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September 24 ST 2015

SCREENING WHEAT GENOTYPES FOR WHOLE GRAIN FLOUR MILLING Juliano Luiz De Almeida =, ° Bram Pareyt =, * Lien R. Gerits =,+ Jan A. Delcour =. °FAPA / Cooperativa Agrária Agroindustrial, Guarapuava, Brazil * Currently at Puratos , Groot-Bijgaarden, Belgium

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September 24 ST 2015

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  1. SCREENING WHEAT GENOTYPES FOR WHOLE GRAIN FLOUR MILLING Juliano Luiz De Almeida =,° Bram Pareyt =,* Lien R. Gerits =,+ Jan A. Delcour= °FAPA / Cooperativa Agrária Agroindustrial, Guarapuava, Brazil * Currently at Puratos, Groot-Bijgaarden, Belgium + Currently at Genzyme, Geel, Belgium = Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Leuven, Belgium September 24ST 2015

  2. INTRODUCTION Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Main benefits of whole grain flour (WGF) consumption. • Rich in wheat bran dietary fiber, antioxidants, vitamins • EFSA approved claims • Wheat bran increases fecal bulk (intestinal problems) • Wheat bran reduces intestinal transit time (increases satiety feeling) • Epidemiological evidence for reduced risk of welfare diseases: type-2 diabetes, cardiovascular disease, obesity, intestinal cancers. • Technological drawbacks • Decreased loaf volume (Campbell et al., 2008) • Modified organoleptic properties (taste, texture, colour, …). • Increases risk of mycotoxin contamination (De Almeida et al., 2015 in press) • Negative impact on shelf life (Tait and Galliard., 1988) 15% bran 7,5% bran 0% bran

  3. INTRODUCTION Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Technological drawbacks – shelf life • Lipids of WGF degrade first by hydrolytic rancidity (diglycerides, monoglycerides and non-esterified fatty acids), which can be followed by oxidative rancidity (hydroperoxides). • Wheat lipase is mostly located in the bran fraction. • Wheat lipoxygenase is located in the germ and bran fractions. Doblado-Maldonado et al., (2012) Journal of Cereal Science 56: 119-126

  4. INTRODUCTION Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Whole grain milling and food industries face challenges to implement WGF in their products, one of the main reasons being a much shorter shelf life of such flour than that of white (patent) flour. • US: • Shelf life of patent flour: 9-15 months • Shelf life of WGF: 3-9 months • Tropical countries or countries under climate change scenarios: • Even more difficulties in producing stable WGF due to high storage temperatures and long travel distances from mill to food manufacturing plants, which favors lipid degradation. • e.g.: in Brazil, shelf life generally ranges from 3 months for WGF to 6 months for patent flour.

  5. INTRODUCTION Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Hypothesis - Objective • To increase WGF shelf life, one may exploit differences in lipolytic activity among wheat cultivars. • Cultivars with less lipolytic activity can be prioritized to produce WGF of adequate shelf life. • Objective: find wheat cultivars with less lipolytic activity in order to increase WGF shelf life.

  6. MATERIAL AND METHODS - BRAZIL Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Small grain field trials were established at three sites in the State of Paraná (Brazil) during the 2011 growing season. • Wheat genotypes were evaluated without Fusarium graminearum inoculation. As this was a field trial, uninoculated plots were likely exposed to natural inoculum. Murakami Guarapuava Pinhao • Experimental design was a split-plot design with three blocks: • Site as a main plot (3). • Small grain cultivar as a sub-plot (20).

  7. MATERIAL AND METHODS - BRAZIL Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Quality determinations of patent (white) and WGF were held at the Coop Agrária Central Laboratory. • Falling number, Alveograph, Farinograph, Gluten content and Minolta colour. • Deoxynivalenol determinations were performed in an UltraPerformance Liquid Chromatography - UPLC equipment, with mass spectrometry detection at the Coop Agrária Central Laboratory.

  8. MATERIAL AND METHODS - BELGIUM Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Grains were milled to WGF using a laboratory mill with water cooling – IKA A10 Basic • WGF moisture content determination • AACC-I Approved Method 44.19-01 (1999) • WGF lipase activity determination (De Almeida, et al., 2012) • WGF (1.0 g) lipase extraction • 2.5 ml deionized water • Shaking (30 min, 150 rpm, room temperature) • Centrifugation (10 min, 550 g, 21ºC) • Lipase activity - UV spectrophotometer • Colorimetrically at 30ºC • p-nitrophenyl palmitate = substrate Murakami Pinhao

  9. MATERIAL AND METHODS - BELGIUM Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Lipase p-nitrophenyl palmitate p-nitrophenol + palmitic acid (substrate) (reaction products) p-nitrophenol (µmol /ml) De Almeida, et al., (2012) Cereal Chemistry 91(4): 321-326 Time (minutes)

  10. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Figure. Correlation among deoxynivalenol in WGF and lipase activityin field experiments conducted at three sites in Brazil, 2011.

  11. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Figure. Correlation among deoxynivalenol in WGF and lipase activityin field experiments conducted at three sites in Brazil, 2011. Among 40 field and quality variables, the strongest correlation was between lipase activity and DON content in WGF!

  12. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Table. Lipase activity of WGF from wheat genotypes at three sites in Brazil during the 2011 crop season.

  13. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Table. Lipase activity of WGF from wheat genotypes at three sites in Brazil during the 2011 crop season. A significant interaction between the main plot “site” and sub-plot “cultivar” was found with regard to WGF lipase activity.

  14. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Table. Lipase activity of WGF from wheat genotypes at three sites in Brazil during the 2011 crop season. 6-fold 6-fold 9-fold

  15. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Table. Deoxynivalenol accumulation in WGF from wheat genotypes at three sites in Brazil during the 2011 crop season. A significant interaction between the main plot “site” and sub-plot “cultivar” was found with regard to WGF DON accumulation.

  16. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Table. Deoxynivalenol accumulation in WGF from wheat genotypes at three sites in Brazil during the 2011 crop season. A significant interaction between the main plot “site” and sub-plot “cultivar” was found with regard to WGF DON accumulation. 11-fold 7-fold 10-fold

  17. RESULTS Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour Table. Deoxynivalenol accumulation in WGF from wheat genotypes at three sites in Brazil during the 2011 crop season. A significant interaction between the main plot “site” and sub-plot “cultivar” was found with regard to WGF DON accumulation. Wheat cultivars with low levels of DON are almost the same cultivars which have lower lipase activity, irrespective of the location. The opposite is also true.

  18. DISCUSSION • They found that the wild type F. graminearum produces lipase enzyme FGL1 during spikelets infection releasing polyunsaturated fatty acids (linoleic and α-linoleic acid) which inhibit callose formation, making spike tissue susceptible to Fusarium Head Blight (FHB). • Callose formation is part of the plant defense mechanism in response to F. graminearum attack. • However they have not yet identified the lipid source in wheat from which the polyunsaturated fatty acids are released in the pathosystem wheat - Fusarium graminearum.

  19. DISCUSSION • One can infer that there are differences among wheat genotypes about composition and quantity of lipids in the grain (although wheat does not produce lipase). Thus, wheat lipids would be the raw material used by the fungus, through lipase, for polyunsaturated fatty acids production (which inhibit callose formation). • Probably differences of composition and quantity of lipids among wheat genotypes can cause different responses of lipase enzyme activity, resulting in different levels of infection and DON production.

  20. DISCUSSION Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Thus, considering the importance of lipase enzyme activity, which is probably related to the composition and amount of lipids in wheat, and also considering DON presence in WGF of different wheat genotypes, we propose : • Cluster analysis of wheat genotypes based on LIPASE and DONWGF variables using PROC CLUSTER of SAS software; • Principal component analysis of wheat genotypes based on LIPASE and DONWGF variables using PROC PRINCOMP SAS software;

  21. DISCUSSION Scientists: Juliano Luiz de Almeida, Bram Pareyt, Lien R. Gerits and Jan A. Delcour • Cluster analysis was made to group the cultivars using two variables, LIPASE and DONWGF through the PROC CLUSTER procedure. Higher LIPASE and DONWGF Lower LIPASE e DONWGF Internal correlation level between variables

  22. DISCUSSION • Principal component analysis (more robust) to confirm cluster analysis results using PROC PRINCOMP procedure. Principal component analysis confirms cluster analysis because cultivars were grouped as much the same way. Lower LIPASE and DONWGF Higher LIPASE and DONWGF

  23. CONCLUSIONS • Most of the entries that showed low lipase activity and low DON content, originated WGF with potential for long shelf life compared to entries with increased lipase activity and high DON contents. • Therefore we propose to consider the use of lipase activity along with DON content, as new parameters (traits) to screen wheat genotypes aiming WGF for extended shelf life. • Also based on the high correlation between lipase activity and DON content, we conclude that this finding could open a new gate to better understand plant resistance to Fusarium graminearum and DON contamination.

  24. ACKNOWLEDGEMENTS • J.L. De Almeida acknowledges the National Council for Scientific and Technological Development - CNPq – Brazil for financial support and FAPA/Cooperativa Agrária Agroindustrial (Guarapuava, Brazil) for the sabbatical leave as a post doc. • B. Pareyt was active as a post doc fellow of the FWO-Vlaanderen (Brussels, Belgium). • J.A. Delcour is WK Kellogg Chair in Cereal Science and Nutrition at KU Leuven. • This research was also part of the Methusalem Program “Food for the Future” (2007-2014) of the KU Leuven.

  25. 44 QUESTIONS?juliano@agraria.com.br With her abundant gifts agriculture nourishes all industries.

  26. Sour PRE-HARVEST MYCOTOXIN REDUCTION STRATEGIES - GENETIC STRATEGY Table 1. Resistance to FHB and DON accumulation in whole grain flour and patent flour in different wheat genotypes during the 2011 and 2012 growing seasons in the field experiments conducted in Guarapuava, Paraná, Brazil. De Almeida, et al., submitted

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