BACTERIAL CELLULOSE FORMULATIONS, METHODS AND USES THEREOF

Abstract

The present disclosure relates to powdered, rehydratable, bacterial cellulose formulations comprising methods of production and uses thereof. In particular the use of the formulation as a colloid stabilizer, foam stabilizer, or as a thickener, as a reinforcer material (as a filler), a dietary fibre, a foodstuff, a cosmetic or pharmaceutical composition, a composite, among others. An aspect of the present subject matter discloses a powdered formulation, comprising bacterial cellulose and an additional component (or third component) selected from the following list: sodium carboxymethyl cellulose, carboxymethyl cellulose, xanthan, methylcellulose, methyl cellulose, hydroxyethyl-cellulose, hydroxyethyl-cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methylcellulose, tylose, glycerol, saccharose, or mixture thereof; wherein the powdered formulation is dispersible in an aqueous media, at 20° C., with low shear mixing.

Claims

1. A powdered formulation, comprising bacterial cellulose; and an additional component selected from the group consisting of: sodium carboxymethyl cellulose, carboxymethyl cellulose, xanthan, methylcellulose, methyl cellulose, hydroxyethyl-cellulose, hydroxyethyl-cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methylcellulose, tylose, glycerol, saccharose, and mixtures thereof; wherein the powdered formulation is dispersible in an aqueous media, at 20° C., with low shear mixing.

2. The powdered formulation according to claim 1, wherein the low shear mixing is at most 1500 rpm.

3. The powdered formulation according to claim 1, wherein the bacterial cellulose is dispersed in an aqueous media in at most 10 min.

4. (canceled)

5. The powdered formulation according to claim 1, comprising particles having a size between 5-500 μm.

6. The powdered formulation according to claim 5, wherein the particles have a size between 80-400 μm.

7. The powdered formulation according to claim 5, wherein the particles have a D50 of 50-300 μm.

8. The powdered formulation according to claim 5, wherein the particles have a D50 of 10-90 μm.

9. The powdered formulation according to claim 5, wherein the bacterial cellulose is dispersed in an aqueous media in at most 2 min and wherein the particles comprise a size between 5-100 μm.

10. The powdered formulation according to claim 1, wherein the mass ratio between the bacterial cellulose and the additional component varies between 1:5 to 1:0.2.

11. The powdered formulation according to claim 1, wherein the additional component is carboxymethyl cellulose, carboxymethyl cellulose and xanthan; carboxymethyl cellulose and hydroxyethyl-cellulose and carboxymethyl cellulose and hydroxypropyl methylcellulose, or mixture thereof.

12. The powdered formulation according to claim 1, wherein the bacterial cellulose is obtained from a bacterium of the following list: Acetobacter, Agrobacterium, Gluconacetobacter, Achromobacter, Alcaligenes, Aerobacter, Azotobacter, Rhizobium, Salmonella, Escherichia, Sarcina, Komagataeibacter, or combinations thereof, preferably the powdered bacterial cellulose is obtained from a bacterium of the following list: Acetobacter, Komagataeibacter, Gluconacetobacter or combinations thereof.

13. A wet bacterial cellulose formulation comprising 5-70% (wt./v) of bacterial cellulose, wherein the bacterial cellulose is dispersed in bundles; wherein the bundles have a size ranging from 20 μm to 2 mm.

14. The wet bacterial cellulose of claim 13, wherein the bacterial cellulose bundle size varies between 20 μm to 2 mm.

15. The wet bacterial cellulose of claim 13, wherein the bacterial bundles have a size ranging from 200 μm to 1 mm.

16. (canceled)

17. The wet bacterial cellulose of claim 13, comprising 5-40% (wt./v) of bacterial cellulose.

18. The wet bacterial cellulose of the claim 17, comprising 10-30% (wt./v) of bacterial cellulose solids.

19. The wet bacterial cellulose of claim 13, wherein the bacterial cellulose is obtained from a bacterium of the following list: Acetobacter, Agrobacterium, Gluconacetobacter, Achromobacter, Alcaligenes, Aerobacter, Azotobacter, Rhizobium, Salmonella, Escherichia, Sarcina, Komagataeibacter, or combinations thereof.

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. A method for producing the powdered formulation of claim 1, the method comprising the following steps of: providing an aqueous suspension of ground bacterial cellulose bundles, with a size between 20 μm to 2 mm; adding a third component at a temperature between 20-80° C.; drying the bacterial cellulose until obtaining at least 60% of solids; and grinding the bacterial cellulose until obtain a particle size between 5-500 μm.

26. The method according to claim 25, wherein the concentration of bacterial cellulose in the ground bacterial cellulose suspension varies between 0.3-10% (wt./v).

27. The method according to the claim 26, wherein the concentration of bacterial cellulose in the ground bacterial cellulose suspension varies between 0.5-5% (wt./v).

28. (canceled)

29. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0085] FIG. 1—Petri dish photographs of bacterial cellulose aqueous suspension, following homogenization with A) Sammic blender, B) Comitrol Processor (2 passages), High Pressure Homogenizer, after C1) 1 passage, C2) 2 passages and C3) 6 passages. White bar scale: 1 mm.

[0086] FIG. 2—Fluorescence photomicrographs of bacterial cellulose aqueous suspension, following homogenization with A) Sammic blender, B) Comitrol Processor (2 passages), High Pressure Homogenizer, after C1) 1 passage C2) 2 passages and C3) 6 passages. BC was stained with Calcofluor white and observed in an Olympus BX51 microscope. Scale: 100 μm.

[0087] FIG. 3—Effect of the addition of bacterial cellulose, plant nanocellulose and hydrocolloids on the overrun of whipped egg whites.

[0088] FIG. 4—Petri dish photographs of bacterial cellulose aqueous suspensions, following homogenization with Sammic blender, Comitrol Processor (6 passages) and High Pressure Homogenizer. Samples were concentrated to 20% solids and diluted and redispersed in water. White bar scale: 1 mm.

[0089] FIG. 5—Fluorescence photomicrographs of bacterial cellulose aqueous suspensions following homogenization, concentration to 20% solids and dispersion dilution in water. BC was stained with Calcofluor white and observed in an Olympus BX51 microscope. Scale: 100 μm.

[0090] FIG. 6—Stability of whipped egg whites over time, as prepared by the addition of bacterial cellulose ground with Sammic blender (at 0.1, 0.2 and 0.3%), xanthan or Bioplus.

[0091] FIG. 7—Classification of the dispersibility of never-dried mixtures of BC and third components. 1—sample is homogeneous and well dispersed; 2—sample contains some very small BC aggregates; 3—sample contains some larger BC aggregates

[0092] FIG. 8—Fluorescence photomicrographs of never-dried BC:CMC aqueous suspension, following homogenization in a A) Sammic blender, B) Comitrol Processor (2 passages) and High Pressure Homogenizer, after E) 6 passages. Following mixing, BC:CMC was dispersed with either I) magnetic stirrer or II) ultraturrax. Samples were stained with Calcofluor white and observed in an Olympus BX51 microscope. Scale: 50 μm.

[0093] FIG. 9—Rheological profile of 0.5% (m/v) BC:CMC samples diluted and dispersed using I) ultraturrax and II) magnetic stirrer.

[0094] FIG. 10—Rheological profile of 0.5% (m/v) BC:CMC samples diluted and dispersed using I) ultraturrax treatment and II) magnetic stirring.

[0095] FIG. 11—Rheological profile of 0.5% (m/v) BC:CMC samples ground at different particle sizes.

[0096] FIG. 12—Examples of % stability.

DETAILED DESCRIPTION OF THE INVENTION

[0097] The present disclosure is also further described, in particular, using embodiments of the disclosure. Therefore, the disclosure is not limited to the descriptions and illustrations provided. These are used so that the disclosure is sufficiently detailed and comprehensive. Moreover, the intention of the drawings is for illustrative purposes and not for the purpose of limitation.

[0098] The present disclosure relates to powdered and wet BC formulations, methods of production and uses thereof.

[0099] The powdered BC formulation of the present disclosure is useful for use in medicine, food, cosmetic, among others.

[0100] The present disclosure concerns with the conditions by which a dried powdered BC formulation, capable of being dispersed in aqueous media under 5 minutes, at room temperature, using low shear mixing, can be obtained. Such formulation preserves the technical properties of the non-dried material, in particular but not only, the potential as a colloid stabilizer.

[0101] The present disclosure also relates to a never-dried BC, methods of production and uses thereof.

[0102] In an embodiment, the never-dried BC (or wet BC) of the present disclosure is useful for use in medicine, food, cosmetic, among others.

[0103] The present disclosure also concerns with the conditions by which a wet BC suspension can be obtained, also capable of quick and low energy dispersion, comprising the comminution of BC to a specific size range, concentrating the aqueous suspension of BC to a final solids content of between 10% by dry mass or more and less than 70% by mass and dispersing it again into an aqueous solution.

[0104] For clarification purposes, the description of the present disclosure is divided into the following sequential steps: [0105] 1. Wet comminution of BC (production of “ground” BC); [0106] 2. Mixing of the ground BC with a third component; [0107] 3. Drying of BC formulations; [0108] 4. Grinding of BC formulations; [0109] 5. Alternatively to the steps 2-4, ground BC may be concentrated without being mixed with a third component.

Example 1—Wet Grinding of Bacterial Cellulose

[0110] In an embodiment, a BC obtained by any means as described in the previous section, in an amount of the range between 0.5% (m/v) and 10% solids (BC) preferably between 0.5% and 5% solids, preferably in the range of 0.7-1% (w/v) was wet ground by three different methods: [0111] A) Using a Sammic fixed speed blender, model TR250 at 9000 Rpm (Samic, S. L.) until a homogeneous pulp was obtained, as observed by visual inspection; [0112] B) By submitting BC to 2 passages through a Comitrol® Processor, Model 1700 (Urschel Laboratories Inc.). In each passage, grinding of BC was done at 900 Rpm, using a labyrinth verycut impeller and a cutting head 160 at 5° inclination; [0113] C) By High Pressure Homogenization (HPH) using a GEA Niro Soavi, model Panther NS3006L, at 600 Bar. Homogenized BC was collected after 1 passage (HPH 1 Pass), 2 passages (HPH 2 Pass) and 6 passages (HPH 6 Pass), through the High-Pressure Homogenizer. Before HPH processing, BC suspensions were first ground described in method A, to facilitate feeding of the suspension to the homogenizer.

[0114] BC suspensions were spread over petri dishes and observed with a SZ40 Zoom Stereo Microscope (Olympus). Photographs were taken with a camera SONY AVC-DSCE and adapter CMA-DSCE, at a magnification from 0.67× to 5× (FIG. 1). BC suspensions were also stained with Calcofluor white and observed in an Olympus BX51 microscope.

[0115] Well-dispersed suspensions are a prerequisite in many industrial applications. BC suspensions are known to exhibit pronounced aggregation in aqueous media, due to strong interfibrillar hydrogen bonds and Van der Waals attraction, an effect that is concentration dependent. These attractive interactions, in combination with the long aspect ratio of the fibres, cause the formation of extended networks when BC is dispersed in water. A ball of threads is obtained irrespective of whether BC is produced in a stirred tank or by static fermentation and then wet grinded. These highly heterogeneous dispersions consist of fibre bundles, flocs, and voids spanning tens to hundreds of micrometres depending on concentration and shear homogenization conditions (FIG. 1 and FIG. 2). In this example, three BC homogenization methods were used, with increasingly shear stress, from a blade blender (A), to high pressure homogenizer (C).

[0116] It was observed that with the increase in the shearing stress (from A to C), an increase in the BC defibrillation/fragmentation occurred. Homogenization of BC with Sammic blender showed larger fibre aggregates and BC fragments within 200 μm to 1-1.5 mm size. BC homogenized with Comitrol Processor and HPH, lower amounts of millimetric fragments were observed, with most of them being in the range of 200-600 μm (Comitrol), 100-200 μm (HPH 1 pass) and 20-100 μm (HPH 6 pass).

[0117] Increasing the number of passes by HPH resulted in the appearance of shorter fibres alongside with their increased separation. Due to their attractive forces and crosslinking, despite extensive deagglomeration, BC fibres always appear to be interconnected to neighbouring fibres, creating a 3D network of very fine fibres and other void areas containing only water. This effect occurred even at low BC concentrations (as low as 0.5% (w/v)) so that stable dispersions of well spread single fibres was never achieved.

Example 2—Effect of Bacterial Cellulose on the Overrun and Foam Stabilization of Whipped Egg Whites

[0118] Many food products are emulsions, foams or whipped emulsions (e.g. milk, cream, ice cream, butter, mayonnaise, finely minced meat like sausage meat, whipped egg white, whipped cream bread, etc). Foam and emulsion properties are strongly influenced by the interfacial properties of the emulsifier in the system. Polysaccharides (e.g. modified starches) and proteins, often play an important role in the formation and stabilization of these colloidal systems. These macromolecules provide the interface with physicochemical and rheological properties (steric hindrance, electrostatic repulsion, viscoelasticity), which determines the resistance to droplet coalescence. During the beating of egg whites, proteins adsorb on the newly-formed interface, denature and form a “gel” network that traps air bubbles and whose rheological properties govern the stability of the foam.

[0119] These non-dairy aerated products pose special concerns and constraints because the bubble wall is thin, relatively weak and unsupported (in contrast to oil droplets, which are supported by the mass of the oil). In these systems, two requirements exist. The first, a physical stabilization of the liquid within the interstitial regions of the foam. The second, a strengthening of the foam walls, accomplished using gums and other ingredients in the mix. Microcrystalline cellulose (MCC) and its derivatives, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), methyl ethylcellulose (MEC) and other polysaccharide gums, such as xanthan have been used for the stabilization of foams.

[0120] In this embodiment, the effect of BC on the overrun and foam stabilization of whipped egg whites was evaluated. For this, BC from different sources (from K. xylinus strain ATCC 700178 and “nata de coco” from HTK Food CO. Ltd. (Vietnam), ground with a Sammic blender as described in Example 1, were used (code samples: 700178, S; Nata de coco, S. BC from ATCC 700178 was further homogenized with Comitrol and High Pressure Homogenization (Example 1) (code samples: BC Comitrol, BC HPH 1 Pass, BC HPH 6 Pass).

[0121] To compare the performance of BC against other hydrocolloids and plant nanocelluloses, several other samples were used in parallel: CelluForce NCC, Arbocel B 600, Arbocel BE 600/30 (from Celluforce Inc.) and Bioplus (from American Process Inc.) as sources of plant nanocellulose; xanthan, carboxymethyl cellulose, carrageenan (from Sigma) as examples of hydrocolloids.

[0122] As previously mentioned, BC suspensions are known to exhibit pronounced aggregation in aqueous media, due to strong interfibrillar hydrogen bonds and Van der Waals attraction, an effect that is more pronounced as the concentration of BC in aqueous media increases. To evaluate if concentrating BC affects the fibres dispersibility and functional properties, all BC samples were concentrated to 20% (wt/v) solids (code samples: 700178, S-P and Nata de coco, S-P), diluted and redispersped in water, under magnetic stirring at 900 Rpm for 2 hr. According to the present embodiment, the BC concentrate may be prepared by dehydration with any known means such as a filter press, belt-press, centrifugation, vacuum filtration or any other method know to those of ordinary skill in the art.

[0123] Dried egg whites (12%, wt/v) were dispersed in water and whipped (using a Silvercrest kitchen blender). During whipping, each BC aqueous suspension or any other plant cellulose or hydrocolloid sample, was added to a final concentration of 0.1-0.3% (wt/v). A control was also done using only dried egg whites. The whipped cream's overrun was determined by measuring the volume increase of the whipped cream, relative to the control.

[0124] As observed in FIG. 3, at 0.3% (wt/v) addition to the mixture, all BC samples ground with Sammic blender (700178, S and Nata de coco, S) showed a remarkable increase in the whipped cream's overrun. Contrarily, to the exception of Celluforce, with a marginal increase of 6% over the control, all plant nanocelluloses and hydrocolloids decreased the whipped cream's overrun. Increasing the extent of the homogenization of BC (HPH), which, as observed, decreases the size of the BC aggregates, decreases the capability of BC to stabilize the air/liquid interface, thus decreasing the cream's overrun. Finally, following compression of BC to 20% (wt/v) solids, low shear dispersion does not allow the full lengthening of the BC fibre aggregates (FIG. 5) when diluted and redispersed in water; however, provided the BC aggregates size is unaffected, compressing BC (FIG. 4) does not affect the capacity to stabilize the foam.

[0125] These results clearly demonstrate the influence of the BC's particle size, as obtained by wet grinding, on the stabilization of protein foam.

[0126] Also, the stability of the foam was registered over time, at room temperature by evaluating, qualitatively, the accumulation liquid at the bottom of the flasks (FIG. 6). Regardless of pressing or not the BC, results also showed that BC ground with the Sammic blender, at 0.3% (wt/v) in the final mixture, results best in the stabilization of whipped egg whites (as no liquid accumulation was observed) as compared to the control, showing better performance than the other materials tested.

Example 3—Mixing Bacterial Cellulose with a Third Component

[0127] In an embodiment, the BC obtained by any of the three different comminution methods described in Example 1 was mixed with either sodium carboxymethyl cellulose, CMC (90 KDa, or 250 KDa or 700 KDa, Sigma), xanthan (Sigma), methylcellulose, MC (Sigma), hydroxyethyl-cellulose, HEC (Sigma), hydroxypropyl methyl cellulose (Sigma), HPMC (Sigma), tylose (Sigma), glycerol (Sigma) and saccharose (Sigma); BC was also mixed with combinations of CMC:Xanthan, CMC:HEC and CMC:HPMC, as described in the following table:

TABLE-US-00001 TABLE 1 Combination of bacterial cellulose and third components Mixture Mass ratio BC:CMC 1:1   1:0.75 1:0.25 BC:Xanthan 1:1   BC:MC BC:HEC BC:HPMC BC:Tylose BC:Saccharose BC:Glycerol 1:1.6  1:1.3  BC:CMC:Xanthan 1:0.25:0.75 CB:CMCHEC l:0.5:0.5 CB:CMC:HPMC 1:0.75:0.25

[0128] In all of these cases, the components to be added to BC were first dissolved in water. Those of ordinary skill in the art can appropriately select the addition amount of the third components, as a redispersing aid, according to the type of substance and the like, usually within 2 to 1,000% (wt/v) by mass of BC.

[0129] To evaluate the dispersibility of the BC mixtures, samples were prepared at 0.5% (wt/v) in water to at room temperature. For this, two dispersion method were used: [0130] I) a high shear dispersion mixing, using an ultraturrax CAT Unidrive 1000D, at 15000 Rpm using a dispersing shaft CAT 20F, also for 5 min and [0131] II) a low mechanical shear dispersion using magnetic stirrer plate (Stuart SD162), at 900 Rpm, for 5 min

[0132] As a control, BC, as obtained by any of methods A, B and C mentioned above (Example 1), was used at the same final solids concentration. The dispersed materials were spread over a petri dish and observed at naked eye. Also, fluorescent microscopy was used to better visualize the BC fibres. As an example, the following table, FIG. 7 and FIG. 8 summarize the main observations pertaining to the BC and CMC mixtures.

TABLE-US-00002 TABLE 2 Dispersibility of never-dried mixtures of BC and third components. Dispersibility Component Dispersion Classification* BC:CMC 90 KDa (1:1): A) Sammic I) UltraTurrax 1 blender B) Comitrol II) Magnetic 2 Processor stirrer HPH C) 1, 2, 6 Pass I) UltraTurrax 1 II) Magnetic 2 stirrer BC:CMC:HPMC 1:0.5:0.5 1) UltraTurrax 2 A) Sammic BC:CMC:HEC 1:0.75:0.25 A) Sammic BC:Xanthan 1:1 B) Comitrol *According to embodiments of paragraphs [0027] and [0066]

[0133] While samples dispersed using an UT showed complete dispersibility (minimum amount of fibre bundles were observed), samples dispersed using a magnetic stirrer still showed fibre bundles and agglomerates (FIG. 8 and Table 2). However, the presence of the third component improves the dispersion of the aggregates, more homogeneous samples being observed (FIG. 8 vs FIG. 2). Also, further homogenization with ultraturrax for up to 60 min, does not change the morphology nor the rheological profile of the BC:CMC mixtures (data not shown).

[0134] It was observed that when CMC or xanthan were added to BC, at preferably a mass ratio of 1:1, a dispersible and stable suspension was obtained, following high shear treatment with ultraturrax. Regarding the control, BC alone, it could not be dispersed by any of the dispersion methods.

[0135] From these results, the water-soluble anionic polyelectrolyte CMC plays a determinant role in ensuring the dispersibility of the BC fibres and allowing their stabilization in aqueous media. The negative charge of CMC may contribute to the improved dispersion of the BC fibres due to steric hindrance. Also, CMC has a strong affinity for water molecules. Non-adsorbed CMC may also prevent the agglomeration of BC fibres due to the creation of a hydration shell in aqueous media, around CMC and BC thus also contributing to the improved dispersibility and stability in aqueous media.

[0136] The BC:CMC samples were further characterized by rheological assays (FIG. 9). For this, stress-strain curves were obtained using a TA instruments rheometer, model 5332-1179 and a disk geometry. Shear rate versus viscosity graphs were drawn in semi-log scale to better visualise the different rheological profiles at low shear rates. From these assays, it may be concluded that the use of higher shear rate stress during wet comminution decreases the dynamic viscosity of the samples (from A to E, FIG. 9). As previously mentioned, this effect is caused by some fragmentation of the fibres with increasing shear stress. Also, to the exception of samples ground with B) Comitrol, which show a coincidental rheological profile, all other samples showed higher rheological profile after dispersion with I) ultraturrax, which suggests that the high shear rates used by ultraturrax, exerted a better dispersion of the BC fibres, without compromising its viscosity—actually improving it.

Example 4—Drying of Bacterial Cellulose Mixtures

[0137] In an embodiment, the BC was wet ground by three different methods, as described in Example 1 (methods A, B and C). The obtained BC was mixed with a third component, as described in Example 3. The obtained BC mixtures can be dried by means of any of the available drying processes and equipment, which include spray drying, drum drying, oven drying, vacuum drying, tunnel drying, infra-red drying, freeze drying. Those skilled in the art may select any other proper drying process and/or combination of methods. In the present disclosure, BC mixtures were dried by four methods:

i) Drying on a hot plate, a fast drying process (within minutes), whereby a thin layer of BC samples was spread over a hot plate (130° C.) of an Ariette Crepes Maker, model 183 and dried; the “dry” state is an absolute dried BC product (100% TS (total solids)), in which there is no residual water in the final product.
ii) Drying in an oven, a slower drying process, in the range of 4-6 hr. For this, samples were placed in aluminium crucibles and dried at 80° C. To evaluate the effect of residual moisture on the dispersibility of BC formulations wet ground with Comitrol Processor, drying was done to a final solid content of a) 80% (m/m) (80% TS) and b) 100% (m/m) (100% TS).
iii) Drum drying, also a fast drying process, was also used. For this, samples were fed to a lab-scale chrome plated cast iron double drum dryer, from Tummer Simon Dryers (drum size 0.3m×0.3m giving a drying surface area of 0.28 m.sup.2 per roll (0.56 m.sup.2 total). Feed was hand fed (‘jugged’) into the nip at room temperature, at a rate was 11.9 Kg/hr. The nip gap was set at 0.1 mm. Drying was done at 2-4 bar steam pressure at a roll speed of 2.5 Rpm. Thin product sheets were obtained at a rate of 0.86 Kg/hr at around 20% residual moisture.
iv) Samples were also dried by spray drying. For this, a sample at 1% (m/v) was fed to a MOBILE MINOR™ R&D Spray Dryer (GEA Group AG) at a flow rate of 240 Kg/h. The inlet and outlet temperatures were set to 220 and 115° C., respectively and a pressure gas nozzle of 3.5 bar was used. In this case, samples were dried to 100% TS.

[0138] All dried material was ground (with a High Power Herb Grain Grinder Cereal Mill Powder Grinding Machine Flour 600G) and sieved (Mat.Mesh:AISI 316 no 1/2871/1 No: 1/2871/1 with an opening of 0.300 mm) to a final particle size preferably in the range of <300 μm. As in the previous examples, the dried powders were dispersed at a final solids concentration of 0.5% in water, using:

I) a high shear dispersion mixing, using an ultraturrax CAT Unidrive 1000D, at 15000 Rpm using a dispersing shaft CAT 20F, for 5 min and
II) a low mechanical shear dispersion using magnetic stirrer plate (Stuart SD162), at 900 Rom, also for 5 min.

[0139] Rheological assays were done using a TA instruments rheometer, model 5332-1179 and a disk geometry. Shear rate versus viscosity graphs were drawn in semi-log scale to better visualise the different rheological profiles at low shear rates.

[0140] The following table summarizes the main observations regarding the aqueous dispersibility of the dried and ground BC:CMC 90 KDa mixture.

TABLE-US-00003 TABLE 3 Dispersibility of BC:CMC mixtures after drying and grinding to a particle size of <300 μm. Dispersibility Homogenization Drying Dispersion Classification* A) Sammic i) Hot plate I) UltraTurrax 1 blender ii) Oven dried II) Magnetic B)Comitrol i) Hot plate stirrer 2 Processor ii) Oven dried  80% TS 100% TS HPH 1, 2 and 6 Pass i) Hot plate I) UltraTurrax 1 II) Magnetic 2 stirrer *According to embodiments of paragraphs [0027] and [0066]

[0141] As was observed for FIG. 8, water dispersion of dried and ground samples under low shear (magnetic stirring) showed a few fibre blundes, whereas with a high shear rate (ultraturrax), the BC was completely dispersed. The highest viscosity profile of BC:CMC was obtained from BC ground with a A) Sammic blender and dried on a hot plate to 100% TS (FIG. 10). The rheological profile was actually higher (higher dynamic viscosity) than the one of non-dried sample. As for the remaining samples, all of the ones dispersed with low shear stress (II—magnetic stirrer) showed a similar rheological profile to those of the never-dried ones. These results suggest that the use of low shear stress dispersion mixing, at room temperature, under 5 minutes is enough to allow for a recovery or even slight increase in the rheological profile of BC:CMC dried formulation. However, the presence of small aggregates of BC in the suspension may contribute to the increase in the apparent viscosity profile. Contrarily to the never-dried samples, dispersion of the dried ones with ultraturrax reduced the rheological profile (as compared to the samples dispersed in the magnetic stirrer), possibly due the reduction of some fibre bundles and agglomerates. Further homogenization for up to 60 min with ultraturrax does not change the rheologic profile of the samples (data not show).

[0142] Contrarily to what has been proposed in other documents, when a dispersing aid is added to BC, such as CMC, drying can be done to the extent of the full removal of water molecules, as dried samples with 80 and 100% TS, showed an identical rheological profile (FIG. 10B). A similar observation was recorded for samples dried using a drum dryer (80 and 100% TS) and spray dryer (100% TS).

[0143] Too extensive wet comminution of the BC suspension such as with HPH (FIG. 10 C, D, E) strongly affected the rheological properties of the dried material, confirming the effect previously observed on the never dried samples.

[0144] A dry control without the addition of CMC was also prepared. Results showed that BC alone is not dispersible, regardless of the homogenization, drying and dispersion method used.

Example 5—Effect of the Particle Size on the Dispersibility and Rheological Profile of Bacterial Cellulose Mixtures

[0145] In the previous example, the dried BC formulations were ground to a particle size <300 μm. It is important, however, to better understand the effect of the particle size in the restauration of the properties of BC mixtures. For this, the BC mixtures obtained in the previous example were further ground and sieved to a final particle size <200 μm (Endecotts, Ltd, aperture 212 μm) and <100 μm (Endecotts, Ltd, aperture 106 μm). Also, samples were fractioned into the following size ranges: 100<x<300 μm, 300<x<500 μm. As in the previous examples, the dried powders were dispersed to a final solids' concentration of 0.5% in water, evaluated by spreading over a petri dish, fluorescent microscopy and through rheological profile.

[0146] As an example, table 4 and FIG. 11 summarize the main observations pertaining to the dispersibility and rheological behaviour of BC:CMC 90 KDa. To simplify the demonstration of the results, only those obtained using hot plate drying are shown. The same profile was observed for samples dried in an oven.

TABLE-US-00004 TABLE 4 Dispersibility of BC:CMC mixtures after drying (in a hot plate) and grinding. Dispersibility Homogenization Particle size Dispersion (Classification)* A) Sammic <100 μm I) UltraTurrax 1 blender II) Magnetic B)Comitrol <100 μm stirrer Processor <200 μm HPH C) 1 Pass <100 μm D) 2 Pass E) 6 Pass A) Sammic 100 < x < 300 μm II) Magnetic 2 blender 300 < x < 500 μm stirrer** 3 *According to embodiments of paragraphs [0027] and [0066] **Stirring for 30 min

[0147] Results show that, decreasing the particle size of the BC:CMC allows for a faster dispersion of BC mixtures (under 1 min in the case of particles ground to <100 μm) at low shear rates (magnetic stirring). Increasing the particle size (from e.g. <100 μm, to 300<x<500 μm), decreases the capability of fully dispersion under low shear mixing conditions, even after mixing for 30 min. However, with the decrease in particle size, a decrease in the viscosity profile was also observed (FIG. 11). In addition, as examples below will show, this impacts the technological performance of BC mixtures.

Example 6—Effect of Particle Size of Dried BC Formulations on the Suspension Stability of Cocoa Beverage

[0148] In an embodiment, the suspension stability of water-insoluble solid particles such as cocoa, powdered green tea, and calcium carbonate are important aspects for the development of certain commercial beverages. In the case of chocolate milk, cocoa particles tend to precipitate soon after the initial mixing.

[0149] In this example, the effect of particle size of dried formulations of BC:CMC 90 KDa, obtained as described in Example 5, on the stabilization of cocoa particles suspensions in a chocolate milk beverage was evaluated. For this, BC:CMC at different particle sizes and final concentrations in the range of 0.075-0.15% and pure cocoa (1.2%) were weighted. Medium-skimmed milk, 15 mL, was added to each BC:CMC concentration. The mixture was stirred in a vortex (2,800 rpm) for 3 min at room temperature and then pasteurized at 75° C. for 15 seconds. Samples were stored at room temperature and the sedimentation of cocoa was evaluated. Further, the effect of ultraturrax on the functional properties of the BC:CMC samples was assessed. For this, BC samples were homogenized with ultraturrax (CAT Unidrive 1000D, at 15000 Rpm using a dispersing shaft CAT 20F) for 4 and 30 min before being added to the milk. In parallel, the same test was done using xanthan, carboxymethyl cellulose, colloidal plant celluloses: Avicel (RT 1133, LM 310 and CM 2159, FMC Biopolymers), Novagel (RCN-10 and RCN-15, FMC Biopolymers), Bioplus Fibrils (a microfibrillated cellulose, not used in food applications, American Process Inc.) and plant nanocelluloses: CelluForce NCC, Arbocel B 600, Arbocel BE 600/30 (from Celluforce Inc.). All Avicel and Novagel celluloses were previously activated, for 30 min, at 23,800 Rpm, according to the specifications sheets. A control assay, where no stabilizers were added, was also made. Also controls with never-dried BC (ground using methods A, B, C and D described in Example 1) to which CMC was added to, were done. In this case, CMC was mixed with BC using I) a low shear dispersion mixing, as described in Example 3. The stabilization of the chocolate drinks was assessed by calculating the percentage of sedimentation, according to the following equation:

Stability (%)=(Milk with chocolate mL×100)/(total milk volume)  (1)

[0150] The higher the stability (%), the more stabilized the suspension is.

[0151] The results from this study are displayed in the following table and FIG. 12, using 0.15% BC:CMC (other concentrations were tested with similar observations).

TABLE-US-00005 TABLE 5 Stabilization of cocoa particles in chocolate milk, with BC:CMC (redispersed under low shear rate unless otherwise specified), added at 0.15%, at room temperature. Particle size Stability/% Homogenization Drying μm 1 h 24 h 4 days A) Sammic i) Hot plate <100 100 100 83 blender <300 100 100 100 Never dried 100 100 100 Never dried (UT)  4 min 100 100 100 30 min 100 100 100 B) Comitrol i) Hot plate <100 100 100 100 Processor <300 100 100 100 Never dried 100 100 100 HPH C) 1 Pass i) Hot plate <100 100 67 50 <300 100 100 100 Never dried 100 100 100 D) 2 Pass i) Hot plate <100 100 67 50 <300 100 100 83 Never dried 100 100 83 E) 6 Pass i) Hot plate <100 100 27 33 <300 100 100 50 Never dried 87 83 67 E) 6 Pass, <100 μm i) Hot plate (UT)  4 min 100 67 50 30 min 30 30 27 Avicel CM 2159 UT 30 min 95 0 0 Novagel RC-15 96 0 0 Novagel RC-10 96 0 0 Bioplus Fibrils 70 40 35 CelluForce NCC 100 100 100 Arbocel B600 30 30 27 Arbocel BE600/30 30 30 27

[0152] These results show that an extensive homogenization through High Pressure Homogenization and extensive grinding of dried material to a particle size <100 μm, reduces the stabilizing effect of BC:CMC. Treatment with UT of never dried BC:CMC processed with the Sammic blender, does not change its stabilizing effect on cocoa particles. However, under extreme processing conditions such as HPH 6 passages, drying and comminution to <100 μm, UT treatment affects the cocoa stability achieved with the BC:CMC.

[0153] To the exception of Celluforce, all colloidal plant celluloses and nanocelluloses showed a very low stabilizing effect on cocoa particles.

[0154] Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term “a polysaccharide” or “the polysaccharide” also includes the plural forms “polysaccharides” or “the polysaccharides,” and vice versa. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0155] Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0156] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0157] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0158] The above described embodiments are combinable.

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