This document discusses how lupin and wheat proteins interact during baking in lupin-wheat bread and how they are extracted during digestion. It finds that most wheat proteins remain extractable after baking but some lose extractability when baked with lupin proteins. Lupin proteins fall into two categories based on extractability - alpha conglutins extract easily while beta, gamma, and delta conglutins do not extract, even under reducing conditions. The document hypothesizes this is due to varying degrees of cross-linking during baking. It provides insights into protein interactions and accessibility during bread production and digestion.

1. Wheat and lupin protein interaction at baking:
modifying extractability from lupin-wheat bread
Shahidul Islam, Guijun Yan, Rudi Appels, Wujun Ma

2. 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

3. Health attributes of lupin-wheat bread
• 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

4. Lupin protein interaction with gluten network
The lupin flour introduces protein molecules into the gluten
network of lupin-wheat bread that are more compact

5. At the consumption of lupin-wheat bread two
complex systems interacting
first stage of
solubilisation
through
chewing and
action of saliva
subsequent
stages of
digestion
bread matrix
- multi-components
- varying degrees of cross-linking

6. We are now able to
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
Methodology used
 Two dimensional gel electrophoresis followed by
MS/MS peptide sequencing
 Direct MALDI-TOF mass spectrometry

7. Wheat flour Wheat bread Wheat bread
100
HMW glutenin
75
50
37
Gliadin
Gliadin Gliadin
25
20
15
10
with reducing extraction with reducing extraction without reducing extraction
Lupin-wheat bread Lupin-wheat bread
HMW
glutenin
Wheat protein’s
response to the
baking process Gliadin

8. 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.

9. Lupin protein extractability from lupin-wheat bread
Under reducing and denaturing condition
4.0------------ -----------------------PI ---------------- -------------------9.0 4.0------------ -------------------------PI ---------------- -------------------9.0 4.0------------ -------------------------PI ---------------- -------------------9.0
100
75
50
37
Wheat flour
25
20
15
10
Lupin flour Lupin-wheat bread Wheat flour
 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

10. Lupin protein extractability from lupin-wheat bread
At milder extraction
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 Lupin-wheat bread protein with
0.5 M NaCl extraction 0.05 M NaCl 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

11. Key points of lupin protein extractability from
lupin-wheat bread
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

12. 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)
first stage of
solubilisation
through
chewing and
action of saliva
subsequent
stages of
digestion

13. Loosing extractability of beta conglutins apparently
indicates decrease of allergenic effect of lupin after baking
4.0------------ -----------------------PI ---------------- -------------------9.0
100
75
50
37
25 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-
20 6377.
15
3
10 1 2 5
4 7 10 11 12
6
Lupin flour
13
9
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.

14. Why lupin proteins behave differently?
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.

15. 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

16. Thank you all

20. Progress in sequencing for alpha conglutins
Peptides of Spot 1 from Peptides of Spot 22 from
An approach to generate protein peptide sequencing by peptide sequencing by
sequence form identified peptides MS/MS MS/MS
AGPVR AGMPK
ASLKVGEEEEEEEAGDGR FYLAGNPEEEYPETQQQR
CAGQGR GDEGQEEEETTTTTEER
CGAKVEFK GGHEEEEVEEER
EQLATFR GGHEEEEVEEERGR
GISILRR GGKDH
1 IRNQEEFEQEIGR GKPSESGPFNLR
22 KPSSPK KYETTEQGR GSVVLSERGDGAAVPR
NKMSVIPYASAIGSIMYAMLCTR HTRGDEGQEEEETTTTTEER
XEEXR IVEFQSNPNTLILPK
KGKPSESGPFNLR
KITMPSSTQGFTY
LLGFGINANENQR
NFLAGSEDNVIR
NNILSGFDPQFLSQALNIDEDTVHK
NTLEATFNTR
NTLEATFNTRYEEIQR
QIIRVEEGLGVISPK
QRVDTYWDLLSPK
RFYLAGNPEEEYPETQQQR
RGQEQSYQDEGVIVR
Targeted two major protein spots of TNRLENLQNYR
alpha conglutins VEEGLGVISPK 20
YQAMKAGPDGEVVSLR

21. Progress in sequencing for alpha conglutins
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
21

22. Progress in sequencing for alpha conglutins
Peptides of Spot 22 from peptide
Sequenced length (509 bp) sequencing
ATTCTATCTAGCTGGTAACCCAGAAGAAGAGTATCCAGAGACCCAGCAACAA
AGGCAGCAGCGACAGCAGCATCAAAGACCTAGTGGACGCAGGCATGGACA AGMPK
ACACCAGAAAGAAGAGGAACAAGAAGGAAAAAACAACATACTGAGTGGATT FYLAGNPEEEYPETQQQR
TGACCCACAGTTTTTATCTCAAGCTCTCAACATAGACGAGGATACAGTACAC GDEGQEEEETTTTTEER
AAACTTCAAAATCCGAATGAACGAATCAAACAAATCATAAGAGTGGAGGAAG GGHEEEEVEEER
GTTTGGGAGTTATCAGCCCCAAGTGGCAAGAACAAGAGGAGGAAGAGGAA
GGHEEEEVEEERGR
GAAAAAGAAGAACCACGCCAGAGAAGACGACGTGAACGAAGGGAGGAAAG
AGAAGAAGAAGAGAAAGAAGAAGAAGATGAACCTCGCGAGTCAAGAAGAC GGKDH
ACCGAGGAGGACATGAAGAAGAGGAGGTGGAGGAAGAGAGAGGAAGAGG GKPSESGPFNLR
AAGAGGAGGTAGTGAATGGAAGAGAACAACACGGCGAAGACATACACGAG GSVVLSERGDGAAVPR
GAG HTRGDEGQEEEETTTTTEER
IVEFQSNPNTLILPK
Retranslation to peptide: KGKPSESGPFNLR
RFYLAGNPEEEYPETQQQRQQRQQHQRPSGRRHGQHQ KITMPSSTQGFTY
KEEEQEGKNNILSGFDPQFLSQALNIDEDTVHKLQNPNERI LLGFGINANENQR
KQIIRVEEGLGVISPKWQEQEEEEEEKEEPRQRRRRERRE NFLAGSEDNVIR
EREEEEKEEEDEPRESRRHRGGHEEEEVEEERGRGRGG NNILSGFDPQFLSQALNIDEDTVHK
SEWKRTTRRRHTR NTLEATFNTR
NTLEATFNTRYEEIQR
QIIRVEEGLGVISPK
Adjustment with peptides from MS/MS:
QRVDTYWDLLSPK
RFYLAGNPEEEYPETQQQRQQRQQHQRPSGRRHGQHQ RFYLAGNPEEEYPETQQQR
KEEEQEGKNNILSGFDPQFLSQALNIDEDTVHKLQNPNER RGQEQSYQDEGVIVR
IKQIIRVEEGLGVISPKWQEQEEEEEEKEEPRQRRRRERR TNRLENLQNYR
EEREEEEKEEEDEPRESRRHRGGHEEEEVEEERGRGRG VEEGLGVISPK
GSEWKRTTRRRHTR YQAMKAGPDGEVVSLR
22