avena genetic resources for quality in human consumption (aveq) aveq first meeting – clermont...

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


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).



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.



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]


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


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


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


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


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


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


-GLUCAN: CHEMICAL STRUCTURE


-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.

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.

-GLUCAN CONTENT IN CEREALS


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)


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)


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)


Sample

Dissolved in buffer, pH 6.5

SUPERNATANT DISCARD

Hydrolysed with lychenase and filtered

Hydrolysed with incubation with -glucosidase


Extraction for 120 min with distilled water

at 38°C

Centrifugation

THE SOLUBLE FRACTION IS CALCULATED FOR

DIFFERENCE,

P6

STARCH: CHEMICAL STRUCTURE


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.


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

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


(GOPOD reagent -Glucose oxidase and peroxidase, λ=510 nm)

PELLET DISCARD

P12

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.

ANALYSIS OF TOTAL DIETARY FIBER Uppsala Method (Theander et al., 1995)


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?


Thank you for your attention!


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