Modeling and biophysical characterisation of the primary gushing mechanism in beer. Interaction between gaseous carbon dioxide and class II hydrophobins

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.Dankwoord i Abstract in Dutch iii Abstract v List of abbreviations and list of symbols vii Literature review 1 The history of primary gushing 3 Aims of this study 17 Chapter I Production of the Class II hydrophobin HFBI 19 I.1. Introduction 21 I.2. Materials and Methods 22 I.2.1. Materials and Chemicals 22 I.2.2. Methods 23 I.3. Results and Discussion 24 I.4. Conclusion 27 Chapter II Nanobubbles and Nanobombs:Why and How? 29 II.1. Introduction 31 II.2. Materials and Methods 39 II.2.1. Location of hydrophobins 39 II.2.2. Molecular Dynamics (MD) 40 II.3. Results 43 II.4. Gushing model 46 II.4.1. Nanobubble formation 46 II.4.2. Nanobomb theory 48 II.5. Conclusion 48 Chapter III Thermodynamic properties of primary gushing 49 III. 1. Introduction 51 III. 2. Theoretical calculation 52 III.3. Conclusion 56 Chapter IV CO2-hydrophobin associated structure responsible for primary gushing: solid structures detectable at atmospheric pressure 57 IV.1. Introduction 59 IV.2. Materials and Methods 66 IV.2.1. Materials and Chemicals 66 IV.2.2. Methods 66 IV.3. Results and Discussion 67 IV.4. Conclusion 72 Chapter V DLS analysis: a new tool to predict the primary gushing potential of barley and malt and to distinguish primary from secondary gushing in beer 73 V.1. Introduction 75 V.2. Materials and Methods 76 V.2.1. Materials 76 V.2.2. Methods 76 V.2.2.1. Characterization of gushing potential of barley and malt by doubly Modified Carlsberg Test followed by DLS 76 V.2.2.2. Characterization of gushing potential of finished beer by DLS 78 V.3. Results and Discussion 78 V.4. Conclusion 81 General conclusions 83 Perspectives 89 References 95 List of publications 105nrpages: 107status: publishe