Avena genetic resources for quality in human consumption (AVEQ)
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Transcript of Avena genetic resources for quality in human consumption (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 - ISCEExperimental Institute for Cereal Research
Bergamo and Roma, Italy
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
Dr. Daniela SgrullettaDr. Ester De StefanisDr. Giulia ScalfatiDr. Rita RedaelliDr. Nicola Berardo
SVALOF WEIBULL
Svalöf, Sweden
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
Dr. Alf Ceplitis
LABORATORY OF QUALITY EVALUATION LABORATORY OF QUALITY EVALUATION OF PLANT MATERIALSOF PLANT MATERIALS
IHAR - Institute of Plant Breeding and Acclimatization, IHAR - Institute of Plant Breeding and Acclimatization, Radzikow, PolandRadzikow, Poland
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
D.Sc. Danuta BorosDr. Malgorzata CyranM.Sc. Magdalena PlochM.Sc. Wioletta DynkowskaM.Sc. Anna FraśTech. Magdalena BodzonTech. Alicja KicińskaMonika Skonieczna
Diagram of activities of WP7 in AVEQ
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
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
Workpackage 7. Milestones
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
MILESTONE DATE
MILESTONE DESCRIPTION
Month 24M7.1. Samples from first field experiment year for quality analysis received by quality labs
Month 32M7.2. Samples from Sweden, France and Bulgaria analysed for total -glucan (P2: 975 analysis)
Month 36M7.3. Samples of 70 accessions from Sweden, France and Bulgaria additionally analysed for soluble -glucan (P6: 210 analysis)
Month 36M7.4. Samples of 70 accessions from Sweden, France and Bulgaria analysed additionally analysed for starch and dietary fibre (P12: 200 analysis)
Workpackage 7. Milestones
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
MILESTONE DATE
MILESTONE DESCRIPTION
Month 38M7.5. Samples from second field experiment year for quality analysis received by quality labs
Month 46M7.6. Samples from Estonia, Italy and Romania analysed for total -glucan (P6: 975)
Month 48M7.7. Samples of 70 accessions from Estonia, Italy and Romania additionally analysed for soluble -glucan (P6: 210 analysis)
Month 48M7.8. 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
CHEMICAL ANALYSES IN WP7
-GLUCANtotalsoluble
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
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
- 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.
PHYSIOLOGICAL ROLE OF -GLUCAN
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
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.
-GLUCAN CONTENT IN CEREALS
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Rimsten et al. Cereal Chem 80(4): 485-490, 2003
% 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
ANALYSIS OF TOTAL -GLUCAN(Analytica-EBC, Method 3.10.2)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Sample
α-amylase digestion at 100°C using thermostable
enzyme Hydrolysed in 0.004M H2S04
Centrifugation
FLUOROMETRIC DETERMINATION OF RELEASED GLUCOSE
(using Calcofluor)PELLET DISCARD
P2
ANALYSIS OF TOTAL -GLUCAN(McCleary and Codd,1991, AOAC reference method 995.16)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Sample
Dissolved in buffer, pH 6.5
Hydrolysed with lychenase and filtered
Hydrolysed with Incubation with -glucosidase
Centrifugation
SPECTROPHOTOMETRIC DETERMINATION OF RELEASED GLUCOSE
(GOPOD reagent -Glucose oxidase and peroxidase, λ=510 nm)
PELLET DISCARD
P6
ANALYSIS OF SOLUBLE ANALYSIS OF SOLUBLE -GLUCAN-GLUCAN(McCleary and Codd,1991, AOAC reference method 995.16, Åman and Graham, 1987)(McCleary and Codd,1991, AOAC reference method 995.16, Åman and Graham, 1987)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Sample
Dissolved in buffer, pH 6.5
SUPERNATANT DISCARD
Hydrolysed with lychenase and filtered
Hydrolysed with incubation with -glucosidase
SPECTROPHOTOMETRIC DETERMINATION OF RELEASED GLUCOSE
Extraction for 120 min with distilled water
at 38°C
Centrifugation
THE SOLUBLE FRACTION IS CALCULATED FOR
DIFFERENCE,
P6
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)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Sample
Extraction of LMW components
80% Et OH, 85ºC
Centrifugation
SUPERNATANT DISCARD
RESIDUE
Incubation at 60°C with thermostable α-amylase (1→4)
Incubation with amyloglucosidase50ºC, overnight (1→6)
Centrifugation
SPECTROPHOTOMETRIC DETERMINATION OF RELEASED GLUCOSE
(GOPOD reagent -Glucose oxidase and peroxidase, λ=510 nm)
PELLET DISCARD
P12
DIETARY FIBRE (AACC 2000)
AVEQ First Meeting – Clermont Ferrand, September 19-21 2007
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.
ANALYSIS OF TOTAL DIETARY FIBER Uppsala Method (Theander et al., 1995)
AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007
Sample Boiling with DMSO, 1 hr Incubation with α-amylase,
boiling water bath
RESIDUE
Hydrolysis of polymers, H2SO4, 1 hr
Determination of uronic acidsby colorimetric method (Scott, 1979)
Derivatization of monosugars and their quantitative analysis by GC(Englyst and Cummings, 1984)
Centrifugation
Precipitation of soluble polymers, mix
with 96% ETOH
Incubation with amylo-glucosidase and proteinases, 40°C overnight
PELLET DISCARD
Determination of Klason lignin(gravimetric method, Theander et al., 1995)
P12
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