Protein Structures as Delivery Vehicles in Foods Presenter. Sung, Jeehye Chapter 5

Contents Introduction Material and Method Results Conclusions Reference

Introduction Lab. of Bioresources and Food Chemistry

Protein Proteins are essentially high-molecular weight linear polymers composed of a chain of amino acids The composition and the sequence of amino acids determine the local secondary structure within the protein overall tertiary structure of the protein In aqueous solution, the folded structure of a protein tends to bury hydrophobic regions within the interior of the molecule and expose hydrophilic regions on the protein surface

Protein Proteins have many different roles in foods They are very important nutritional components of the diet. The proteins that have surface active are widely used in food systems where a surface-active component is required  milk, dairy product Specific active proteins exist that can be developed and applied to give new functionalities and opportunities HYDROPHOBINS

Hydrophobin A negative aspect of hydrophobins is that they are produced by fungi when growing on grains(e.g. barley) and during grain treatment Barley is a very important vegetal food with high economic value as it is used in the production of beer and other drinks The presence of hydrophobins leads to gushing, which has many economic drawbacks for brewing industries Consequently, improvement in rapid and early detection methods for hydrophobins is required

Hydrophobins Low molecular mass secreted proteins of fungi Be found on the outside of the fungi The proteins are all about 10kDa in size and contain a large proportion of hydrophobic amino acids The main unifying feature is the presence of 8 Cys residues Very little sequence conservation in general, apart from the idiosyncratic pattern of eight Cys residues implicated in the formation of four disulfide bridge

Hydrophobins These proteins are able to assemble spontaneously into amphipathic monolayers at hydrophobic-hydrophilic interfaces Figure 1. The role of hydrophobins in fungal hyphae growth through the airwater interface as shown by Wösten et al. (1999). As the hyphae grow submerged in aqueous medium they produce hydrophobins into the medium. Hydrophobins adsorb to the air-water interface and lower the water surface tension, thus enabling the hyphae to penetrate the air-water interface and grow into the air. (According to Wösten et al. (1999).) These proteins are able to assemble spontaneously into amphipathic monolayers at hydrophobic-hydrophilic interfaces

Hydrophobin Due to the distribution of the cysteines and the clustering of hydrophobic and hydrophilic residues, hydrophobins are divided into two classes; SC3 and EAS HSBI and HFBII Class I hydrophobins asseble into highly insoluble polymeric monolayers composed of fibrillar structures known as rodlets Class II hydrophobins lack the fibrillar rodlet morphology and can be solubilized with organic solvents and detergents

Hydrophobin Due to the distribution of the cysteines and the clustering of hydrophobic and hydrophilic residues, hydrophobins are divided into two classes; Class I hydrophobins : SC3 and SC4 of schizophyllum commune ABH1 of agaricus bisporus Class II hydrophobins : cerato-ulmin(CU) of oph iostoma ulmi cryparin(CRP) of cryphonectria parasitica HFBI and HFBII of trichoderma ressei

Hydrophobin Hydrophobins were consequently first suggested for a number of applications involving the modification of surfaces properties leading to improving biocompatibility, reducing friction, or providing specific sites for protein immoblization

Materials and methods Lab. of Bioresources and Food Chemistry

Class II hydrophobins Cultivation conditions T. reesei strain IMI ii 200 rpm at 29 ℃ (1)Inoculation of 50-mL medium in 250- mL flask using spores from a week old culture grown on YM agar (2)Transferring of the inoculum, after three days of fermentation, into the 2-L shake flask containing 200-mL of medium for six days further growth Cultivation media used contained (g/L): lactose(20.0), peptone(4.0), yeast(1.0), KH 2 PO 4 (4.0), (NH4) 2 SO 4 (2.8), MgSO 4 ∙7H 2 O(0.6), CaCl 2 ∙2H 2 O(0.8), etc.

Class II hydrophobins Hydrophobin rich extract preparation and characterization Culture supernatant(500mL) (Hydrophobin fraction) Foam was allowed to accumulate on liquid surface until it nearly filled the remaining space in the funnel The liquid layer was then drained away Only the foam layer remained The foam was collapse and washed with 80% ethanol  evaporating freeze-drying  The dried, hydrophobin rich, foam was re-suspended in cold 100% trifluoroacetic acid(TFA) on ice for 90 min

Class II hydrophobins Hydrophobin rich extract preparation and characterization Undissolved fragments were removed by centrifugation TFA supernatant was evaporated off under a steam of nitrogen  re-suspended overnight in water (hydrophobin rich fraction,HRE) The presence of hydrophobin was checked at different stages by running SDS-PAGE

Class II hydrophobins Preparation of air-filled emulsion with an HFBII coat (AFE) Hydrophobin rich extract was placed in a jacketed vessel The solution was sonicated at 20kHz, 50% amplitude for 3 min at 54 ℃ while sparging with air at 60mL/min Produce a working stock of concentrated air cells (68% by volume)

Class II hydrophobins Preparation of oil/water emulsions (O/W) The oil-in-water emulsion contained  20wt% sunflower oil  0.2wt% iota Carrageenan(stabiliser): add to the water phase and stirred containously at 60 ℃ until totally dispersed  0.5% wt% Tween 60: clled to room temperature before adding the Tween rpm for 4 min The oil-in-water emulsion mixture

Class II hydrophobins Preparation of the tri-phasic air/oil/water (A/O/W) emulsion 3000 rpm for 7 min + O/W emulsion AFE A/O/W emulsion Well-mixed tri-phasic emulsions with 28%, 36%, 52% or 68% of total included phase volume