Synthetic Multiphase Systems

Abstract

A synthetic multiphase product comprising BsIA is presented. Methods of producing a synthetic multiphase product comprising BsIA, and applications of BsIA in synthetic multiphase products are also presented.

Claims

1. A synthetic multiphase product comprising BslA.

2. A synthetic multiphase product according to claim 1, wherein the synthetic multiphase product is a multiphase food product.

3. (canceled)

4. A synthetic multiphase product according to claim 1, wherein the synthetic multiphase product is a personal care product.

5. A synthetic multiphase product according to claim 1, wherein BslA has a sequence according to one of SEQ ID NO. 1 to 4, and SEQ ID NO:18 to 29, and variants thereof.

6. A synthetic multiphase product according to claim 1, wherein the synthetic multiphase product comprises at least 0.005 wt % BslA.

7. A synthetic multiphase product according to claim 1, further comprising at least one co-surfactant.

8. (canceled)

9. (canceled)

10. A synthetic multiphase product according to claim 1 comprising at least three or more intimately mixed phases of matter.

11. A synthetic multiphase product according to claim 10, wherein the synthetic multiphase product comprises a pharmaceutical active agent and the synthetic multiphase product is a pharmaceutical composition or a pharmaceutical product.

12. A method of manufacture of a synthetic multiphase product according to claim 1, comprising the steps of: a providing the one or more components of the synthetic multiphase product; b adding BslA to the one or more components of the synthetic multiphase product; and c mixing the one or more components to form the synthetic multiphase product.

13. A method according to claim 12, wherein the synthetic multiphase product is a multiphase food product.

14. A method according to claim 13, wherein the synthetic multiphase product is a frozen multiphase food product.

15. A method according to claim 12, wherein the synthetic multiphase product is a personal care product.

16. A method according to claim 12, wherein at least one of the one or more components may comprise one or more co-surfactants.

17. (canceled)

18. (canceled)

19. (canceled)

20. A composition of particles of a first material, the particles comprising a coating of BslA over at least a portion of the surface of the particles, wherein the particles within the composition of particles are more hydrophilic than particles of the first material that do not comprise a coating of BslA over the surface of the particles.

21. A composition according to claim 20, wherein the first material is an intimate mixture of different chemical compounds formulated into particles.

22. A composition according to claim 20, wherein the first material is hydrophobic.

23. (canceled)

24. (canceled)

25. (canceled)

26. A frozen synthetic multiphase product comprising BslA.

27. A frozen synthetic multiphase product according to claim 26 comprising at least one co-surfactant.

28. A frozen synthetic multiphase product according to claim 27, wherein the at least one co-surfactant is a protein surfactant.

29. A frozen synthetic multiphase product according to claim 26, comprising one or more additional components, wherein the one or more additional components comprises one or more of milk proteins, sugars, carbohydrates, egg proteins and fats.

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0188] FIG. 1 (a) Interfacial tension profiles of a droplet WT-BslA (0.02 mg.Math.mL.sup.−1) in air (black line) and in a glyceryl trioctanoate (grey line). (b) A 50 μL droplet of WT-BslA (0.03 mg.Math.mL.sup.−1) on HOPG after 0 (left) and 30 (right) minutes (c) A 25 μL droplet of WT-BslA (0.02 mg.Math.mL.sup.−1) in air before and after compression, (d); A 40 μL droplet of WT-BslA (0.2 mg.Math.mL.sup.−1) in oil (triglyceride) before and after compression.

[0189] FIG. 2 is a plot of Regime I times versus concentration of WT-BslA (black diamonds) and BslA-L77K (circles). The dashed line represents the predicted time to reach a surface coverage of 1.57 mg.Math.m.sup.−2 for monomers with a diffusion coefficient of 9.87×10.sup.−7 cm.sup.2.Math.s.sup.−1 using Equation 1;

[0190] FIG. 3 (a) CD spectra of WT-BslA (black line) and BslA-L77K (grey line) in 25 mM phosphate buffer (pH 7). (b) CD spectra of refractive index matched emulsions stabilised by WT-BslA (black line) and BslA-L77K (grey line). The raw data is presented as semi-transparent dotted lines, whereas data smoothed using Savitzky-Golay smoothing is represented by solid lines;

[0191] FIG. 4 shows TEM images of (a) WT-BslA and (b) BslA-L77K stained with uranyl acetate. Scale bar=100 nm. Insets: FFTs of (i) The entire TEM image, (ii) the selected square area in each image. The numbers in (a)(ii) correspond to the Miller indices of the 2D lattice structure;

[0192] FIG. 5 shows (a) the entire BslA decamer from the crystal structure with chains A-H displayed in light grey and chains I and J displayed in dark grey. The hydrophobic caps are displayed as surface representations, while the rest of the chains are displayed as cartoon backbone representations. (b) A depiction of the hydrophobic core of the decamer with the hydrophobic caps of chains A-H in light grey and the hydrophobic caps of chains I and J in dark grey. The hydrophobic caps comprise residues 75-81 (CAP1), 119-126 (CAP2), and 153-155 (CAP3). (c) A depiction of chain C, showing the hydrophobic residues (black) oriented outwards as opposed to (d) chain I, in which the hydrophobic residues have no particular orientation. Images were generated using Visual Molecular Dynamics.sup.26 with PDB file 4BHU.sup.1;

[0193] FIG. 6 is a schematic of BslA adsorption. When unbound (U), the conformation of the hydrophobic cap of WT-BslA orients the hydrophobic residues away from the aqueous medium, slowing the rate of adsorption (indicated by a small arrow). The L77K mutation removes the adsorption barrier by exposing some or all of the hydrophobic residues within the hydrophobic cap, increasing the rate of adsorption (indicated by a bold arrow). Once adsorbed onto the interface, the surface-bound WT-BslA (S) refolds to a conformation rich in β-sheet and is able to form strong lateral interactions with adjacent molecules, forming an organised lattice (S*) that under normal circumstances will not be removed from the interface (indicated by the crossed arrow). Surface bound BslA-L77K (S) forms a less well-organised lattice and can be removed from the interface with only minimal energy, such as droplet compression;

[0194] FIG. 7 shows a the percentage of spherical droplets that are observed when WT-BslA is co-emulsified with other surfactants (both BslA and surfactant at 0.1 mg/mL), where a low percentage of spherical droplets is indicative of the presence of BslA at the droplet interface;

[0195] FIG. 8 is a bar chart showing the percentage of spherical droplets remaining in emulsions made with 0.1 mg/mL of an alternative surfactant and then re-emulsified with 1 mg/mL WT-BslA;

[0196] FIG. 9 is a bar chart showing the percentage of spherical droplets remaining in emulsions made with WT-BslA and mixed with an alternative surfactant;

[0197] FIG. 10 is a bar chart showing the percentage of spherical droplets remaining in emulsions made with WT-BslA and re-emulsified with an alternative surfactant;

[0198] FIG. 11 shows a series of Images of emulsions prepared by emulsifying decane in the presence of WT-BslA and re-emulsified in the presence of excess (a) CTAB, (b) SDS, (c) Pluronic F127, (d) Tween-20, (e) Sodium caseinate and (f) Whey protein isolate. Scale bars=100 μm;

[0199] FIG. 12 shows an image of a water in oil (sunflower oil) emulsion made using WT-BslA;

[0200] FIG. 13 shows multiple emulsion droplets stabilised by emulsification of sunflower oil with WT-BslA in a single step in a rotor-stator. Note the asphericity of the droplets;

[0201] FIG. 14 shows emulsions after addition of (a) CTAB or (b) SDS at high (>10 mg/mL) concentrations, the asphericity of the emulsion droplets disappears, indicating that both CTAB and SDS were able to bind to the outer oil-water interface. However, the inner water-oil droplets remained, demonstrating that multiple emulsions created by stabilisation with WT-BslA are stable against the presence of competitive surfactants;

[0202] FIG. 15 shows (a) Decane and (b) sunflower oil emulsions stabilised by BslA-L77K;

[0203] FIG. 16 shows BslA stabilised foam prepared using 0.4 mg/mL WT-BslA at 0, 1, 12 and 25 hours;

[0204] FIG. 17 shows foam stabilised by WT-BslA and sodium caseinate with a total concentration of surfactant of 0.4 mg/mL at 0, 1, 12 and 25 hours: (a) 75% WT-BslA, 25% sodium caseinate; (b) 50% WT-BslA, 50% sodium caseinate; (c) 25% WT-BslA, 75% sodium caseinate; (d) 100% sodium caseinate;

[0205] FIG. 18 shows the relative volume of foams formed with varying ratios of WT-BslA to sodium caseinate over time;

[0206] FIG. 19 shows foam stabilised by WT-BslA and Pluronic F127 with a total concentration of surfactant of 0.4 mg/mL at 0, 1, 12 and 25 hours: (a) 75% WT-BslA, 25% Pluronic F127; (b) 50% WT-BslA, 50% Pluronic F127; (c) 25% WT-BslA, 75% Pluronic F127; (d) 100% Pluronic F127;

[0207] FIG. 20 shows the relative volume of foams formed with varying ratios of WT-BslA to Pluronic F127 over time;

[0208] FIG. 21 shows foam stabilised by WT-BslA and Tween-20 with a total concentration of surfactant of 0.4 mg/mL at 0, 1, 12 and 25 hours: (a) 75% WT-BslA, 25% Tween-20; (b) 50% WT-BslA, 50% Tween-20; (c) 25% W-BslA, 75% Tween-20; (d) 100% Tween-20;

[0209] FIG. 22 shows the relative volume of foams formed with varying ratios of WT-BslA to Tween-20 over time;

[0210] FIG. 23 shows foam stabilised by WT-BslA and CTAB with a total concentration of surfactant of 0.4 mg/mL at 0, 1, 5 and 10 hours: (a) 75% WT-BslA, 25% CTAB; (b) 50% WT-BslA, 50% CTAB; (c) 25% WT-BslA, 75% CTAB; (d) 100% CTAB;

[0211] FIG. 24 shows foam stabilised by WT-BslA and SDS with a total concentration of surfactant of 0.4 mg/mL at 0, 1, 5 and 10 hours: (a) 75% WT-BslA, 25% SDS; (b) 100% SDS;

[0212] FIG. 25 shows foam stabilised by WT-BslA and BslA-L77K with a total concentration of surfactant of 0.4 mg/mL at 0, 1, 12 and 25 hours: (a) 75% WT-BslA, 25% BslA-L77K; (b) 50% WT-BslA, 50% BslA-L77K; (c) 25% WT-BslA, 75% BslA-L77K; (d) 100% BslA-L77K;

[0213] FIG. 26 shows the relative volume of foams formed with varying ratios of WT-BslA to BslA-L77K over time;

[0214] FIG. 27 shows foams stabilised by WT-BslA, A, 0.05 mg/mL; B, 0.1 mg/mL; C, 0.2 mg/mL; D, 0.3 mg/mL; E, 0.4 mg/mL; and F, 1 mg/mL. G shows a graph of relative foam volume against time for the foams from 0.2 mg/mL to 1 mg/mL;

[0215] FIG. 28 Left, ice cream mix aged with Tween 60 (0.3 wt %) for four hours at 4° C. Right, the same mix after heating to 38° C. for 10 minutes;

[0216] FIG. 29 Left, ice cream mix aged with Tween 60 (0.3 wt %) and WT-BslA (0.5 mg/mL) for four hours at 4° C. Right, the same mix after heating to 38° C. for 10 minutes;

[0217] FIG. 30 Left, ice cream mix aged with Tween 60 (0.03 wt %) for 18 hours at 4° C. Right, the same mix after heating to 38° C. for 10 minutes;

[0218] FIG. 31 Left, ice cream mix aged with WT-BslA (0.5 mg/mL) (no Tween-60) for 18 hours at 4° C. Right, the same mix after heating to 38° C. for 10 minutes;

[0219] FIG. 32 Left, ice cream mix aged with Tween 60 (0.03 wt %) and WT-BslA (0.5 mg/mL) for 18 hours at 4° C. Right, the same mix after heating to 38° C. for 10 minutes;

[0220] FIG. 33 Height of foams produced from four different ice cream mix compositions at different incubation times. Samples were incubated in a rotating wheel at 4° C.;

[0221] FIG. 34 The same air stability experiment as performed in FIG. 34, except the vessel size and thus air reservoir size was considerably smaller, increasing the longevity of the bubbles;

[0222] FIG. 35 Left, a representative cryoSEM image of ice cream without BslA, imaged on the same day as it was prepared. The image on the right is the same with highlighted regions which outline the measured ice crystals (or ice crystal cross-sectional areas). Measurements were made on five images at a magnification of 250×;

[0223] FIG. 36 A representative cryoSEM image of ice cream without BslA, imaged after 28 days of storage at −20° C. As is expected of an Ostwald ripened system, there are very few small ice crystals (<1000 μm.sup.2) compared to the same sample after 0 days (FIG. 35). Measurements were made on eight images at a magnification of 250×;

[0224] FIG. 37 A representative cryoSEM image of ice cream containing BslA at 0.05 wt %, imaged on the same day as it was prepared. Measurements were made on five images at a magnification of 250×;

[0225] FIG. 38 A representative cryoSEM image of ice cream containing BslA at 0.05 wt %, imaged after 28 days of storage at −20° C.;

[0226] FIG. 39 Size distribution histograms of measured ice crystal cross-sectional areas for ice creams with and without BslA after 0 days and 28 days stored at −20° C.;

[0227] FIG. 40 A) Arithmetic mean of ice cream samples with and without BslA after 0 and 28 days. B) Geometric mean of ice cream samples with and without BslA after 0 and 28 days;

[0228] FIG. 41 A representative cryoSEM image of ice cream containing no BslA, imaged after 24 hours of storage at −20° C.;

[0229] FIG. 42 A representative cryoSEM image of ice cream containing no BslA, imaged after 24 hours of storage at −5° C.;

[0230] FIG. 43 A representative cryoSEM image of ice cream containing BslA at 0.05 wt %, imaged after 24 hours of storage at −20° C.;

[0231] FIG. 44 A representative cryoSEM image of ice cream containing BslA at 0.05 wt %, imaged after 24 hours of storage at −5° C.;

[0232] FIG. 45 Size distributions of ice crystal cross-sectional areas in (top left) −BslA ice cream stored at −20° C., (top right) −BslA ice cream stored at −5° C., (bottom left) +BslA ice cream stored at −20° C. and (bottom right)+BslA ice cream stored at −5° C.;

[0233] FIG. 46 Top, Arithmetic mean of ice crystal cross-sectional area in ice cream samples with and without BslA stored for 24 hours at −20° C. and −5° C. Bottom, Geometric mean of ice crystal cross-sectional area in ice cream samples with and without BslA stored for 24 hours at −20° C. and −5° C.;

[0234] FIG. 47 CD spectra of 0.1 mg/ml solutions of AxA-BslA (solid black line) and WT-BslA (dashed black line);

[0235] FIG. 48 Typical data from pendant drop tensiometry experiments on unfractionated AxA-BslA (solid black line) and monomeric WT-BslA (dashed black line) droplets in air. The concentration used in each experiment was 0.03 mg/ml. (a) IFT curves. The dotted grey line is a marker to indicate 72 mN/m. (b) Laplace fit error curves corresponding to the IFT curves in (a);

[0236] FIG. 49 TEM images of the rectangular lattice structure formed by (a) WT-BslA and (b) AxA-BslA;

[0237] FIG. 50 Contact angle images of a 5 μL droplet of water on (a) a hydrophobically functionalised, (b) unfractionated WT-BslA modified, (c) unfractionated AxA-BslA modified and (d) sodium caseinate modified glass cover slips;

[0238] FIG. 51 Left, AxA-BslA single emulsion prepared with decane. Right, AxA-BslA double emulsion prepared with glyceryl trioctanoate;

[0239] FIG. 52 Co-emulsification of 0.1 mg/mL (a,b,c) CTAB and (d,e,f) SDS with 0.1 mg/mL (a,d) AxA-BslA, (b,e) dimeric WT-BslA and (c,f) monomeric WT-BslA (dimeric WT-BslA incubated in 1 mM DTT overnight). These images represent columns 2, 3, and 4 in Table 4. Scale: Each image is 400 μm in width;

[0240] FIG. 53 Emulsions prepared by emulsifying 10% decane into AxA-BslA (0.1 mg/mL) mixed with 0.1 mg/mL of (a) CTAB, (b) SDS, (c) Pluronic F127, (d) Tween-20, (e) Sodium caseinate, and (f) Whey protein isolate. Scale: Each image is 400 μm in width;

[0241] FIG. 54 Stability of AxA-BslA control foams over 24 hours;

[0242] FIG. 55 Stability of AxA-BslA/sodium caseinate composite foams over 24 hours;

[0243] FIG. 56 Stability of AxA-BslA/Pluronic F127 composite foams over 24 hours;

[0244] FIG. 57 Stability of AxA-BslA/L77K-BslA composite foams over 24 hours;

[0245] FIG. 58 Stability of AxA-BslA/Tween-20 composite foams over 24 hours;

[0246] FIG. 59 Regime I Times for BslA Orthologues;

[0247] FIG. 60 Relaxation of BslA Orthologue Elastic Films. B. amyloliquefaciens (circles), B. licheniformis (squares), B. pumilis (triangles), YweA (diamonds), WT-BslA (hexagon), and L77K BslA (stars); and

[0248] FIG. 61 Circular Dichroism of BslA orthologues. (A) Solution state circular dichroism spectra of BslA orthologues: B. amyloliquefaciens, B. licheniformis, B. pumilus, YweA, and WT-BslA. (B) Circular dichroism spectra of RIMEs: B. amyloliquefaciens, B. licheniformis, B. pumilis, and YweA. The spectra of YweA and the orthologues produced by B. licheniformis and B. pumilus are consistent with large scale β-sheet structure. However, the orthologue produced by B. amyloliquefaciens dimers from the other samples and has a double minimum at 213 and 217 nm. Comparing (A) and (B), it is clear that all the orthologues undergo a structural transition when bound to an interface. Note since the determination of amount of protein present within the RIME is undetermined, we have normalised the spectra by the HT value at 218 nm.

SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0249] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0250] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

Expression of WT-BslA and BslA-L77K

[0251] The method of expressing a truncated form of WT-BslA (SEQ ID NO:2) and the BslA mutant BslA-L77K is described in Hobley et al..sup.1 which is incorporated herein by reference. References to “WT-BslA” in the examples given below refer to the truncated form of the wild type BslA minus a signal sequence (also known as BslA.sub.42-181). References to BslA-L77K in the examples given below refer to the truncated form of the wild-type BslA minus a signal sequence and comprising a point mutation (at position 77, numbered relative to the full length BslA sequence).

[0252] The nucleotide sequences used to encode the various BslA proteins are given below.

BslA Reduces the Surface Tension of Water

[0253] Pendant drop tensiometry was performed on aqueous droplets of BslA to observe the change in interfacial tension over time. In this technique, the shape of a drop is fitted to the Young-Laplace equation to measure the interfacial tension (IFT) at the droplet surface.sup.27, 28, which usually decreases as the interface is populated by surface active species..sup.29 An increase in the error of the fit to the Young-Laplace equation indicates that a viscoelastic film has formed at the interface, and since a solid layer now separates the two liquid phases the concept of interfacial tension no longer applies. FIG. 1a shows the change in IFT of droplets of WT-BslA suspended in air and in oil. Typically, the interfacial tension of the water-air or water-oil interface drops after a lag period during which the population of protein at the interface is increasing. The magnitude of the decrease in IFT caused by BslA was consistently smaller than the typical drop in IFT observed for the class II fungal hydrophobin HFBII at similar concentrations and time scales..sup.30 For example, at 0.02 mg.Math.mL.sup.−1 and 300 s, BslA decreases the apparent IFT to 70.8±1 mN.Math.m.sup.−1, whereas HFBII decreases the IFT to ˜56 mN.Math.m.sup.−1 under the same conditions.30 However, despite this comparatively small decrease in IFT, the increase in the error of the Laplace fit indicates that a BslA film has already formed by 300 s, whereas HFBII must lower the IFT to at least 50 mN.Math.m.sup.−1 before the error of the Laplace fit increases..sup.30

[0254] WT-BslA does not deform sessile drops at 0.01, 0.03 and 0.1 mg.Math.mL.sup.−1 after thirty minutes, even though visual inspection confirmed the formation of a viscoelastic film in each case (FIG. 1b). The formation of such a film was additionally confirmed at water-air or water-oil interfaces by the appearance of persistent wrinkles on the surface of pendant drops following compression..sup.1 FIG. 1c shows a WT-BslA droplet suspended in air before and after compression, while the WT-BslA droplet depicted in FIG. 1d was suspended in triglyceride oil. Taken together our results indicate that BslA forms interfacial films at lower protein densities than the class II fungal hydrophobins, and that the resulting films, while very stable, can form without causing a significant deformation in droplet shape.

[0255] Pendant drop tensiometry with drop shape analysis was performed on BslA solutions at concentrations between 0.01 and 0.1 mg.Math.mL.sup.−1. At low protein concentrations, the IFT initially remains unchanged for a lag time that is designated “Regime I”.sup.31, 32 (FIG. 1a). During this period the interface becomes occupied by protein to a critical surface coverage above 50%,.sup.31 and provides a measure of the rate at which the protein partitions to the interface. During Regime II, the IFT decreases steeply until the interface is saturated with adsorbed protein. Following saturation, the IFT levels off (Regime III), although a shallow gradient often indicates rearrangement of the protein layer. Although these characteristics can be seen in typical BslA dynamic interfacial tension response curves, the fit error of the Young Laplace equation to the droplet increased at some point during most experiments, indicating the formation of a viscoelastic layer..sup.30

[0256] The time (t) it takes for a particle to adsorb onto an interface via diffusion can be predicted by Equation 1.sup.33:

[00001]$\begin{array}{cc}\Gamma \left(t\right)=2.Math..Math.C_b.Math.\sqrt{\frac{\mathrm{Dt}}{\pi }}& \left(1\right)\end{array}$

where Γ is surface concentration, C.sub.b is bulk concentration and D is the diffusion coefficient of the particle. Equation 1 assumes that C.sub.b is unchanging and that there is no back diffusion from the interface..sup.33 We can estimate Γ.sub.max(for 100% surface coverage) to be 1.57 mg.Math.m.sup.−2 from TEM images of the BslA 2D lattice (FIG. 4a), while D was measured to be 9.87×10.sup.−7 cm.sup.2.Math.s.sup.−1 for monomeric BslA using dynamic light scattering (DLS). In cases where the error of the Laplace fit increased before a decrease in IFT was observed, then the onset time of any increase in the error of the Laplace fit was used.

[0257] WT-BslA vs BslA-L77K

[0258] FIG. 2 shows a plot of Regime I time against BslA concentration for WT-BslA (black diamonds) and BslA-L77K (black circles) as well as the “ideal” Regime I times calculated from Equation 1 (dashed line). The results clearly demonstrate that WT-BslA takes more time to decrease the interfacial tension of a droplet (or increase the error of Laplace fit) in air than would be expected for a system that did not exhibit an adsorption barrier or back diffusion. In contrast, the BslA-L77K mutant reduced the interfacial tension of the droplet within the maximum calculated time for particles of equivalent size with no adsorption barrier. Under diffusion-limiting conditions, as determined by Equation 1, BslA at a concentration of 0.03 mg.Math.mL.sup.−1 should take 22 s to reach a surface concentration of 1.57 mg.Math.m.sup.−2. As the IFT will begin to decrease at a surface coverage below 100%, BslA should require less than 22 s to reduce the IFT of a droplet. At 0.03 mg.Math.mL.sup.−1 the Regime I time for WT-BslA was 97±18 s, compared to 12±4 s for BslA-L77K, confirming that BslA-L77K adsorption is purely diffusion-limited, whereas WT-BslA faces an additional barrier to adsorption. As the protein concentration was increased or decreased, the corresponding Regime I times followed the power law predicted by Equation 1.

BslA Undergoes Conformational Change at Interface to a Structure Enriched in Beta-Sheet (CD Data) WT+L77K

[0259] To study the conformation of BslA in aqueous solution and at an oil-water interface, circular dichroism (CD) spectroscopy of WT-BslA and the L77K mutant was performed in refractive index matched emulsions (RIMEs)..sup.34 Refractive index matching enables the generation of oil-in-water emulsions without the light scattering that interferes with spectroscopic measurements. The folding of WT-BslA and BslA-L77K was very similar at pH 7 in phosphate buffer, with both curves exhibiting a maximum at ˜205 nm, a minimum at ˜212 nm and a shoulder at ˜226 nm (FIG. 3a). The minimum at ˜212 nm is consistent with some β-sheet structure, whereas the minimum at <200 nm suggests a significant contribution from random coil. On binding to the interface of decane-water emulsions, the CD spectra of both WT-BslA and BslA-L77K are altered substantially (FIG. 3b), exhibiting a positive signal below 200 nm and a minimum at 215-218 nm. Such features indicate a structural change to a form enriched in β-sheet conformation..sup.35

BslA Forms Uniform Rectangular Lattice (TEM Data) WT Vs L77K

[0260] Transmission electron microscopy (TEM) of WT-BslA stained with uranyl acetate indicates that the protein forms a highly ordered rectangular lattice (FIG. 4a). Multiple domains of the WT-BslA lattice could be observed in any location on the grid. The observed domain areas varied from as small as 1000 nm.sup.2 (˜50 BslA molecules) up to 200000 nm.sup.2 (>10000 BslA molecules). Less ordered “inter-domain” areas were also observed. Performing a Fast Fourier Transform (FFT) on TEM images of WT-BslA (FIG. 4a, insets) revealed a rectangular lattice (α=β=90°, a≠b) with dimensions of d(10)=3.9 nm and d(01)=4.3 nm. TEM images of BslA-L77K revealed a predominantly disorganised arrangement of protein, which nonetheless contained patches of rectangular packed protein (FIG. 4b). The largest BslA-L77K domain size observed was approximately 20000 nm.sup.2 (1250 BslA molecules). FFT on ordered domains of BslA-L77K revealed that the lattice parameters (d(10)=3.9 nm, d(01)=4.3 nm, α=β=90°) were identical to the WT-BslA lattice (FIG. 4b, insets).

Crystal Structure Shows Two Distinct Forms

[0261] Although the crystal structure of WT-BslA features a large hydrophobic cap that allows the molecule to become anchored to a hydrophobic interface, kinetic measurements using the pendant drop method indicated that WT-BslA must overcome an energy barrier prior to or during adsorption (FIG. 2). The fact that WT-BslA exhibits an adsorption barrier suggests that the hydrophobic residues in the cap region are not optimally oriented outwards in solution. Moreover, CD spectroscopy indicates a secondary structure change between the stable, monomeric form of the protein in aqueous solution, and the protein self-assembled at an interface. Analysis of the X-ray crystal structure.sup.1 reveals two substantially different cap configurations in the decameric repeat unit Eight of the ten subunits are positioned with their caps in close proximity to each other in a micelle-like arrangement. In these proteins, the cap regions are in a β-sheet configuration with the hydrophobic residues oriented outwards from the protein (FIG. 5c), creating the oily core of the micelle. The remaining two subunits (chains I and J) are further away from the centre of the decamer (FIG. 5a-b) and the cap regions are in a random coil configuration with many of the hydrophobic residues oriented inwards towards the protein (FIG. 5d). This difference highlights the ability of the cap region to undergo substantial rearrangement in different solvent environments. The introduction of a positively charged amine would hinder this shielding mechanism as the lysine would orient outwards, forcing neighbouring hydrophobic residues to be exposed at the surface.

Emulsion Formation and Stability

[0262] WT-BslA stabilised and alternative surfactant (CTAB, SDS, Pluronic F127, Tween-20, sodium caseinate or whey protein isolate) stabilised emulsions were prepared by placing 900 μL of 0.1 mg/mL WT-BslA or 0.1 mg/mL surfactant and 100 μL of decane in a vial before mixing the two phases using a rotor-stator at Level 6 (˜30000 rpm) for 20 seconds. Emulsions prepared by co-emulsifying in the presence of two stabilisers (WT-BslA and each of the six surfactants, each at a concentration of 0.1 mg/mL) were mixed by vortexing 180 μL of aqueous phase and 20 μL of decane at top speed for 30 seconds. Re-emulsification of WT-BslA stabilised emulsions was performed via vortexing in the presence of an excess concentration (>2 mg/mL) of each of the six surfactants mentioned above. Re-emulsification of surfactant stabilised emulsions in WT-BslA was performed via vortexing in the presence of 1 mg/mL WT-BslA.

[0263] Images and video recordings (not included here) were captured using an Olympus optical microscope and QCapture Pro software.

WT-BslA

[0264] Creating emulsions using WT-BslA as a stabiliser results in the formation of a population of aspherical droplets within the emulsion. The emulsification method used changes the proportion of aspherical droplets and also the extent of asphericity. The two methods used were the rotor-stator method and the vortexing method. WT-BslA stablilsed emulsions prepared using a rotor-stator have fewer examples of anisotropic droplets and the extent of anisotropy is less than observed using the vortexing method.

[0265] WT-BslA stabilised emulsions were assessed by mixing and vortexing with surfactant additives. The surfactants chosen were CTAB (positively charged small molecule surfactant), SDS (negatively charged small molecule surfactant), Pluronic F-127, Tween-20 (both non-ionic, polymeric surfactants), sodium caseinate and whey protein isolate (protein and protein mixture commonly used as surfactants in food). If the anisotropic morphology of the droplets was removed (i.e. the droplets became spherical), then it was concluded that the surfactant had adsorbed onto the droplet interface and potentially displaced the WT-BslA surface layer, although the experiments performed here do not provide direct evidence of BslA displacement. Videos of WT-BslA stabilised emulsion droplets becoming exposed to high concentrations of each of the six surfactants were recorded. Addition of CTAB and SDS caused the emulsion droplets to become spherical, whereas addition of Pluronic F-127, Tween-20, sodium caseinate and whey protein isolate had no effect on droplet morphology.

[0266] When WT-BslA stabilised emulsions were re-emulsified by vortexing in the presence of an excess concentration of CTAB or SDS, all droplets became isotropic. Although most droplets became isotropic and spherical upon re-emulsiication with non-ionic surfactants, examples of anisotropic droplets were still present. Re-emulsification in the presence of the protein samples sodium caseinate and whey protein isolate did not result in the formation of a large proportion of isotropic droplets.

[0267] In addition to assessing the stability of WT-BslA stabilised emulsions, emulsions were prepared in the presence of both WT-BslA and a surfactant additive at a 1:1 mass ratio. FIG. 7 shows the percentage of droplets in the emulsion that were spherical, and anisotropic droplets could be identified in all samples. The proportion of spherical droplets observed in the CTAB and SDS samples was higher than observed in the other four samples.

[0268] Examples of non-spherical droplets is shown in FIG. 9 for emulsions prepared by co-emulsifying decane in the presence of a 1:1 mass ratio of WT-BslA and an additional surfactant.

[0269] Emulsions stabilised by each of the six surfactants were prepared and re-emulsified in the presence of 1 mg.Math.mL.sup.−1 WT-BslA (FIG. 8). In every case, examples of non-spherical droplets were observed, indicating that WT-BslA could bind to the oil-water interface despite the presence of surfactant at the interface. This does not mean that WT-BslA could actively displace the surfactant. Instead, it is most likely that co-adsorption occurred due to WT-BslA binding to freshly exposed oil-water interface during emulsification. Furthermore, the presence of non-spherical droplets means that some sort of elastic film has formed at the interface.

[0270] Creating a WT-BslA stabilised emulsion with sunflower oil using a rotor-stator creates a multiple (water-in-oil-in-water) emulsion (FIG. 10). Despite the presence of internal droplets, the outer droplets are still often anisotropic. When mixed with excess CTAB or SDS (>100:1 mass ratio), the outer droplets became spherical, indicating that as with the single emulsions, the CTAB or SDS replaced (or coadsorbed with) the WT-BslA at the interface (FIG. 11). However, the surfactants did not disrupt the internal droplets, which remained present even after the outer droplets had become spherical. This property suggests that WT-BslA could be utilised to introduce stable internal droplets even when surfactants that remove WT-BslA from the interface are present.

BslA-L77K

[0271] BslA-L77K is a point mutant of WT-BslA that exhibits different interfacial properties to the wildtype. Specifically, the BslA-L77K interfacial film is able relax after compression, unlike WT-BslA. This is likely due to reduced level of 2D lattice formation observed in BslA-L77K samples relative to WT-BslA. Despite the ability of the film to relax, emulsions stabilised by BslA-L77K have the same properties as emulsions stabilised by WT-BslA-droplets are aspherical and multiple emulsions can be formed in a single step by emulsification of sunflower oil (FIG. 12).

[0272] As an example, of the stability of emulsions formed using WT-BslA, a mixture of glyceryl trioctanoate and water stabilised by WT-BslA was observed to be stable for up to 18 months. In comparison, a mixture of glyceryl trioctanoate and water stabilised by BslA-L77K had fully separated out into the constituent phases after 18 months, thereby showing that WT-BslA is the superior emulsion stabiliser over the mutant, and is an effective emulsion stabiliser over long time scales.

Foam Formation and Stability

Preparation of Foams

[0273] To form foams, 500 μL of “foaming solution” (0.4 mg/mL total surfactant containing 0-100% WT-BslA and 0-100% co-surfactant, in water) was placed in 1×1 cm cuvette. In a separate experiment, discussed below, foams were prepared using solutions of WT-BslA at concentrations between 0.05 mg/mL and 1 mg/mL.

[0274] A modified 25 gauge needle was connected to a 60 mL syringe and placed through a small hole at the bottom of the cuvette. The syringe was placed in a syringe pump and air was pumped through the “foaming solution”. The syringe pump was set to pump at a rate of 5 mL/min. Once enough air had been passed through the foaming solution to form a foam, a cap was placed on top of the cuvette and wrapped in Parafilm. The needle was removed from the base of the cuvette and the hole was sealed with hot candle wax.

Imaging of Foams

[0275] The foams were placed in an incubator at 22° C. and imaged at a rate of 12 frames per hour for 25 hours. Foam volume was measured using ImageJ software by measuring foam height, while accounting for any cavities that developed within the foam.

[0276] Foams containing 0, 25, 50 and 75% WT-BslA and 100, 75, 50 and 25% surfactant respectively were created by injection of air into the foaming solution (500 μL) via a fine needle. The total concentration of subphase surfactants (including BslA) was 0.4 mg.Math.mL.sup.−1. The lifetime of the foams was monitored by imaging every five minutes for 10 hours or longer.

[0277] FIG. 13 shows a WT-BslA control foam at 0 hours, 1 hour, 12 hours and 25 hours. WT-BslA foams did not collapse or disproportionate significantly within the timeframe of the experiment (25 hours).

Mixed Surfactant Foam Data

[0278] Mixed WT-BslA/sodium caseinate foams are shown in FIGS. 14 and 15, the foams having, 75% WT-BslA, 25% sodium caseinate (a); 50% WT-BslA, 50% sodium caseinate (b); 25% WT-BslA, 75% sodium caseinate (c); 100% sodium caseinate (d). The foams are shown at 0 hours, 1 hour, 12 hours and 25 hours. The sodium caseinate control foam (100% sodium caseinate) had mostly collapsed after 25 hours. In contrast, all of the foams that contained WT-BslA did not collapse beyond what was observed in the WT-BslA control foam. However, increased disproportionation and/or coalescence was observed with increasing sodium caseinate concentration.

[0279] As well as enhancing the stability of protein (sodium caseinate) foams, the stability of non-ionic surfactant foams (Pluronic F127 and Tween-20) was also improved by the addition of WT-BslA (FIGS. 18 and 17 for Pluronic F127, FIGS. 18 and 19 for Tween-20). Control Pluronic F127 and Tween-20 foams had collapsed by 10 hours and 5 hours respectively. The mixed 25/75 WT-BslA/Pluronic F127 foam remained stable for significantly longer than the control Pluronic F127 foam and had not completely collapsed by the end of the experiment (25 hours). The foams containing a higher WT-BslA content (50 and 75%) collapsed earlier than the foam containing only 25% WT-BslA, despite the onset of disproportionation occurring later.

[0280] Mixed WT-BslA/Tween-20 foams with 50 or 75% WT-BslA remained stable for far longer than the control Tween-20, but the 25/75 WT-BslA/Tween-20 foam had collapsed after 4 hours, earlier than the control Tween-20 foam.

[0281] Foams prepared using the positively charged surfactant CTAB were not enhanced by the presence of WT-BslA. In the experiments shown in FIG. 20, the presence of WT-BslA destabilised CTAB foams.

[0282] As SDS crystallises with phosphate buffer, WT-BslA was dialysed into pure water for foaming experiments with CTAB and SDS. WT-BslA in pure water does not foam, due to the low ionic strength. Despite this, the 75/25 WT-BslA/SDS mixture did create a foam that remained stable for 10 hours (FIG. 21), more than 10 times longer than the control SDS foam. 50/50 and 25/75 WT-BslA/SDS mixtures did not foam.

[0283] As BslA-L77K lacks a barrier to adsorption and reduces surface tension more readily than WT-BslA, it foams more effectively. FIGS. 22 and 23 shows mixed WT-BslA/BslA-L77K foams and a control BslA-L77K foam. The 75/25 and 50/50 WT-BslA/BslA-L77K foams were not formed effectively, but the foams remained relatively stable for the duration of the experiment (25 hours). Some of the shrinkage was observed in those foams may have been due to drying. The 25/75 WT-BslA/BslA-L77K and the BslA-L77K control foams were formed well and although they had not completely collapsed by the end of the experiment, significant collapse and disproportionation had begun in both samples.

BslA as Sole Surfactant, Foam Data

[0284] As mentioned above, experiments were undertaken to analyse the effect of concentration of BslA on foaming and foam stability, where BslA was the sole surfactant. This complements the data discussed above in which BslA was assessed in combination with co-surfactants.

[0285] Foams were prepared using 500 μL of “foaming solution”, i.e. a solution of WT-BslA in water at concentrations from 0.05 mg/mL to 1 mg/mL (0.005 wt % to 0.1 wt %). The same foaming procedure and other experimental protocols as discussed above were used.

[0286] FIG. 28 shows the results of this experiment, with A (0.05 mg/mL) and B (0.1 mg/mL) showing essentially no foam formation, C (0.2 mg/mL) and D (0.3 mg/mL) demonstrating good foam formation, and E (0.4 mg/mL) and F (1 mg/mL) demonstrating excellent foam formation. The data shows that a stable foam could be formed at 0.2 mg/mL (0.02 wt %), but not at 0.1 mg/mL (0.01 wt %). At lower concentrations of BslA, e.g. up to 0.3 mg/mL, relatively large bubbles were formed within the foam, and this would suggest that higher concentrations of BslA are required to stabilise the bubbles quickly enough to keep them from coalescing to some extent during foam formation. At higher concentrations, e.g. 0.4 mg/mL and 1 mg/mL, much smaller bubbles, and hence a much finer foam, was formed, with the foam at 1 mg/ml being both very fine and highly consistent.

[0287] FIG. 28G shows a graph of relative foam volume (i.e. volume compared with time 0) against time for the foams from 0.2 mg/mL to 1 mg/mL BslA. All of the foams demonstrated significant stability over 24 hours. A foam formed with BslA at 1 mg/mL (0.1 wt %) was extremely stable.

Air Bubbles Stabilised by BslA

[0288] Air bubbles stabilised by BslA were formed by vigorously shaking, by hand, a 2 mg/ml solution of wt-BslA in 25 mM phosphate buffer for 90 seconds. The sample was then placed on a glass cover slip and imaged using an optical microscope. The morphology of the resulting air bubbles is typically non-spherical. The stability of BslA stabilised air bubbles were mixed in the presence of co-surfactants by applying an excess of six different surfactants: CTAB, SDS, Pluronic F-127, Tween-20, Sodium Caseinate, and Whey Protein Isolate. The stability of the BslA stabilised air bubbles was determined by observing whether the air bubbles transformed from non-spherical to spherical in the presence of the co-surfactant.

Frozen Multiphase Products

Ice Cream Composition and Preparation

[0289] The composition of the ice creams prepared in each experiment reported here is as follows: [0290] 14 wt % coconut oil (melted) [0291] 12 wt % skimmed milk powder (SMP: 50% lactose, 35% milk proteins) [0292] 14 wt % sucrose [0293] 60 wt % water [0294] Optional additives used: [0295] 0.03 wt % Tween 60 (standard) or 0.3 wt % Tween 60 [0296] 0.05 wt % WT-BslA

[0297] As the ice cream composition is identical in all experiments apart from the two additives, the following shorthand was used to describe the samples: [0298] −Tween-60, −BslA=no additives [0299] +Tween-60=only Tween-60 added [0300] +BslA=only BslA added [0301] +Tween-60, +BslA=both additives present

[0302] To prepare the Ice cream, SMP and sucrose were dissolved in water and melted coconut oil was pipetted on top of the solution. The mix was then sheared using a rotor-stator for 30 seconds. At this stage, the mix was split into four parts and any additives required (Tween and/or BslA) were added. Each aliquot was then re-homogenised three to four times in the rotor-stator for 20 seconds with 20 second pauses in between cycles. This homogenisation process was adjusted for the air stabilisation experiment to reduce the time between homogenising the first and last aliquots. In that experiment, the initial mix was homogenised four times for 30 seconds with a 30 second pause between cycles. The aliquots were then re-homogenised in the presence of additives for 30 seconds.

[0303] After the homogenisation step, the mixture was aged for sixteen hours (unless stated otherwise) at 4° C. in a slowly rotating wheel. After aging, the samples were placed in a Perspex insert in an aluminium bowl at −20° C. and manually stirred for 5 minutes. This simultaneously froze and aerated the mix, creating ice cream. In each experiment, the sample mass, freezing onset time and total “churn time” were monitored and recorded.

Measurement of Fat Droplet Stability

[0304] To demonstrate that BslA had successfully adsorbed onto the surface of the fat droplets in an ice cream mix, aged mixes, in which the fat droplets had partially coalesced, were first imaged using an optical microscope. The samples on the slide were then warmed to 38° C. to melt the fat. The samples were imaged again using optical microscopy to identify whether the partially coalesced droplets had retained their morphology. Retention of morphology demonstrated that BslA was present and stabilising the partially coalesced structure.

Measurement of Air Bubble Stability

[0305] To measure the stability of air bubbles in ice cream, the mixes were studied before the aging and freezing processes. As the mixing process incorporates some air into the ice cream mixture (prior to the simultaneous freezing and aeration step), it is possible to determine the longevity of those air bubbles in the mixture. Simply allowing the mix to cream and monitoring the stability of the resultant foam does not work as the aqueous phase quickly drains away, leaving a solid fat stabilised foam. Instead the mixtures were incubated at 4° C. on a rotating wheel (to prevent creaming). At various time points, the samples were removed from the rotating wheel and allowed to cream. The height of the foam was then imaged and measured to establish the air content of the sample at that moment in time. The samples were then returned to the rotating wheel to continue incubation. This process was repeated to gather data at several time points.

Measurement of Ice Crystal Stability

[0306] CryoSEM was used to study ice crystal stability against long term storage (4 weeks) at −20° C. and against temperature abuse (1 day stored at ˜−5° C.). To ensure that ice cream samples loaded onto the cryoSEM sample stage had not melted, the samples were cut out using a narrow straw to produce a cylindrical “core” of ice cream. The cylinder of ice cream was placed onto a dab of cooled Tissue-Tek glue on a cooled sample stage. The stage and adhered ice cream were then immediately plunged into nitrogen slush (−210° C.) and subsequently placed into a precooled prechamber (−170° C.) attached to the SEM instrument. Maintenance of the cylindrical shape indicated the ice cream had not melted. A scalpel built in to the prechamber was used to fracture the ice cream cylinders revealing the structural features of the ice cream interior. At this stage, the prechamber was warmed to −90° C. for 10 minutes to etch the ice crystals embedded into the protein-sugar matrix. After re-cooling to −170° C., the samples were sputter-coated in gold-palladium before the sample was inserted into the cryoSEM chamber, which was also held at −170° C.

Results

Stabilisation of Partially Coalesced Fat Droplets

[0307] To improve the stability of ice cream, milk protein stabilised emulsions are aged for four hours at 4° C. in the presence of an “emulsifier” such as Tween 60. By undergoing this process, the emulsion droplets begin to partially coalesce as a result of fat crystallisation and Tween 60 weakening the droplet interface. FIG. 28 shows a typical ice cream mix with a high concentration of Tween 60 (0.3 wt %) after incubation at 4° C. for four hours. The presence of anisotropic droplets indicated that partial coalescence had occurred. After heating this solution to 38° C., the coconut oil (MP≈24° C.) melted and the partially coalesced droplets returned to a spherical shape. Addition of WT-BslA (0.5 mg/mL) to the ice cream mix did not prevent partial coalescence of the droplets due to the action of Tween 60. However, after heating to 38° C., the anisotropic partially coalesced droplets were left intact as BslA at the interface formed a rigid film, preventing droplet relaxation (FIG. 29).

[0308] This experiment was repeated with samples that allowed the partial coalescence to proceed further. In certain cases, it was possible to image the same partially coalesced regions before and after heating. Without BslA present, the large partially coalesced structures melted into large spherical oil droplets (FIG. 30). The overall structure of the partially coalesced aggregates was retained, although the individual fat droplets appeared to coalesce after melting (FIG. 31 and FIG. 32). Interestingly, FIG. 31 demonstrates that BslA can help to instigate partial coalescence even without an emulsifier such as Tween-60 present, although partial coalescence was limited compared to Tween-60 samples. These images also show how partially coalesced droplets can surround and stabilise air bubbles in the mix.

Stabilisation of Air Phase

[0309] Four ice cream mixes containing either no additives, “Tween-60”, “BslA” and “Tween-60+BslA” were foamed using a rotor-stator. The lifetime of the air bubbles was studied as described in the Experimental. From the data shown in FIG. 33, it is clear that addition of BslA stabilises the air bubbles as the sample still produced a foam after two hours incubating whereas the addition of Tween-60 in the absence of BslA caused almost immediate destabilisation of the bubbles. BslA helped to stabilise bubbles in the presence of Tween-60 although bubbles in the absence of both BslA and Tween survived a little longer. Performing the same experiment on samples in smaller vessels (with a much smaller air reservoir) caused both BslA-stabilised and control bubbles to survive for over 20 hours (FIG. 34). The disparity in survival time is likely a consequence of the mechanism of bubble destruction—disproportionation—which is accelerated in the presence of a large air reservoir. The addition of Tween-60 introduces a different form of destabilisation called coalescence. Coalescence is not possible in ice cream as the air bubbles are static, meaning that BslA should stabilise air bubbles in ice cream, even in the presence of an emulsifier.

Stabilisation of Ice Phase

[0310] CryoSEM was utilised to monitor ice crystal coarsening in ice creams with and without BslA. The ice creams studied in this section all contained Tween-60.

[0311] Samples were prepared for cryoSEM by cutting out a cylindrical section of ice cream and placing the cylinder onto cold Tissue-Tek glue on a chilled sample stage. The stage and sample were then immediately plunged into nitrogen “slush” at −210° C., freezing the sample onto the stage. The stage was then quickly transferred into a cold (−180° C.) prechamber under vacuum. At this point, visual inspection of the ice cream shape confirmed that the sample had not melted. The cylinder was then fractured using a scalpel (built into the prechamber) and the sample was “etched” by heating to −90° C. for 10 minutes. Then, the fractured and etched sample was coated in gold and platinum in preparation for imaging. At this point, the sample was moved into the main SEM chamber and imaging could begin.

CryoSEM Imaging of Ice Cream

[0312] The fractured sample morphologies revealed three primary distinctive structures: Ice crystals, air bubbles and the sugar-protein matrix. Ice crystals were identified by the presence of a flat surface at the bottom of a basin. This pitting is caused by the etching process, which causes sublimation of the ice. Air bubbles were observed as inward or outward facing large spherulites. The matrix was the material in between the ice crystals and air bubbles. Some oversized fat droplets could be seen embedded in the matrix and on the surface of air bubbles.

[0313] Ice crystals were identified by the flat surface at the bottom of the feature. The cross-sectional area was measured using ImageJ software. In instances where it was not clear whether the feature was an air bubble cavity or an ice crystal depression, the feature was ignored. Ice crystals were also ignored if they overlapped with the edge of the image. All of the samples were imaged at 250× magnification. The ice crystal areas were analysed by plotting the data as histograms and also taking the arithmetic and geometric means.

[0314] Two separate types of experiment were performed to study whether BslA had an effect on ice crystal coarsening during storage. In the first experiment, ice cream samples were studied under cryoSEM when fresh (on the same day as freezing and aeration occurred) and also after 28 days of being stored in a freezer at −20° C. In the second experiment, fresh ice cream samples were stored overnight at either −20° C. or at approximately −5° C. By “temperature-abusing” the sample, the rate of ice crystal coarsening is increased.

Effect of Storage at −20° C.

[0315] Analysis of cryoSEM images of fractured ice creams (FIG. 35-FIG. 38) revealed that the size distribution of ice crystals, which are measured as ice crystal cross-sectional areas, increases with storage time as Ostwald ripening of the ice occurs through the viscous, liquid sugar-rich matrix. After 28 days, the size distribution of ice crystals increased in both “−BslA” and “+BslA” samples compared to the same samples imaged at 0 days (FIG. 39). However, the coarsening was limited by the presence of BslA, as indicated by a comparison of both the arithmetic (Table 1 and FIG. 40, Right) and geometric means (Table 1 and FIG. 40, Left). The geometric mean limits the effect of large outliers in the data, so the relative values are not as affected by limited statistical analysis.

Data Summary

[0316] The average size and standard deviations of the data sets were:

TABLE-US-00001 TABLE 1 Summary of average crystal sizes in ice creams with and without BsIA stored at −20° C. for 0 and 28 days. Minus BsIA Plus BsIA Arithmetic Geometric Arithmetic Geometric Age mean mean Age mean mean (days) (μm.sup.2) (μm.sup.2) (days) (μm.sup.2) (μm.sup.2) 0 1273.62 1010.61 0 1299.14 897.17 28 2380.53 1683.57 28 1838.58 1359.97

“Temperature Abused” Ice Creams

[0317] In this experiment, two ice cream samples with Tween-60 were prepared with and without BslA. The ice cream samples were split into two parts, with one part being stored at −20° C. overnight and the second part being stored at approximately −5° C. overnight. Analysis of cryoSEM images (FIG. 41-FIG. 44) revealed the size distribution of the ice crystals in both temperature abused increased markedly in comparison to the control samples stored at −20° C. (FIG. 45). Significantly, the ice crystals in the BslA-containing ice cream coarsened less than in the control sample both when comparing the arithmetic mean and the geometric mean (Table 2 and FIG. 46).

Data Summary

[0318]

TABLE-US-00002 TABLE 2 Summary of average crystal sizes in ice creams with and without BsIA stored for 24 hours at −20° C. and −5° C. Minus BsIA Plus BsIA Temperature Arithmetic Geometric Arithmetic Geometric (° C.) mean (μm.sup.2) mean (μm.sup.2) mean (μm.sup.2) mean (μm.sup.2) −20 1156.10 836.45 1206.90 877.38 −5 2663.39 1887.02 2268.78 1580.39

“AxA” Mutant BslA

[0319] Although BslA has a hydrophobic cap that is resistant to self-assembly in aqueous media, the C-terminal region contains two cysteine (C) residues at residue positions 178 and 180 that are capable of forming intermolecular disulfide bonds, thus allowing dimers, tetramers, hexamers and potentially higher order oligomers to form. Although dimers can still stabilise an air-water or oil-water interface, tensiometry experiments demonstrated that they bind via only one cap, leaving the second cap pendant in the aqueous phase. Thus, the presence of dimers will alter the surface chemistry of BslA-stabilised emulsions and foams and also reduce the effective concentration of adsorbable BslA in solution. By adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol), it is possible to reduce WT-BslA dimers into its constituent monomers, but such reducing agents won't be usable in every application. To avoid the use of reducing agents while maintaining a functional, monomeric BslA solution, a mutant was developed that replaced the cysteine residues with alanine (A) residues. The mutations were carried out using primers such as SEQ ID NO:12-17. The resultant double mutant is given the shorthand name “AxA”, as WT-BslA would be “CxC”. The “x” represents any amino acid, although it is an alanine (A) in the experiments performed in this work. The results in this section demonstrate that a solution of unfractionated AxA-BslA functions in exactly the same way as a solution of monomeric WT-BslA, except the ability to cross-link into dimers has been removed.

AxA-BslA Forms a Stable. Monomeric Solution in Aqueous Media

[0320] When WT-BslA solutions are passed through a size-exclusion column, two peaks can clearly be resolved that multiangle laser light scattering confirms are attributed to a mixed population of monomers and dimers. Applying the same separation technique to the AxA mutant reveals only one peak that corresponds to a pure population of monomers. Performing SDS-PAGE chromatography on WT-BslA and AxA-BslA yields the same result—AxA-BslA exists as a pure population of monomers.

The Conformation of AxA-BslA is the Same as WT-BslA in Solution and at an Interface

[0321] Circular dichroism spectroscopy (CD) confirmed that AxA-BslA is conformationally identical to WT-BslA in solution (25 mM phosphate buffer, pH 7), exhibiting the same maximum at ˜205 nm, a minimum at ˜212 nm and a shoulder at ˜226 nm (FIG. 47). Upon binding to an oil-water interface, both WT-BslA and AxA-BslA undergo a folding change to a conformation richer in β-sheet. This was confirmed by measuring the CD spectra of WT-BslA and AxA-BslA when adsorbed to an oil-water interface in a refractive index matched emulsion. In each case, a positive at or below 200 nm was observed as well as a minimum between 215-218 nm.

The Kinetics to AxA-BslA Binding to an Air-Water Interface is Identical to WT-BslA Monomers

[0322] Pendant drop tensiometry was used to study how long it takes for WT-BslA and AxA-BslA to bind to an air-water interface. In this case, the lag time (“Regime I time”) before the interfacial tension (IFT) begins to drop was monitored and compared between monomeric WT-BslA and AxA-BslA samples at 0.03 mg/mL. The average Regime I time for monomeric WT-BslA was 97 s, whereas the average Regime I time for AxA-BslA was 102 s. FIG. 48a shows typical IFT curves for monomeric WT-BslA and AxA-BslA. In addition, the lag times before an increase in Laplace fit error in each sample were very similar (FIG. 48b) indicating that the viscoelastic films formed at the air-water interface at the same time (˜100 s).

Both WT-BslA and AxA-BslA Form a Rectangular Lattice Upon Binding to an Interface

[0323] Transmission electron microscopy of monomeric WT-BslA and AxA-BslA adsorbed onto a carbon film revealed that there is no difference in the two-dimensional arrangement of BslA molecules on the substrate (FIG. 49). Further, the lattice spacing of WT-BslA and AxA-BslA films was very similar, with d(10)=3.9 nm and d(01)=4.1-4.3 nm.

Wrinkles Formed by Both WT-BslA and AxA-BslA Film Compression do not Relax

[0324] Once a film has formed around a pendant drop of BslA solution submerged in oil, withdrawing a small amount (5 μL) of the droplet (total initial volume=40 μL) causes the film to compress and wrinkles to form. WT-BslA is known to form wrinkles that are incapable of relaxing as the WT-BslA molecules cannot be removed from the interface by such compression. Wrinkles formed in AxA-BslA films were also found to be incapable of relaxing as the wrinkles did not dissipate after formation due to compression.

Unfractionated AxA-BslA can Modify the Surface Hydrophilicity of a Hydrophobic Surface More Efficiently than Unfractionated WT-BslA

[0325] Coating a surface with BslA can reverse the surface's hydrophobicity. To demonstrate this, Circular glass cover slips (diameter=10 mm) were first cleaned in 2% Hellmanex for 3 hours, before rinsing in Milli-Q water. They were then further cleaned in 1M HCl (in 50% ethanol) for 3 hours and then thoroughly rinsed in Milli-Q water again. The clean cover slips were then incubated in octadecyltrimethoxysilane for 24 hours before being cleaned in acetone, then ethanol and finally Milli-Q water. The hydrophobic cover slips were then dried at 50° C. for 1 hour.

[0326] The hydrophobic cover slips were coated in three different protein samples (unfractionated WT-BslA, unfractionated AxA-BslA and sodium caseinate) using the Langmuir-Blodgett technique. Briefly, hydrophobic cover slips were submerged in 0.05 mg/mL adsorbent solutions for five minutes and withdrawn vertically through air-water interface at a withdrawal rate of 5 mm/min. Excess solution was wicked from the edge of the cover slips, which were then left to dry on filter paper. Imaging and contact angle measurements were performed using a Krüss EasyDrop tensiometer.

[0327] Contact angle experiments revealed the contact angle of a 5 μL droplet of Milli-Q water against the cover slip surface. FIG. 21 shows images of the droplets of water against each glass cover slip. Table 3 summarises the measured contact angles after two minutes equilibration.

TABLE-US-00003 TABLE 3 Contact angles measured on each different surface. Cover slip type Contact angle/° Hydrophobic control 96.6 Unfractionated WT-BsIA 48.6 Unfractionated AxA-BsIA 33.8 Sodium caseinate 86.8

[0328] The contact angle against the hydrophobic control cover slip was 96.6°. Functionalising with unfractionated WT-BslA, a mixture of monomers and dimers, reduced the contact angle to 48.6°. A further reduction in contact angle to 33.8° was achieved by using an unfractionated solution of AxA-BslA, which cannot form disulfide bridged dimers due to a lack of cysteine residues. FIG. 50 shows the images of the water drops against each cover slip. Sodium caseinate only reduced the contact angle to 86.8°, demonstrating the reversal of hydrophobicity achieved by BslA is not a general effect of binding proteins to hydrophobic surfaces.

AxA-BslA Emulsions

[0329] Just like observed with WT-BslA previously, AxA-BslA will make a single emulsion when prepared with decane and a double emulsion when prepared with a triglyceride oil like glyceryl trioctanoate (FIG. 51). These emulsions were prepared using an AxA-BslA concentration of 0.2 mg/mL at an oil volume fraction of 0.2 using a rotor-stator.

Resistance of Emulsions to Surfactants

[0330] The behaviour of AxA-BslA stabilised emulsions against the effect of surfactants is very similar to WT-BslA. The primary difference was the observation that AxA-BslA (purely monomeric BslA) is resistant to displacement by sodium dodecyl sulfate (SDS), whereas it was previously observed that WT-BslA was not. This is likely due to the presence of dimers in the latter sample, as resistance to SDS could be recovered by the addition of dithiothreitol (DTT) to the primarily dimeric WT-BslA solution, reducing the dimers to monomers.

[0331] Another difference between AxA-BslA and dimeric WT-BslA was the observation that emulsions stabilised with dimeric WT-BslA were resistant to displacement by cetyl trimethylammonium bromide (CTAB) during co-emulsification, whereas the purely monomeric AxA-BslA was not. Previously, it was noted that WT-BslA was not resistant to CTAB, presumably as it contained a mixture of monomers and dimers. The overall conclusion is that BslA monomers (either WT or AxA) can maintain a presence at the interface in the presence of SDS, but not CTAB, whereas WT-BslA dimers can remain at the interface in the presence of CTAB, but not SDS. The observations regarding resistance to SDS and CTAB are summarised in Table 4 and FIG. 52.

TABLE-US-00004 TABLE 4 Summary of emulsion drop shapes after co-emulsification between BsIA and CTAB or SDS (all at 0.1 mg/mL). WT-BsIA WT-BsIA WT-BsIA dimers + (previous) AxA-BsIA dimers DTT CTAB S S NS S SDS S NS S NS The “S” denotes that all droplets were spherical, indicating that BsIA had no influence on drop morphology. “NS” denotes that non-spherical droplets were present, confirming that BsIA was at the interface and causing the trapped anisotropic droplet shapes.

Co-Emulsification of AxA-BslA and Surfactants

[0332] Co-emulsified emulsions were prepared by vortexing AxA-BslA (90 uL, 0.2 mg/mL), surfactant (90 uL, 0.2 mg/mL) and decane (20 uL) in a PCR tube for 1 minute. Non-spherical emulsion droplets were observed in all co-emulsified samples except for the AxA-BslA/CTAB emulsion (FIG. 52).

Addition of Excess Surfactant to BslA Emulsions

[0333] The stability of preformed AxA-BslA emulsions to a high concentration of surfactant (5 mg/mL) was monitored by gently mixing the surfactant solution (10 mg/mL) at a 1:1 volume ratio with a vortexed AxA-BslA emulsion prepared using 0.1 mg/mL AxA-BslA. The results were similar to WT-BslA, except SDS (as with co-emulsification) was unable to displace AxA-BslA from the oil-water interface with gentle mixing (FIG. 53).

Stability of AxA-BslA Foams and AxA-BslA-Surfactant Composite Foams

[0334] AxA-BslA foams behaved similarly to the WT-BslA foams studied previously. The composite foams prepared with BslAL77K, sodium caseinate, Pluronic F127 and Tween-20 also demonstrated similar stability. The concentration of AxA-BslA and the surfactants in the composite foams was 0.4 mg/ml. They were mixed at different ratios to provide the different compositions. Foams were created by pushing air from a syringe through a fine hole (<100 μm diameter) into 1 mL of BslA and/or surfactant solution. The time course graphs for each foam are shown in FIGS. 54-58.

Hydrophobic Sand

[0335] Hydrophobic sand was produced in house by functionalising sand with dichloro-dimethyl silane. The hydrophobised sand was then incubated in a 0.2 mg/ml WT-BslA solution overnight. The following day, the sand was placed in a drying oven at 50° C. and allowed to dry for 2 hours. The sand was placed in a thin layer on a cavity slide. A 20 μL sessile drop of MilliQ water was placed on the layer of sand and imaged using the Krüss Easy Drop.

[0336] The drop of water was observed to sit on top of the layer of hydrophobic sand, but was adsorbed into the layer of hydrophobic sand that had been treated with WT-BslA. Accordingly, this result showed that the treatment of the hydrophobic sand with BslA increased the hydrophilicity of the hydrophobic sand such that the water was able to wet the sand and thereby be absorbed by it.

BslA Orthologues

[0337] We performed pendant drop tensiometry on BslA orthologues produced by three different organisms: B. amyloliquefaciens, B. licheniformis, and B. pumilis along with the protein YweA (B. subtilis). Samples were prepared by diluting each protein in phosphate buffer to a concentration of 0.03 mg ml.sup.−1. Droplets were expelled in air and the interfacial tension was measured using standard techniques. As was the case with BslA produced by B. subtilis, once an elastic film forms around the droplet the measured interfacial tension becomes a meaningless quantity. A good indication of when the film forms is by monitoring the fit error. Regime I times were then extracted one of two ways: (1) the transition time between regimes I and II when the fit error was still low (<0.4 μm); or (2) when the fit error increased to a threshold value (>0.75 μm). Each reported Regime I time is the average of 4 experiments. The results can be found in FIG. 59. We find that the Regime I times of B. amyloliquefaciens BslA and B. pumilus BslA are within error of B. subtilis BslA. However, the Regime I time of B. licheniformis BslA is nearly twice as long as the other samples. The Regime I time for YweA was faster by 25%.

[0338] We also measured the relaxation of the elastic films formed by the orthologues. Samples were diluted to a concentration of 0.2 mg ml.sup.−1 in phosphate buffer. A droplet (40 μL) was then expelled into glyceryl trioctanoate and allowed to equilibrate for 30 minutes. After equilibration, the droplet was compressed by retraction of 5 μL. A video (2 fps for 10 minutes) was recorded of the wrinkles formed in the elastic film. Film relaxation was measured by loss of wrinkles as measured by the reduction in normalised grey scale values. The results are shown in FIG. 60. We find that B. amyloliquefaciens and B. licheniformis BslA exhibit very similar behaviour, showing very slow relaxation over the time window of the experiment. In contrast, YweA relaxes extremely rapidly in less than 5 seconds; B. pumilus BslA relaxes within a minute. For comparison, YweA relaxes more quickly than L77K BslA (FIG. 60).

[0339] Circular dichroism (CD) spectroscopy was used to study the conformation of the BslA orthologues in aqueous solution and at an oil-water interface. Solution state CD measurement were performed on samples diluted to a concentration of 0.03 mg ml.sup.−1 and measured in a 1 cm path length quartz cuvette. Results are shown in FIG. 61A. Qualitatively, the spectra are reminiscent of the solution state CD spectrum for WT-BslA. One distinction can be found for the orthologue produced by B. pumilus where there is no apparent minimum between 210-218 nm, as can be found for the other proteins.

[0340] In order to investigate the conformation of the proteins at an interface we performed Circular Dichroism (CD) on oil in water emulsions made from the orthologues. Typically, emulsions would be opaque and strongly scatter light in the far UV. To solve this problem we use refractive indexed matched emulsions (RIMEs) to obtain the spectra. We make a standard water in oil emulsion using a protein solution of 0.5 mg ml.sup.−1 mixed with a 20% decane (by volume). The emulsions are prepared by rotor stator for 5 minutes. The emulsions are allowed to cream and we introduce a washing step in order to remove any protein still present in solution by removing the supernatant and replacing it with fresh buffer. The sample is then emulsified and allowed to cream again. We remove supernatant and replace it with glycerol to a final amount of 59% by mass. Finally, we emulsify this glycerol solution. The addition of the glycerol index matches the emulsion droplets allowing for light to pass through the sample. We measure CD spectra using a 0.01 cm path length quartz cuvette. The results are shown in FIG. 61B. Comparing FIG. 60A to FIG. 61B, it is clear that the orthologues undergo a structural transition when adsorbed to the interface. The spectra of YweA and the orthologues produced by B. licheniformis and B. pumilus are consistent with large scale n-sheet structure and is very similar to what we observe for WT-BslA. However, the orthologue produced by B. amyloliquefaciens differs from the other samples and has a double minimum at 213 and 217 nm.

TABLE-US-00005 TABLE 5 B. B. B. BsIA licheniformis amyloliquifaciens pumilus YweA Regime I WT Slow WT WT Fast Film WT WT WT Fast Very Relaxation Fast Solution WT WT Weak min. No min. WT CD RIMES β-sheet β-sheet α-helix? β-sheet β-sheet CD TEM crystal crystal crystal domains crystal crystal domains

Sequences Relevant to Present Invention

[0341]

TABLE-US-00006 Full length WT-BslA (SEQ ID NO: 1) MKRKLLSSLA ISALSLGLLV SAPTASFAAE STSTKAHTES TMRTQSTASL FATITGASKT EWSFSDIELT YRPNTLLSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPCGC N WT-BslA truncated, BslA.sub.42-181 (SEQ ID NO: 2) MRTQSTASL FATITGASKT EWSFSDIELT YRPNTLLSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPCGC N Full length BslA-L77K (SEQ ID NO: 3) MKRKLLSSLA ISALSLGLLV SAPTASFAAE STSTKAHTES TMRTQSTASL FATITGASKT EWSFSDIELT YRPNTLKSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPCGC N BslA-L77K truncated (SEQ ID NO: 4) MRTQSTASL FATITGASKT EWSFSDIELT YRPNTLKSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPCGC N DNA sequence used by Bacillus subtilis to encode full length wild type BslA protein. (SEQ ID NO: 5) ATGAAACGCAAATTATTATCTTCTTTGGCAATTAGTGCATTAAGTCTC GGGTTACTCGTTTCTGCACCTACAGCTTCTTTCGCGGCTGAATCTACA TCAACTAAAGCTCATACTGAATCCACTATGAGAACACAGTCTACAGCT TCATTGTTCGCAACAATCACTGGCGCCAGCAAAACGGAATGGTCTTTC TCAGATATCGAATTGACTTACCGTCCAAACACGCTTCTCAGCCTTGGC GTTATGGAGTTTACATTGCCAAGCGGATTTACTGCAAACACGAAAGAC ACATTGAACGGAAATGCCTTGCGTACAACACAGATCCTCAATAACGGG AAAACAGTAAGAGTTCCTTTGGCACTTGATTTGTTAGGAGCTGGCGAA TTCAAATTAAAACTGAATAACAAAACACTTCCTGCCGCTGGTACATAT ACTTTCCGTGCGGAGAATAAATCATTAAGCATCGGAAATAAATTTTAC GCAGAAGCCAGCATTGACGTGGCTAAGCGCAGCACTCCTCCGACTCAG CCTTGCGGTTGCAACTAA

GST-TEV-BslA Construct Sequences

[0342] These are the sequences of constructs used to express and then purify BslA (BslA.sub.42-181, truncated form) and the L77K variant from E. coli.

TABLE-US-00007 (SEQ ID NO: 6): Nucleotide sequence  ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATA TCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGT TTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCT ATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGA GATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAG ACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGT TTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCT TGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAAC GTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAG GGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGAAGTTCTGTTCCAGGG [00001] TCACTGGCGCCAGCAAAACGGAATGGTCTTTCTCAGATATCGAATTGACTTACCGTCCAAACACGCTT CTCAGCCTTGGCGTTATGGAGTTTACATTGCCAAGCGGATTTACTGCAAACACGAAAGACACATTGAA CGGAAATGCCTTGCGTACAACACAGATCCTCAATAACGGGAAAACAGTAAGAGTTCCTTTGGCACTTG ATTTGTTAGGAGCTGGCGAATTCAAATTAAAACTGAATAACAAAACACTTCCTGCCGCTGGTACATAT ACTTTCCGTGCGGAGAATAAATCATTAAGCATCGGAAATAAATTTTACGCAGAAGCCAGCATTGACGT GGCTAAGCGCAGCACTCCTCCGACTCAGCCTTGCGGTTGCAACTAATAA (SEQ ID NO: 7): Protein sequence - BslA (42-181 truncated form) linked to GST  MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ [00002] LSLGVMEFTLPSGFTANTKDTLNGNALRTTQILNNGKTVRVPLALDLLGAGEFKLKLNNKTLPAAGTY TFRAENKSLSIGNKFYAEASIDVAKRSTPPTQPCGCN (SEQ ID NO: 8). GST-TEV-BslA (L77K) construct sequences  ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATA TCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGT TTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCT ATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGA GATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAG ACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGT TTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCT TGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAAC GTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAG GGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGAAGTTCTGTTCCAGGG [00003] TCACTGGCGCCAGCAAAACGGAATGGTCTTTCTCAGATATCGAATTGACTTACCGTCCAAACACGCTT AAAAGCCTTGGCGTTATGGAGTTTACATTGCCAAGCGGATTTACTGCAAACACGAAAGACACATTGAA CGGAAATGCCTTGCGTACAACACAGATCCTCAATAACGGGAAAACAGTAAGAGTTCCTTTGGCACTTG ATTTGTTAGGAGCTGGCGAATTCAAATTAAAACTGAATAACAAAACACTTCCTGCCGCTGGTACATAT ACTTTCCGTGCGGAGAATAAATCATTAAGCATCGGAAATAAATTTTACGCAGAAGCCAGCATTGACGT GGCTAAGCGCAGCACTCCTCCGACTCAGCCTTGCGGTTGCAACTAATAA [00004]

[0343] The nucleotides encoding the L to K substitution are in underlined

TABLE-US-00008 (SEQ ID NO: 9): Protein sequence -BslA-L77K (42-181 truncated form) linked to GST  MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ [00005] KSLGVMEFTLPSGFTANTKDTLNGNALRTTQILNNGKTVRVPLALDLLGAGEFKLKLNNKTLPAAGTY TFRAENKSLSIGNKFYAEASIDVAKRSTPPTQPCGCN

[0344] The L to K substitution is in underlined.

Primers

[0345]

TABLE-US-00009 L77K (SEQ ID NO: 10) CCGTCCAAACACGCTTAAAAGCCTTGGCGTTATGG L77K (SEQ ID NO: 11) CCATAACGCCAAGGCTTTTAAGCGTGTTTGGACGG C178A NSW1906 (SEQ ID NO: 12) TCCTCCGACTCAGCCTgcaGGTTGCAACTAATAAC

[0346] The region for mutation of the DNA is in lower case.

TABLE-US-00010 C178A NSW1907 (SEQ ID NO: 13) GTTATTAGTTGCAACCtgcAGGCTGAGTCGGAGGA

[0347] The region for mutation of the DNA is in lower case.

TABLE-US-00011 C180A NSW1908 (SEQ ID NO: 14) GACTCAGCCTTGCGGTgcaAACTAATAACTCGAGC

[0348] The region for mutation of the DNA is in lower case.

TABLE-US-00012 C180A NSW1909 (SEQ ID NO: 15) GCTCGAGTTATTAGTTtgcACCGCAAGGCTGAGTC

[0349] The region for mutation of the DNA is in lower case.

TABLE-US-00013 C178A NSW1910 (SEQ ID NO: 16) TCCGACTCAG CCTgcaGGTg caAACTAAT AACTCG

[0350] The region for mutation of the DNA is in lower case.

TABLE-US-00014 C178A NSW1911 (SEQ ID NO: 17) CGAGTTATTAGTTtgcACCtgcAGGCTGAGTCGGA

[0351] The region for mutation of the DNA is in lower case.

TABLE-US-00015 Full length BslA mutant (SEQ ID NO: 18) MKRKLLSSLA ISALSLGLLV SAPTASFAAE STSTKAHTES TMRTQSTASL FATITGASKT EWSFSDIELT YRPNTLLSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPXGX N

[0352] X is a non-sulfur containing residue.

TABLE-US-00016 BslA mutant truncated (SEQ ID NO: 19) MRTQSTASL FATITGASKT EWSFSDIELT YRPNTLLSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPXGX N

[0353] X is a non-sulfur containing residue.

TABLE-US-00017 Full length AxA-BslA mutant (SEQ ID NO: 20) MKRKLLSSLA ISALSLGLLV SAPTASFAAE STSTKAHTES TMRTQSTASL FATITGASKT EWSFSDIELT YRPNTLLSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPAGA N

[0354] C to A substitution is underlined.

TABLE-US-00018 AxA-BslA truncated (SEQ ID NO: 21) MRTQSTASL FATITGASKT EWSFSDIELT YRPNTLLSLG VMEFTLPSGF TANTKDTLNG NALRTTQILN NGKTVRVPLA LDLLGAGEFK LKLNNKTLPA AGTYTFRAEN KSLSIGNKFY AEASIDVAKR STPPTQPAGA N

[0355] C to A substitution is underlined.

TABLE-US-00019 Truncated (amino acids 40-179) B. licheniformis BslA (SEQ ID NO: 22) YRPAASASLY SVITGASKQE WSFSDIELTY RPNSILALGT VEFTLPSGFS ATTKDTVNGR ALTTGQILNN GKTVRLPLTI DLLGIAEFKL VLANKTLPAA GKYTFRAENR VLGLGSTFYA ESSIEVQKRA TPPTQPCNCK Truncated (amino acids 42-181) B. amyloliquefaciens BslA (SEQ ID NO: 23) MSTKATATLF AKYTGASQQE WSFSDIELTY RPNTILSLGV MEFTLPSGFA ATTKDTVNGH ALRERQILNN GKTVRLPLNI DLLGAAEFKL SLNNKTLPAA GTYKFRAENK SLSIGSKFYA EDTIVVQKRS TPPTQPCNCK Truncated (amino acids 37-177) B. pumilus BslA (SEQ ID NO: 24) STNARPAELY AKITGTSKQE WSFSDIELTY RPNSVLSLGA IEFTLPAGFQ ATTKDIFNGK ALKDSYILNS GKTVRIPARL DLLGISQFKL QLSHKVLPAA GTYTFRAENR ALSIGSKFYA EDTLDIQTRP VVVTPPDPCG C Full length B. licheniformis BslA (SEQ ID NO: 25) MKMKHKFFST VMASLFGLVL LLSLPTASFA AESSSTVHEP EMSTKATATL FAKYTGASQQ EWSFSDIELT YRPNTILSLG VMEFTLPSGF TATTKDTVNG HALRERQILN NGKTVRLPLN IDLIGAAEFK LSLNNKTLPA AGTYKFRAEN KSLSIGSKFY AEDTIVVQKR STPPTQPCNC K Full length B. amyloliquefaciens BslA (SEQ ID NO: 26) MLKRMYRSKL SILAVSLVMM VSIFLPSFQA SAQTTKTESV YRPAANASLY ATITGASKQE WSFSDIELTY RPNSILALGT VEFTLPSGFS ATTKDTVNGR ALTTGQILNN GKTVRLPLTI DLLGIAEFKL VLANKTLPAA GKYTFRAENR VLGLGSTFYA ESSIEVQKRA TPPTQPCNCK Full length B. pumilus BslA (SEQ ID NO: 27) MKKTWTMIMM GMLTLVMALS VPIAASAEGA TQEGKASTNA RPAELYAKIT GTSKQEWSFS DIELTYRPNS VLSLGAIEFT LPAGFQATTK DIFNGKALKD SYILNSGKTV RIPARLDLLG ISQFKLQLSH KVLPAAGTYT FRAENRAISI GSKFYAEDTL DIQTRPVVVT PPDPCGC Full length B. subtilis YweA (SEQ ID NO: 28) MLKRTSFVSS LFISSAVLLS ILLPSGQAHA QSASIEAKTV NSTKEWTISD IEVTYKPNAV LSLGAVEFQF PDGFHATTRD SVNGRTLKET QILNDGKTVR LPLTLDLLGA SEFDLVMVRK TLPRAGTYTI KGDVVNGLGI GSFYAETQLV IDPR Truncated B. subtilis YweA (SEQ ID NO: 29) QSASIEAKTV NSTKEWTISD IEVTYKPNAV LSLGAVEFQF PDGFHATTRD SVNGRTLKET QILNDGKTVR LPLTLDLLGA SEFDLVMVRK TLPRAGTYTI KGDVVNGLGI GSFYAETQLV IDPR

REFERENCES

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