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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.

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Avena genetic resources for quality in human consumption (AVEQ)

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  1. Avena genetic resources for quality in human consumption (AVEQ) AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  2. Workpackage 7. Analysis for -glucan and dietary fibre AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  3. 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

  4. 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

  5. Workpackage 7. Analysis for -glucan and dietary fibre WP Leader: Partner P6, CRA – ISCE Responsible Rita REDAELLI, rita.redaelli@entecra.it Partner P2, SVALÖF WEIBULL AB, Sweden Responsible Alf CEPLITIS, alf.ceplitis@swseed.com Partner P12, IHAR, Poland Responsible Malgorzata CYRAN, m.cyran@ihar.edu.pl AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  6. CRA - ISCEExperimental Institute for Cereal Research Bergamo and Roma, Italy Dr. Daniela Sgrulletta Dr. Ester De Stefanis Dr. Giulia Scalfati Dr. Rita Redaelli Dr. Nicola Berardo AVEQ First Meeting – Clermont Ferrand, September 19-21 2007

  7. SVALOF WEIBULL Svalöf, Sweden Dr. Alf Ceplitis AVEQ First Meeting – Clermont Ferrand, September 19-21 2007

  8. LABORATORY OF QUALITY EVALUATION OF PLANT MATERIALSIHAR - 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

  9. Diagram of activities of WP7 in AVEQ AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  10. Workpackage 7. Milestones AVEQ First Meeting – Clermont Ferrand, September 19-21 2007

  11. Workpackage 7. Milestones AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  12. CHEMICAL ANALYSES IN WP7 • -GLUCAN total soluble • STARCH • DIETARY FIBRE AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  13. -GLUCAN: CHEMICAL STRUCTURE AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  14. -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 environmentalfactors 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

  15. 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

  16. -GLUCAN CONTENT IN CEREALS Rimsten et al. Cereal Chem 80(4): 485-490, 2003 AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  17. 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 FLUOROMETRIC DETERMINATION OF RELEASED GLUCOSE(using Calcofluor) PELLET DISCARD AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  18. ANALYSIS OF TOTAL -GLUCAN(McCleary and Codd,1991, AOAC reference method 995.16) P6 Sample Hydrolysed with lychenase and filtered Dissolved in buffer, pH 6.5 Hydrolysed with Incubation with -glucosidase Centrifugation SPECTROPHOTOMETRIC DETERMINATION OF RELEASED GLUCOSE(GOPOD reagent -Glucose oxidase and peroxidase, λ=510 nm) PELLET DISCARD AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  19. ANALYSIS OF SOLUBLE -GLUCAN(McCleary and Codd,1991, AOAC reference method 995.16, Åman and Graham, 1987) Extraction for 120 min with distilled water at 38°C Centrifugation Sample SUPERNATANT DISCARD Dissolved in buffer, pH 6.5 P6 Hydrolysed with lychenase and filtered Hydrolysed with incubation with -glucosidase THE SOLUBLE FRACTION IS CALCULATED FOR DIFFERENCE, SPECTROPHOTOMETRIC DETERMINATION OF RELEASED GLUCOSE AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  20. STARCH: CHEMICAL STRUCTURE AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  21. - 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. STARCH: CHARACTERISTICS AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  22. 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 Incubation at 60°C with thermostable α-amylase (1→4) RESIDUE Incubation with amyloglucosidase50ºC, overnight (1→6) Extraction of LMW components80% Et OH, 85ºC Centrifugation Centrifugation PELLET DISCARD SUPERNATANT DISCARD SPECTROPHOTOMETRIC DETERMINATION OF RELEASED GLUCOSE(GOPOD reagent -Glucose oxidase and peroxidase, λ=510 nm) P12 AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  23. 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

  24. ANALYSIS OF TOTAL DIETARY FIBERUppsala Method (Theander et al., 1995) Incubation with α-amylase, boiling water bath Sample Boiling with DMSO, 1 hr Precipitation of soluble polymers, mix with 96% ETOH Incubation with amylo-glucosidase and proteinases, 40°C overnight Determination of Klason lignin (gravimetric method, Theander et al., 1995) Centrifugation RESIDUE PELLET DISCARD P12 Hydrolysis of polymers, H2SO4, 1 hr Derivatization of monosugars and their quantitative analysis by GC (Englyst and Cummings, 1984) Determination of uronic acids by colorimetric method (Scott, 1979) AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  25. NOTES • Reproducibility of β-glucan quantification by P2 and P6 • De-hulling of samples? AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

  26. Thank you for your attention! AVEQ First Meeting – Clermont Ferrand, September 19-21, 2007

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