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Purification is a critical step to obtain hydrophobin HFBII for use in positive applications. In this study, hydrophobin HFBII was produced by Trichoderma reesei via submerged fermentation. Using the CO2-foam fractionation method yielded a fourfold increase in protein concentration. The foamate (αL-HFBII) was dried using a nano spray-dryer under optimal temperature. The gushing activity of the dried foamate (αS-HFBII) decreased. Addition of Tween 80 to the foamate before the drying process partially prevented the deactivation of hydrophobin HFBII. The purity of the powder was enhanced based on the theory of CO2-nanobubbles in a CO2-rich environment. The collected CO2-nanobubbles were added to an apolar–polar system and the interface of these two phases was collected. After evaporation of the apolar phase, the purity of the hydrophobins assembled on the surface of the liquid was significantly improved.

Gushing has been recognized as a disastrous phenomenon for contaminated carbonated beverages. Recent advances highlighted the ability of the class II hydrophobin films to interact via the hydrophob...

Hydrophobins have exceptional surface activity with potential applications for food and pharmaceutical industry. In this study, the effect of Class II hydrophobin HFBII on the retention of a volatile compound, ocimene in a water phase was investigated. We observed a negative gushing when 200 μ gp ure HFBII (κ-grade) and 40 μg ocimene (assay ≥ 90%) was added to 1 L sparkling water with CO2 concentration of 7 g/L. The droplet size analysis of κ-HFBII, ocimene and the mixture of them showed that the ocimene was emulsified in a solution containing κ-HFBII. The stability of this emulsion was reduced in function of time and this effect was intensified by increasing the temperature and the presence of CO2. Additionally, the retention of ocimene in a HFBII rich solution was measured by SPME-GC-MS. When 0.26±0.03 mg HFBII/mL is added to ocimene solution, 43% of the original ocimene concentration (i.e. 5.9 × 10 � 6 mg/mL) remained in the solution after 3 days, and 32% after 7 days. In contrast, original ocimene concentration in solutions without HFBII was only retained 20% and 14%, respectively. It showed the potential of HFBII to incor- porate insoluble flavour compounds in beverages and control their release. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher' sw eb site

Production and purification of hydrophobin HFBII has recently been the subject of intensive research, but the yield of production needs to be further improved for a generic use of this molecule at industrial scale. In a first step, the influence of different carbon sources on the growth of Trichoderma reesei and the production of HFBII was investigated. The optimum productivity was obtained by using 40 g/L lactose. Carbon starvation and excretion of extracellular enzyme were determined as two main conditions for the production of HFBII. In the second phase, and according to the physiological mechanisms observed during the screening phase, a bioreactor set up has been designed and two modes of cultures have been investigated, i.e. the classical submerged fermentation and a fungal biofilm reactor. In this last set-up, the broth is continuously recirculated on a metal packing exhibiting a high specific surface. In this case, the fungal biomass was mainly attached to the metal packing, leading to a simplification of downstream processing scheme. More importantly, the HFBII concentration increased up to 48.6 ± 6.2 mg/L which was 1.8 times higher in this reactor configuration and faster than the submerged culture. X-ray tomography analysis shows that the biofilm overgrowth occurs when successive cultures are performed on the same packing. However, this phenomenon has no significant influence on the yield of HFBII, suggesting that this process could be operated in continuous mode. Protein hydrolysis during stationary phase was observed by MALDI-TOF analysis according to the removal of the last amino acid from the structure of HFBII after 48 h from the beginning of fermentation in biofilm reactor. Hopefully this modification does not lead to alternation of the main physicochemical properties of HFBII.

Food industries are interested in finding highly stable foaming agents for product texture improvements. Hydrophobins have been mentioned many times in that context. For the production and purification of hydrophobin HFBII from Trichoderma reesei we found that foam fractionation of the fungal fermentation media is an interesting first step. After this step, the biophysical properties of the obtained foamate were studied. The foam generated by CO2 as the only sparging gas into a HFBII enriched solution in a closed environment was more stable than the foam generated by sparging with air. The equilibrium surface tension of a foamate containing 100 mg/L HFBII was measured by CAMTEL CDCA-100 tensiometry as equal to 40 mJ/m2, and was confirmed by the maximum bubble pressure tensiometer. Besides, it was found that for a long term foam preservation, the foam created by the HFBII-SDS combination is more stable than that of SDS.

There are several possible uses of the Class II hydrophobinHFBII in clinical applications. To fully understand and exploit thispotential however, the antioxidant activity and ACE-inhibitory potentialof this protein need to be better understood and have not been previouslyreported. In this study, the Class II hydrophobin HFBII was produced bythe cultivation of wild type Trichoderma reesei. The crude hydrophobinextract obtained from the fermentation process was purified usingreversed-phase liquid chromatography and the identity of the purifiedHFBII verified by MALDI-TOF (molecular weight: 7.2 kDa). Subsequently theantioxidant activity of different concentrations of HFBII (0.01-0.40mg/mL) were determined. The results show that for HFBII concentrations of0.04 mg/mL and upwards the protein significantly reduced the presence ofABTS+ radicals in the medium, the IC50 value found to be 0.13 mg/mL.Computational modeling highlighted the role of the amino acid residueslocated in the conserved and exposed hydrophobic patch on the surface ofthe HFBII molecule and the interactions with the aromatic rings of ABTS.The ACE-inhibitory effect of HFBII was found to occur from 0.5 mg/mL andupwards, making the combination of HFBII with strong ACE-inhibitorsattractive for use in the healthcare industry

J. Am. Soc. Brew. Chem. 72(1):12-21, 2014 Gushing is the vigorous overfoaming of carbonated beverages when the bottle is opened. Primary gushing in beer is mostly caused by a group of proteins called hydrophobins secreted by filamentous fungi, which contaminate CO2 gaseous molecules during carbonation and form nanobubbles. The influence of hop oil antifoam on primary gushing showed a complete suppressing effect in sparkling water, a decreasing effect in wort, and no influence on gushing-positive beers. This shows the importance of the critical point of addition of this product in the brewing process. GC and GC-MS analysis show that commercial available lipophilic hop extract comprises fatty acids, either saturated fatty acids (SFA) or unsaturated fatty acids (UFA) (both free and incorporated in mono-, di-, and triglycerides), waxes (long-chain alkanes), and steroid compounds. Gushing analysis of each compound showed that SFA and UFA behave in a different manner regarding gushing. In contrast to SFA and trans-form UFA, cis-form UFAs do not induce gushing. Long-chain alkanes provide sufficient hydrophobic structures to interact with gaseous CO2 molecules and induce gushing. Because hop antifoam molecules are hydrophobic, they interact with hydrophobins and prevent interaction with CO2 and, consequently, inhibit the possibilities of development of the explosive nanobubble structure.