Title: Upscaling the high-grade purification of hydrophobin HFBII fromTrichoderma reesei for carbonated beverages purposes: Behavior of thehydrophobic patch
Other Titles: Opschaling van de hoogwaardige opzuivering van hydrofobine HFBII vanTrichoderma reesei voor gebruik in koolzuurhoudende dranken: Gedrag vande hydrofobe patch
Authors: Khalesi, Mohammadreza
Issue Date: 11-Feb-2015
Abstract: Hydrophobins are a family of low molecular weight proteins consisting of four disulfide bridges and an extraordinary hydrophobic patch. Under typical condition, the latter can interact with gaseous CO2 resulting in the formation of the stable CO2 nanobubbles covered by an elastic membrane in carbonated beverages, providing the required energy for ocurring primary gushing. Hydrophobins include exceptional surface active properties with potential applications in several scientific and technological fields. A number of applications for Class II hydrophobins have been proposed to use it as biosurfactant and where high grade purity is needed, like where studying the compatibility between gaseous CO2 and other hydrophobic molecules for interaction with the hydrophobins, as it is necessary for the flavor and beverages industry. Therefore, it becomes necessary to develop a large scale procedure for their production and purification.
A protocol to produce Class II hydrophobin HFBII, a protein with a molecular weight (MW) of 7.2 kDa consisting of 71 amino acids, from Trichoderma reesei (T. reesei) through fermentation process was established. After that, the procedure of purification via chromatography was carried out. The highly pure HFBII (kappa-grade) was identified by MALDI-TOF. Fundamental understanding of the mechanism of primary gushing was established after obtaining kappa-HFBII and using it for gushing tests. The possible interaction between this molecule and gaseous CO2 was further investigated. The obtained kappa-HFBII was added to the carbonated water and the standard gushing test was carried out. The CO2 nanobubbles observed in gushed liquid with critical diameter at atmospheric pressure (i.e. around 110-120 nm) was considered to be an essential pre-condition for providing sufficient energy to provoke the primary gushing.
Several operative parameters are involved for production of HFBII by the wild type T. reesei. The production of HFBII occurred when the major part of lactose was taken up. This observation led to the design of a biofilm reactor, promoting the growth of the fungal biomass in a solid state physiology, and the excretion of hydrophobin. The use of biofilm has led to a significant increase of HFBII production in shorter time by comparison with a classical submerged bioreactor. Upscaling  the  biofilm  reactor  was  effectively realized to a 10 liter working volume fermenter. X-ray tomography illustrated that the biofilm overgrowth occurred when successive cultures were performed on the same packing. However, this phenomenon did not influence the yield of HFBII, suggesting that the procedure could be operated in continuous mode.
After production of HFBII, it is required to extract it from the growth media of T. reesei. For this, the physico-chemical properties (e.g. amphiphilic nature and high elasticity) of Class II hydrophobins were used by applying a CO2 foam fractionation system. After foam fractionation, a foamate was obtained which is in fact an aqueous solution enriched by HFBII (namely alpha-HFBII). The enrichment value was calculated to be 4.6 (based on the volumetric concentration) and 3.75 (based on the protein content). The physico-chemical characteristics of alpha-HFBII were also investigated. The gushing activity of alpha-HFBII after spray-drying was diminished to 31% of the gushing activity of the sample before spray-drying, suggesting the occurrence of protein rearrangement during the process. It was shown that addition of an auxiliary biosurfactant besides hydrophobin during spray-drying could partially avoid the deactivation of dried alpha-HFBII. Next step was upscaling the purification using a 30RPC liquid chromatography. The quality and the quantity of the final obtained kappa-HFBII with this last set-up was comparable to that of the 15RPC lab-scale column.
The retention of a dispersed volatile compound by HFBII in a liquid solution for a prolonged time was studied at the end of this project. For the proof of principle, at first the binding between HFBII and ocimene, a typical monoterpene in hops, was studied. Inhibition of gushing by addition of ocimene to bottles of carbonated water contaminated by kappa-HFBII confirmed the hypothesis. It was proven that ocimene in a liquid solution was emulsified in the presence of HFBII, since new droplets with smaller size were observed to be generated. Besides, the migration of HFBII to the interface of the liquid in the presence of ocimene was postponed. It was concluded that HFBII and ocimene bind together. The retention of ocimene in a solution by addition of HFBII was measured by SPME-GC-MS. The results showed that when HFBII (0.26 ± 0.03 mg/mL) was present in a solution of ocimene (5.9 × 10-6 mg/mL), 43% of the ocimene remained after 3 days. This value was twice as high as for the control sample and indicated that the bond between HFBII and ocimene slowed down the release of ocimene to the gas phase, probably due to the better dispersion of the ocimene in the liquid.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre for Food and Microbial Technology
Centre for Surface Chemistry and Catalysis

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