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L'inflorescence du houblon (Humulus lupulus L.) appelée aussi cônes est presqu'exclusivement utilisée dans le domaine de la brasserie comme l'un des principaux ingrédients pour parfumer et conserver la bière. La tendance actuelle des brasseries artisanales d'houblonnage à cru conséquent conduit parfois à une production de profils aromatique incontrôlée et aberrante. Le but de ce travail est de déterminer si une partie de la teneur enzymatique du houblon, à savoir l'α-amylase et la β-amylase, pourrait influencer le profil aromatique de la bière houblonnée à crû consécutivement à la fermentation par la levure des hydrates de carbone produits par ces enzymes. Pour ce faire, des méthodes spectrophotométriques de quantification de l'activité enzymatique ont été élaborées pour évaluer le contenu au sein du houblon. De plus, une méthode de chromatographie liquide (HPLC-ELSD) a été utilisée pour déterminer l'impact sur le profil des sucres de la bière par la production par ces enzymes de glucose et de maltose à partir de sucres de plus haut degré de polymérisation. En outre, des techniques de chromatographie en phase gazeuse (GC-ECD/FID) ont été utilisées pour évaluer la métabolisation éventuelle par la levure en utilisant des cétones vicinales (butane/pentane dione) comme marqueurs de la fermentation. Enfin, une analyse en composantes principales évaluant le changement global en surveillant la concentration en esters (éthyle et acétate), alcools supérieurs et aldéhydes démontre l'impact sur le profil aromatique de cette interaction levure-houblon.

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The gushing of carbonated beverages is defined as a spontaneous wild and uncontrolled overfoaming upon opening of non-shaken bottles. As overfoaming is detected after bottling this results in significant economic and image losses to the producer. A mechanism referred to as secondary gushing is well understood and is due to the presence of external inorganic material or technical shortcomings. On the other hand, the very disastrous primary gushing mechanism is still under discussion although it is known it is related to the presence of fungal proteins, the Class II hydrophobins. Despite 25 years of research, several parts of the puzzle are still missing. Recent reviews on the phenomenon indicate that hydrophobins may stabilize gaseous CO2 bubbles without explaining however how this occurs and how coated bubbles are made and gain stability.We studied how the two main factors, Class II hydrophobins and CO2 interact to induce gushing. Class II hydrophobin HFBI was extracted from the mycelium of Trichoderma reesei MUCL 44908 with a Tris/HCl buffer, purified by RP-HPLC, detected by MALDI-TOF and identified by Edman amino acid sequencing. Sparkling water was used as source of CO2. Considering the physico-chemical properties of both Class II hydrophobins and CO2 and by performing molecular dynamics simulation, it was observed that CO2 molecules interact with the hydrophobic patch of Class II hydrophobin and a detailed mechanism formation of coated CO2 bubbles, the stabilized nanobubbles, was proposed. It occurs in several steps : migration of hydrophobins to the liquid-gas interface, contamination by contact with pure CO2 atmosphere, concentration followed by crystallization, shaking causing imbalance of Henry’s equilibrium in the closed recipient, re-equilibrium resulting in the nanobubble formation by closing due to the lateral forces (Young-Laplace forces). Finally, the closed bottle contains gaseous CO2 nanobubbles stabilized by a crystalline layer of Class II hydrophobin. These solid structures are characterized by a consistent geometric value as their volume is determined by the critical diameter of CO2 at the pressure in the bottle. The explosion of the bubble, i.e. the “nanobomb effect”, can be described by Avogadro’s law and even by Boyle-Mariotte’s law since temperature has a small influence. Nevertheless, primary gushing behavior in function of temperature does not fit with this theory in practice. Indeed, much more liquid than could be predicted is expelled from the bottle by increasing the liquid temperature at the opening. This indicates that beside the chemical binding, a lot of CO2 is linked by low energy bonds that can be broken just by agitation energy.Since Class II hydrophobins are able to stabilize gaseous CO2 bubbles, which must have a critical diameter at atmospheric pressure a method based on the dynamic light scattering was developed. This novel method allows to distinguish primary (hydrophobin) from secondary (kieselghur) gushing. It is based on the presence of 100 nm particles which areformed in the overfoamings after rest under saturated CO2 (1 bar) only when hydrophobins are present. The 100 nm diameter corresponds to the critical diameter of CO2 at atmospheric pressure. The method clearly indicates whether the gushing of bottled beer is either primary or secondary. This method was also applied to predict the gushing potential of barley and malt and can distinguish contaminated from non-contaminated raw materials. The target is to further propagate this method in order to confirm at industrial scale the good results obtained.Finally, the work describes some suggestions in order to prevent and/or to cure primary gushing.

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