BACKGROUND OF THE INVENTION

Field of the Invention

This disclosure generally relates to a stabilized colloidal dispersion of inorganic particles in water and to a method for stabilizing a colloidal dispersion of inorganic particles in water.

Description of the Related Art

The stability of particles dispersed in a liquid {i.e. a colloidal dispersion) is an important factor in any application that is based on colloidal dispersions. Such colloidal dispersions contain suspended micro or nano- scale particles and serve a wide variety of purposes. Colloidal dispersions can be raw materials for nanomaterials, composites, and other materials. They are used in inks, paints, and other emulsions, pharmaceutical products, and are found naturally in clays, biological fluids such as blood, natural organic matter colloids, and petroleum and geological processes. The stability of colloidal dispersions during transport through porous media is important in separation processes and the spread of contaminants, nutrients, and bio- solids through soil. In all applications it is necessary to be able to maintain the particles in a colloidal dispersion, wherein the particles are suspended as individual particles and with minimal amounts of flocculation, agglomeration, coagulation and/or sedimentation.

A particular class of inorganic particles for which stable colloidal dispersions are desired is the class of silica particles. There are several different types of silica particles, for example mesoporous silica, fumed silica particles and silica gel. When one or more of these silica particles are dispersed in liquid, colloidal silica (a colloidal dispersion or sol) is formed. Fumed silica, which is a very fine particulate form of silicon dioxide, is prepared by burning tetrachlorosilane in an oxygen rich hydrocarbon flame to produce a "smoke" of silicon dioxide. Fumed silica particles and/or their colloids find applications as anticaking agent, as a desiccant, and in cosmetics for its light-diffusing properties. In addition, they are used as abrasives, fillers in paints, coatings and adhesives, and in the production of cat box filler. Applications are also found in medical areas, including drug delivery.

In recent years, significant effort has been devoted to develop techniques which increase the amount of oil or gas extracted from oil or gas wells. There currently are several different methods of Enhanced Oil

Recovery (EOR) including steam flood, water flood injection and hydraulic fracturing. In all these methods, a pressurized mobile phase is used to press out the oil from the oil containing soil or rock. The publication "The Potential of Hydrophilic Silica Nanoparticles for EOR Purposes; A literature review and an experimental study; Bjornar Engeset; May 2012" discloses that nano-scale silica particles can be used for enhanced oil recovery, increasing the viscosity of the pressurized mobile phase and thus the displacement of the liquid.

However, the publication "Stability of aqueous silica nanoparticle dispersions; J Nanopart Res (201 1 ) 13:839-850; Cigdem O. Metin et al.; September 2010" discloses that using silica particles creates a problem in the storage and process of drilling and/or pumping due to aggregation, flocculation or sedimentation, and that stabilization of the silica particles is needed.

The publication "Stabilization of weakly charged microparticles using highly charged nanoparticles; Master's thesis; David J. Herman; August, 201 1 " discloses the use of latex nanoparticles for stabilizing dispersions of silica microspheres. However, the stabilization effect is very weak, and in addition, latex cannot be used for a large number of applications. Also, the latex nanoparticles permanently modify the surface of the silica particles, thereby modifying the silica particles' properties which is not always desired.

Also, the publication "Design and screening of synergistic blends of silicon dioxide nanoparticles and surfactants for enhanced oil recovery in high-temperature reservoirs; Nhu Y Thi Le et al.; Adv. Nat. Sci.: Nanosci. Nanotechnol. 2 (201 1 ) 035013 (6pp)" discloses surfactants in combination with silica particles which can be used to help improving oil displacement for EOR. This publication however does not mention whether the addition of surfactants improves the stability and whether the proposed material solves the problem of flocculation, agglomeration, coagulation and/or sedimentation during the drilling process and storage.

Silica particles in aqueous media form a colloidal dispersion which becomes unstable and therefore unusable at high temperatures and high salt concentrations.

It is an object of the present invention to provide novel strategies for improving the stability of colloidal dispersions of inorganic particles compared to known colloidal dispersions of inorganic particles.

BRIEF SUMMARY OF THE INVENTION

This disclosure is directed to a colloidal dispersion of inorganic particles in water that is stabilized with soy particles, whey particles, collagen nanoparticles and/or collagen-derived nanoparticles. It is further directed to the use of soy particles, whey particles, collagen nanoparticles and/or collagen-derived nanoparticles for increasing the stability of a colloidal dispersion of inorganic particles in water.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to a composition comprising inorganic particles, in particular silica nanoparticles, and protein nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles. The composition is in particular a colloidal dispersion in an aqueous medium {i.e. water), wherein at least the inorganic particles form an aqueous colloidal dispersion. The nanoparticles of soy, whey, collagen and collagen-derived material are usually also present as an aqueous colloidal dispersion.

The inorganic particles may in principle be any inorganic particle. In particular, they are selected from the group of silica particles, sand particles, mineral particles including calcium phosphate and calcium apatite, titanium dioxide particles, aluminum trioxide particles, ceramic particles, metal particles including gold and silver particles, and particles of semi-conductor material.

In case the inorganic particles are silica particles, the silica

nanoparticles are selected from the group of mesoporous silica particles, fumed silica particles and silica gel.

For the purpose of the invention, by nanoparticles are meant particles with a size in the range of 1 -1000 nm. By the size is in particular meant the mean cross-section of the particles. The mean particle size of the particles used in the invention is measured by dynamic light scattering (DLS) according to Z-average.

The inorganic particles may have a size of 100 μιη or less, 50 μιη or less, 20 μιη or less, 10 μιη or less or 1 μιη or less. In in particular, the inorganic particles are nanoparticles. More in particular, their size is in the range of 1 -900 nm, even more in particular 5-800 nm. Usually, their size is

750 nm or less, 500 nm or less, 250 nm or less, 100 nm or less, 50 nm or less or 25 nm or less. Their size may be 1 nm or more, 2 nm or more, 4 nm or more, 7 nm or more, 10 nm or more, 20 nm or more, 40 or more, 70 nm or more or 100 nm or more.

The nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived

nanoparticles usually have a size in the range of 1 -900 nm, in particular in the range of 5-800 nm, more in particular in the range of 40-600 nm, and even more in particular in the range of 80-500 nm. Usually, their size is 750 nm or less, 600 nm or less, 550 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 50 nm or less, 25 nm or less or 10 nm or less. Their size may be 1 nm or more, 2 nm or more, 4 nm or more, 7 nm or more, 10 nm or more, 20 nm or more, 40 or more, 70 nm or more or 100 nm or more.

By collagen nanoparticles are meant nanoparticles that consist of collagen or that contain a certain component of collagen. The collagen may for example be present in amount of at least 20 wt%, at least 35 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, at least 95 wt%, at least 98 wt% or at least 99 wt%.

By collagen-derived nanoparticles are meant nanoparticles that consist of collagen-derived material or that contain a certain component of collagen-derived material. The collagen-derived material may for example be present in amount of at least 20 wt%, at least 35 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, at least 95 wt%, at least 98 wt% or at least 99 wt%.

By the term "collagen" (as in collagen nanoparticles) is meant the natural collagen as it can be obtained from an organism without substantial modification, as well as recombinant collagen that can be obtained by genetically modifying the collagen-producing organism. The term "collagen- derived" is meant to include material that is derived from the naturally obtained collagen by modifying it, for example by hydrolyzing it. Collagen- derived material for example includes gelatin or gelatin hydrolysates. It also includes material derived from recombinant collagen, such as recombinant gelatin.

The collagen and the collagen-derived material are usually obtained from an animal source, for example from pig, bovine, poultry and fish.

The whey is usually obtained from bovine milk.

The nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived

nanoparticles (such as gelatin nanoparticles) can be readily produced using methods disclosed in WO2013004370.

In a composition of the invention, the soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles (such as gelatin nanoparticles) may be stabilized by a cross linking agent, for example selected from the group of dialdehydes, formaldehyde, isocyanates, diisocyanates, carbodiimides, alkyl dihalides and glutaraldehyde.

The gel strength of the collagen or collagen-derived raw material that is used for preparing the corresponding nanoparticles may be in the range of 50-350 g Bloom, preferably it is in the range of 200-300 g Bloom.

In case a composition of the invention is a dispersion, the content of inorganic particles may be in the range of 0.05 to 50 wt%, usually it is in the range of 0.1 to 25 wt%. The solids content may in particular be 0.05 wt% or more, 0.07 wt% or more, 0.1 wt% or more, 0.2 wt% or more, 0.5 wt% or more, 0.7 wt% or more, 1 wt% or more, 2 wt% or more, 5 wt% or more, 7 wt% or more, 10 wt% or more, 15 wt% or more, 20 wt% or more, 25 wt% or more, 30 wt% or more, 35 wt% or more, 40 wt% or more or 50 wt% or more.

The inorganic particles (a) and the nanoparticles (b) selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles are usually present in a mass ratio (a) : (b) that lies in the range of 0.1 : 10 to 10 : 0.1 . In particular, the mass ratio is in the range of 0.5 : 1 to 5 : 1 . Usually, the mass ratio is 1 : 1 or more, 1 : 1 .5 or more, 1 : 2 or more, 1 : 3 or more, 1 : 4 or more or 1 : 6 or more.

The invention in particular relies on the surprising effect that nanoparticles of soy, whey, collagen or collagen-derived material are capable of stabilizing an aqueous colloidal dispersion of inorganic particles, in particular of silica nanoparticles.

This in particular means that such nanoparticles prevent or delay precipitation, flocculation and/or aggregation of the inorganic nanoparticles. For example, when a known aqueous colloidal dispersion of inorganic particles is exposed to a change in an environmental condition {e.g. a change in pH, a change in temperature, contacted with a higher salt concentration), precipitation, flocculation and/or aggregation may occur. In the presence of the protein nanoparticles in the claimed composition (nanoparticles of soy, whey, collagen or collagen-derived material) however, the aqueous colloidal dispersion of inorganic particles remains stable when exposed to the same change in an environmental condition. The examples, demonstrate for example that aqueous colloidal dispersions according to the invention remain stable at 60 °C during a certain amount of time, while in aqueous colloidal dispersions that lack the protein nanoparticles that are exposed to the same conditions, a precipitate is formed during the same amount of time.

Accordingly, in an embodiment, the inorganic particles in a

composition of the invention are present as a colloidal dispersion in water. Accordingly, the invention also relates to a stabilized aqueous colloidal dispersion of inorganic particles, the dispersion comprising inorganic particles and nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles.

In an embodiment, the collagen-derived nanoparticles in the stabilized aqueous colloidal dispersion are gelatin nanoparticles. In a preferred embodiment, the inorganic particles are silica particles, in particular silica nanoparticles. It appeared that an aqueous colloidal dispersion of silica particles, especially silica nanoparticles, is particularly well stabilized by gelatin nanoparticles. In addition, also soy nanoparticles, whey nanoparticles and collagen nanoparticles are capable of stabilizing aqueous colloidal dispersions of inorganic particles, in particular silica particles, more in particular silica nanoparticles.

In another embodiment, the stabilized aqueous colloidal dispersion has a content of inorganic particles of at least 0.05 wt%.

In yet another embodiment, the stabilized aqueous colloidal dispersion has a pH in the range of 3.0-14.0, in particular of 3.0-9.0, more in particular in the range of 8.0-9.0. It may also have a pH in the range of 5.0- 12.0 or of 6.0-10.0.

An effect of the invention is that the shelf-life of the dispersion is increased. Another effect is that the dispersion remains stable {i.e. it remains a colloidal dispersion) when it is applied for a particular purpose, for example in an environment with a higher temperature, a high salt concentration and/or an extreme pH. Such environment may be found in geological formations such as underground oil and/or gas reservoirs.

Therefore, the invention further relates to the use of a composition as described herein (and in particular to the use of a stabilized aqueous colloidal dispersion of inorganic particles as described herein) as a mobile phase in the recovery of oil and/or gas from underground oil and/or gas reservoirs.

A stabilized aqueous colloidal dispersion of inorganic particles according to the invention may also be used for increased rheology control, suspension and stability behavior, adsorbent, free-flow of powders, anti- settling, anti-sagging, anti-blocking, reinforcement, pigment stabilization & dispersion, print definition, anti-setoff, mechanical/optical properties improvement, thixotropy, thickening, hydrophobicity control, and improved processability.

A stabilized aqueous colloidal dispersion of inorganic particles according to the invention may in particular be applied in adhesives, sealants, plastics, inks, paints, coatings, defoamers, greases, toner, silicone rubber, agriculture, cable gels, food, fire extinguishers, polyester resins and cosmetics.

The invention is in particular suitable for stabilizing an aqueous colloidal dispersion of silica.

A stabilized dispersion of the invention may be prepared in several ways. It can be prepared by mixing water with a dry composition of the inorganic particles and the nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen- derived nanoparticles. It can also be prepared by mixing an aqueous colloidal dispersion of the inorganic particles with the dry nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles. It can also be prepared by mixing an aqueous colloidal dispersion of the nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen- derived nanoparticles with the dry inorganic particles. It can also be prepared by mixing an aqueous colloidal dispersion of the nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles with an aqueous colloidal dispersion of the inorganic particles.

The colloidal dispersion containing silica and gelatin particles is usually formed by mixing a silica solution and a gelatin particle solution. Surprisingly, it was found that when gelatin nanoparticles were added to a silica solution, flocculation and sedimentation of the silica was prevented, which is different from the addition of dissolved bulk gelatin, which results in a slow precipitation of the silica. In addition, this effect was observed when a composition of the inorganic particles and the nanoparticles was kept at an increased temperature and/or an increased salt concentration.

Gelatin particles may be added to the colloidal dispersion of inorganic particles. In a preferred embodiment, the gelatin particles are added as a powder. In another preferred embodiment, the gelatin particles are added as a colloidal dispersion.

Sometimes, it is necessary to optimize the conditions in the preparation of the protein nanoparticles in order to obtain sufficient

stabilization of the inorganic particles and so create a sufficiently stabilized aqueous colloidal suspension. Such optimization is performed by routine experimentation known to the skilled person. For example, it may be necessary to prepare and isolate the dispersion of protein nanoparticles as described in WO2013004370 under conditions with a modified pH. Usually, a composition of the invention, wherein the inorganic particles are present as a colloidal dispersion in water, has a pH in the range of 3.0-14.0, in particular of 3.0-9.0, more in particular in the range of 8.0-9.0. It may also have a pH in the range of 5.0-12.0 or of 6.0-10.0.

In an embodiment of the invention, the whey, collagen or collagen- derived material may be charged or have a net neutral charge. The type of protein particles is chosen and prepared such that the net charge is positive, negative or neutral at the pH dictated by the application.

The invention further relates to a stabilized dispersion obtainable by a method as given hereinabove.

The invention further relates to a composition comprising inorganic particles and protein nanoparticles wherein the protein is selected from the group of soy protein, zein protein, whey protein and recombinant protein.

The invention is in particular based on the realization that the formulation of a mixture of an inorganic particles dispersion (such as a silica dispersion) with a dispersion of particular nanoparticles (nanoparticles of soy, whey, collagen and collagen-derived material such as gelatin) in an aqueous medium results in a stable colloidal dispersion in which the concentration of the nanoparticles {e.g. gelatin particles) is high enough to prevent the inorganic particles {e.g. silica) to flocculate and sediment.

Furthermore, the colloidal dispersion remains stable at elevated temperatures and increased salt concentration. For example, it is possible to stabilize the silica in a salted water solution for at least several days at 60 °C.

The stability is tested by subjecting a sample of a dispersion with a certain composition to a stable temperature during at least 15 minutes followed by evaluating the sample by eye. If the sample is still a

homogeneous, turbid or nearly clear liquid, the specific dispersion is deemed to be stable at the specific temperature. If there is a precipitation at the bottom, if flocks are visible or if any other inhomogeneity is observed by eye, the dispersion is deemed to be unstable.

The effect of the stabilization (and the apparent absence of aggregation) was further investigated by measuring the hydrodynamic particle size of the inorganic particles as a function of time. This was performed by making use of DLS. For the stabilization of silica nanoparticles by gelatin, this is demonstrated in Figures 1 -2.

Each of Figures 1 a-1 c shows a graphic result of a particle size distribution after 5, 10 and 15 minutes. Figure 1 a shows the particle size distribution of silicon dioxide particles in aqueous medium (mixture B of the Examples); Figure 1 b shows the particle size distribution of gelatin

nanoparticles in aqueous medium (FCPGP-RD450 of the Examples); Figure 1 c shows the particle size distribution of the mixture of 1 ) mixture B and 2) the FCPGP-RD450 in aqueous medium.

The results show that the silicon dioxide in mixture B aggregates and forms multiple peaks (Figure 1 a). After 15 minutes all silicon dioxide is precipitated and the size could not be analyzed anymore. Secondly, the gelatin nanoparticles appear to undergo a slight shift in peak distribution, but they remain in a stable dispersion (Figure 1 b). Finally, it is demonstrated that the particle size distribution remains unchanged when the silicon dioxide particles and the gelatin nanoparticles are in one mixture (Figure 1 c). This means that substantially no agglomeration of particles has occurred, which is strong support for the observation that the silicon dioxide particles are stabilized after the addition of the gelatin.

The effect of gelatin nanoparticles on the stability of a dispersion of silicon dioxide particles is further visualized in Figure 2, wherein average hydrodynamic particle sizes (DLS nanosizer) as a function of time are displayed. The solid line corresponds to the average hydrodynamic particle size of silicon dioxide particles in aqueous medium (mixture B of the

Examples); the dotted line shows the average hydrodynamic particle size of gelatin nanoparticles in aqueous medium (FCPGP-RD450 of the Examples); the dashed line shows the average hydrodynamic particle size of the mixture of 1 ) mixture B and 2) the FCPGP-RD450 in aqueous medium.

The results demonstrate that the average hydrodynamic particle size in solution B increases rapidly after 10 minutes (solid line), while it remains stable when the silicon dioxide particles and the gelatin nanoparticles are in one mixture. This means that substantially no agglomeration of particles has occurred, which is again strong support for the observation that the silicon dioxide particles are stabilized after the addition of the gelatin.

The invention further relates to a method for stabilizing a colloidal dispersion of inorganic particles in water, comprising mixing the colloidal dispersion with nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived

nanoparticles.

The invention further relates to the use of nanoparticles selected from the group of soy nanoparticles, whey nanoparticles, collagen nanoparticles and collagen-derived nanoparticles for improving the stability of a colloidal dispersion of inorganic particles in water. EXAMPLES

Materials and Methods Stock solutions:

A) 0.2 wt% solution: 2 mL S1O2 (dispersion) in 100 mL Artificial Marine

Water.

B) 0.2 wt% Ssolution: 1 .67 mL S1O2 (Aerosdisp® W1824, 200 nm, pH 5.0- 6.0, 24 %wt, phosphate stabilized) in 133 mL Artificial Ocean Water (ASTM) and diluted with 65 mL demineralized water.

C) 0.2 wt% Solution: 3.33 mL S1O2 (CAB-O-SPERSE ® 4012K, <200 nm, pH 10.1 -10.5, 12 %wt, potassium hydroxide stabilized) in 10 mL Artificial Ocean Water (ASTM) and diluted with 187 mL demineralized water.

D) 0.2 wt% Solution: 3.33 mL S1O2 fibers (Snowtex ® ST-OUP, 40-100 nm, pH 2.0-4.0, 15.0-16.0 %wt, O type stabilized) in 10 mL Artificial Ocean Water (ASTM) and diluted with 187 mL demineralized water.

E) 0.2 wt% Solution: 0.8 mL ZnO (Zinc(ll) oxide, <100 nm, pH = 6.0-8.0, 45.0-55.0 %wt) in 13 mL Artificial Ocean Water (ASTM) and diluted with 184 mL demineralized water.

F) 0.2 wt% Solution: 1 .1 14 mL T1O2 (Tianium(IV) oxide, <150 nm, 33.0- 37.0 %wt) in 2 mL Artificial Ocean Water (ASTM) and diluted with 197 mL demineralized water.

G) 0.2 wt% Solution: 1 .6 mL AI2O3 (Aerosdisp® W925, 1 10 nm, pH 3.0-5.0, 25 %wt) in 198.4 mL demineralized water. H) 0.2 wt% Solution: 4.0 mL CeO2 (Cerium(IV) oxide, <25 nm, 10.0 %wt) in 0.5 mL Artificial Ocean Water (ASTM) and diluted with 195.5 mL demineralized water.

Salt solutions:

1 ) Artificial Marine Water (for stock solution A) was prepared by adding the amounts of the salts as provided in table 1 to 1 L of demineralized water.

Table 1. Contents of Artificial Marine Water

2) Artificial Ocean Water (ASTM D 1 141 - 98 (2003) salted water) (for stock solutions B-H) was prepared by adding the amounts of the salts as provided in table 2 to 1 L of demineralized water.

Table 2. Contents of Artificial Ocean Water (ASTM D 1141 - 98 (2003))

Salt type Amount (g)

NaCI 24.534

Na2S04 4.094

MgCI2.6H20 11.11

CaCI2 1.158

SrCI2.6H20 0.0422

KCI 0.694

NaHC03 0.201

KBr 0.1006

H3B03 0.0272

NaF 0.003 Protein dispersions

Protein dispersions are prepared according to the technology described in: EP2726068 (A1 ) - CONTINUOUS FLOW PRODUCTION OF GELATIN NANOPARTICLES

Stock solutions in an aqueous solution are prepared for the different protein particle dispersions. Mean particle size of the protein particles is measured by DLS according to Z-average.

Proteins used for protein nanoparticle formation:

Gelatin type B (Bloom = 272): Gelita Gelatin SI

Gelatin type A (Bloom = 300): Sigma Aldrich Porcine Gelatin G-2500

Gelatin type A (Bloom = 90-1 10): Sigma Aldrich Porcine Gelatin G-6144

Gelatin type B (Bloom = 225): Sigma Aldrich Bovine Gelatin G-9391

Gelatin type B (Bloom = 75): Sigma Aldrich Bovine Gelatin G-6650

Gelatin type A (Bloom = 100): Trobas Porcine Gelatin G1

Gelatin type A (Bloom = 180): Trobas Porcine Gelatin ZS800

Zein: Sigma Aldrich Zein Z3625

Whey: Whey Davisco - JE 063-4-420

Collagen: Geltech bovine collagen

Soy protein isolate: krachtshop.nl, soy protein isolate neutral

Method 1 : Stability testing protocol 1

To a 20 imL test tube, 3 imL of inorganic particle dispersion A-H (2 g/L) was added. 3 imL of protein particle solution (10 g/L, pH = 3.5) was added. The mixture was homogenized by vortexing for 10 seconds. The mixtures were observed for precipitation (+/-). Next, the tubes were immersed into an oil bath for 24 and 48 hours that was set at 60 °C. Again, the mixtures were observed for precipitation (+/-). Method 2: accelerated stability testing protocol 1

To a 20 ml_ test tube, 3 ml_ of inorganic particle dispersion A-H (2 g/L) was added. 3 imL of solution protein particle solution (10 g/L, pH = 3.5) was added. The mixture was homogenized by vortexing for 10 seconds. The mixtures were observed for precipitation (+/-). Next, the tubes were immersed into an oil bath for 15 and 60 minutes that was set at 60 °C. Again, the mixtures were observed for precipitation (+/-).

Method 3: accelerated stability testing protocol 2

To a 20 mL test tube, 3 imL of inorganic particle dispersion A-H (2 g/L) was added. 3 mL of solution protein particle solution (10 g/L, pH = 8) was added. The mixture was homogenized by vortexing for 10 seconds. The mixtures were observed for precipitation (+/-). Next, the tubes were immersed into an oil bath for 15 and 60 minutes that was set at 60 °C. Again, the mixtures were observed for precipitation (+/-).

Results

Experiment 1 : effect of gelatin nanoparticles onto stabilization of Si0 ₂

For the experiment, the method 1 was used. Solution A was mixed with a protein particle solution at pH = 3.5 as depicted in Table 3. Stabilization of the dispersion is depicted as + in Table 3.

Table 3. Results of experiment 1

Experiment 2: silica dispersion to gelatin particle ratio onto stabilization of Si0 ₂

Idem as for experiment 1 , but with the difference that the mass ratio of inorganic particles and protein particles was varied according to Table 4. Stabilization of the dispersion is depicted as + in Table 4.

Table 4. Results of experiment 2

Experiment 3: effect of temperature onto stabilization of Si0 ₂

Idem as for experiment 1 , but with the difference that the samples were stored at different temperatures according to Table 5. Stabilization of the dispersion is depicted as + in Table 5.

Table 5. Results of experiment 3

Example Inorganic Protein particle Temperature Stable particle type (°C) dispersion at dispersion t = 24 h, 60 °C

3-1 A Gelatin type B 20 +

(Bloom = 272)

3-2 A Gelatin type B 60 +

(Bloom = 272)

3-3 A Gelatin type B 70 +

(Bloom = 272)

3-4 A Gelatin type B 80 - (Bloom = 272)

3-5 A Gelatin type B 90 - (Bloom = 272) Experiment 4: effect of pH onto stabilization of S1O2

Idem as for experiment 1 , but with the difference that he pH of the solution was varied. Stabilization of the dispersion is depicted as + in Table 6.

Table 6. Results of experiment 4

Experiment 5: effect of Na+ ions onto stabilization of S1O2

Idem as for experiment 1 , but with the difference that an aqueous solution of NaCI was used instead of artificial marine water. Stabilization of the dispersion is depicted as + in Table 7.

Table 7. Results of experiment 5

Example Inorganic Protein particle Na ⁺ Stable

particle type Concentration dispersion at dispersion t = 24 h, 60 °C

5-1 A Gelatin type B 0 g/L +

(Bloom = 272)

5-2 A Gelatin type B 10.6 g/L +

(Bloom = 272) Experiment 6: effect of Mg2+ ions onto stabilization of S1O2

Idem as for experiment 1 , but with the difference that an aqueous solution of MgCl2 was used instead of artificial marine water. Stabilization of the dispersion is depicted as + in Table 8.

Table 8. Results of experiment 6 Experiment 7: effect of different protein particles onto stabilization of

For the experiment the method 2 was used. Solution B was mixed with a protein particle solution (pH = 3.5) as depicted in Table 9. Stabilization of the dispersion is depicted as + in the Table 9.

Table 9. Results of experiment 7

Ex Inorganic Protein Code Mean Stable Stable am particle particle type particle dispersion at dispersion at pie dispersion size (nm) t = 15 (min) t = 60 (min)

Ref B - - - - -

7-1 B Gelatin type B FCPGP- 1 22 + +

(Bloom = 272) D166

7-2 B Gelatin type A FCPGP- 1 51 + +

(Bloom = 300) RD440

7-3 B Gelatin type A FCPGP- 1 15 + +

(Bloom = 300) RD442

7-4 B Gelatin type A FCPGP- 93 + +

(Bloom = 300) RD444

7-5 B Gelatin type A FCPGP- 225 + +

(Bloom = 300) RD448

7-6 B Gelatin type A FCPGP- 1 34 + +

(Bloom = 300) RD450

7-7 B Gelatin type B FCPGP- 238 + +

(Bloom = 225) RD452

7-8 B Gelatin type B FCPGP- 227 + +

(Bloom = 225) RD454

7-9 B Gelatin type B FCPGP- 1 31 + +1- (Bloom = 225) RD456

7-10 B Gelatin type B FCPGP- 21 6 + +

(Bloom = 75) RD458

7-11 B Gelatin type B FCPGP- 21 0 + +

(Bloom = 75) RD460

7-12 B Gelatin type B FCPGP- 1 28 + +

(Bloom = 75) RD462

7-13 B Gelatin type A FCPGP- 1 1 0 + +

(Bloom = 100) RD474

7-14 B Gelatin type A FCPGP- 1 01 + +

(Bloom = 100) RD478

7-15 B Gelatin type A FCPG P- 78 + +

(Bloom = 100) RD482

7-16 B Gelatin type A FCPG P- 100 + +

(Bloom = 180) RD486

7-17 B Gelatin type A FCPG P- 98 + +

(Bloom = 180) RD490

7-18 B Gelatin type A FCPG P- 72 + +

(Bloom = 180) RD494 Experiment 8: Reference testing

For the reference experiment the method 3 was used. Solution A-H were mixed with demineralized water (pH = 8 - 9) as depicted in Table 10. Stabilization of the dispersion is depicted as + in Table 10.

Table 10. Results of experiment 8

Experiment 9: effect of different protein particles onto stabilization of different inorganic particles

Table 1 1 lists the stabilization of S1O2 dispersions by different protein particles, which is depicted as + in Table 1 1 .

Table 11. Results of experiment 9

Experiment 10: effect of different protein particles onto stabilization of different inorganic particles

For the experiment the method 3 was used. Different inorganic particles are stabilized with protein particles at pH = 8 - 9 as depicted in Tables 1 2A - 12E. Stabilization of the dispersion is depicted as + in the tables.

Table 12A: Stabilization of elongated Si0 ₂ particle dispersion D

Table 12C: Stabilization of Ti0 ₂ particle dispersion F