Wheat and lupin protein interaction at baking: modifying extractability from lupin-wheat bread

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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

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International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

Transcript of Wheat and lupin protein interaction at baking: modifying extractability from lupin-wheat bread

Page 1: Wheat and lupin protein interaction at baking: modifying extractability from lupin-wheat bread

Wheat and lupin protein interaction at baking:

modifying extractability from lupin-wheat bread

Shahidul Islam, Guijun Yan, Rudi Appels, Wujun Ma

Page 2: Wheat and lupin protein interaction at baking: modifying extractability from lupin-wheat bread

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

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• 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

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The lupin flour introduces protein molecules into the gluten

network of lupin-wheat bread that are more compact

Lupin protein interaction with gluten network

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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

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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

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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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Page 8: Wheat and lupin protein interaction at baking: modifying extractability from lupin-wheat bread

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

Page 10: Wheat and lupin protein interaction at baking: modifying extractability from lupin-wheat bread

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

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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

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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)

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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

711

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.

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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?

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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

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Thank you all

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An approach to generate protein

sequence form identified peptides

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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

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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