Transcript Protein Structures as Delivery Vehicles in Foods

Chapter 5
Protein Structures as Delivery
Vehicles in Foods
Presenter. Sung, Jeehye
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
Be found on the outside of the fungi
Low molecular mass secreted proteins of 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
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).)
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
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
HSBI and HFBII
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 192656ii
200 rpm at 29℃
(1) Inoculation of 50-mL medium in 250mL 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), KH2PO4(4.0),
(NH4)2SO4(2.8),
MgSO4∙7H2O(0.6),
CaCl2∙2H2O(0.8), etc.
Class II hydrophobins
Hydrophobin rich extract preparation and characterization
Foam was allowed to
accumulate on liquid
surface until it nearly filled
the remaining space in the
funnel
Culture supernatant(500mL)
(Hydrophobin fraction)
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
TFA supernatant was evaporated off
under a steam of nitrogen
 re-suspended overnight in water
(hydrophobin rich fraction,HRE)
Undissolved fragments were
removed by centrifugation
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)
6000 rpm for 4 min
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 60
The oil-in-water
emulsion mixture
Class II hydrophobins
Preparation of the tri-phasic air/oil/water (A/O/W) emulsion
+
AFE
A/O/W emulsion
O/W emulsion
Well-mixed tri-phasic emulsions
with 28%, 36%, 52% or 68% of
total included phase volume
3000 rpm for 7 min