the study of El Nour et al. (1998) and
Duodu et al. (2002).
Fig. 1a shows a typical non-reduced karin electrophoretic
prole with spots characteristic of high molecular- weight (HMW)
aggregates 45 kDa oligomers, g-and a-monomers and b-monomer.
b-karin disappears after 24 h germination conrming the results
reported by Mazhar and Chandrashekar (1993). With regard to the
spot attributed to g-and a-karin proteins, it slightly decreases in
intensity upon germination supporting the ndings from Taylor
and Evans (1989). When the karin was analyzed under reducing
conditions, the electrophoretic proles underwent some visible
changes: disappearance of the HMW aggregate oligomer electrophoretic spots whereas a-and g-monomer electrophoretic spots
(Fig. 1b) are unchanged. However there is visually an increase in
intensity of the latter up to 24 h germination after which it
decreases. Reducing conditions cause SeS bond cleavage, which
leads to disruption of the oligomers and the appearance of their
constituents (Correia et al., 2008). In the case of Feterita, formation
of complexes between tannins and karin proteins might be

Fig. 1. SDS PAGE under non-reducing conditions (a) and reducing conditions (b) of
karin fraction extracted from sorghum seed germinated at 0, 24, 48 and 72 h.

induced (Taylor et al., 2007). Indeed under reducing conditions,

novel sites on the surface of the karin proteins might have been
exposed, favoring interactions between tannins and karin
monomers. Consequently formation of aggregates might interfere
during the gel separation which might not give a true picture of the
changes in karin. Additionally a-amylase activity during malting
results in modications in grain starch (Belton and Taylor, 2004),
inducing the lack of cohesion due to starch digestion within the
endosperm. Consequently, extraction reagents will have easy
access to the proteins. But this could also promote more complex
formation between tannins and karin as earlier explained. In
addition, carbohydrate respiration can also promote prolamin
concentration in malted sorghum. Germination also caused
a decrease in the concentration of b-karin (Fig. 1a). Under nonreducing conditions, the observed weak decrease in prolamin
electrophoretic spot areas after 2 or 3 days of germination might be
due to prolamin hydrolysis as reported by Correia et al. (2008).
Taylor (1983) and Subramanian et al. (1995) reported that malting
results in the production of proteolytic enzymes that hydrolyze
a proportion of prolamins and glutelins. Mazhar and
Chandrashekar (1993) reported that b- and g-karins were the
rst proteins to be degraded during sorghum germination. The
authors concluded that because protein degradation occurs from
the surface, the maximal breakdown of b- and g-karins may be
explained by their peripheral location. Germination studies in corn
led to similar conclusions (Torrent et al., 1989). In the case of
Feterita it is clear that SDS PAGE results show some variation but
the inuence of tannins via the formation of complexes with the
karin proteins cannot be ignored.
3.2. TGA
Fig. 2a shows the change in weight of the karin extracted from
variously germinated sorghum seeds as a function of temperature.
All the specimens show a slight decrease in weight below 100
C.
This also is accompanied by a maximum at 70
C seen in Fig. 2b.
This weight change is commonly associated to water loss as earlier
described by Geneau-Sbarta et al. (2008) who identied the
dehydration of sunower protein isolates at similar temperature.
This is also supported by the work of Aluigi et al. (2007) who found
a similar event in wool keratin. The results from weight loss then
associated to the water content were extracted from Fig. 2a. The
water content lies around 3% (based on the initial weight content)
with the karin extracted from the 72 h germinated sorghum seeds
having the lowest water content though there is no statistical
difference for p < 0.05.
Further upon heating the weight change signicantly passes
through a sharp decrease just above 250
C (Fig. 2a). The decrease is
sudden for karin originated from ungerminated seeds whereas
a gradual decrease in weight starting from 100
C until 250
C
occurs for the 24, 48 and 72 h germination with a decrease in slope
with increasing germination time. After 250
C the decrease in the
change of weight is sharp and is accompanied by a maximum at
around 300
C in the rst derivative (Fig. 2b). The values for this
maximum are 309.9
C (0.21), 303.5
C (1.98), 315.0
C (0.87)
and 319.0
C (0.96) for ungerminated, germinated for 24 h, 48 h
and 72 h, respectively. This change in weight is characteristic of
thermal degradation as earlier reported by Wang et al. (2009) who
studied the thermal degradation of variously extracted karin. This
is also supported by the work on gluten (Mohamed et al., 2006). In
the present study, this change in thermal degradation could be
reected by a difference in the chemical composition of the karin
proteins as earlier detected in SDS PAGE analysis which showed
a decrease in b-karin with germination time. It appears that the
extent of karin thermal degradation markedly depends on the

Please cite this article in press as: Georget, D.M.R., et al., Structural changes in karin extracted from a white type II tannin sorghum during
germination, Journal of Cereal Science (2011), doi:10.1016/j.jcs.2011.10.007

D.M.R. Georget et al. / Journal of Cereal Science xxx (2011) 1e6

a
100

Change in weight / % w.w.b

90
80
70
60
50

0hr
24hrs
48hrs
72hrs

40
30
20
0

50

100

150

200

250

300

350

400

250

300

350

400

Temperature / C

b 1.200
0 hr
24 hrs
48 hrs
72 hrs

First derivative / %/ C

1.000

0.800

0.600

0.400

0.200

assignment in the amide I region that is between 1700 cm1 and

1600 cm1 can be based on the work from Elkhalifa et al. (2009). A
tentative assignment of the valleys is presented in Fig. 3 based on
the work from Georget and Belton (2006). In the amide I region, the
valleys are detected at 1692 cm1 due to turns or b-hairpins,
1677 cm1 assigned to b-turns followed by 1650 cm1 associated to
random coils and a-helices and another valley is also discerned at
1620 cm1 attributed to intermolecular b-sheets. It is interesting to
note that a shoulder occurs approximately at 1639 cm1 and could
thus be attributed to intramolecular b-sheets as reported earlier in
glycinin (Mills et al., 2003). The three major absorbances associated
to b-turns, random coils/a-helices and intermolecular b-sheets
were studied in more details (Elkhalifa et al., 2009). The respective
intensities on the initial FT-IR spectra were determined and their
relative contributions to the total intensity of these three absorbances were calculated. This helps to present a clearer picture of
the relative change in the conformation of the karin proteins. The
results are shown in Fig. 4a. The level of b-turns remains constant
during 48 h of germination after which it slightly decreases. The
reason for that is unclear. It is also much lower than that of random
coils/a-helices and intermolecular b-sheets. The random coils and
a-helices signicantly increases after 48 h (p < 0.05) whereas the
relative proportion of intermolecular b-sheets is unaffected. This
might implicitly be related to the change in the relative content of
karin proteins. Indeed upon germination the level of a-karin was
greater than that of b-karin based on the SDS PAGE results. Published data describing the conformation of a-zein proteins very
similar to a-karin proteins in their amino acid sequences tend to
suggest that the conformation of a-karin proteins would mimic

0.000
0

50

100

150

200

Temperature / C

Fig. 2. TGA changes in weight (a) and 1st derivative (b) for karin extracted from
sorghum seeds germinated at 0, 24, 48 and 72 h (w.w.b: wet weight basis).

ratio a-karin/b-karin with the lowest ratio conferring more

thermally stable karin proteins.
3.3. FT-IR analysis
In Fig. 3, the FT-IR analysis of the amide I of karin extracted
from ungerminated sorghum seeds is represented. The amide I
region relies on conformational change and is very often used as
a good indicator. After Fourier self-deconvolution of the initial FT-IR
spectrum, the second derivative was determined. Each valley
corresponds to a specic conformation of the karin protein and its

Fig. 3. Analysis of a typical FT-IR spectrum in the amide I region for karin extracted
from ungerminated sorghum seeds.

Fig. 4. Change in karin b-turns (black), random coils and a-helices (gray) and intermolecular b-sheets (white) based on FT-IR analysis of the amide I region (a) and
change in karin a-helices (black), b-sheets (gray) and tyrosine (white) based on FT-IR
analysis of amide II region (b) as a function of germination time. For a given conformation, means sharing the same letter were not signicantly different (p < 0.05).

Please cite this article in press as: Georget, D.M.R., et al., Structural changes in karin extracted from a white type II tannin sorghum during
germination, Journal of Cereal Science (2011), doi:10.1016/j.jcs.2011.10.007

D.M.R. Georget et al. / Journal of Cereal Science xxx (2011) 1e6

that of a-zein proteins (Forato et al., 2003). Based on their work,

a-helices would govern the secondary structure of a-karin
proteins though random coils could also contribute in this absorbance which might be due to the extraction process. As for the
b-karin proteins very little is known on their conformation per se.
When compared with a-zeins, the work from Forato et al. (2003)
suggested that the conformation of b-zeins was mainly governed
by b-sheets. It is possible to assume that b-karin proteins resemble
in conformation to b-zeins. Consequently, b-sheets might be the
basis of b-karin secondary structure. It is well established that
from the work of Mazhar and Chandrashekar (1993) and Oria et al.
(2000), a-karin proteins form the core of the protein bodies of
sorghum seeds coated by b-and g-karins. Upon germination,
knowing that the sorghum variety under investigation is a soft
endosperm cultivar, more b-and g-karins will be readily digested
when compared to a hard endosperm sorghum, subsequently
exposing gradually a-karins as germination progresses (Mazhar
and Chandrashekar, 1993). However tannins might also interact
with the karin proteins. This relative increase in random coils and
mainly a-helices could originate from this phenomenon. However,
at this stage, the constant level of b-sheets remains unexplained.
Possible interactions between the karin proteins upon extraction
might favor b-sheets. This study indicates some relative changes
occurring during germination; that is, an increase in a-helices. This
might also explain the thermal analysis described earlier; that is,
a-helices might confer to the proteins a certain degree of thermal
stability perhaps via a more energetically stable packing of the
proteins.
The amide II region of the FT-IR spectrum has been investigated.
The amide II absorbance peak mainly originates from CN and NH
vibration and is mainly sensitive to weak interactions in the NH
environment (Bandekar, 1992). After the Fourier self-deconvolution

and determination of the second derivative, three peaks predominate. These occur at 1545 cm1 assigned to a-helices, at 1530 cm1
attributed to b-sheets and at 1515 cm1 due to tyrosine (Duodu
et al., 2001). Calculation of their relative intensities based on their
total sum was performed and the results are presented in Fig. 4b. As
conrmed by the analysis of the amide I, upon germination time,
the level of a-helices signicantly increases whereas statistically
b-sheets remain constant. A similar effect is detected for the tyrosine peak. FT-IR analysis demonstrates that after 72 h germination
the level of a-helices increases.
3.4. SEM analysis
Fig. 5 represents SEM micrographs of karin proteins extracted
from variously germinated sorghum seeds. In Fig. 5a, the karin
proteins extracted from ungerminated sorghum seeds are detected
as globules with size diameter ranging from 5 to 10 mm. The particle
shape is spherical and regular. The particle geometry remains
unaltered with increasing germination time (Fig. 5b, c and d). The
microscopic visualization of the extract is very similar to the ndings from Elkhalifa et al. (2009), Elkhalifa et al. (2006) and Rom
et al. (1992). The protein `globules observed on the micrographs
are signicantly larger than the protein bodies reported by Elkhalifa
et al. (2009). This would suggest that upon the extraction procedure, aggregation of the karin proteins might have occurred. The
fusion between the karin globules seems to occur irrespective of
the germination time. It is interesting to note that bridges between
protein globules led to formation of agglomerates better seen in
Fig. 5c and d. Upon the protein isolation, the protein bodies very
likely lost their integrity. Solvation of these proteins after extraction
and evaporation of the solvent upon drying might result in a newly
packed protein system. Exposure of novel sites of the proteins

Fig. 5. SEM of karin extracted from (a) ungerminated sorghum, (b) 24 h germinated, (c) 48 h germinated and (d) 72 h germinated sorghum. Arrows indicate bridges.

Please cite this article in press as: Georget, D.M.R., et al., Structural changes in karin extracted from a white type II tannin sorghum during
germination, Journal of Cereal Science (2011), doi:10.1016/j.jcs.2011.10.007

D.M.R. Georget et al. / Journal of Cereal Science xxx (2011) 1e6

might result in more interactions between the karin proteins,

perhaps via weak interactions.
4. Conclusion
Karin proteins were extracted from sorghum seeds at different
stages of germination. Using SDS PAGE, a decrease in b-karin
proteins was mainly detected. Based on the structure of sorghum
protein bodies, b-karin proteins together with g-karin proteins
form the coating of the protein bodies with the core being rich in
a-karin. Upon digestion of the protein bodies, the periphery will
be the rst site of enzymatic activity. However, given that Feterita
contains tannins, interactions with karin proteins might also take
place.
FT-IR analysis also shows that the resulting change upon
germination is an increase in a-helices whereas the level of bsheets remains constant. This has a signicant effect on their
thermal properties. Indeed with the water content of karin
extracts being similar, increase in germination time led to protein
thermal decomposition occurring earlier. This might suggest that
the protein conformation might inuence the thermal decomposition of karin. The microstructure is that of particles ranging from
5 to 10 mm in size with aggregation. This study has demonstrated
the changes in the karin composition with germination employing physical techniques. Further this sheds some evidence on the
fate of these proteins and this signicantly would improve our
knowledge to better understand the processing of sorghum grains
from a white type II tannin cultivar.
References
Aluigi, A., Zoccola, M., Vineis, C., Tonin, C., Ferrero, F., Canetti, M., 2007. Study on the
structure and properties of wool keratin regenerated from formic acid. International Journal of Biological Macromolecules 41, 266e273.
Axtell, J.D., Kirleis, A.W., Hassen, M.M., Mason, N.D., Mertz, E.T., Munck, L., 1981.
Digestibility of sorghum proteins (rat assay/pepsin assay/temperature effects/
fermentation). Proceedings of the National Academy of Sciences of the United
States of America 78, 1333e1335.
Bandekar, J., 1992. Amide modes and protein conformation. Biochimica et Biophysica Acta 1120, 123e143.
Belton, P.S., Delgadillo, I., Halford, N.G., Shewry, P.R., 2006. Karin structure and
functionality. Journal of Cereal Science 44, 272e286.
Belton, P.S., Taylor, J.R.N., 2004. Sorghum and millets: protein sources for Africa.
Trends in Food Science and Technology 15, 94e98.
Carter, R., Reck, D.R., 1970. Low temperature solvent extraction process for
producing high purity zein. US Patent 3,535,305.
Correia, I., Nunes, A., Barros, A.S., Delgadillo, I., 2008. Protein prole and malt
activity during sorghum germination. Journal of the Science of Food and
Agriculture 88, 2598e2605.
Dirar, H.A., 1993. Indigenous Fermented Foods of the Sudan. CAB International,
Wallingford, UK.
Duodu, K.G., Nunes, A., Delgadillo, I., Parker, M.L., Mills, E.N.C., Belton, P.S.,
Taylor, J.R.N., 2002. Effect of grain structure and cooking on sorghum and maize
in vitro protein digestibility. Journal of Cereal Science 35, 161e174.
Duodu, K.G., Tang, H., Grant, A., Wellner, N., Belton, P.S., Taylor, J.R.N., 2001. FTIR and
solid state 13C NMR spectroscopy of proteins of wet cooked and popped
sorghum and maize. Journal of Cereal Science 33, 261e269.
Elkhalifa, A.O., 1998. Effect of cooking on the digestibility of sorghum karins and its
improvement. Ph.D. thesis, Faculty of Agriculture, University of Khartoum,
Sudan.
Elkhalifa, A.E.O., Bernhardt, R., Bonomi, F., Iametti, S., Pagani, M.A., Zardi, M., 2006.
Fermentation modies protein/protein and protein/starch interactions in
sorghum dough. European Food Research Technology 222, 559e564.
Elkhalifa, A.E.O., Schifer, B., Bernhardt, R., 2004. Selected physicochemical properties of starch isolated from fermented sorghum our. Starch/Strke 56,
582e585.
Elkhalifa, A.E.O., Georget, D.M.R., Barker, S.A., Belton, P.S., 2009. Study of the
physical properties of karin during the fabrication of tablets for pharmaceutical applications. Journal of Cereal Science 50, 159e165.
Elkhalifa, A.O., El Tinay, A.H., 1994. Effect of fermentation on protein fractions and
tannin content of low- and high-tannin cultivars of sorghum. Food Chemistry
49, 265e269.
Elmaki, H.B., Babiker, E.E., El Tinay, A.H., 1999. Changes in chemical composition,
grain malting, starch and tannin contents and protein digestibility during
germination of sorghum cultivars. Food Chemistry 64, 331e336.

El Nour, I.N.A., Peruffo, A.D.B., Curioni, A., 1998. Characterisation of sorghum karins
in relation to their cross-linking behaviour. Journal of Cereal Science 28,
197e207.
Forato, L.A., Bicudo, T.D.C., Colnago, L.A., 2003. Conformation of a-zeins in solid state
by Fourier Transform IR. Biopolymers (Biospectroscopy) 72, 421e426.
Gao, C., Taylor, J., Wellner, N., Byaruhanga, Y.B., Parker, M.L., Mills, E.N.C., Belton, P.S.,
2005. Effect of preparation conditions on protein secondary structure and
biolm formation of karin. Journal of Agricultural and Food Chemistry 53,
306e312.
Geneau-Sbarta, C., Leyris, J., Silvestre, F., Rigal, L., 2008. Sunower cake as a natural
composite: composition and plastic properties. Journal of Agricultural and Food
Chemistry 56, 11198e11208.
Georget, D.M.R., Belton, P.S., 2006. Effects of temperature and water content on the
secondary structure of wheat gluten studied by FTIR spectroscopy. Biomacromolecules 7, 469e475.
Hamaker, B.R., Mohamed, A.A., Habben, J.E., Huang, C.P., Larkins, B.A., 1995. Efcient
procedure for extracting maize and sorghum kernel proteins reveals higher
prolamin contents than the conventional method. Cereal Chemistry 72,
583e588.
Hassan, I.A.G., El Tinay, A.H., 1995. Effect of fermentation on tannin content and invitro protein and starch digestibilities of two sorghum cultivars. Food Chemistry
53, 149e151.
Herald, T.J., Smith, D.M., 1992. Heat-induced changes in the secondary structure of
hen egg S-ovalbumin. Journal of Agricultural and Food Chemistry 40,
1737e1740.
Izquierdo, L., Godwin, I.D., 2005. Molecular characterization of a novel methionine
rich delta-karin seed storage protein gene in sorghum (Sorghum bicolor L.).
Cereal Chemistry 82, 706e710.
Mazhar, H., Chandrashekar, A., 1993. Differences in karin composition during
endosperm development and germination in sorghum cultivars of varying
hardness. Cereal Chemistry 70, 667e671.
Mertz, E.T., Hassen, M.M., Cairns-Wittern, C., Kirleis, A.W., Tu, L., Axtell, J.D., 1984.
Pepsin digestibility of proteins in sorghum and other major cereals. Proceedings of the National Academy of Sciences of the United States of America 81,
1e2.
Mills, E.N.C., Marigheto, N.A., Wellner, N., Fairhurst, S.A., Jenkins, J.A., Mann, R.,
Belton, P.S., 2003. Thermally induced structural changes in glycinin, the 11S
globulin of soya bean (Glycine max) e an in situ spectroscopic study. Biochimica
et Biophysica Acta 1648, 105e114.
Mohamed, A.A., Gordon, S.H., Carriere, C.J., Kim, S., 2006. Thermal characteristics of
polylactic acid/wheat gluten blends. Journal of Food Quality 29, 266e281.
Nout, M.J.R., Ngoddy, P.O., 1997. Technological aspects of preparing affordable fermented complementary foods. Food Control 8, 279e287.
Oria, M.P., Hamaker, B.R., Axtell, J.D., Huang, C.P., 2000. A highly digestible sorghum
mutant cultivar exhibits a unique folded structure of endosperm protein bodies.
Proceedings of the National Academy of Sciences of the United States of
America 97, 5065e5070.
Oumarou, H., Ejoh, R., Ndjouenkeu, R., Tanya, A., 2005. Nutrient content of
complementary foods based on processed and fermented sorghum, groundnut,
spinach, and mango. Food and Nutrition Bulletin 26, 385e392.
Rom, D.L., Shull, J.M., Chandrashekar, A., Kirleis, A.W., 1992. Effects of cooking and
treatment with sodium bisulte on in vitro protein digestibility and microstructure of sorghum our. Cereal Chemistry 69, 178e181.
Shem, M.N., Lekule, F.P., Zakayo, G.Z., Eggum, B.O., 1990. Nutritive value of germinated and ungerminated high tannin sorghum for growing pigs. Acta Agriculturae Scandinavica, 253e258.
Shull, J.M., Watterson, J.J., Kirleis, A.W., 1991. Proposed nomenclature for the
alcohol-soluble proteins (karins) of Sorghum bicolor (L. Moench) based on
molecular weight, solubility, and structure. Journal of Agricultural and Food
Chemistry 39, 83e87.
Subramanian, V., Rao, N.S., Jambunathan, R., Murty, D.S., Reddy, B.V.S., 1995. The
effect of malting on the extractability of proteins and its relationship to diastatic
activity in sorghum. Journal of Cereal Science 21, 283e289.
Taylor, J.R.N., 1983. Effect of malting on the protein and free amino nitrogen
composition of sorghum. Journal of the Science of Food and Agriculture 34,
885e892.
Taylor, J., Bean, S.R., Ioerger, B.P., Taylor, J.R.N., 2007. Preferential binding of sorghum
tannins with g-karin and the inuence of tannin binding on karin digestibility and biodegradation. Journal of Cereal Science 46, 22e31.
Taylor, J.R.N., Evans, D.J., 1989. Action of sorghum proteinase on the protein bodies
of sorghum starchy endosperm. Journal of Experimental Botany 40, 763e768.
Taylor, J.R.N., Novellie, L., Liebenberg, N.W., 1985. Protein body degradation in the
starchy endosperm of germinating sorghum. Journal of Experimental Botany
36, 1287e1295.
Torrent, M., Geli, M.I., Ludevid, M.D., 1989. Storage-protein hydrolysis and proteinbody breakdown in germinated Zea mays L. seeds. Planta 180, 90e95.
Wang, Y.D., Fields, M.L., 1978. Germination of corn and sorghum in the home to
improve nutritive value. Journal of Food Science 44, 456e459.
Wang, Y., Tilley, M., Bean, S., Sun, S., Wang, D., 2009. Comparison of methods for
extracting karin proteins from sorghum distillers dried grains with soluble.
Journal of Agricultural and Food Chemistry 57, 8366e8372.
Wellner, N., Belton, P.S., Tatham, A.S., 1996. Fourier transform IR spectroscopic study
of hydration-induced structure changes in the solid state of u-gliadins.
Biochemical Journal 319, 741e747.

Please cite this article in press as: Georget, D.M.R., et al., Structural changes in karin extracted from a white type II tannin sorghum during
germination, Journal of Cereal Science (2011), doi:10.1016/j.jcs.2011.10.007