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, Partner P2, SVALÖF WEIBULL AB, Sweden Responsible Alf CEPLITIS, Partner P12, IHAR, Poland Responsible Malgorzata CYRAN, AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

CRA - ISCE Experimental Institute for Cereal Research Bergamo and Roma, Italy AVEQ First Meeting – Clermont Ferrand, September Dr. Daniela Sgrulletta Dr. Ester De Stefanis Dr. Giulia Scalfati Dr. Rita Redaelli Dr. Nicola Berardo

SVALOF WEIBULL Svalöf, Sweden AVEQ First Meeting – Clermont Ferrand, September Dr. Alf Ceplitis

LABORATORY OF QUALITY EVALUATION OF PLANT MATERIALS IHAR - Institute of Plant Breeding and Acclimatization, Radzikow, Poland AVEQ First Meeting – Clermont Ferrand, September 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

Diagram of activities of WP7 in AVEQ AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007 Year 1Year 2Year 3Year 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 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)

Workpackage 7. Milestones AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007 MILESTONE DATE 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 48M7.9. All analysis results entered into EADB

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 AVEQ First Meeting – Clermont Ferrand, September 19-21, 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): , 2003 % d.m. BARLEY3.6 BARLEY MALT0.5 RYE2.0 RYE BRAN0.9 WHEAT1.0 WHEAT BRAN0.9 OAT3.4 OAT BRAN8.3

ANALYSIS OF TOTAL  -GLUCAN (Analytica-EBC, Method ) AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007 Sample α-amylase digestion at 100°C using thermostable enzyme Hydrolysed in 0.004M H 2 S0 4 Centrifugation FLUOROMETRIC DETERMINATION OF RELEASED GLUCOSE (using Calcofluor) PELLET DISCARD P2

ANALYSIS OF TOTAL  -GLUCAN (McCleary and Codd,1991, AOAC reference method ) 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  -GLUCAN (McCleary and Codd,1991, AOAC reference method , 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 , 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 amyloglucosidase 50ºC, 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 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, H 2 SO 4, 1 hr Determination of uronic acids by 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