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Wheat and lupin protein interaction at baking:
modifying extractability from lupin-wheat bread
Shahidul Islam, Guijun Yan, Rudi Appels, Wujun Ma
Nutritional status of lupin
Unique combination of
• High protein
• High dietary fibre
• Low oil
• Negligible starch content
• Low Glycaemic Index (G.I.)
Lupin has high lysine content
that is low in wheat
• Increases satiety and reduces energy intake
• Lowers cholesterol
• Decreases blood glucose level
• Lowers blood pressure
• Decreases the risk of cardiovascular diseases
• Hall, R. S., et al. (2005) Asia Pacific J. Clinical Nutrition 14: 91-97.
• Lee et. al. (2006) Am J Clin Nutr 84: 975– 80.
• Lee et. al. (2009) Am J Clin Nutr 89: 1-7
Health attributes of lupin-wheat bread
The lupin flour introduces protein molecules into the gluten
network of lupin-wheat bread that are more compact
Lupin protein interaction with gluten network
At the consumption of lupin-wheat bread two
complex systems interacting
bread matrix
- multi-components
- varying degrees of cross-linking
first stage of
solubilisation
through
chewing and
action of saliva
subsequent
stages of
digestion
Directly examine the proteins in the baked product:
• new quality assurance tools
• investigate attributes of wheat and lupin protein
regarding accessibility to the overall digestion process
We are now able to
Methodology used
Two dimensional gel electrophoresis followed by
MS/MS peptide sequencing
Direct MALDI-TOF mass spectrometry
Lupin-wheat bread
with reducing extraction
Wheat flour
without reducing extraction
Wheat bread Wheat bread
Lupin-wheat bread
Wheat protein’s
response to the
baking process
HMW glutenin
Gliadin Gliadin Gliadin
HMW
glutenin
Gliadin
with reducing extraction
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Key points of wheat protein extractability
after baking
Most of the wheat proteins, including HMW glutenins are extractable.
LMW wheat proteins are even extractable at milder extraction buffer
(non-reducing and non-denaturing) while the HMW are not.
Some wheat proteins loose their extractability as interaction with lupin
proteins in baking.
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Lupin flour Lupin-wheat bread
Wheat flour
4.0------------ -----------------------PI ---------------- -------------------9.0 4.0------------ -------------------------PI ---------------- -------------------9.0 4.0------------ -------------------------PI ---------------- -------------------9.0
Lupin protein extractability from lupin-wheat bread
The alpha conglutins are extractable
The beta, gamma and delta conglutins become intimately bound
within the bread matrix so that they are difficult to extract Islam et. al. (2011) journal of agriculture and food chemistry 59:6696-6704
Wheat flour
Under reducing and denaturing condition
Alpha conglutins are readily extractable from lupin-wheat bread even at
very mild condition such as 0.5 M NaCl
Lupin-wheat bread protein with
0.5 M NaCl extraction
Lupin-wheat bread protein with
0.05 M NaCl extraction
Lupin protein extractability from lupin-wheat bread
At milder extraction
High resolution study of proteins by direct MALDI-TOF also confirmed the
recovery of lupin protein from lupin-wheat bread under 0.5 M NaCl extraction
The conglutins (lupin proteins) fall into two very clear categories:
• the alpha group (both high and low molecular weight) which is
readily extracted even under mild conditions (0. 5M NaCl)
• the beta, delta and gamma groups that cannot be extracted, some
even under reducing/denaturing conditions
Key points of lupin protein extractability from
lupin-wheat bread
first stage of
solubilisation
through
chewing and
action of saliva
subsequent
stages of
digestion
We postulate that this readily extracted class of lupin
protein (alpha-conglutins) are solubilised early in the
chewing process and may be significant in accounting
for some effects on health attributes (for example: blood
pressure regulation)
Loosing extractability of beta conglutins apparently
indicates decrease of allergenic effect of lupin after baking
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Lupin flour
4.0------------ -----------------------PI ---------------- -------------------9.0
Goggin et. al. 2008: Proteomic analysis of lupin seed proteins to identify conglutin β
as an allergen, Lup an 1. Journal of Agricultural and Food Chemistry 56, 6370-
6377.
4 6 1012
1 23
5
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913
Islam et. al. 2012. Comparative proteome analysis of seed storage and allergenic
proteins among four narrow-leafed lupin cultivars. Food Chemistry;
dx.doi.org/10.1016/j.foodchem.2012.05.081.
We believe the different lupin protein classes have varying degrees of
cross-linking with the gluten matrix.
The alpha-conglutins are generally a class of protein that is more
thermally stable than beta-conglutins and we consider that may help to
maintain their independent status during the baking process.
Sirtori, et al. Food Chemistry, 2010. 120: p. 496-504.
Why lupin proteins behave differently?
Conclusions
Lupin and wheat proteins in the baked products have been
surprisingly straight-forward to identify
Most of the wheat proteins, including HMW glutenins are
extractable after baking although some are not extractable after the
addition of lupin protein in the flour mix for baking
Lupin proteins (conglutins) are divided into two distinct groups in
terms of extractability from bread matrix
• The alpha conglutins are readily extracted
• The beta, delta and gamma conglutins cannot be extracted, in
some cases even under reducing/denaturing conditions
Thank you all
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An approach to generate protein
sequence form identified peptides
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22
Peptides of Spot 1 from peptide sequencing by MS/MS
Peptides of Spot 22 from peptide sequencing by MS/MS
AGPVR
ASLKVGEEEEEEEAGDGR
CAGQGR
CGAKVEFK
EQLATFR
GISILRR
IRNQEEFEQEIGR
KPSSPK KYETTEQGR
NKMSVIPYASAIGSIMYAMLCTR
XEEXR
AGMPK
FYLAGNPEEEYPETQQQR
GDEGQEEEETTTTTEER
GGHEEEEVEEER
GGHEEEEVEEERGR
GGKDH
GKPSESGPFNLR
GSVVLSERGDGAAVPR
HTRGDEGQEEEETTTTTEER
IVEFQSNPNTLILPK
KGKPSESGPFNLR
KITMPSSTQGFTY
LLGFGINANENQR
NFLAGSEDNVIR
NNILSGFDPQFLSQALNIDEDTVHK
NTLEATFNTR
NTLEATFNTRYEEIQR
QIIRVEEGLGVISPK
QRVDTYWDLLSPK
RFYLAGNPEEEYPETQQQR
RGQEQSYQDEGVIVR
TNRLENLQNYR
VEEGLGVISPK
YQAMKAGPDGEVVSLR
Progress in sequencing for alpha conglutins
Targeted two major protein spots of
alpha conglutins
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Primer design based on the peptides
Forward primer
R F Y L A G N P E E E Y P E T Q Q Q R
Reverse translation
CGATTCTATCTAGCTGGTAACCCAGAAGAAGAATATCCGGAAACCCA
GCAGCAGCGT
Primer (5’-3’): ATTCTATCTAGCTGGTAACCC
Reverse primer
H T R G D E G Q E E E E T T T T T E E R
Reverse translation
CATACACGAGGAGATGAAGGACAAGAGGAGGAAGAAACAACAACA
ACAACCGAAGAGCG
Primer (5’-3’): CTCTTCGGTTGTTGTTGTTG
Progress in sequencing for alpha conglutins
22
Sequenced length (509 bp) ATTCTATCTAGCTGGTAACCCAGAAGAAGAGTATCCAGAGACCCAGCAACAA
AGGCAGCAGCGACAGCAGCATCAAAGACCTAGTGGACGCAGGCATGGACA
ACACCAGAAAGAAGAGGAACAAGAAGGAAAAAACAACATACTGAGTGGATT
TGACCCACAGTTTTTATCTCAAGCTCTCAACATAGACGAGGATACAGTACAC
AAACTTCAAAATCCGAATGAACGAATCAAACAAATCATAAGAGTGGAGGAAG
GTTTGGGAGTTATCAGCCCCAAGTGGCAAGAACAAGAGGAGGAAGAGGAA
GAAAAAGAAGAACCACGCCAGAGAAGACGACGTGAACGAAGGGAGGAAAG
AGAAGAAGAAGAGAAAGAAGAAGAAGATGAACCTCGCGAGTCAAGAAGAC
ACCGAGGAGGACATGAAGAAGAGGAGGTGGAGGAAGAGAGAGGAAGAGG
AAGAGGAGGTAGTGAATGGAAGAGAACAACACGGCGAAGACATACACGAG
GAG
Retranslation to peptide:
RFYLAGNPEEEYPETQQQRQQRQQHQRPSGRRHGQHQ
KEEEQEGKNNILSGFDPQFLSQALNIDEDTVHKLQNPNERI
KQIIRVEEGLGVISPKWQEQEEEEEEKEEPRQRRRRERRE
EREEEEKEEEDEPRESRRHRGGHEEEEVEEERGRGRGG
SEWKRTTRRRHTR
Adjustment with peptides from MS/MS:
RFYLAGNPEEEYPETQQQRQQRQQHQRPSGRRHGQHQ
KEEEQEGKNNILSGFDPQFLSQALNIDEDTVHKLQNPNER
IKQIIRVEEGLGVISPKWQEQEEEEEEKEEPRQRRRRERR
EEREEEEKEEEDEPRESRRHRGGHEEEEVEEERGRGRG
GSEWKRTTRRRHTR
Peptides of Spot 22 from peptide sequencing
AGMPK
FYLAGNPEEEYPETQQQR
GDEGQEEEETTTTTEER
GGHEEEEVEEER
GGHEEEEVEEERGR
GGKDH
GKPSESGPFNLR
GSVVLSERGDGAAVPR
HTRGDEGQEEEETTTTTEER
IVEFQSNPNTLILPK
KGKPSESGPFNLR
KITMPSSTQGFTY
LLGFGINANENQR
NFLAGSEDNVIR
NNILSGFDPQFLSQALNIDEDTVHK
NTLEATFNTR
NTLEATFNTRYEEIQR
QIIRVEEGLGVISPK
QRVDTYWDLLSPK
RFYLAGNPEEEYPETQQQR
RGQEQSYQDEGVIVR
TNRLENLQNYR
VEEGLGVISPK
YQAMKAGPDGEVVSLR
Progress in sequencing for alpha conglutins