Thixotropic α-lactalbumin hydrogels, method for preparing same and uses thereof

Abstract

The present invention relates to shear-thinning α-lactalbumin hydrogels, which have a threshold and are thixotropic, to a method for preparing same and to the use thereof.

Claims

1. process of preparing an α-lactalbumin hydrogel from an aqueous suspension of α-lactalbumin at a concentration C.sub.a-La of between 5 and 60 mg/ml, comprising the following steps: (a) suspending the α-lactalbumin in an acidic aqueous solution having an ionic strength of an added salt of less than or equal to 60 mM; said suspending consisting in: (a1) preparing an acidic aqueous solution having a concentration of protons expressed in mM determined by the sum: (numerical value of C.sub.a-La expressed in g/l) +10; (a2) suspending the α-lactalbumin in said acidic aqueous solution; and (a3) adjusting the pH to a value of between 1.5 and 2.5; (b) forming the gel from said α-lactalbumin suspension obtained at the end of step a); said forming of the gel is carried out under the following conditions; at a temperature below 60° C.; with stirring having a strength defined by a Reynolds number of between 37 and 1000; for 10 hours to 1 week, and in the absence of evaporation of water from said α-lactalbumin suspension, wherein said α lactalbumin hydrogel is an α lactalbumin shear-thinning hydrogel which has a yield point and is thixotropic, having an α lactalbumin content of between 5 and 60 mg/ml, a pH of between 1.5 and 2.5 and an ionic strength of an added salt of less than or equal to 60 mM.

2. The process according to claim 1, wherein the Reynolds number is between 300 and 500.

3. The process according to claim 1, wherein the temperature is between 35 and 55° C.

4. The process according to claim 1, wherein the pH is between 1.8 and 2.2.

5. The process according to claim 1, wherein the α-lactalbumin suspension obtained at the end of step (a) is filtered.

6. An α-lactalbumin shear-thinning hydrogel obtained according to the process of claim 1.

7. An α-lactalbumin shear-thinning hydrogel which has a yield point and is thixotropic, having an α-lactalbumin content of between 5 and 60 mg/ml, a pH of between 1.5 and 2.5and an ionic strength of less than or equal to 60 mM.

8. A food product comprising at least one α-lactalbumin shear-thinning hydrogel of claim 6.

9. A dressing comprising at least one α-lactalbumin shear-thinning hydrogel of claim 6, and optionally at least one active compound, such as a healing agent or an antimicrobial agent.

10. A cosmetic composition comprising at least one α-lactalbumin shear-thinning hydrogel of claim 6.

11. A paint comprising at least one α-lactalbumin shear-thinning hydrogel of claim 6.

12. A method of treating a wound in a subject in need thereof comprising administering to the wound the dressing according to claim 9.

13. An α-lactalbumin shear-thinning hydrogel which has a yield point and is thixotropic, having an α-lactalbumin content of between 5 and 60 mg/ml and an ionic strength of an added salt of less than or equal to 60 mM.

14. A method of enhancing texture of a food product comprising adding the αlactalbumin shear-thinning hydrogel produced by the process of claim 1 to the food product.

15. A method of preparing a cosmetic comprising adding the α-lactalbumin shear-thinning hydrogel produced by the process of claim 1 to the cosmetic.

16. A method of preparing paint comprising adding the α-lactalbumin shear-thinning hydrogel produced by the process of claim 1 to the paint.

Description

FIGURES

(1) FIG. 1 illustrates the behavior of the thixotropic fluids of which the viscosity decreases over time when a constant shear rate is applied thereto, and which, reversibly, increases again if the shear is interrupted.

(2) FIG. 2 represents the monitoring of the destructuring and of the restructuring at 15° C. over time of a hydrogel of a-La at 20 mg/ml and 30 mM of NaCl in small strains in harmonic shear. Various procedures for monitoring the change in the elastic modulus G′ (solid circle) and viscous modulus G″ (open circle) as a function of time and of the applied strain (crosses), were applied; the stresses applied at each step are detailed in example 2 which follows.

(3) FIG. 3 represents the monitoring of the restructuring at 15° C. over time of a hydrogel of a-La at 20 mg/ml and 30 mM of NaCl after large strain destructuring at a shear rate of 10 s.sup.−1; the elastic modulus G′ is represented as solid circles and the viscous modulus G″ as open circles.

(4) FIG. 4 illustrates the end of the monitoring of the restructuring at 15° C. over time of a hydrogel of α-La at 20 mg/ml and 30 mM of NaCl after large strain destructuring at a shear rate of 10 s.sup.−1 (step 6), then step 7 of harmonic shear destructuring at increasing strain amplitude, and finally step 8 of monitoring the small strain recovery in consistency.

(5) FIG. 5 is a comparison of the kinetics of restructuring at 15° C. over time of a hydrogel of a-La at 20 mg/ml and 30 mM of NaCl (step 6) after large strain destructuring at a shear rate of 10 s.sup.−1 (G′ is represented as solid triangles and G″ as open triangles) and (step 8) after destructuring in small strains in harmonic shear at increasing strain amplitude (G′ is represented as solid circles and G″ as open circles).

(6) FIG. 6 represents the monitoring of the destructuring in harmonic shear at increasing strain amplitude at 15° C. over time of a hydrogel of a-La at 20 mg/ml and 30 mM of NaCl: identification of the critical strain γ.sub.c of approximately 0.2 beyond which the sol-gel transition begins to appear (step 7).

(7) FIG. 7 represents the monitoring of the destructuring and of the restructuring at 15° C. over time of a hydrogel of a-La in small strains in harmonic shear; 20 mg/ml-0 mM NaCl.

(8) FIG. 8 represents the monitoring of the destructuring and of the restructuring at 15° C. over time of a hydrogel of a-La in small strains in harmonic shear; 20 mg/ml-60 mM NaCl.

(9) FIG. 9 represents the monitoring of the destructuring and of the restructuring at 15° C. over time of a hydrogel of a-La in small strains in harmonic shear; 40 mg/ml-60 mM NaCl.

(10) FIG. 10 compares the levels of the viscoelastic moduli G′ and G″ as a function of the ionic strength of NaCl and of the concentration of a-La: the modulus G′ for a hydrogel at 20 mg/ml of a-La is represented by solid disks, the modulus G″ for a hydrogel at 20 mg/ml of a-La is represented by open disks, the modulus G′ for a hydrogel at 40 mg/ml of a-La is represented by solid triangles and the modulus G′ for a hydrogel at 40 mg/ml of a-La is represented by open triangles. The levels were compared at the same 500 s restructuring time of step 3 corresponding to the slow restructuring kinetics zone.

(11) FIG. 11 is a graph which compares the levels of critical strain γ.sub.c as a function of the ionic strength of NaCl and of the concentration of a-La (open disks for an a-La concentration of 20 mg/ml and solid disks for an a-La concentration of 40 mg/ml). For each condition, the destructuring phenomenon is very reproducible, which demonstrates the high capacity of the system to undergo various shear stresses without undergoing physicochemical modification or any denaturation.

(12) FIG. 12 represents the monitoring of the destructuring and of the restructuring at 15° C. over time of two hydrogels of a-La at 20 mg/ml and 0 mM of NaCl (one prepared from purified a-La, the second from whey with a 45% enrichment in a-La) in small strains in harmonic shear. Various procedures for monitoring the change in the elastic moduli G′ (solid circle for the hydrogel prepared from purified a-La; solid triangle for the hydrogel prepared from whey with a 45% enrichment in a-La) and viscous moduli G″ (open circle for the hydrogel prepared from purified α-La; open triangle for the hydrogel prepared from whey with a 45% enrichment in a-La) as a function of time and of the applied strain (crosses), were applied; the stresses applied during steps 1 to 3 are detailed in example 2 which follows.

(13) FIG. 13 comprises two images of Eppendorf tubes comprising, on the one hand, a hydrogel prepared according to the protocol of the thesis of C. Blanchet (B) and, secondly, a hydrogel according to the invention (A). The image on the left shows these two hydrogels after they had been prepared (both are in the conical end of the tubes); the image on the right shows these two hydrogels after stirring; the hydrogel (A) according to the invention is in the bottom part of the Eppendorf tube, whereas the hydrogel (B) has remained in the top part (conical end).

EXAMPLE 1

Determination of the Reynolds Number Value Range for the Stirring Strength for Implementing the Process According to the Invention

(14) The Reynolds number represents the stirring strength; in a stirred reactor, it is equal to

(15) $\mathrm{Re}=\frac{\rho \mathrm{Vd}}{\mu }$
where: ρ is the density of the fluid mixed, in Kg.Math.m.sup.−3, V is the rotational velocity of the magnetic bar (in m/s), μ is the viscosity of the fluid mixed (in Pa.Math.s), and d is the size of the stirring tool (for example, the length for the case of a magnetic bar) (in m).

(16) The rotational velocity N of the magnetic bar is defined by the velocity applied by the stirrer, for a velocity range of from 0 to 300 rpm.

(17) The relationship between the velocity V in m/s and the rotational velocity N in rpm is the following:

(18) $V=\frac{\pi d}{30}N$

(19) That is to say, it is possible to evaluate a Reynolds number:

(20) $\mathrm{Re}=\frac{\mathrm{\rho \pi }{d}^2}{30\mu }N.$

(21) The fluids mixed (alpha-lactalbumin powder+aqueous suspension) have a viscosity very close to that of water because the concentration of alpha-lactalbumin powder is sufficiently low to not greatly modify the viscosity of the suspension when it is introduced into the water: consequently, during the initial mixing of the suspension, the viscosity and the density of the suspension stirred will be taken to be equal to that of water, i.e.: ρ=1000 K/μ.sup.3, μ=10.sup.−3 Pa.Math.s.

(22) The size of the magnetic bar used is d=6×10.sup.−3 m in length.

(23) The table below groups together examples of Reynolds number values suitable for the mixture recommended for obtaining thixotropic gels from suspensions of alpha-lactalbumin:

(24) TABLE-US-00001 N (rpm) V (m/s) = (2 × 10.sup.−4 π) × N Re = 1.2 πN  10 2π × 10.sup.−3  12π = 37.699  100 2π × 10.sup.−2 120π = 376.99  300 2π × 0.03 360π = 1130.973 $\mathrm{Re}=\frac{1000,\pi ,{\left(6.Math.{10}^{-3}\right)}^2}{30.Math.{10}^{-3}}N=1.2\pi N$

EXAMPLE 2

Preparation of α-Lactalbumin Hydrogels According to the Invention

(25) 2.1. Hydrogel Prepared from Purified a-La

(26) The purified protein “α-lactalbumin from bovine milk Type III, calcium depleted, 85%” which is sold under catalog reference L6010 by Sigma and is lyophilized is resuspended in an aqueous solution of HCl optionally containing NaCl.

(27) The HCl concentration depends on the final concentration of a-La. It is calculated in mM by adding 10 to the numerical value of the desired concentration of a-La.

(28) For example, if it is desired to have 40 mg/ml of a-La, the HCl concentration for resuspending it will be 40+10=50 mM.

(29) First of all, it is necessary to prepare the HCl solution at the predetermined concentration and then to add thereto NaCl at between 0 and 60 mM. Next, it is necessary to weigh out the required amount of a-La. This amount depends on the final concentration of protein and on the volume of gel to be prepared. The a-La concentrations used range from 5 to 60 mg/ml.

(30) The protein is dissolved in the defined volume of HCl solution and then the pH is adjusted to 2.0±0.1 with a few microliters of 1M HCl. The solution is subjected to magnetic stirring using a magnetic bar and incubated overnight at a temperature which can range from 37 to 45° C. The following day, i.e. approximately 16 h later, the gel is formed.

(31) 2.2. Hydrogel Prepared from Whey with a 45% Enrichment in a-La

(32) The preceding protocol is reproduced using a lactoserum with a 45% by weight enrichment in a-La, supplied by the company Armor Protéines.

EXAMPLE 3

Determination of the Viscoelastic Characteristics of the a-La Hydrogels by Rheology

(33) Notions of Rheology

(34) Rheology is a branch of physics which studies the flow or the strain of bodies under the effect of the stresses which are applied thereto, taking into account the rate of application of these stresses or more generally their variation over time.

(35) At a high concentration of α-lactalbumin, the formation of amyloid fibers is accompanied by an increase in viscosity of the solution. When the fibers are formed, they interact with one another to form a gel. This increase in viscosity is monitored by rheometry. The sample placed in a rheometer will be subjected to a certain stress (τ) which is dependent on the shear rate ({dot over (γ)}) applied. The stress τ varies with the shear rate {dot over (γ)} and the ratio between the two makes it possible to determine the viscosity (η) of the fluid studied. When τ is proportional to {dot over (γ)}, then η is a constant and the fluid is Newtonian, whereas if τ is not proportional to) {dot over (γ)}, then the fluid is non-Newtonian and may be of various natures: if the viscosity η decreases when τ and {dot over (γ)} increase, then the fluid is a shear-thinning fluid; conversely, if η increases when τ and {dot over (γ)} increase, then the fluid is a shear-thickening fluid.

(36) Thixotropic fluids are shear-thinning, their viscosity decreases under the same stress over time because of a destructuring of the material. These fluids are reversible since, when the stress is stopped, the material becomes restructured and regains its initial viscoelastic characteristics.

(37) The viscoelastic characteristics of a material are obtained by determining the dynamic viscosity moduli according to Hooke's law: τ=G{dot over (γ)} where G has two components, G′ and G″, which serve to quantify the viscous or elastic behavior of materials. G′ is the storage (elastic) modulus and G″ is the loss (viscous) modulus. When the elastic nature dominates, G′>>G″ and, conversely, when the viscous nature dominates, G′<<G″.

(38) Rheometric Behavior of the Gels

(39) 3.1. Rheometric Measurements

(40) The characterization of the behavior under shear flow of the a-La gels was carried out by rotary rheometry. The measurements were carried out using an applied-torque rotary rheometer (ARG2, TA Instruments, 78 Guyancourt, France). The geometries used are titanium cone-plate geometries (angle 4°, diameter 20 mm, truncation 113 μm). In order to avoid evaporation of the sample during the measurements, the atmosphere was saturated with water around the sample. For the measurements in harmonic shear, a preliminary study made it possible to define the levels of strain and of optimum frequency for which the measurements are part of the linear regime range. In this range, the applied harmonic shear stress does not modify the rheological behavior of the suspensions, and merely probes the viscoelastic moduli of the gels without disrupting them. The frequency of 0.1 Hz was defined as being part of the linear regime regardless of the applied strain and the restructuring time of the samples. All the measurements in harmonic shear will therefore be carried out at this frequency of 0.1 Hz. In the monitoring of the restructuring, a strain γ of 0.01 was also defined as not disrupting the measurement G′ and G″, and will be systematically used for monitoring the restructuring of the samples.

(41) 3.2. Temporal Monitoring of Destructuring-Restructuring of the Gels Under Shear

(42) 3.2.1. Small Strain Behavior

(43) A procedure for monitoring consistency by harmonic shear was set up and used systematically for various samples under given concentration and ionic strength conditions.

(44) FIG. 2 shows a succession of harmonic shears for an a-La gel (20 mg/ml a-La-30 mM NaCl) according to the strain amplitude conditions recorded in table I below:

(45) TABLE-US-00002 TABLE I Strain conditions applied during the procedure for harmonic shear destructuring-restructuring of the a-La gels Step {dot over (γ)} 1- Temporal monitoring of the restructuring 0.01 following placement of the gel in the gap in the tools. 2- Strain ramp for destructuring the gel 0.01 to 10 3- Temporal monitoring of the restructuring 0.01 4- Strain ramp for destructuring the gel 0.01 to 10 5- Temporal monitoring of the restructuring 0.01

(46) During steps 1, 3 and 5 at constant strain amplitude, the recovery in consistency of the gel corresponding to its restructuring can be demonstrated.

(47) During steps 2 and 4, the gradual increase in the strain amplitude makes it possible to monitor the destructuring of the gel brought about by the preceding shear. At increasing strain amplitude, the elastic modulus G′ and viscous modulus G″ decrease regularly until a critical strain γ.sub.c beyond which the levels drop greatly, which demonstrates the destructuring of the gel and the change from an elastic behavior to a viscous behavior (G′ becomes less than G″).

(48) The recovery in consistency at the beginning of steps 3 and 5, characterized by the increase in G′ and G″ over time, clearly demonstrates the thixotropic behavior of the gel. During the restructuring, it can be noted that there is a short time Tr1 of restructuring with a strong recovery in consistency which is about 300 s, followed by a longer time Tr2 during which the increases in G′ and G″ follow slower kinetics.

(49) The same observations can be made when applying the first three steps described above to the hydrogel prepared from whey with a 45% by weight enrichment in a-La; the gel obtained is therefore itself also thixotropic.

(50) 3.2.2. Large Strain Behavior

(51) In order to demonstrate the thixotropic behavior on strains of larger amplitudes, a large strain simple shear was applied, followed by a small strain harmonic shear in order to monitor the recovery in consistency of the gel over time. In order to demonstrate the effect of the shear rate on the level of destructuring reached and also on the restructuring kinetics, various large strain shear rates were applied.

(52) TABLE-US-00003 TABLE II Strain conditions applied during the procedures for large strain destructuring and for monitoring restructuring of the a-La gels in harmonic shear Steps {dot over (γ)} 6 - Temporal monitoring of the restructuring 0.01 following large strain destructuring at 10 s.sup.-1 7 - Strain ramp for destructuring the gel 0.01 to 10 8 - Temporal monitoring of the restructuring 0.01

(53) Represented in FIG. 3 is the recovery in consistency (step 6) after a large strain shear at a shear rate of 10 s.sup.−1 for 300 s. It can again be noted that there is a first period Tr1 over which the elastic and viscous moduli cross strongly with time, and a second period Tr2 for which restructuring kinetics are much slower.

(54) Following lengthy monitoring of restructuring over 1000 min (more than 16 h), a procedure for destructuring in harmonic shear at increasing strain amplitude is again applied (step 7) (see FIG. 4), followed by a recovery in consistency at small strain (step 8) (see FIG. 4).

(55) FIG. 5 shows a comparison of the two recoveries in consistency either after a large strain simple shear (step 6), or after a harmonic shear at increasing strain amplitude (step 8). The results demonstrate different restructuring kinetics according to these two destructuring modes employed. The increasing strain kinetics following a large strain simple shear are much slower than during increasing-amplitude small strain harmonic shear. Indeed, the large strain shear manages to destructure the sample at a higher level than that obtained during a small strain shear. This result again demonstrates the importance of the type of stress and of its strength on the level of destructuring reached in the sample during shearing thereof, which is an indication of the behavior of thixotropic systems.

(56) Represented in FIG. 6 is the change in the viscoelastic moduli as a function of strain, measured during a procedure for destructuring (step 7) in harmonic shear at increasing strain amplitude. It is demonstrated that the critical strain γ.sub.c beyond which the gel begins to flow, identified by the crossing of G′ and G″, is about 0.2.

(57) 3.3. Effect of Ionic Strength on the Thixotropic Behavior of the a-La Gels

(58) In order to evaluate the differences in restructuring-destructuring kinetics of the a-La gels and also the levels of consistency reached as a function of NaCl ionic strength, a procedure identical to that presented in table I was carried out on various suspensions of a-La. The results are given in FIGS. 7 to 9.

(59) Represented in FIG. 10 is the change in the viscoelastic moduli G′ and G″ as a function of the NaCl ionic strength and of the a-La concentration. The levels were compared at the same 500 s restructuring time of step 3 corresponding to the slow restructuring kinetics zone, i.e. on the “plateau” reached during the restructuring. It is demonstrated that the levels of G′ and G″ decrease when the ionic strength increases, which corresponds to a reduction in the consistency of the gel. The increase in the a-La concentration leads to an increase in the viscoelastic moduli.

(60) FIG. 11 shows a comparison of the destructurings applied to various samples; these results demonstrate that the critical strain moves toward higher levels when the ionic strength increases or when the protein concentration decreases. Successive destructurings of one and the same sample exhibit critical strains of the same order of magnitude (not represented in the figure), which demonstrates the very good stability of the system with respect to undergoing successive destructuring-restructuring actions, and also a very good stability over time by virtue of the reproducibility of the G′ and G″ measurements.

EXAMPLE 4

Preparation of an α-Lactalbumin Hydrogel According to the Conditions Described in the Thesis of C. Blanchet

(61) The purpose of this test is to reproduce an a-La suspension which can be obtained by means of the protocol described by C. Blanchet et al. and then to characterize its rheological properties.

(62) The experiments were carried out under the conditions and according to the protocol described on page 203 of the thesis: the a-La proteins (10 mg/ml with 30 mM of NaCl) are placed in suspension, the pH of this suspension is then adjusted to 2 and then the suspension is placed in an Eppendorf tube; the tube is stirred at 40° C. (the stirring conditions are those used for preparing the hydrogels according to the invention).

(63) In parallel, a hydrogel containing 10 mg/ml of a-La with 30 mM of NaCl is prepared by means of the process according to the invention.

(64) The image on the left in FIG. 13 illustrates the appearance of the hydrogels thus obtained ((B) according to the thesis and (A) according to the invention): the hydrogel (B) has a less homogeneous appearance than the hydrogel (A).

(65) It is also observed that these two hydrogels do not have the same behavior when they are stirred: the image on the right in FIG. 13 shows these two hydrogels after stirring. Because of its thixotropic behavior, the viscosity of the hydrogel (A) according to the invention decreased during the stirring and it flowed into the bottom part of the Eppendorf tube; conversely, the stirring did not bring about any flow of the hydrogel (B), which remained in the top part of the Eppendorf tube (conical end).

EXAMPLE 5

Preparation of an α-Lactalbumin Hydrogel at pH 7 and 80° C.

(66) The purpose of this test is to reproduce the hydrogel described by Kavanagh, G. M., A. H. Clark, et al. (2000). “Heat-induced gelation of beta-lactoglobulin/alpha-lactalbumin blends at pH 3 and pH 7.” Macromolecules 33(19): 7029-7037, and then to characterize its rheological properties.

(67) Conditions Described in the Article:

(68) a-La concentration of 15% (w/w), i.e. 150 mg/ml T° C.=80° C. Solvent=deionized water pH=7.0 The gels are observed after 1 to 2 h at 80° C.
Conditions Implemented: a-La concentration of 15% (w/w), i.e. 150 mg/ml T° C.=80° C. for 1 h Solvent=deionized water pH=7.2.

(69) The a-La concentration is verified by measuring the absorbance at 280 nm of the solution using a Nanodrop® ND-1000 spectrophotometer (LabTech): the measurements are carried out on three solutions of proteins each diluted five-fold: A1=61; A2=62; A3=61. The a-La concentration is determined according to the Beer-Lambert law A=εC l where ε=27 880 L.Math.mol.sup.−1.Math.cm.sup.−1 and l=1 cm. The molar concentration C of the a-La solution is therefore 10.9 mM, which corresponds to a concentration by weight of 154 mg/ml (the molar mass of a-La is 14 150 g.Math.mol.sup.−1).

(70) The a-La solution prepared is separated into two tubes of 200 μl. One tube is placed at 80° C. for 1 h without stirring and the other tube is kept at ambient temperature as a control.

(71) In less than one hour, a hydrogel formed in the tube placed at 80° C. The gel obtained is hard, a tip cannot be pushed into it, but shows a certain elasticity. It is also possible to remove it from the mold while retaining the shape of the tube, which is not the case with the hydrogels according to the invention, which have a softer consistency.

(72) The hydrogel thus obtained also has a more transparent appearance, whereas the hydrogels according to the invention are translucent (they allow a scattered light to pass through, but objects cannot be distinguished through these hydrogels). Finally, if it is vigorously shaken, it does not change shape, it is irreversible.

(73) The hydrogel produced here thus has neither the appearance nor the physical properties of the thixotropic gels prepared according to the process of the invention.

LITERATURE

(74) Akkermans, C., P. Venema, et al. (2006). “Peptides are building blocks of heat-induced fibrillar protein aggregates of beta-lactoglobulin formed at pH 2.” Biomacromolecules 9(5): 1474-1479. Audic, J. L., B. Chaufer, et al. (2003). “Non-food applications of milk components end dairy co-products: A review.” Lait 83(6): 417-438. Chen, H. (1995). “Functional properties and applications of edible films made of milk proteins.” Journal of Dairy Science 78(11): 2563.2583. Gosal, W. S., A. H. Clark, et al. (2004). “Fibrillar bet-lactoglobulin gels: Part 1. Fibril formation and structure.” Biomacromolecules 5(6): 2408-2419. Gosal, W. S., A. H. Clark, et al. (2004). “Fibrillar beta-lactoglobulin gels: Part 2. Dynamic mechanical characterization of heat-set systems.” Biomacromolecules 5(6): 2420-2429. Gosal, W. S., A. H. Clark, et al. (2004). “Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical of solvent-induced systems.” Biomacromolecules 5(6): 2430-2438. Graveland-Bikker, J. F., R. Ipsen, et al. (2004). “Influence of calcium on the self-assembly of partially hydrolyzed alpha-lactalbumin.” Langmuir 20(16): 6841-6846. Gunasekaran, S., S. Ko, et al. (2007). “Use of whey proteins for encapsulation and controlled delivery applications.” Journal of Food Engineering 83(1): 31-40. Heidebach, T., P. Forst, et al. (2009). “Microencapsulation of probiotic cells by means of rennet-gelation of milk proteins.” FOOD HYDROCOLLOIDS 23(7): 1670-1677. Heidebach, T., P. Forst, et al. (2009). “Transglutaminase-induced caseinate gelation for the microencapsulation of probiotic cells.” INTERNATIONAL DAIRY JOURNAL 19(2): 77-84. Hines, M. E. and E. A. Foegeding (1993). “INTERACTIONS OF ALPHA-LACTALBUMIN AND BOVINE SERUM-ALBUMIN WITH BETA-LACTOGLOBULIN IN THERMALLY INDUCED GELATION.” Journal of Agricultural and Food Chemistry 41(3): 341-346. Holmes, T. C., S. de Lacalle, et al. (2000). “Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds.” Proceedings of the National Academy of Sciences of the United States of America 97(12): 6728-6733. Ipsen, R. and J. Otte (2007). “Self-assembly of partially hydrolysed alpha-lactalbumin.” BIOTECHNOLOGY ADVANCES 25(6): 602-605. Ipsen, R., J. Otte, et al. (2001). “Molecular self-assembly of partially hydrolysed alpha-lactalbumin resulting in strong gels with a novel microstructure.” Journal of Dairy Research 68(2): 277-286. Kamau, S. M., S. C. Cheison, et al. (2010). “Alpha-Lactalbumin: Its Production Technologies and Bioactive Peptides.” Comprehensive Reviews in Food Science and Food Safety 9(2): 197-212. Kavanagh, G. M., A. H. Clark, et al. (2000). “Heat-induced gelation of beta-lactoglobulin/alpha-lactalbumin blends at pH 3 and pH 7.” Macromolecules 33(19): 7029-7037. Kavanagh, G. M., A. H. Clark, et al. (2000). “Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH beta-lactoglobulin gels.” International Journal of Biological Macromolecules 28(1): 41-50. Kopecek, J. and J. Yang (2009). “Peptide-directed self-assembly of hydrogels.” Acta Biomater 5(3): 805-816. Kyle, S., A. Aggeli, et al. (2009). “Production of self-assembling biomaterials for tissue engineering.” Trends in Biotechnology 27(7): 423-433. Le Tien, C. C. Vachon, et al. (2001). “Milk protein coatings prevent oxidative browning of apples and potatoes,” Journal of Food Science 66(4): 512-516. Livney, Y. D. (2010). “Milk proteins as vehicles for bioactives.” Current Opinion in Colloid & Interface Science 15(1-2): 73-83. Loveday, S. M., M. A. Rao, et al. (2009). “Factors Affecting Rheological Characteristics of Fibril Gels: The Case of beta-Lactoglobulin and alpha-Lactalbumin.” Journal of Food Science 74(3): R47-R55. Madureira, A. R., C. I. Pereira, et al. (2007). “Bovine whey proteins—Overview on their main biological properties” Food Research International 40(10): 1197-1211. Mewis, J. (1979). “THIXOTROPY—GENERAL-REVIEW.” Journal of Non-Newtonian fluid Mechanics 6(1): 1-20. Oboroceanu, D., L. Wang, et al. (2010). “Characterization of β-Lactoglobulin Fibrillar Assembly Using Atomic Force Microscopy, Polyacrylamide Gel Electrophoresis, and in Situ Fourier Transform Infrared Spectroscopy.” Journal of Agricultural and Food Chemistry. Paulsson, M., P. O. Hegg, et al. (1986). “HEAT-INDUCED GELATION OF INDIVIDUAL WHEY PROTEINS A DYNAMIC RHEOLOGICAL STUDY.” Journal of Food Science 51(1): 87-90. Pek, Y. S., A. C. A. Wan, et al. (2008). “A thixotropic nanocomposite gel for three-dimensional cell culture.” Nature Nanotechnology 3(11): 671-675. Permyakov, E. A. and L. J. Berliner (2000). “alpha-Lactalbumin: structure and function.” Febs Letters 473(3): 269-274. Piau, J. M. (2007). “Carbopol gels: Elastoviscoplastic and slippery glasses made of individual swollen sponges Meso- and macroscopic properties, constitutive equations and scaling laws.” Journal of Non-Newtonian Fluid Mechanics 144(1): 1-29. Pignon, F., A. Magnin, et al. (1998). “Thixotropic behavior of day dispersions: Combinations of scattering and rheometric techniques.” Journal of Rheology 42(6): 1349-1373. Restani, P., C. Ballabio, et al. (2009). “Molecular aspects of milk allergens and their role in clinical events.” ANALYTICAL AND BIOANALYTICAL CHEMISTRY 395(1): 47-56. Rodrigues, M. M. A., A. R. Simioni, et al. (2009). “Preparation, characterization and in vitro cytotoxicity of BSA-based nanospheres containing nanosized magnetic particles and/or photosensitizer.” Journal of Magnetism and Magnetic Materials 321(10): 1600-1603. Semo, E., E. Kasselman, et al. (2007). “Casein micelle as a natural nano-capsular vehicle for nutraceuticals.” Food Hydrocolloids 21(5-6): 936-942. Song, F., L. Zhang, et al. (2009). “Genipin-crosslinked casein hydrogels for controlled drug delivery.” INTERNATIONAL JOURNAL OF PHARMACEUTICS 373(1-2): 41-47. Tokpavi, D. L., P. Jay, et al. (2009). “Experimental study of the very slow flow of a yield stress fluid around a circular cylinder.” Journal of Non-Newtonian Fluid Mechanics 164(1-3): 35-44. van der Linden, E. and P. Venema (2007). “Self-assembly and aggregation of proteins.” Current Opinion in Colloid & Interface Science 12(4-5): 158-165. Vintiloiu, A. and J.-C. Leroux (2008). “Organogels and their use in drug delivery—A review.” JOURNAL OF CONTROLLED RELEASE 125(3): 179-192. Yan, H., H. Frielinghaus, et al. (2008). “Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway.” Soft Matter 4(6): 1313-1325. Yan, H., A. Saiani, et al. (2006). “Thermoreversible Protein Hydrogel as Cell Scaffold.” Biomacomolecues 7(10): 2776-2782. Yantian, Y., K. Ulung, et at. (2009). “Designer self-assembling peptide nanomaterials.” Nano Today 4(2): 193-210. Zhang, S. (2008). Designer self-assembling peptide nanofiber scaffolds for study of 3-0 cell biology and beyond. Advances in Cancer Research. Vol 99. San Diego, Elsevier Academic Press Inc. 99: 335-+.