Identification of factors involved in the gushing of beer in order to develop a technique for early detection of "gushing risk"

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1 Identification of factors involved in the gushing of beer in order to develop a technique for early detection of "gushing risk" ThesisdirectorIFBM: Marc Schmitt Patrick Boivin ThesisdirectorLRGP: Ivan Marc Romain Kapel Julien BILLARD Phd student

2 Outline of this presentation I Context II Aim of thesis III State of art IV Strategy V Solid phase fermentation of infected barley seed VI Purification of extracted compounds by RPC VII Conclusion VIII Prospect 2

3 I II III IV V VI VII VIII I. Context Overfoaming of beer without vigorous shaking Primary: caused by fungal contamination of barley and malt Secondary: caused by foreign particule in beer Lossup to 50 % of the product Harmfulleffecton the image of the product 3

4 II. Aimof thesis Identification of biochemical compounds, isolatedor in combination, whichare responsible of gushing Mechanism of beer gushing Measurement method Identification of key stage process 4

5 I II III IV V VI VII VIII III. State of art a. Mechanism of gushing- nanobomb theory Compounds with amphiphile properties and good liaison with carbon dioxide Hydrophobicpart CO 2 CO 2 Hydrophilic part CO 2 CO 2 CO 2 CO 2 CO 2 Modeling and biophysical characterisation of the primary gushing mechanism in beer. Sylvia Deckers Dynamic light scattering (DLS) as a tool to detect CO 2 -Hydrophobin structures and study the primary gushing potential of beer. Deckerset al CO 2 -hydrophobin structures acting as nano bombs. Deckerset al

6 Structure III. State of art b. Hydrophobins hypothesis Classe I Promote gushing Classe II Function Barley seed producedby fungus(10 15 kda) needed for the aerial growth Hyphae adhering to solid suface Hydrophobins, beer foaming and gushing. Shokribousjein et al Hydrophobins: Proteins that self assemble at interfaces. Linder,

7 III. State of art c. Production and purification of hydrophobins Strain Production Extraction Purification T. reesei Fermentor Saccharomyces cerevisiae(modified) F. poae, T. reesei, Nigrospora Fed-batch fermentation Shake flasks Foam fractionation from media Overfoaming of media From mycelium and foam fractionation of media Trichoderma reesei Fermentor From mycelium(tris-hcl+sds) T. reesei Fed-batch fermentation From mycelium(tris-hcl+sds) 15 RPC HPLC HPLC C4 vydac Preparative RP C4 Hydrophobicinteraction and anion exchange column RPC HPLC C4 A novel method for hydrophobin extraction using CO2 foam fractionation system. Khalesi et al Characterisation of HFBII biosurfactant production and foam fractionation with and without antifoaming agents. Winterburn et al Fungal hydrophobin as predictors of the gushing activity of malt. Sarlin et al Overproduction, purification and characterization of T. reesei hydrophobin. Askolin et al Modeling and biophysical characterization of the primary gushing mechanism in beer. Sylvie Deckers

8 IV. Strategy G+: gushing positive G-: gushing negative G+ / G- industrial malt Artificiallycontaminatedbarleyby fungi involved in gushing Molecular cartography Fractionationand screening of fraction Comparisonof compounds in sample Reverse phase HPLC SDS-PAGE 2D electrophoresis MALDI-TOF Separation of compounds Analysis of gushing potential Identification of compounds 8

9 V. Solid phase fermentation of barleyseedfor the extraction of compounds involved in beer gushing Previous work at IFBM Production of gushing positive beer with barley artificially infected 2 phenomenaobserved: gushing on barley gushingon a mix of barleyand negativegushingmalt Is it possible to extract compounds directly from the surface of barley? 9

10 V. Solid phase fermentation of barleyseedfor the extraction of compounds involved in beer gushing 1. Solid phase fermentation of barley Isolating of Fungi strains from malting barley Microdochium nivale Fusarium graminearum Fusarium tricinctum Alternaria Production of spores on Agar Autoclaved malting barley F. tricinctum Infection of sterile barley by fungi spores Incubation to 1 2 weeks at 25 C 10

11 V. Solid phase fermentation of barleyseedfor the extraction of compounds involved in beer gushing 2. Water extraction of compounds on barley artificially infected by fungus Problems: Studythe possibilityto extractthe compounds involvedin gushingdirectlyon the surface of barley seeds Experimentation: Extraction of compounds in surface of barley with ultrapure water Analysis of gushing potential according to the modified Carlsberg test in beer 11

12 V. Solid phase fermentation of barleyseedfor the extraction of compounds involved in beer gushing 2. Water extraction of compounds on barley artificially infected by fungus 1: Alternaria 2: F. tricinctum 3: F. graminearum 4: M. nivale Extraction of compounds involved in gushing is possible directly from surface of barley artificially infected by fungus 12

13 V. Solid phase fermentation of barleyseedfor the extraction of compounds involved in beer gushing 3. Mix with gushing negative malt extract Water extraction on F. tricinctum barley Production of gushingnegativemalt extract according to the modified Carlsberg test Boiling of F. tricinctum extract with gushing negative malt extract Filtration Gushing analysis of extract on beer Phenomena close to IFBM previous experiments 13

14 V. Solid phase fermentation of barleyseedfor the extraction of compounds involved in beer gushing Conclusion Twophenomenaobserved: production of gushingextractfromsurface of M. nivale and F. graminearum barley association /modification of compounds occuredwithf. tricinctumand malt negative extract Extraction of compounds involvedin gushingas been possible directly from surface of barley artificially infected by fungus especially by Microdochium nivale and Fusarium graminearum 14

15 I II III IV V VI VII VIII VI. Purification of extracted compounds by RPC 1. Fractionation and analysis of gushing potential Objectives: fractionation of compounds from M. nivale extract and identification of a gushing fraction Experiment: extraction of compounds from barley artificially contaminated by M. nivale fractionation of compounds by reverse phase HPLC concentration of fractions analysis of fraction(electrophoresis, gushing test) 15

16 I II III IV V VI VII VIII VI. Purification of extracted compounds by RPC 1. Fractionation and analysis of gushing potential Pool 1: whichcontainscarbohydrate and non-protein nitrogen Pool 2: which contains proteins(with high absorbance) Pool 3: which contains proteins(with low absorbance) Pool 1 Pool 1 Pool 2 Pool 3 Pool 2 Pool

17 VI. Purification of extracted compounds by RPC 1. Fractionation and analysis of gushing potential Identification of gushing fraction 1: raw extract 2: pool 1 3: pool 2 4: pool 3 20

18 VI. Purification of extracted compounds by RPC 3. Characterization of gushing fraction Problems: No information about proteins from pool 3 Objectives: Visualization, purification and identification of proteins from pool 3 Strategy: Production of M. nivale extract Fractionation of M. nivale extract and recovering of pool 3 Analysis by 2D electrophoresis 18

19 I II III IV V VI VII VIII VI. Purification of extracted compounds by RPC 3. Characterization of gushing fraction tris-tricinegel with15 % of acrylamide coloration with colloidal blue G-250 Decrease of complexity of sample Spot can be analyzed for their identification 22

20 I II III IV V VI VII VIII VI. Purification of extracted compounds by RPC 4. Identification by Peptide Mass Fingerprint Spot excision Trypsine digestion MALDI-TOF analysis Database: MASCOT Peptide Mass Fingerprint provides a first idea of the proteins present in sample Spots separate by 2DE has been identified as proteins from Microdochium 20

21 CONCLUSION Solid phase fermentation of barley seed Extraction of compounds involved in gushing as been possible directly from grains surface artificially infected by fungus especially by Microdochium nivale Fractionation of compounds by reverse phase chromatography Preparative fractionation provides a good separation of compounds with preservation of gushing power Identification of a gushing pool (3) Characterization of gushing fraction Identification of proteins from Microdochium which have gushing capacity 21

22 Prospect G+ / G- industrial malt Artificiallycontaminatedbarleyby fungi involved in gushing F. graminearum F. tricinctum Alternaria Molecular cartography Fractionationand screening of fraction Comparisonof compounds in sample Reverse phase HPLC SDS-PAGE 2D electrophoresis MALDI-TOF Separation of compounds Analysis of gushing potential Identification of compounds Developmentof a simple test (ELISA?) Apply the purification strategy by reverse phase HPLC to: barley artificially contaminated by other fungus involved in gushing Industrial malt gushing positive and negative Compare protein profil of each fungus and malt sample Identify common compounds 22

23 Acknowledgment Qualmalt UMT: Thesis commitee: Guy Derdelinckx KU Leuven Associate professor Center for food and microbial technology Didier Marion INRA Nantes Associate professor Biopolymère, Interactions, Assemblages Jean-Hughes Renault Université de Reims Associate professor Isolement et structure (IS) IFBM team : LRGP team : 23

24 Contact: IFBM Julien Billard: Thanks to the support of: Thank you for your attention Malteurs de France & Brasseurs de France 24

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