Transcript aveq

Avena genetic
resources for quality in
human consumption
(AVEQ)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Workpackage 7.
Analysis for -glucan and
dietary fibre
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Workpackage 7.
Analysis for -glucan and dietary fibre

Characterise carbohydrates (starch, dietary fibre,
-glucan ) in genetic resources of Avena.

Evaluate -glucan content in a number of European
oat genotypes (wild Avena species, landraces,
traditional and obsolete and modern cultivars), when
grown under contrasting growing conditions all over
Europe from the Nordic countries to South (Italy) and
South East (Bulgaria, Romania).
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Workpackage 7.
Analysis for -glucan and dietary fibre

Describe the distribution of -glucan contents in
relation to the structure of the collection.

Describe correlations of -glucan content with
other traits relevant from a qualitative and
agronomic point of view, especially those related
to Fusarium infection, mycotoxin formation and
cold tolerance in the working collection.
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Workpackage 7.
Analysis for -glucan and dietary fibre
WP Leader: Partner P6, CRA – ISCE
Responsible Rita REDAELLI, [email protected]
Partner P2, SVALÖF WEIBULL AB, Sweden
Responsible Alf CEPLITIS, [email protected]
Partner P12, IHAR, Poland
Responsible Malgorzata CYRAN, [email protected]
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
CRA - ISCE
Experimental Institute for Cereal Research
Bergamo and Roma, Italy
Dr.
Dr.
Dr.
Dr.
Dr.
Daniela Sgrulletta
Ester De Stefanis
Giulia Scalfati
Rita Redaelli
Nicola Berardo
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
SVALOF WEIBULL
Svalöf, Sweden
Dr. Alf Ceplitis
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
LABORATORY OF QUALITY EVALUATION
OF PLANT MATERIALS
IHAR - Institute of Plant Breeding and Acclimatization,
Radzikow, Poland
D.Sc. Danuta Boros
Dr. Malgorzata Cyran
M.Sc. Magdalena Ploch
M.Sc. Wioletta Dynkowska
M.Sc. Anna Fraś
Tech. Magdalena Bodzon
Tech. Alicja Kicińska
Monika Skonieczna
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
Diagram of activities of WP7 in AVEQ
Year 1
Year 2
Year 3
Year 4
WP 1
WP 2
WP 3
WP 4
WP 5
WP 6
WP 7
WP 8
WP 9
WP
10
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Workpackage 7. Milestones
MILESTONE
DATE
MILESTONE DESCRIPTION
Month 24
M7.1. Samples from first field experiment year for quality
analysis received by quality labs
Month 32
M7.2. Samples from Sweden, France and Bulgaria
analysed for total -glucan (P2: 975 analysis)
Month 36
M7.3. Samples of 70 accessions from Sweden, France and
Bulgaria additionally analysed for soluble -glucan
(P6: 210 analysis)
Month 36
M7.4. Samples of 70 accessions from Sweden, France and
Bulgaria analysed additionally analysed for starch and
dietary fibre (P12: 200 analysis)
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
Workpackage 7.
MILESTONE
DATE
Milestones
MILESTONE DESCRIPTION
Month 38
M7.5. Samples from second field experiment year for quality
analysis received by quality labs
Month 46
M7.6. Samples from Estonia, Italy and Romania analysed for
total -glucan (P6: 975)
Month 48
M7.7. Samples of 70 accessions from Estonia, Italy and
Romania additionally analysed for soluble -glucan
(P6: 210 analysis)
Month 48
M7.8. Samples of 70 accessions from Estonia, Italy and
Romania additionally analysed for starch and dietary fibre
(P12: 200 analysis)
Month 48
M7.9. All analysis results entered into EADB
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
CHEMICAL ANALYSES IN WP7

-GLUCAN
total
soluble

STARCH

DIETARY FIBRE
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
-GLUCAN: CHEMICAL STRUCTURE
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
-GLUCAN: LOCATION ETC
- Mixed-linked (1-3),(1-4)--D-glucans are frequently present
in endosperm cell walls of cereals.
- High contents of -glucan have been reported for barley
(2-10%) and oats (2-6%).
- Both genetic and environmental factors affect the total
content of -glucan.
- Both soluble and insoluble -glucans are present in cereals;
in oats the soluble fraction is larger, up to 80% of total glucan.
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
PHYSIOLOGICAL ROLE OF -GLUCAN
It can lower the cholesterol levels in blood.
It has effects on the glucose metabolism, by reducing
the post-prandial increase of glucose.
In 1997 the Food and Drug Administration established
an “health claim” which states that a 3 g daily intake
of oat soluble fibre (-glucan) can reduce the risks of
cardiovascular diseases.
This claim was then confirmed in 2002.
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
-GLUCAN CONTENT IN CEREALS
% d.m.
BARLEY
3.6
BARLEY MALT
0.5
RYE
2.0
RYE BRAN
0.9
WHEAT
1.0
WHEAT BRAN
0.9
OAT
3.4
OAT BRAN
8.3
Rimsten et al. Cereal Chem 80(4): 485-490, 2003
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
ANALYSIS OF TOTAL -GLUCAN
(Analytica-EBC, Method 3.10.2)
P2
Sample
α-amylase
digestion at 100°C
using thermostable
enzyme
Hydrolysed in 0.004M H2S04
Centrifugation
PELLET
DISCARD
FLUOROMETRIC DETERMINATION OF
RELEASED GLUCOSE
(using Calcofluor)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
ANALYSIS OF TOTAL -GLUCAN
(McCleary and Codd,1991, AOAC reference method 995.16)
P6
Hydrolysed with lychenase and
filtered
Sample
Dissolved in buffer,
pH 6.5
Hydrolysed with Incubation with
-glucosidase
Centrifugation
PELLET
DISCARD
SPECTROPHOTOMETRIC DETERMINATION
OF RELEASED GLUCOSE
(GOPOD reagent -Glucose oxidase and
peroxidase, λ=510 nm)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
ANALYSIS OF SOLUBLE -GLUCAN
(McCleary and Codd,1991, AOAC reference method 995.16, Åman and Graham, 1987)
Sample
Extraction for 120
min with distilled
water at 38°C
Centrifugation
SUPERNATANT
DISCARD
Dissolved in buffer,
pH 6.5
P6
Hydrolysed with lychenase and
filtered
Hydrolysed with incubation with
-glucosidase
SPECTROPHOTOMETRIC
DETERMINATION OF
RELEASED GLUCOSE
THE SOLUBLE FRACTION
IS CALCULATED FOR
DIFFERENCE,
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
STARCH: CHEMICAL STRUCTURE
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
STARCH: CHARACTERISTICS
- Oat starch content ranges from 40 to 60%.
- Significant genotypic and environmental effects were
observed on the content of total starch.
- It cannot be easily separated from the other components
of the grain.
- Oat starch is associated to a larger lipid content (1-3%)
than the starch of other cereals.
- The average size of individual oat starch granules varies
from 3 to 10 μm.
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
ANALYSIS OF TOTAL STARCH
(Amyloglucosidase/α-Amylase method)
(Megazyme Assay Procedure, AOAC Method 996.11, AACC Method 76.13, ICC Standard Method No. 168)
Sample
RESIDUE
Extraction of LMW
components
80% Et OH, 85ºC
Centrifugation
Incubation at 60°C with
thermostable α-amylase (1→4)
Incubation with amyloglucosidase
50ºC, overnight (1→6)
Centrifugation
PELLET
DISCARD
SUPERNATANT
DISCARD
P12
SPECTROPHOTOMETRIC DETERMINATION
OF RELEASED GLUCOSE
(GOPOD reagent -Glucose oxidase and
peroxidase, λ=510 nm)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
DIETARY FIBRE (AACC 2000)
Dietary fibre is the edible part of plants or analogous
carbohydrates that are resistant to digestion and
absorption in the human small intestine with complete or
partial fermentation in the large intestine.
Dietary fibre includes polysaccharides, oligosaccharides,
lignin and associated plant substances.
Dietary fibres promote beneficial physiological effects
including laxation, and/or blood cholesterol attenuation,
and/or blood glucose attenuation.
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
ANALYSIS OF TOTAL DIETARY FIBER
Uppsala Method (Theander et al., 1995)
Incubation with α-amylase,
boiling water bath
Boiling with DMSO, 1 hr
Sample
Determination of
Klason lignin
(gravimetric
method, Theander
et al., 1995)
Incubation with amyloglucosidase and proteinases,
40°C overnight
Precipitation of
soluble polymers,
mix with 96% ETOH
Centrifugation
RESIDUE
PELLET DISCARD
P12
Hydrolysis of polymers, H2SO4, 1 hr
Determination of uronic acids
by colorimetric method (Scott, 1979)
Derivatization of monosugars and
their quantitative analysis by GC
(Englyst and Cummings, 1984)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
NOTES
Reproducibility of β-glucan quantification by P2
and P6
 De-hulling of samples?

AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Thank you for your attention!
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007