AU2006250004B2 - Bacterial cellulose-containing formulations and method of producing effective bacterial cellulose-containing formulations - Google Patents

Abstract

New formulations of bacterial cellulose and a new method to produce formulations of bacterial cellulose that exhibit improved viscosity-modifying properties particularly with low energy applied to effectuate viscosity changes therewith is provided. Such a method includes the novel co-precipitation with a water soluble co-agent that permits precipitation in the presence of excess alcohol to form an insoluble fiber that can than be utilized as a thickener or suspension aid without the need to introduce high energy mixing. Such bacterial cellulose properties have been available in the past but only through highly labor and energy intensive processes. Such an inventive method as now proposed thus provides a bacterial cellulose-containing formulation that exhibits not only properties that are as effective as those for previous bacterial celluloses, but, in some ways, improvements to such previous types. Certain end-use compositions and applications including these novel bacterial cellulose-containing formulations are also encompassed within this invention.

Description

WO 2006/127810 PCT/US2006/020080 BACTERIAL CELLULOSE CONTAINING FORMULATIONS AND METHOD OF PRODUCING EFFECTIVE BACTERIAL CELLULOSE CONTAINING FORMULATIONS Field of the Invention The present invention relates generally to a new method to produce formulations of bacterial cellulose that exhibit improved viscosity-modifying properties particularly with low energy applied to effectuate viscosity changes therewith. Such a method includes the novel co precipitation with a water soluble co-agent that permits precipitation in the presence of excess alcohol to form an insoluble fiber that can than be utilized as a thickener or suspension aid without the need to introduce high energy mixing. Such bacterial cellulose properties have been available in the past but only through highly labor and energy intensive processes. Such an inventive method as now proposed thus provides a bacterial cellulose-containing formulation that exhibits not only properties as effective as those for previous bacterial celluloses, but, in some ways, improvements to such previous types. Certain end-use compositions and applications including these novel bacterial cellulose-containing formulations are also encompassed within this invention. Background of the Invention Bacterial cellulose is a broad- category of polysaccharides that exhibit highly desirable properties, even though such compounds are essentially of the same chemical structure as celluloses derived from plant material. As the name purports, however, the source of these polysaccharides are bacterial in nature (produced generally by microorganisms of the WO 2006/127810 PCT/US2006/020080 Acetobacter genus) as the result of fermentation, purification, and recovery thereof. Such bacterial cellulose compounds are comprised of very fine cellulosic fibers having very unique dimensions and aspect ratios (diameters of from about 40 to 100 nm each and lengths of from 0.1 to 15 microns) in bundle form (with a diameter of 0.1 to 0.2 microns on average). Such an 5 entangled bundle structure forms a reticulated network structure that facilitates swelling when in aqueous solution thereby providing excellent three-dimensional networks. The three dimensional structures effectuate proper and desirable viscosity modification as well as suspension capabilities through building a yield-stress system within a target liquid as well as excellent bulk viscosity. Such a result thus permits highly effective suspension of materials 10 (such as foodstuffs, as one example) that have a propensity to settle over time out of solution, particularly aqueous solutions. Additionally, such bacterial cellulose formulations aid in preventing settling and separation of quick-preparation liquid foodstuffs (i.e., soups, chocolate drinks, yogurt, juices, dairy, cocoas, and the like), albeit with the need to expend relatively high amounts of energy through mixing or heating to initially reach the desired level of suspension for 15 such foodstuffs. The resultant fibers (and thus bundles) are insoluble in water and, with the capabilities noted above, exhibit polyol- and water-thickening properties. One particular type of bacterial cellulose, microfibrillated cellulose, is normally provided in an uncharged state and exhibits the ability to associate without any added influences. However, without any such extra additives to 20 effectuate thickening or other type of viscosity modification, it has been realized that the resultant systems will themselves exhibit high degrees of instability, particularly over time periods associated with typical shelf life requirements of foodstuffs. As a result, certain co agents, like carboxymethylcellulose (CMC), also known as cellulose gum, have been introduced 2 WO 2006/127810 PCT/US2006/020080 to bacterial cellulose products through adsorption to the fibers thereof, following by spray drying (without any co-precipitation steps) in order to provide stabilization and dispersion improvements, most likely through the presence of negative charges on the CMC transferred to the bacterial cellulose fibers themselves. Such charges thus appear to provide repulsion 5 capabilities to prevent the fiber bundles from relaxing the network formed. Even with such a possibility, the selection of a proper CMC has been known to greatly affect the resultant rheological properties of the target bacterial cellulose due to the salt and acid sensitivities of certain CMC products. As such, although improvements in bacterial cellulose utilization have been provided with such CMC inclusions in the past, great care must be taken to ensure the 10 proper level of pH and salt conditions are suitable for the overall formulation. For this reason, further improvements to permit more reliability of bacterial cellulose use in myriad applications are of great interest to the target industries. Additionally, although such bacterial celluloses are of great interest and importance in providing effective rheological modifications within liquid-based foodstuffs, for the reasons 15 mentioned above, the costs associated with producing such cellulosic materials has proven very high, particularly in terms of necessary labor and waste issues resulting therefrom. Fermentation of such materials initially yields very low amounts. Generally, the production method of purifying and recovering such bacterial cellulose materials entails a cumbersome series of steps after fermentation is complete in order to produce a wet cake with a sufficient amount of 20 bacterial cellulose product in terms of efficiency from initial fermentation. Further spray drying may also affect the final recovery yield of the bacterial cellulose during powder production. Such excessive steps are not only labor and energy intensive but also result in large amounts of waste water and waste materials that require disposal and handling. As such, the 3 costs for production of bacterial cellulose (in particular microfibrillated cellulose) have proven excessively high relative to other gums, thus restricting the utilization of such a product within certain desirable end-uses. To date, there has been no effective method developed that has remedied these problems, not to mention a method that ultimately 5 provides a bacterial cellulose material that exhibits certain improved properties within target applications as compared with the materials produced through the aforementioned traditional production method. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common 10 general knowledge in the field. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Brief Description of the Invention According to a first aspect, the present invention provides a method for the 15 production of a bacterial cellulose-containing formulation comprising the steps of a) providing a bacterial cellulose product that includes a reticulated network of bacterial cellulose fibres; b) optionally lysing the bacterial cells from the bacterial cellulose product; c) mixing said bacterial cellulose product of either step "a" or step "b" with 20 a polymeric thickener selected from the group consisting of at least one charged cellulose ether, at least one precipitation agent, and any combination thereof to provide a coating of the polymeric thickener on at least a portion of the surface of said bacterial cellulose product; and d) co-precipitating the mixture of step "c" with a water-miscible non 25 aqueous liquid to recover the coated bacterial cellulose product. According to a second aspect, the present invention provides a method for the production of a bacterial cellulose-containing formulation comprising the steps of a) providing a bacterial cellulose product that includes a reticulated network of bacterial cellulose fibres; 30 b) optionally lysing the bacterial cells from the bacterial cellulose product; c) mixing said resulting bacterial cellulose product of either step "a" or step "b" with at least one precipitation agent selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, 4 carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof to provide a coating of the precipitation agent on at least a portion of the surface of said bacterial cellulose product; and 5 d) co-precipitating the mixture of step "c" with a water-miscible non aqueous liquid to recover the coated bacterial cellulose product. According to a third aspect, the present invention provides a method for the production of a bacterial cellulose-containing formulation comprising the steps of a) providing a bacterial cellulose product that includes a reticulated network 10 of bacterial cellulose fibres; b) mixing said bacterial cellulose product with at least one precipitation agent selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any 15 mixtures thereof to provide a coating of the precipitation agent on at least a portion of the surface of said bacterial cellulose product; c) co-lysing the mixture of step "b" to remove bacterial cells therefrom; and d) co-precipitating the mixture of step "c" with a water-miscible non aqueous liquid to recover the coated bacterial cellulose product. 20 According to a fourth aspect, the present invention provides a bacterial cellulose containing formulation comprising at least one reticulated network of bacterial cellulose fibres that are at least partially coated with at least one polymeric thickener selected from the group consisting of at least one charged cellulose ether, or at least one precipitation agent, or any mixtures thereof, wherein said formulation exhibits a 25 viscosity capability of at least 300 cps and a yield stress measurement of 1.0 dyne/cm when introduced in an amount of at most 0.36% by weight of a 500 mL sample of water and after application of at most 2 passes at 1500 psi in an extensional homogenizer. According to a fifth aspect, the present invention provides a bacterial cellulose containing formulation when produced by the method according to any one of the first to 30 third aspects. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an 4a inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Accordingly, this invention encompasses a method for the production of a bacterial cellulose-containing formulation comprising the steps of a) providing a 5 bacterial cellulose product through fermentation; b) optionally lysing the bacterial cells from the resultant bacterial cellulose product; c) mixing said resulting bacterial cellulose of either step "a" or "b" product with a polymeric thickener selected from the group consisting of at least one charged cellulose ether, at least one precipitation agent, and any combination thereof; and d) co-precipitating the mixture of step "c" with a water 10 miscible nonaqueous liquid (such as, as one non-limiting example, an alcohol). The possible charged cellulose ether of step "c" is a compound utilized to disperse and stabilize the reticulated network in the final end-use compositions to which such a bacterial cellulose-containing formulation is added. The charged compounds facilitate, as alluded to above, the ability to form the needed network of fibers through the 15 repulsion of individual fibers. The possible precipitation agent of step "c" is a compound utilized to preserve the functionality of the reticulated bacterial cellulose fiber during drying and milling. Examples of such charged cellulose ethers include such cellulose based compounds that exhibit either an overall positive or negative and include, without limitation, any sodium carboxymethylcellulose 4b WO 2006/127810 PCT/US2006/020080 (CMC), cationic hydroxyethylcellulose, and the like. The precipitation (drying) agent is selected from the group of natural and/or synthetic products including, without limitation, xanthan products, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean 5 gum, and the like. Preferably, though not necessarily, for reasons associated with the ability to reactivate the bacterial cellulose after spray drying and prior to incorporation within a target liquid to be rheologically modified therewith, a precipitation (drying) agent is included. Thus, one more specific method encompassed within this invention comprises the steps of a) providing a bacterial cellulose product through fermentation; b) optionally lysing the bacterial cells from 10 the bacterial cellulose product; c) mixing said resulting bacterial cellulose product of either step "a" or step "b" with a biogum (which if incorporated as a fermentation broth has had the bacterial cells preferably lysed there from); and d) co-precipitating the mixture of step "c" with a water-miscible nonaqueous liquid. Alternatively, such a specific method may comprise the steps of a) providing a bacterial cellulose product through fermentation; b) mixing said bacterial 15 cellulose product with a biogum; c) co-lysing the mixture of step "b" to remove bacterial cells therefrom; and d) co-precipitating the mixture of step "c" with a water-miscible nonaqueous liquid. The resultant coprecipitated product will be in the form of a presscake that can then be dried and the particles obtained thereby may then be milled to a desired particle size. Furthermore, for certain applications, the particles may then be blended with another 20 hydrocolloid, such as carboxymethylcellulose (CMC), to provide certain properties. Additionally, an inventive product of this development would be defined as a bacterial cellulose containing formulation comprising at least one bacterial cellulose material and at least one polymeric thickener selected from the group consisting of at least one charged cellulose ether, at 5 WO 2006/127810 PCT/US2006/020080 least precipitation agent selected from the group consisting of xanthan products, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and the like, and any mixtures thereof, wherein said formulation exhibits a viscosity capability of at least 300 5 cps and a yield stress measurement of 1.0 dyne/cm 2 when introduced in an amount of at most 0.36% by weight of a 500 mL sample of water and after application of at most 2 passes at 1500 psi in an extensional homogenizer. As one potentially preferred embodiment, the formulation of bacterial cellulose and xanthan produced thereby has the distinct advantage of facilitating activation without any labor 10 or energy-intensive activation required. Another distinct advantage of this overall method is the ability to collect the resultant bacterial cellulose-containing formulation through precipitation with isopropyl alcohol, whether with a charged cellulose ether or a precipitation (drying) agent present therein. Thus, since the bacterial cellulose is co-precipitated in the manner described above, the alcohol-insoluble polymeric thickener (such as xanthan or sodium CMC) appears, 15 without intending on being bound to any specific scientific theory, to provide protection to the bacterial cellulose by providing a coating over at least a portion of the resultant formed fibers thereof. In such a way, it appears that the polymeric thickener actually helps associate and dewater the cellulosic fibers upon the addition of a nonaqueous liquid (such as preferably a lower alkyl alcohol), thus resulting in the collection of substantial amounts of the low-yield 20 polysaccharide during such a co-precipitation stage. The avoidance of substantial amounts of water during the purification and recovery steps thus permits larger amounts of the bacterial cellulose to be collected ultimately. With this novel process, the highest amount of fermented bacterial cellulose can be collected, thus providing the high efficiency in production desired, as 6 WO 2006/127810 PCT/US2006/020080 well as the avoidance of, as noted above, wastewater and multiple passes of dewatering and re slurrying typically required to obtain such a resultant product. Furthermore, as noted previously, the presence of a drying agent, in particular, as one non-limiting example, a xanthan product, as a coating over at least a portion of the bacterial cellulose fiber bundles, appears to provide the 5 improvement in activation requirements when introduced within a target end use composition. Surprisingly, there is a noticeable reduction in the energy necessary to effectuate the desired rheological modification benefits accorded by this inventive bacterial cellulose-containing formulation as compared with the previously practiced products of similar types. As well, since bacterial cellulose (i.e., microfibrillated cellulose, hereinafter referred to as "MFC") provides 10 unique functionality and rheology as compared to a soluble polymeric thickener alone, the resultant product made via this inventive method permits a lower cost alternative to typical processes with improvements in reactivation requirements, resistance to viscosity changes during high temperature food processing, and improved suspension properties during long term shelf storage. 15 Detailed Description of the Invention For purposes of this invention, the term "bacterial cellulose-containing formulation" is intended to encompass a bacterial cellulose product as produced by the inventive method and thus including xanthan product coating at least of the portion of the resultant bacterial cellulose 20 fiber bundles. The term "formulation" thus is intended to convey that the product made therefrom is a combination of bacterial cellulose and xanthan produced in such a manner and exhibiting such a resultant structure and configuration. The term "bacterial cellulose" is intended to encompass any type of cellulose produced via fermentation of a bacteria of the genus 7 WO 2006/127810 PCT/US2006/020080 Acetobacter and includes materials referred popularly as microfibrillated cellulose, reticulated bacterial cellulose, and the like. As noted above, bacterial cellulose may be used as an effective rheological modifier in various compositions. Such materials, when dispersed in fluids, produce highly viscous, 5 thixotropic mixtures possessing high yield stress. Yield stress is a measure of the force required to initiate flow in a gel-like system. It is indicative of the suspension ability of a fluid, as well as indicative of the ability of the fluid to remain in situ after application to a vertical surface. Typically, such rheological modification behavior is provided through some degree of processing of a-mixture of the bacterial cellulose in a hydrophilic solvent, such as water, polyols 10 (e.g., ethylene glycol, glycerin, polyethylene glycol, etc.), or mixtures thereof. This processing is called "activation" and comprises, generally, high pressure homogenization and/or high shear mixing. The inventive bacterial cellulose-containing formulations of the invention, however, have been found to activate at low energy mixing. Activation is a process in which the 3 dimensional structure of the cellulose is modified such that the cellulose imparts functionality to 15 the base solvent or solvent mixture in which the activation occurs, or to a composition to which the activated cellulose is added. Functionality includes providing such properties as thickening, imparting yield stress, heat stability, suspension properties, freeze-thaw stability, flow control, foam stabilization, coating and film formation, and the like. The processing that is followed during the activation process does significantly more than to just disperse the cellulose in base 20 solvent. Such processing "teases apart" the cellulose fibers to expand the cellulose fibers. The bacterial cellulose-containing formulation may be used in the form of a wet slurry (dispersion) or as a dried product, produced by drying the dispersion using well-known drying techniques, such as spray-drying or freeze-drying to impart the desired rheological benefits to a target fluid 8 WO 2006/127810 PCT/US2006/020080 composition. The activation of the bacterial cellulose (such as MFC or reticulated bacterial cellulose) expands the cellulose portion to create a reticulated network of highly intermeshed fibers with a very high surface area. The activated reticulated bacterial cellulose possesses an extremely high surface area that is thought to be at least 200-fold higher than conventional 5 microcrystalline cellulose (i.e., cellulose provided by plant sources). The bacterial cellulose utilized herein may be of any type associated with the fermentation product of Acetobacter genus microorganisms, and was previously available, as one example, from CPKelco U.S. under the tradename CELLULON@. Such aerobic cultured products are characterized by a highly reticulated, branching interconnected network of fibers 10 that are insoluble in water. The preparation of such bacterial cellulose products are well known. For example, U.S. Pat. No. 5,079,162 and U.S. Pat. No. 5,144,021, both of which are incorporated by reference herein, disclose a method and media for producing reticulated bacterial cellulose aerobically, under agitated culture conditions, using a bacterial strain of Acetobacter aceti var. xylinum. Use 15 of agitated culture conditions results in sustained production, over an average of 70 hours, of at least 0.1 g/liter per hour of the desired cellulose. Wet cake reticulated cellulose, containing approximately 80-85% water, can be produced using the methods and conditions disclosed in the above-mentioned patents. Dry reticulated bacterial cellulose can be produced using drying techniques, such as spray-drying or freeze-drying, that are well known. 20 Acetobacter is characteristically a gram-negative, rod shaped bacterium 0.6-0.8 microns by 1.0-4 microns. It is a strictly aerobic organism; that is, metabolism is respiratory, not fermentative. This bacterium is further distinguished by the ability to produce multiple poly p 1,4-glucan chains, chemically identical to cellulose. The microcellulose chains, or microfibrils, 9 WO 2006/127810 PCT/US2006/020080 of reticulated bacterial cellulose are synthesized at the bacterial surface, at sites external to the cell membrane. These microfibrils generally have cross sectional dimensions of about 1.6 nm by 5.8 nm. In contrast, under static or standing culture conditions, the microfibrils at the bacterial surface combine to form a fibril generally having cross sectional dimensions of about 3.2 nm by 5 133 nm. The small cross sectional size of these Acetobacter-produced fibrils, together with the concomitantly large surface and the inherent hydrophilicity of cellulose, provides a cellulose product having an unusually high capacity for absorbing aqueous solutions. Additives have often been used in combination with the reticulated bacterial cellulose to aid in the formation of stable, viscous dispersions. 10 The aforementioned problems inherent with purifying and collecting such bacterial cellulose have led to the determination that the method employed herein provides excellent results to the desired extent. The first step in the overall process is providing any amount of the target bacterial cellulose in fermented form. The production method for this step is described above. The yield for such a product has proven to be very difficult to generate at consistently 15 high levels, thus it is imperative that retention of the target product be accomplished in order to ultimately provide a collected product at lowest cost. Purification is well known for such materials. Lysing of the bacterial cells from the bacterial cellulose product is accomplished through the introduction of a caustic, such as sodium hydroxide, or any like high pH (above about 12.5 pH, preferably) additive in an amount to 20 properly remove as many expired bacterial cells as possible from the cellulosic product. This may be followed in more than one step if desired. Neutralizing with an acid is then typically followed. Any suitable acid of sufficiently low pH and molarity to combat (and thus effectively neutralize or reduce the pH level of the product as close to 7.0 as possible) may be utilized. 10 WO 2006/127810 PCT/US2006/020080 Sulfuric acid, hydrochloric, and nitric acid are all suitable examples for such a step. One of ordinary skill in the art would easily determine the proper selection and amount of such a reactant for such a purpose. Alternatively, the cells may be lysed and digested through enzymatic methods (treatment with lysozyme and protease at the appropriate pH). 5 The lysed product is then subjected to mixing with a polymeric thickener in order to effectively coat the target fibers and bundles of the bacterial cellulose. The polymeric thickener must be insoluble in alcohol (in particular, isopropyl alcohol). Such a thickener is either an aid for dispersion of the bacterial cellulose within a target fluid composition, or an aid in drying the bacterial cellulose to remove water therefrom more easily, as well as potentially aid in dispersing 10 or suspending the fibers within a target fluid composition. Proper dispersing aids (agents) include, without limitation, CMC (of various types), cationic HEC, etc., in essence any compound that is polymeric in nature and exhibits the necessary dispersion capabilities for the bacterial cellulose fibers when introduced within a target liquid solution. Preferably such a dispersing aid is CMC, such as CEKOL@ available from CP Kelco. Proper precipitation aids 15 (agents), as noted above, include any number of biogums, including xanthan products (such as KELTROL@, KELTROL T@, and the like from CP Kelco), gellan gum, welan gum, diutan gum, rhamsan gum, guar, locust bean gum, and the like, and other types of natural polymeric thickeners, such as pectin, as one non-limiting example. Preferably, the polymeric thickener is a xanthan product and is introduced and mixed with the bacterial cellulose in a broth form. 20 Basically, the commingling of the two products in broth, powder or rehydrated powder form, allows for the desired generation of a xanthan coating on at least a portion of the fibers and/or bundles of the bacterial cellulose. In one embodiment, the broths of bacterial cellulose and xanthan are mixed subsequent to purification (lysing) of both in order to remove the residual 11 WO 2006/127810 PCT/US2006/020080 bacterial cells. In another embodiment, the broths may be mixed together without lysing initially, but co-lysed during mixing for such purification to occur. The amounts of each component within the method may vary greatly. For example, the bacterial cellulose will typically be present in an amount from about 0.1% to about 5% by weight 5 of the added polymeric thickener, preferably from about 0.5 to about 3.0%, whereas the polymeric thickener may be present in an amount form 10 to about 900% by weight of the bacterial cellulose. After mixing and coating of the bacterial cellulose by the polymeric thickener, the resultant product is then collected through co-precipitation in a water-miscible nonaqueous 10 liquid. Preferably, for toxicity, availability, and cost reasons, such a liquid is an alcohol, such as, as most preferred, isopropyl alcohol. Other types of alcohols, such as ethanol, methanol, butanol, and the like, may be utilized as well, not to mention other water-miscible nonaqeuous liquids, such as acetone, ethyl acetate, and any mixtures thereof. Any mixtures of such nonaqueous liquids may be utilized, too, for such a co-precipitation step. Generally, the co 15 precipitated product is processed through a solid-liquid separation apparatus, allowing for the alcohol-soluble components to be removed, leaving the desired bacterial cellulose-containing formulation thereon. From there, a wetcake form product is collected and then transferred to a drying apparatus and subsequently milled for proper particle size production. Further co-agents may be 20 added to the wetcake or to the dried materials in order to provide further properties and/or benefits Such co-agents include plant, algal and bacterial polysaccharides and their derivatives along with lower molecular weight carbohydrates such as sucrose, glucose, maltodextrin, and the like. Other additives that may be present within the bacterial cellulose-containing formulation 12 WO 2006/127810 PCT/US2006/020080 include, without limitation, a hydrocolloid, polyacrylamides (and homologues), polyacrylic acids (and homologues), polyethylene glycol, poly(ethylene oxide), polyvinyl alcohol, polyvinylpyrrolidones, starch (and like sugar-based molecules), modified starch, animal-derived gelatin, and non-charged cellulose ethers (such as carboxymethylcellulose, 5 hydroxyethylcellulose, and the like). The bacterial cellulose-containing formulations of this invention may then be introduced into a plethora of possible food compositions, including, beverages, frozen products, cultured dairy, and the like; non-food compositions, such as household cleaners, fabric conditioners, hair conditioners, hair styling products, or as stabilizers or formulating agents for asphalt emulsions, 10 pesticides, corrosion inhibitors in metal working, latex manufacture, as well as in paper and non woven applications, biomedical applications, pharmaceutical excipients, and oil drilling fluids, etc. The fluid compositions including this inventive formulation, prepared as described above, may include such bacterial cellulose-containing formulations in an amount from about 0.01% to about 1% by weight, and preferably about 0.03% to about 0.5% by weight of the total weight of 15 the fluid composition. The ultimately produced bacterial cellulose-containing formulation should impart a viscosity modification to water sample of 500 mL (when added in an amount of at most 0.36% by weight thereof) of at least 300 cps as well as a yield stress measurement within the same test sample of at least 1.0 dynes/cm 2 . 20 Preferred Embodiments of the Invention The following non-limiting examples provide teachings of various methods that are encompassed within this invention. 13 WO 2006/127810 PCT/US2006/020080 Example 1 MFC was produced in a 1200 gal fermentor with final yield of 1.49 wt%. The broth was 5 treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan gum broth (MFC/XG=2/1, dry basis) and the resultant mixture was then precipitated with isopropyl alcohol (85%) to form a press cake. A portion of the press cake was then dried in an oven at 70'C for 2 hrs and milled in a Brinkmann Mill to 60 mesh. The powdered 10 formulation was then introduced into a standard tap water (STW, 2.782 g of CaCl 2 .2H 2 0 and 18.927 g of NaCl are dissolved into 5 gal of de-ionized water) solution (500 mL) in an amount of about 0.36% by weight thereof, with 20% by weight of carboxymethyleellulose (CMC) added simultaneously (resulting in amounts of 0.288% of MFC/Xanthan and 0.072% of CMC), and the composition was then mixed with a Silverson mixer at 8000 rpm for 10 15 min. The product viscosity (measured via Brookfield viscometer, 61 Spindle at 5 rpm for 1 min) and yield stress was 1176 cP and 4.91 dynes/cm2, respectively. Subsequently, 210 mL of the resultant activated MFC solution (0.36%) was then mixed with 15.5 grams of graded sand (through 60 mesh but on 80 mesh) to one beaker and mixed for 1 minute. To a separate beaker, another 210 mL sample of the resultant activated MFC 20 solution was then also mixed with 15.5 grams of fine CaCO 3 and mixed for 1 minute. The contents of each beaker was then poured into separate 100 mL graduated cylinders and diluted to the 100 mL mark in each cylinder. In each case, the solutions exhibited excellent 14 WO 2006/127810 PCT/US2006/020080 suspension properties and the solids (either sand or calcium carbonate) exhibited no precipitation from the target solution. The graduated cylinders were then each stored at room temperature (22-25'C) for 24 hours to determine if precipitation occurred during that period of time. In each sample, after the 24 hours were completed, the phase separations for 5 samples from either the top or the bottom were less than 10% (through visual estimation), thus indicating excellent long-term suspension properties. Example 2 MFC was produced in a 1200 gal fermentor with final yield of 1.49 wt%. The broth was 10 treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan gum broth (MFC/XG=3/1, dry basis) under high shear and the resultant mixture was then precipitated with IPA (85%) to form a press cake. The press cake was dried and milled as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of 15 about 0.36% by weight thereof, with 20% by weight of CMC added simultaneously, and the composition was then mixed with a Silverson mixer at 8000 rpm for 10 min. The product viscosity and yield stress were 709 cP and 1.96 dynes/cm2, respectively. Example 3 15 WO 2006/127810 PCT/US2006/020080 MFC was produced in a 1200 gal fermentor with final yield of 1.49 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan gum broth (MFC/XG=4/1, dry basis) under high shear and the resultant mixture was then 5 precipitated with IPA (85%) to form a press cake. The press cake was dried and milled as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, with 20% by weight of CMC added simultaneously, and the composition was then mixed with a Silverson mixer at 8000 rpm for 10 min. The product viscosity and yield stress were 635 cP and 1.54 dynes/cm 2 , respectively. 10 Example 4 MFC was produced in a 1200 gal fermentor with final yield of 1.49 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan 15 gum broth (MFC/XG=3/1, dry basis) and the resultant mixture was then precipitated with IPA (85%) to form a press cake. The press cake was then dried and milled as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, with 10% CMC added simultaneously, and the composition was then mixed with a Silverson mixer at 8000 rpm for 10 min. The product viscosity and yield stress were 1242 cP 20 and 4.5 dynes/cm 2 , respectively. 16 WO 2006/127810 PCT/US2006/020080 Example 5 5 MFC was produced in a 1200 gal fermentor with final yield of 1.49 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan gum broth (MFC/XG=3/1, dry basis) and the resultant mixture was then precipitated with IPA (85%) to form a press cake. The press cake was then dried and milled as in Example 1. The 10 powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, with 20% of CMC added simultaneously, and the composition was then mixed with a Silverson mixer at 8000 rpm for 10 min. The product viscosity and yield stress were 1242 eP and 4.5 dynes/cm 2 , respectively. 15 Example 6 MFC was produced in a 1200 gal fermentor with final yield of 1.49 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan gum broth (MFC/XG=3/1, dry basis) and the resultant mixture was then precipitated with IPA 20 (85%) to form a press cake. The press cake was then dried and milled as in Example 1. The 17 WO 2006/127810 PCT/US2006/020080 powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, with 20% by weight of CMC added simultaneously, and the composition was then activated with an extensional homogenizer at 1500 psi for 2 passes. The product viscosity and yield stress measurements were 1010 cP and 1.76 dynes/cm 2 , respectively. 5 Example 7 MFC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan gum broth (MFC/XG=3/1, dry basis) and the resultant mixture was then precipitated with IPA 10 (85%) to form a press cake. The press cake was then dried and milled as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, with 20% CMC added simultaneously, and the composition was then mixed with a Silverson mixer at 8000 rpm for 5 min. The product viscosity and yield stress were 690 cP and 2.19 dynes/cm 2 , respectively. 15 Example 8 MFC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of xanthan 20 gum broth and CMC solution (MFC/XG/CMC=3/1/1, dry basis) and the resultant mixture was then precipitated with IPA (85%) to form a press cake. The press cake was then dried and milled 18 WO 2006/127810 PCT/US2006/020080 as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, and the composition was then mixed with a Silverson mixer at 8000 rpm for 5 min. The product viscosity and yield stress were 1057 cP and 3.65 dynes/cm 2 , respectively. 5 Example 9 MFC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of pectin solution (MFC/Pectin=6/1, dry basis) and the resultant mixture was then precipitated with IPA 10 (85%) to form a press cake. The press cake was dried and milled as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, with 20% CMC added simultaneously, and the composition was then mixed with a Silverson mixer at 8000 rpm for 5 min. The product viscosity and yield stress were 377 cP and 1.06 dynes/cm 2 , respectively. 15 Example 10 MFC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of CMC 20 solution (MFC/CMC=3/1, dry basis) and the resultant mixture was then precipitated with IPA (85%) to form a press cake. The press cake was dried and milled as in Example 1. The 19 WO 2006/127810 PCT/US2006/020080 powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, and the composition was then mixed with a Silverson mixer at 8000 rpm for 5 min. The product viscosity and yield stress were 432 cP and 1.39 dynes/cm 2 , respectively. 5 Example 11 MFC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 194 ppm of protease. A portion of the treated MFC broth was mixed with a given amount of pectin and CMC solutions (MFC/Pectin/CMC=6/1/2, dry basis) and the resultant mixture was then 10 precipitated with IPA (85%) to form a press cake. The press cake was dried and milled as in Example 1. The powdered formulation was then introduced into a STW sample in an amount of about 0.36% by weight thereof, and the composition was then mixed with a Silverson mixer at 8000 rpm for 5 min. The product viscosity and yield stress were 552 cP and 1.74 dynes/cm 2 , respectively. 15 Example 12 MFC was produced in a 1200 gal fermentor with final yield of 1.51 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC 20 broth was mixed with a given amount of xanthan gum broth (MFC/XG=2/1, dry basis), then 20 WO 2006/127810 PCT/US2006/020080 preuipuateat wim wA (6r/o), and dried and milled as in Example 1. The powdered formulation was then introduced into a STW solution in an amount of about 0.2% by weight thereof, with 10% CMC added simultaneously, and the composition was then activated with an extensional homogenizer at 1500 psi for 2 passes. The product viscosity at 6 rpm was 377 cP. 5 Example 13 MFC was produced in a 1200 gal fermentor with final yield of 1.6 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC 10 broth was mixed with a given amount of xanthan gum broth (MFC/XG=2/1, dry basis), then precipitated with IPA (85%), and dried and milled as in Example 1. The powdered formulation was then introduced into a deionized water solution, a STW solution and 0.25% CaCl 2 solution, respectively, in an amount of about 0.2% by weight thereof, with 10% by weight of CMC added simultaneously, and the composition was then activated with an extensional homogenizer at 15 1500 psi for 2 passes. The product viscosities were 512 cP, 372 cP and 358 cP, in de-ionized water, STW and 0.25% CaCl 2 solution, respectively. Analogous to the test performed in Example 1, with this sample about 20 3.2 mm diameter nylon beads (exhibiting a density each of about 1.14 g/mL) were dropped into each of the solutions (in de-ionized water, STW or 0.25% CaC 2 solution) and the solutions were left at 20 room temperature for 24 hours. None of the beads settled down to the bottom of the beakers after the time period expired, thus indicating excellent long-term suspension properties. 21 WO 2006/127810 PCT/US2006/020080 Example 14 MFC was produced in a 1200 gal fermentor with final yield of 1.51 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC 5 broth was mixed with a given amount of xanthan gum broth (MFC/XG=2/1, dry basis), then precipitated with IPA (85%), and dried and milled as in Example 1. The powdered formulation was then introduced into a deionized water sample in an amount of about 0.2% by weight thereof, with 10% by weight of CMC added simultaneously, and the composition was then activated with a propeller mixer at 2500 rpm for 10 min. The product viscosity was 185 cP. 10 Example 15 MFC was produced in a 1200 gal fermentor with final yield of 1.4 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC 15 broth was mixed with a given amount of xanthan gum broth and pre-hydrated CMC solution (MFC/XG/CMC = 6/3/1, dry basis), then precipitated with IPA (85%), and dried and milled as in Example 1. The powdered formulation was then introduced into a STW solution and 0.25% CaCl2 solution in an amount of about 0.2% by weight thereof, respectively, and the composition was then activated with an extensional homogenizer at 1500 psi for 2 passes. The product 20 viscosities at 6 rpm were 343 cP and 334 cP in STW and 0.25% CaCl 2 solutions, respectively. About 20 3.2 mm diameter nylon beads (1.14 g/mL) were dropped into each of the solutions (in 22 WO 2006/127810 PCT/US2006/020080 STW or 0.25% CaCl 2 solution) and the solutions were left at room temperature for 24 hrs. None of the beads settled down to the bottom of the beakers after the 24-hour time period. 5 Example 16 MFC was produced in a 1200 gal fermentor with final yield of 1.6 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC broth was mixed with a given amount of pre-hydrated pectin and CMC solutions 10 (MFC/Pectin/CMC = 6/3/1, dry basis), then precipitated with IPA (85%), and dried and milled as in Example 1. The powdered formulation was then introduced into a STW solution and 0.25% CaCl2 solution in an amount of about 0.2% by weight thereof, respectively, and the composition was then activated with an extensional homogenizer at 1500 psi for 2 passes. The product viscosities at 6 rpm were 306 cP and 293cP in STW and 0.25% CaCl 2 solutions, respectively. 15 About 20 3.2 mm diameter nylon beads (1.14 g/mL) were dropped into each of the solutions (in STW or 0.25% CaCl 2 solution) and the solutions were left at room temperature for 24 hours. None of the beads settled down to the bottom of the beakers after the 24-hour time period. Example 17 23 WO 2006/127810 PCT/US2006/020080 MFC was produced in a 1200 gal fermentor with final yield of 1.6 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC broth was mixed with a given amount of pre-hydrated CMC solution (MFC/CMC = 3/1, dry 5 basis), then precipitated with IPA (85%), and dried and milled as in Example 1. The powdered formulation was then introduced into a STW solution and 0.25% CaCl2 solution in an amount of about 0.2% by weight thereof, respectively, and the composition was then activated with an extensional homogenizer at 1500 psi for 2 passes. The product viscosities at 6 rpm were 206 cP and 202 cP in STW and 0.25% CaCl2 solutions, respectively. About 20 3.2 mm diameter nylon 10 beads (1.14 g/mL) were dropped into each of the solutions (in STW or 0.25% CaCl 2 solution) and the solutions were left at room temperature for 24 hours. None of the beads settled down to the bottom of the beakers after the 24-hour time period. Example 18 15 MFC was produced in a 1200 gal fermentor with final yield of 1.54 wt%. The broth was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm of lysozyme and 350 ppm of protease followed with another 350 ppm of hypochlorite. A portion of the treated MFC broth was mixed with a given amount of Diutan broth (MFC/Diutan=2/1, dry basis), then precipitated with IPA (85%), and dried and milled as in Example 1. The powdered formulation 20 was then introduced into a de-ionized water solution in an amount of about 0.2% by weight thereof, with 10% CMC added simultaneously, and the composition was then activated with an extensional homogenizer at 1500 psi for 2 passes. The product viscosity at 6 rpm was 214 cP. 24 WO 2006/127810 PCT/US2006/020080 Each sample exhibited excellent and highly desirable viscosity modification and yield stress results. In terms of bacterial cellulose products, such results have been heretofore unattainable with bacterial cellulose materials alone and/or with the low complexity methods followed herein. 5 While the invention will be described and disclosed in connection with certain preferred embodiments and practices, it is in no way intended to limit the invention to those specific embodiments, rather it is intended to cover equivalent structures and all alternative embodiments and modifications as may be defined by the scope of the appended claims and equivalence thereto. 10 15 20 25

Claims (33)

1. A method for the production of a bacterial cellulose-containing formulation comprising the steps of a) providing a bacterial cellulose product that includes a reticulated network of bacterial cellulose fibres; b) optionally lysing the bacterial cells from the bacterial cellulose product; c) mixing said bacterial cellulose product of either step "a" or step "b" with a polymeric thickener selected from the group consisting of at least one charged cellulose ether, at least one precipitation agent, and any combination thereof to provide a coating of the polymeric thickener on at least a portion of the surface of said bacterial cellulose product; and d) co-precipitating the mixture of step "c" with a water-miscible non aqueous liquid to recover the coated bacterial cellulose product.

2. The method according to Claim 1 wherein said polymeric thickener of step "c" is a charged cellulose ether.

3. The method according to Claim 2 wherein said charged cellulose ether is selected from the group consisting of sodium carboxymethylcellulose, cationic hydroxyethylcellulose, and any mixtures thereof

4. The method according to Claim 1 wherein said polymeric thickener of step "c" is a precipitation agent.

5. The method according to Claim 4 wherein said precipitation agent is selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof

6. The method according to Claim I wherein said bacterial cellulose product is a microfibrillated cellulose.

7. The method according to Claim 6 wherein said polymeric thickener of step "c" is a charged cellulose ether. 26

8. The method according to Claim 7 wherein said charged cellulose ether is selected from the group consisting of sodium carboxymethylcellulose, cationic hydroxyethylcellulose, and any mixtures thereof.

9. The method according to Claim 6 wherein said polymeric thickener of step "c" is a precipitation agent.

10. The method according to Claim 9 wherein said precipitation agent is selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, diutan gum, welan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof.

11. The method according to Claim 10 wherein said precipitation agent is selected from the group consisting of xanthan, pectin, diutan gum, and any mixtures thereof

12. A method for the production of a bacterial cellulose-containing formulation comprising the steps of a) providing a bacterial cellulose product that includes a reticulated network of bacterial cellulose fibres; b) optionally lysing the bacterial cells from the bacterial cellulose product; c) mixing said resulting bacterial cellulose product of either step "a" or step "b" with at least one precipitation agent selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof to provide a coating of the precipitation agent on at least a portion of the surface of said bacterial cellulose product; and d) co-precipitating the mixture of step "c" with a water-miscible non aqueous liquid to recover the coated bacterial cellulose product.

13. The method according to Claim 12 wherein said precipitation agent is selected from the group consisting of xanthan, pectin, diutan gum, and any mixtures thereof 27

14. A method for the production of a bacterial cellulose-containing formulation comprising the steps of a) providing a bacterial cellulose product that includes a reticulated network of bacterial cellulose fibres; b) mixing said bacterial cellulose product with at least one precipitation agent selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof to provide a coating of the precipitation agent on at least a portion of the surface of said bacterial cellulose product; c) co-lysing the mixture of step "b" to remove bacterial cells therefrom; and d) co-precipitating the mixture of step "c" with a water-miscible non aqueous liquid to recover the coated bacterial cellulose product.

15. The method according to Claim 14 wherein said precipitation agent is selected from the group consisting of xanthan, pectin, diutan gum, and any mixtures thereof

16. A bacterial cellulose-containing formulation comprising at least one reticulated network of bacterial cellulose fibres that are at least partially coated with at least one polymeric thickener selected from the group consisting of at least one charged cellulose ether, or at least one precipitation agent, or any mixtures thereof, wherein said formulation exhibits a viscosity capability of at least 300 cps and a yield stress measurement of 1.0 dyne/cm when introduced in an amount of at most 0.36% by weight of a 500 mL sample of water and after application of at most 2 passes at 1500 psi in an extensional homogenizer.

17. The formulation according to Claim 16 wherein said polymeric thickener is at least one charged cellulose ether.

18. The formulation according to Claim 17 wherein said charged cellulose ether is selected from the group consisting of at least one sodium carboxymethylcellulose, at least one cationic hydroxyethylcellulose, and any mixtures thereof

19. The formulation according to Claim 16 wherein said polymeric thickener is a precipitation agent. 28

20. The formulation according to Claim 19 wherein said precipitation agent is selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof.

21. The formulation according to Claim 16 wherein said bacterial cellulose product is microfibrillated cellulose.

22. The formulation according to Claim 21 wherein including both said charged cellulose ether and said precipitation agent.

23. The formulation according to Claim 22 wherein said precipitation agent is selected from the group consisting of a xanthan product, pectin, alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust bean gum, and any mixtures thereof

24. The formulation according to Claim 23 wherein said charged cellulose ether is a sodium carboxymethylcellulose.

25. The formulation according to Claim 24 wherein said precipitation agent is selected from the group consisting of xanthan, pectin, diutan gum, and any mixtures thereof

26. The formulation according to Claim 25 wherein said precipitation agent is xanthan.

27. The formulation according to Claim 25 wherein said precipitation agent is pectin.

28. The formulation according to Claim 25 wherein said precipitation agent is diutan gum.

29. The formulation according to Claim 20 wherein said precipitation agent is xanthan. 29

30. The formulation according to Claim 20 wherein said precipitation agent is pectin.

31. The formulation according to Claim 20 wherein said precipitation agent is diutan gum.

32. A bacterial cellulose-containing formulation when produced by the method according to any one of claims 1 to 15.

33. A method for the production of a bacterial cellulose-containing formulation, a bacterial cellulose-containing formulation when produced by the method, or a bacterial cellulose-containing formulation substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 30