Title: Interaction of class II hydrophobins with hydrophobic interfaces as a basis for solving primary gushing problems in the brewing industry
Other Titles: Interactie van hydrofobines van klasse II met hydrofobe interfaces als basis voor een oplossing bij primaire gushing in de brouwerijindustrie
Authors: Shokribousjein, Zahra
Issue Date: 27-Aug-2014
Abstract: Hydrophobins are small surface active proteins produced by filamentous fungi. These proteins are famous for their potential to induce primary gushing. Primary gushing is an economically disastrous phenomenon which is provoked by the simultaneous presence of hydrophobins and gaseous CO2 molecules in carbonated beverages. Although hydrophobins are well known for causing problems in the beverage industry, they could be useful in other technological domains and used as surfactants or emulsifiers, or for the immobilization of proteins and enzymes on solid surfaces, for surface coating of biomaterials and modification of electrodes. Based on the large numbers of applications of class II hydrophobins, the objective of this work is to study and analyse, both the positive and negative potentials of hydrophobins. First part of this study was to characterize the two class II hydrophobins: one is not yet being studied (HFB-2a-2 from Trichoderma harzianum) and the other is being much investigated (HFBI from Trichoderma reesei). The second part of this study was to focus especially on gushing and the mechanisms behind solving gushing problem.As the new class II hydrophobin, HFB-2a-2 from Trichoderma harzianum was selected and the characteristics of this hydrophobin were compared to those of HFBI from Trichoderma reesei. Both hydrophobins were extracted and purified from the mycelium of the fungi. Their surface activity and their ability to adhere to solid surfaces were studied. In both cases, HFB-2a-2 was stronger than HFBI. Changes of the water contact angle were studied further for their adherence to the solid surfaces. HFB-2a-2 showed higher ability to change the angle than HFBI. In order to analyse the differences between hydrophobins, their structure and self-assemblies were studied by Rosetta docking simulation. The results showed that HFB-2a-2 has two hydrophobic patches while HFBI has only one. This indicates that such differences in the structure of these proteins result in different characteristics and it was concluded that HFB-2a-2 is a better choice than HFBI to be used as a surface active molecule or for the covering of solid surfaces.Until now, not many class II hydrophobins have been characterized. In our group, the Centre for Microbial Malting Technology (M2T), KU Leuven, the characterization of new hydrophobins is in process and the objective is to develop a database with the different characteristics of these proteins. Such a database will be useful for the researchers to select the best hydrophobins for particular applications.Second part of this thesis was concentrating on the negative aspect of hydrophobins in the brewing industry. Since hydrophobins cause gushing, it is of high importance to solve this problem. First, the mechanism by which gushing is inhibited was further investigated. To this end, identified model substances were selected to study the mechanism of gushing inhibiting. For this, it was referred to the mechanism of gushing whichwas already explained in details by Deckers et al., 2012. They have demonstrated that gushing happens because of the interaction of gaseous CO2 molecules with the hydrophobic patch of hydrophobins. Therefore, it was decided to select non-polar molecules in our study which will interact with hydrophobic patch (es) of hydrophobins. Through this interaction, gaseous CO2 can no longer interact with the hydrophobins and consequently, gushing will be inhibited. This theory was confirmed by the decrease/ inhibition of gushing when specific non-polar molecules were added to hydrophobin HFBI. It was observed that the addition of some linear saturated and unsaturated hydrocarbons with the same length as the hydrophobic patch of hydrophobins, inhibits gushing. In order to understand the mechanism of gushing inhibition and to characterize the interaction between hydrophobins and gushing inhibitors following techniques were used: Fourier Transform Infrared spectroscopy (FTIR), Fiber Optic Surface Plasmon Resonance (FO-SPR), Quartz Crystal Microbalance (QCM) and particle size analysis. FTIR demonstrated that hydrophobins interact with non-polar molecules through non-bonded interactions. Moreover, the immobilization of hydrophobins on a solid surface using FO-SPR and QCM and subsequent interaction with gushing inhibitors indicated very clearly that there is a tendency between these molecules to interact together. Based on FTIR results, this interaction is not of a bonded type. Furthermore, particle size analysis revealed that non-polar molecules attach to hydrophobins and form larger particles which were not present before the addition.Since the interaction of hydrophobins with gaseous CO2 is mainly responsible for gushing and these results indicate that non-polar molecules interfere with this interaction, the use of non-polar molecules is a solution for solving gushing problem. The effective non-polar molecules against gushing are the ones that are not potentially gushing inducers and have enough hydrophobicity to interact with hydrophobins.The next part of the study was concentrated on the application part of gushing inhibitors. A lipophilic hop extract antifoam was used to analyze the influence on gushing of beer. First, a hop extract antifoam was characterized and its influence on gushing provoked by HFBI was studied. Then the process for using this extract was optimized. The analysis of the hop extract antifoam by GC and GC-MS showed the presence of non-polar molecules, mainly long linear hydrocarbons and fatty acids. The saturated long linear hydrocarbons were gushing inducers while most of the fatty acids were unsaturated and did not induce gushing. Addition of the hop extract antifoam to sparkling water or wort containing hydrophobins, showed that this extract inhibited gushing of water and decreased gushing of wort. The addition to gushing beer had some influence. This indicates that when the hop extract antifoam is used to reduce gushing, the extract must be added before the interaction of hydrophobins and CO2.After the hydrohobin-CO2 complexes are formed, the hop extract antifoam is less active. The decreased gushing by hop extract antifoam is explained by the formation of an interface to which hydrophobins attach so that they are no longer available to interact with CO2 and therefore, gushing is decreased.Furthermore, the hop extract antifoam was also evaluated in order to increase its interaction with hydrophobins. Since this extract provides a surface for hydrophobins to attach, by increasing the specific surface of the extract, higher numbers of hydrophobins will be attached and therefore, less gushing will occur. Better dispersion of the hop extract antifoam will increase the specific surface area. Further research was done to analyse the effect of better dispersion on the effect of hop extract antifoam against gushing. This was evaluated by using (1) high temperature, at the time of the addition of hop extract antifoam during mashing of malt, and (2) use of a magnetic field for dispersion. The addition of hop extract antifoam in the mashing step changed its properties which then became a gushing inducer. The high temperature in mashing breaks the hop extract antifoam matrix containing gushing inducer and inhibitor molecules. Since gushing inducers are the majority, they become available to physically interact with CO2 molecules and induce gushing. As a result, addition of this extract should be limited to addition in a cold step of brewing.The magnetic field is used in some companies to disperse the hop extract antifoam, before entering the fermenters. The gushing potential of hydrophobins is decreased when the hop extract antifoam is first treated by magnet and then added to the wort. The best result is then obtained when only a low amount of the hop extract antifoam is treated and then added to the wort.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre for Surface Chemistry and Catalysis
Centre for Food and Microbial Technology

Files in This Item:
File Status SizeFormat
Whole thesis after preliminary for examination committee.pdf Published 4527KbAdobe PDFView/Open Request a copy

These files are only available to some KU Leuven Association staff members


All items in Lirias are protected by copyright, with all rights reserved.