Patent Description:
The powdered bacterial cellulose formulations of the present disclosure are useful in medicine, cosmetic, food, detergents, polymers and composites industries, among others.

Bacterial cellulose (BC) is an exopolysaccharide synthesized by certain Gram-negative, obligate aerobic, acetic acid bacteria, the Komagataeibacter genus being the most important due to the high cellulose yield obtained. Morphologically BC is comprised of randomly assembled ribbon-shaped fibrils less than <NUM> wide and composed of aggregated bundles of elementary nanofibrils; these fibrils have a lateral size of <NUM>-<NUM> and several micrometres in length. As Komagataeibacter microorganisms are mandatory aerobes, under static conditions, BC is synthesized at the air/liquid interface of the culture medium [<NUM>-<NUM>].

For bulk commercial applications, dried BC formulations offer several advantages over aqueous suspensions, such as a decrease in the size and mass, thus lower storage space and transportation costs, improved storage stability, lower risk of contamination. It is known that, as with plant cellulose, the properties of BC are mostly lost upon drying. As water evaporates, the fine fibres from BC aggregates, establishing hydrogen bonds that are not easily broken upon resuspension in water [<NUM>]. Several attempts have been made to prepare dried BC formulations, some of these, addressing the restauration of the cellulose properties after aqueous dispersion, as will be reviewed below.

Document <CIT> [<NUM>] is related to the use of BC as a dietary fibre, to absorb/bind cholesterol (in vitro). BC, alone or in combination with sucrose, was either freeze-dried or frozen and cryomilled. While reasonably effective in preventing hydrogen bonding between cellulose fibres, these are rather costly solutions for industrial implementation. In this document, a comparison of the properties of BC, before and after drying, was made only in terms of the effect on cholesterol adsorption. Also, the authors recognize that, regardless of the drying method, drying BC alone strongly reduced the cholesterol adsorption properties of BC, as compared to the never-dried BC suspension. The addition of sucrose only partially mitigated such limitation.

Document <CIT> [<NUM>] describes a drying method for BC. High pressure homogenization and thin screw extrusion, in both cases with temperature treatment, were studied for the homogenization of BC. The BC was then dried by drum drying, spray drying, extrusion drying, oven drying and frying in oil. BC was also dried in the form of sheets and ground-milled. Regardless of the different drying method, maximum BC swelling took from <NUM> to up to <NUM> hr. This is unpractical for industrial applications; also, this document did not establish whether the properties of the swelled BC are equivalent to those of the never-dried one.

Document <CIT>[<NUM>] proposes the addition of a third component to an aqueous BC suspension before drying, through several methods, such as oven drying, drum drying, infra-red drying. In this document, disintegration of BC was done with a kneader extruder at maximum speed, for up to <NUM>, before drying. Examples of the tested third component included calcium carbonate, at <NUM> times the mass of BC, to obtain a dispersible product (under <NUM> sec, stirring with a spatula). A mixture of BC with calcium carbonate at <NUM> times the amount of BC and heat treated (<NUM>, <NUM>) after mixing in water with a spatula, results in a dispersible suspension. When glycerine was used as a third component, at a mass ratio BC:glycerine of <NUM>:<NUM>, a dispersion could be achieved also by first heating the aqueous mixture to <NUM>, for <NUM> hr. Without heating, glycerine, at <NUM> times the amount of BC was required to allow obtaining a dispersible product. However, only when glycerine was added at <NUM> times the amount of BC (or <NUM> times if previous heating was used), nearly <NUM>% of the initial product viscosity could be recovered. In all these cases, the stability of the dispersed products (as assessed by the precipitation degree following centrifugation) was low. BC was also mixed with carboxymethyl cellulose (CMC) and dried. A mixture composed of a mass ratio of BC:CMC of <NUM>:<NUM> was dispersible, with <NUM>% of the initial product viscosity recovery and <NUM>% stability recovery. Other polysaccharides (such as dextrin, xanthan, soluble starch) and combinations of polysaccharides and glycerin or polysaccharides and CMC, were tested as the "third" component, to be added to BC aqueous suspensions before drying. To the exception of BC:dextrin at <NUM>:<NUM> mass ratio, all mixtures required an excess of the third component, to allow for an almost complete viscosity and stability recovery, following aqueous dispersion. One example of that document, related to the preparation of a BC/dextrin mixture, addresses the grinding of the dried product, to a final particle size of <NUM> or less. Aqueous dispersion, at <NUM>%, of the powder, with magnetic stirring, took <NUM> seconds, with complete recovery of the viscosity, as compared to the non-dried mixture. The dispersion time was defined as the time until large macroscopic lumps were no longer observable to the naked eye. However, no details were provided on the mass ratio of BC and dextrin, in this specific mixture. In all these cases, after stirring in water, the dried material was submitted to heat treatment at <NUM>, for <NUM>. Clearly, heat treatment (a time consuming and costly solution for industrial implementation) and excess of a third component (resulting actually in a small amount of BC in the final product) are required to obtain a dispersible product.

Document <CIT> [<NUM>] proposes several drying methods of BC alone (i.e. without the need of adding a third component to the aqueous BC suspension), allowing the re-stauration of its properties. According to this document, during drying and dehydrating of BC fibres, tensile stress is exerted on both the crystalline and amorphous parts of the fibres. Since elasticity is higher in the crystalline parts than in the amorphous ones, a degree of strain for the tensile stress is higher in the amorphous parts. As bonds in the amorphous parts of the cellulose fibres consist of hydrogen bonds between and within cellulose molecules, the distance between the hydrogen bonds will be enlarged due to the high degree of strain, allowing water molecules to penetrate. As a result, the hydrogen bonds between the BC fibrils with the high degree of strain will be easily broken upon the addition of water and the hydrogen bonds between BC and water molecules, which existed before dehydration and drying, will be easily re-formed. In such document, the "dry" state means a state in which water is present in an amount of about <NUM>% or less on the basis of dry BC mass. To demonstrate the dispersibility of dried BC, a BC homogenate (blended at <NUM> Rpm, for <NUM>, with a blender) was dried by pouring the BC homogenate into trays and using either air drying, oven drying, infra-red drying; a BC homogenate was also freeze dried and dried under vacuum or in a drum dryer. Then, a BC aqueous suspension was prepared by further homogenization of the dried BC, using an ultraturrax (Physcotron), at maximum speed (<NUM> Rpm) for <NUM>. At such rotational speeds, ultraturrax exerts quite a substantial shear stress, thus most likely inducing extensive BC fibre breakdown and/or defibrillation. This is further reinforced by a noticeable decrease in the mechanical properties of dried BC sheets that were previously submitted to ultraturrax treatment, as compared to BC that was only ground with a blender (control). Further, the authors claim a good recovery of the dispersibility and viscosity of dried BC (to the exception of spray-drying), but the never-dried BC samples (controls) were only submitted to homogenization at <NUM> Rpm for <NUM>, not ultraturrax. The obtained results are therefore not comparable. From the above, regardless of the drying method, a high shear force is most likely responsible for the dispersion of the BC fibres. For industrial applications, this implies added capital investment (the need for a high shear mixing equipment) and operating costs.

Documents, <CIT> and <CIT> [<NUM>, <NUM>], describe the addition of polysaccharide hydrocolloids as co-agents, to BC, before drying. Dispersion of the dried material could be done by high shear mixing (e.g. high pressure homogenization at <NUM> psi [<NUM>] or <NUM> rpm for <NUM> with a Silverson mixer, <NUM> psi for <NUM> passes with an extensional homogenizer and <NUM> rpm for <NUM> [<NUM>]), with or without the addition of additional co-agents. These documents propose the use of alcoholic solutions, for the co-precipitation of the BC-hydrocolloid mixture; this is followed by concentration, drying and milling. However, for large-scale implementation, very high alcohol volumes are required for the co-precipitation. As such, the use of alcohols, the inevitable product losses (from the non-precipitated material), the need for alcohol recovery equipment, all contribute significantly to the total cost of production. The use of such organic solvents also poses additional safety and environmental concerns. In these documents, the fermented broth containing BC is treated with hypochlorite, glycosidases and proteases (to lyse the cells). Hydrocolloids are mixed and then organic solvents are added. However, co-precipitation of culture media components and bacterial cell hydrolysates will also occur, thus limiting the degree of purity (hence the quality) of the final product. The use of charged hydrocolloids further aggravates the co-precipitation of unwanted substances, due to ionic interactions.

Another document, <CIT> [<NUM>], discloses a process for drying reticulated (crosslinked) BC without co-agents: this involves dispersing a BC suspension in an organic solvent using high shear mixing and milling, concentrating the dispersion to substantially remove the solvent, and drying. Optionally, the dried material may be ground. Exemplary organic solvents useful in this disclosure include hydrocarbons, alkyl alcohols, alkyl sulfoxides, mixtures thereof or aqueous mixtures thereof. Preferably, the cellulose material is dried using a multi-step process comprising alternating drying and grinding processes. As with the previous documents, here too, a strong limitation exists regarding the use of organic solvents. The proposed method also presents superfluous unpractical steps for use at industrial scale, namely the addition followed by removal of the organic solvent and the multi-stage drying and grinding. Also, in all examples, co-agents (one or more) such as xanthan, CMC, sucrose, were mixed with dried BC before aqueous dispersion. Further, no details on the particle size, required time and energy requirements for dispersion of the dried material (only one example of such document describes the milling of dried BC to <NUM> mesh size (< <NUM>)). Finally, a comparison of the properties of the BC before and after drying was not presented.

From the above, several approaches have been considered to obtain redispersible BC formulations, some of which claiming the restoration of its properties, as compared to those of the never-dried formulations. However, the redispersion methods involve the use of high energy mixing and/or high temperature and/or long dispersion times and/or the use of excess amounts of third components, still with limited results regarding the recovery of the properties of the original material. Further, these energy and time intensive processes represents high capital and operating costs, when considering industrial applications.

Most of the mentioned documents do not fully demonstrate the recovery of the BC technological properties upon redispersion, being mainly focused on demonstrating the BC's redispersibility, which does not necessarily imply the recovery of the technological properties of the never-dried material. This is also the case with document <CIT> [<NUM>], that, instead of a dried product, provides a method of producing concentrated BC having improved properties such as dispersibility, suspensibility and viscosity, encompassing the homogenization (wet grinding) and concentration of BC. As observed for the other documents, the claimed restauration of the BC properties is limited to demonstrating the redispersibility as obtained by different concentration methods, different targeted BC final concentrations and different redispersing methods. No information was provided on the particle size range of the ground material, nor on the functional properties of the redispersed one.

As will be observed in the present disclosure, the apparent (macroscopic) BC redispersion or the recovery of a viscosity similar to never dried samples (a complex and potentially misleading property when dealing with heterogeneous systems) does not fully demonstrate the restauration of its properties. The technical properties, i.e., the functional properties of BC (e.g. the stabilization of multiphasic systems), provide more detailed information.

The invention is defined by a powdered formulation comprising bacterial cellulose and an additional component as defined in the claims <NUM>-<NUM>, the use of powdered bacterial cellulose formulation as defined in claim <NUM> and a method for producing a bacterial cellulose powdered formulation as defined in claim <NUM>.

The present disclosure relates to dried powdered and rehydratable bacterial cellulose (BC) 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.

The present disclosure concerns with the conditions by which dried powdered BC formulations, capable of being dispersed in aqueous media under <NUM> minutes, at room temperature, using low shear mixing, can be obtained. Such formulations preserve the technical properties of the non-dried material, in particular but not only, as a colloid stabilizer.

The present disclosure concerns with the conditions by which dried powdered BC formulations, capable of being dispersed in aqueous media under <NUM> minutes, at room temperature (<NUM>-<NUM>), using low shear mixing (at most <NUM> rpm, <NUM> rpm), can be obtained. Such formulation preserves the technical properties of the non-dried material, in particular, but not only, its performance as a colloid stabilizer. More specifically, the level of grinding involved in the processing of the BC formulations must be controlled in such a way that full redispersion is possible, while not compromising the functional properties. The smaller the particle size, the better the redispersion. However, the comminution conditions required for optimal restoration of the BC technical properties may be achieved at non-optimal conditions concerning redispersion.

In an embodiment, the full restoration of the BC technical properties requires the addition of a third component but may be achieved regardless of the drying technology used or the amount of residual water in the dried formulation.

In an embodiment, the presence of a third component (or an additional component) may not always be desired, due to changes in the functional properties of native BC. However, dried BC (without the added third component) cannot be redispersed in aqueous media. Alternatively, a concentrated BC suspension can be prepared, also providing the full redispersion and restauration of the functional properties; however, the wet comminution of BC performed before the concentration step must also be controlled in such a way that the technical properties of BC are not compromised.

This disclosure encompasses the conditions by which dried BC formulations (containing preferably from <NUM>% (w/w) to <NUM>%(w/w) of water) can be obtained. In both cases, the BC formulations disperse quickly in aqueous media, under low shear stress, at room temperature. Once dispersed, the properties of the dried material are equivalent to those of the never dried one.

For the purpose of the present invention, BC may be produced from a bacterium. The genus of the bacterium may be Acetobacter, Agrobacterium, Gluconacetobacter, Rhizobium, Achromobacter, Alcaligenes, Aerobacter, Azotobacter, Rhizobium, Salmonella, Escherichia and Sarcina. Preferably, BC is obtained using pure strains belonging to the genus Acetobacter or Komagataeibacter (formerly Gluconacetobacter), as these are the most productive, but any other cellulose-producing microbial strain, natural or mutated, may be used. The aerobic fermentation of BC can be done by any know static or agitated/aerated conditions. Any known culture fermentation method such as batch, fed batch, repeated batch or continuous fermentation may be used. Several feed stocks can be used for bacterial growth and cellulose production, provided they are free of contaminants (exogenous microorganisms or toxicants). For instance, carbon sources may be individual sugars or mixtures thereof, such as glucose and fructose, disaccharides such as sucrose, and mixtures of monosaccharide and disaccharides. In addition, the carbon source can be supplied as a complex mixture of sugars such as molasses, or plant biomass hydrolysates such as wood hydrolysate, straw hydrolysate, corn stalk hydrolysate, sorghum hydrolysate or other biomass wastes. Alternative carbon sources such as ethanol, glycerol or other sugar alcohols, alone or in combination with the previously mentioned, can also be used. Also, several nitrogen sources, organic or inorganic ones, can be used, alone or combined, such as - but not limited to - ammonium sulphate, ammonium chloride, ammonium phosphate, urea, sodium nitrate, yeast extract, corn steep liquor, whey hydrolysate, peptone, casein hydrolysate. Trace amounts of organic nutrients may be further added, including amino acids, vitamins, fatty acids, nucleic acids, <NUM>,<NUM>,<NUM>-tricarboxy-<NUM> pyrrolo [<NUM>,<NUM>,<NUM>]-quinoline-<NUM>,<NUM>-dione, ethanol, sulphite pulp waste liquor, lignin sulfonic acid and the like. Preferentially, the effective pH range of the culture media is in the range of <NUM>-<NUM>. Optionally, during fermentation, the pH may be controlled using buffer solutions, such as, but not limited to, citrate or <NUM>,<NUM>-dimethylglutaric acid, or the addition of base or acid to the medium in sufficient amounts, to maintain the pH within the desired range. Preferentially, the temperature for inoculum development and fermentation is in the range of <NUM>-<NUM>. Following fermentation, bacteria and remnants from the fermentation media can be removed by a variety of protocols. These include washing with water, dilute acid and/or alkali, bleaching with sodium hypochlorite or hydrogen peroxide, lysing the bacteria with lytic enzymes such as lysozyme, treatment with surfactants such as sodium lauryl sulphate or sodium deoxycholate. Washing procedures can be done at a temperature range between room temperature and <NUM>, with any combination of the above treatments. Those skilled in the art may optionally select other fermentation and purification conditions.

In an embodiment, BC obtained by any of the means described above, at a concentration in the range between <NUM>% (m/v) and <NUM>% solids (BC) preferably between <NUM>% and <NUM>% solids, is submitted to the following sequential steps, to yield a dried powdered formulation:.

In an embodiment, both wet and dry comminution must be controlled in such a way that full redispersion is possible while not compromising the functional properties. Thus, the restoration of the BC technical properties may be achieved at non-optimal conditions concerning redispersion. Indeed, the smaller the particle size, the better the redispersion, but the same does not apply to the technical properties.

The invention as defined in the claims relates with powdered formulations comprising BC dispersible in an aqueous solution, at <NUM> using low shear mixing, and an additional (third) component selected from the following list: carboxymethyl cellulose, xanthan, methylcellulose, hydroxyethyl-cellulose, hydroxypropyl methylcellulose, tylose, or mixture thereof.

The invention as defined in the claims discloses a powdered formulation comprising.

The measurement of the dispersibility of the BC powdered formulation may be carried out in various ways. In the present disclosure, samples are prepared at <NUM>% (m/v) in water at room temperature (i.e. within the range of <NUM>). For this, a low mechanical shear dispersion using magnetic stirrer plate (Stuart SD162), at <NUM> Rpm, for up to <NUM>. The dispersed materials are spread over a petri dish and observed at naked eye. Dispersibility of the formulations is classified as follows: <NUM>-sample is homogeneous and no visible particles or aggregates are observable; <NUM> - sample contains some very small particles or aggregates; <NUM> - the sample contains some larger particles or aggregates; <NUM> - the water remains transparent and the well separated particles or aggregates are observed.

In an embodiment, for better results, the powdered BC formulation may be dispersed in an aqueous media in at most <NUM>; preferably in at most <NUM>; more preferably in at most <NUM>.

In an embodiment, for better results, the powdered BC formulation is dispersed in an aqueous media in at most <NUM>; preferably in at most <NUM>. In particular, it may use particles of at least <NUM>.

In an embodiment, for better results, the particles may comprise a size between <NUM> - <NUM>, more preferably <NUM>-<NUM>. These dimensions are particularly suitable for foodstuff or for use as, more in particular beverages, dairy based beverages, in particular chocolate milk.

In an embodiment, for better results, the D50 of the particle is between <NUM>-<NUM>, preferably the D50 of the particle is between <NUM>-<NUM>, more preferably the D50 of the particle is between <NUM>-<NUM>. These dimensions are particularly suitable for foodstuff or for use as, more in particular beverages, dairy based beverages, in particular chocolate milk.

In an embodiment for better results, the powdered BC formulation is dispersed in an aqueous media in at most <NUM> and wherein the particles comprise a size between <NUM>-<NUM>; more preferably <NUM>-<NUM>. These dimensions are particularly suitable for composite reinforcement.

In an embodiment, for better results, the D50 of the particle is between <NUM>-<NUM>, preferably the D50 of the particle is between <NUM>-<NUM>, more preferably the D50 of the particle is between <NUM>-<NUM>. These dimensions are particularly suitable for composite reinforcement.

The measurement of the particle size may be carried out in various ways, in this disclosure the measurement was carried out on the basis of the standard granulometry analysis by mechanical sieving. In particular concerning the sizes of the particles obtained by the sieves specified: Mat. Mesh:AISI <NUM> nº <NUM>/<NUM>/<NUM> Nº: <NUM>/<NUM>/<NUM> with an opening of <NUM>, Endecotts, Ltd, aperture <NUM> and Endecotts, Ltd, aperture <NUM>.

In an embodiment, for better results, wherein the mass ratio between BC and the additional component varies between <NUM>:<NUM> to <NUM>:<NUM>.

In an embodiment, for better results, the additional component (third component) may be selected from a list consisting in: carboxymethyl cellulose, carboxymethyl cellulose and xanthan; carboxymethyl cellulose and hydroxyethyl-cellulose and carboxymethyl cellulose and hydroxypropyl methylcellulose, or mixtures thereof.

In an embodiment, the powdered BC formulation 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 Acetobacter, Komagataeibacter, Gluconacetobacter or combinations thereof.

Another aspect of the present invention relates to the use of the powdered BC formulation described in the present disclosure as a thickener. In particular, the particles may comprise a size between <NUM>-<NUM>, preferably between <NUM>-<NUM>, more preferably <NUM>-<NUM>. Preferably, the formulation may be used in foodstuff, more in particular in beverages, dairy base beverages, even more in particular chocolate milk.

Another aspect of the present invention relates to the use of the powdered BC described in the present disclosure as a reinforcer material, namely as filler. Preferably, by the use of a powdered BC formulation wherein the particles comprise a size between <NUM>-<NUM>; more preferably <NUM>-<NUM>.

Another aspect of the present disclosure relates to a pharmaceutical or a cosmetic composition comprising at least an active ingredient and the powdered BC formulation described in the present disclosure.

Another aspect of the present disclosure relates to a dietary fibre or a foodstuff comprising an effective amount of powdered BC formulation.

Another aspect of the present disclosure relates to a composite comprising an effective amount of powdered BC formulation.

Method for producing a BC formulation of dried powder comprising the following steps of:.

In an embodiment, the step of providing a suspension of BC bundles (i) comprises wet comminution.

In an embodiment, the BC powdered formulation comprises a size between <NUM>-<NUM>.

In an embodiment, the concentration of BC in the ground BC suspension varies between <NUM> - <NUM> % (wt.

In an embodiment, the concentration of BC in the ground BC suspension varies between <NUM>-<NUM> % (wt.

In an embodiment, the additional (third) component may be selected from a list consisting of: xanthan gum, carboxymethyl cellulose, methylcellulose, hydroxypropylcellulose, ethylcellulose, tylose, or mixture thereof.

Another aspect of the present invention, relates to a method for producing a BC powdered formulation described in the present disclosure comprising the following steps of:.

In an embodiment, the concentration of BC in the ground BC suspension may vary between <NUM>-<NUM> % (wt. Preferably the concentration of BC in the ground bacterial cellulose suspension varies between <NUM>-<NUM> % (wt.

In an embodiment, the step of homogenizing the BC suspension may comprise wet comminution.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

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

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.

The present disclosure relates to powdered, methods of production and uses thereof.

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

The present disclosure concerns with the conditions by which a dried powdered BC formulation, capable of being dispersed in aqueous media under <NUM> 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.

For clarification purposes, the description of the present disclosure is divided into the following sequential steps:.

In an embodiment, a BC obtained by any means as described in the previous section, in an amount of the range between <NUM>% (m/v) and <NUM>% solids (BC) preferably between <NUM>% and <NUM>% solids, preferably in the range of <NUM>-<NUM>% (w/v) was wet ground by three different methods:.

BC suspensions were spread over petri dishes and observed with a SZ40 Zoom Stereo Microscope (Olympus). Photographs were taken with a camera SONY AVC-D5CE and adapter CMA-D5CE, at a magnification from <NUM>. 67x to 5x (<FIG>). BC suspensions were also stained with Calcofluor white and observed in an Olympus BX51 microscope.

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> and <FIG>). In this example, three BC homogenization methods were used, with increasingly shear stress, from a blade blender (A), to high pressure homogenizer (C).

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 <NUM> to <NUM>-<NUM> size. BC homogenized with Comitrol Processor and HPH, lower amounts of millimetric fragments were observed, with most of them being in the range of <NUM>-<NUM> (Comitrol), <NUM>-<NUM> (HPH <NUM> pass) and <NUM>-<NUM> (HPH <NUM> pass).

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 <NUM>%(w/v)) so that stable dispersions of well spread single fibres was never achieved.

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.

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 etylcellulose (MEC) and other polysaccharide gums, such as xanthan have been used for the stabilization of foams.

In this embodiment, outside the subject-matter of the claims but useful for understanding the disclosure, 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 <NUM> and "nata de coco" from HTK Food CO. (Vietnam), ground with a Sammic blender as described in Example <NUM>, were used (code samples: <NUM>, S; Nata de coco, S. BC from ATCC <NUM> was further homogenized with Comitrol and High Pressure Homogenization (Example <NUM>) (code samples: BC Comitrol, BC HPH <NUM> Pass, BC HPH <NUM> Pass).

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

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 <NUM>%(wt/v) solids (code samples: <NUM>, S-P and Nata de coco, S-P), diluted and redispersped in water, under magnetic stirring at <NUM> Rpm for 2hr. 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.

Dried egg whites (<NUM>%, 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 <NUM>-<NUM>% (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.

As observed in <FIG>, at <NUM>%(wt/v) addition to the mixture, all BC samples ground with Sammic blender (<NUM>, 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 <NUM>% 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 <NUM>%(wt/v) solids, low shear dispersion does not allow the full lengthening of the BC fibre aggregates (<FIG>) when diluted and redispersed in water; however, provided the BC aggregates size is unaffected, compressing BC (<FIG>) does not affect the capacity to stabilize the foam.

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

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>). Regardless of pressing or not the BC, results also showed that BC ground with the Sammic blender, at <NUM>%(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.

In an embodiment, the BC obtained by any of the three different comminution methods described in Example <NUM> was mixed with either sodium carboxymethyl cellulose, CMC (<NUM> KDa, or <NUM> KDa or <NUM> 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:.

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 <NUM> to <NUM>,<NUM>%(wt/v) by mass of BC.

To evaluate the dispersibility of the BC mixtures, samples were prepared at <NUM>% (wt/v) in water to at room temperature. For this, two dispersion method were used:.

As a control, BC, as obtained by any of methods A, Band C mentioned above (Example <NUM>), 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> and <FIG> summarize the main observations pertaining to the BC and CMC mixtures.

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> and Table <NUM>). However, the presence of the third component improves the dispersion of the aggregates, more homogeneous samples being observed (<FIG> vs <FIG>). Also, further homogenization with ultraturrax for up to <NUM>, does not change the morphology nor the rheological profile of the BC:CMC mixtures (data not shown).

It was observed that when CMC or xanthan were added to BC, at preferably a mass ratio of <NUM>:<NUM>, 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.

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.

The BC:CMC samples were further characterized by rheological assays (<FIG>). For this, stress-strain curves were obtained using a TA instruments rheometer, model <NUM>-<NUM> 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>). 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.

In an embodiment, the BC was wet ground by three different methods, as described in Example <NUM> (methods A, B and C). The obtained BC was mixed with a third component, as described in Example <NUM>. 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:.

All dried material was ground (with a High Power Herb Grain Grinder Cereal Mill Powder Grinding Machine Flour <NUM>) and sieved (Mat. Mesh:AISI <NUM> nº <NUM>/<NUM>/<NUM> Nº: <NUM>/<NUM>/<NUM> with an opening of <NUM>) to a final particle size preferably in the range of <<NUM>. As in the previous examples, the dried powders were dispersed at a final solids concentration of <NUM>% in water, using:.

Rheological assays were done using a TA instruments rheometer, model <NUM>-<NUM> 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.

The following table summarizes the main observations regarding the aqueous dispersibility of the dried and ground BC:CMC <NUM> KDa mixture.

As was observed for <FIG>, 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 <NUM>%TS (<FIG>). 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 <NUM> 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 <NUM> with ultraturrax does not change the rheologic profile of the samples (data not show).

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 <NUM> and <NUM>%TS, showed an identical rheological profile (Figure 10B). A similar observation was recorded for samples dried using a drum dryer (<NUM> and <NUM>%TS) and spray dryer (<NUM>%TS).

Too extensive wet comminution of the BC suspension such as with HPH (Figure <NUM> C, D, E) strongly affected the rheological properties of the dried material, confirming the effect previously observed on the never dried samples.

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.

In the previous example, the dried BC formulations were ground to a particle size <<NUM>. 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 <<NUM> (Endecotts, Ltd, aperture <NUM>) and <<NUM> (Endecotts, Ltd, aperture <NUM>). Also, samples were fractioned into the following size ranges: <NUM><x<<NUM>, <NUM><x<<NUM>. As in the previous examples, the dried powders were dispersed to a final solids' concentration of <NUM>% in water, evaluated by spreading over a petri dish, fluorescent microscopy and through rheological profile.

As an example, table <NUM> and <FIG> summarize the main observations pertaining to the dispersibility and rheological behaviour of BC:CMC <NUM> 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.

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

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.

In this example, the effect of particle size of dried formulations of BC:CMC <NUM> KDa, obtained as described in Example <NUM>, 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 <NUM>-<NUM>% and pure cocoa (<NUM>%) were weighted. Medium-skimmed milk, <NUM>, was added to each BC:CMC concentration. The mixture was stirred in a vortex (<NUM>,<NUM> rpm) for <NUM> at room temperature and then pasteurized at <NUM> for <NUM> 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 <NUM> Rpm using a dispersing shaft CAT 20F) for <NUM> and <NUM> before being added to the milk. In parallel, the same test was done using xanthan, carboxymethyl cellulose, colloidal plant celluloses: Avicel (RT <NUM>, LM <NUM> and CM <NUM>, FMC Biopolymers), Novagel (RCN-<NUM> and RCN-<NUM>, FMC Biopolymers), Bioplus Fibrils (a microfibrillated cellulose, not used in food applications, American Process Inc. ) and plant nanocelluloses: CelluForce NCC, Arbocel B <NUM>, Arbocel BE <NUM>/<NUM> (from Celluforce Inc. All Avicel and Novagel celluloses were previously activated, for <NUM>, at <NUM>,<NUM> 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 <NUM>) 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 <NUM>. The stabilization of the chocolate drinks was assessed by calculating the percentage of sedimentation, according to the following equation: <MAT>.

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

The results from this study are displayed in the following table and <FIG>, using <NUM>% BC:CMC (other concentrations were tested with similar observations).

These results show that an extensive homogenization through High Pressure Homogenization and extensive grinding of dried material to a particle size < <NUM>, 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 <NUM> passages, drying and comminution to <<NUM>, UT treatment affects the cocoa stability achieved with the BC:CMC.

Claim 1:
A powdered formulation, comprising
bacterial cellulose;
and an additional component selected from the following list: carboxymethyl cellulose, xanthan, methylcellulose, hydroxyethyl-cellulose, hydroxypropyl methylcellulose, tylose, or mixture thereof;
wherein the powdered formulation has a particle size between <NUM>-<NUM>, with methods of particle size determination as disclosed in the description;
wherein the mass ratio between bacterial cellulose and the additional component varies between <NUM>:<NUM> to <NUM>:<NUM>, preferably <NUM>:<NUM> to <NUM>:<NUM>;
wherein the powdered formulation is dispersible in an aqueous media, at <NUM>, with low shear mixing.