Stable solid compositions with high content of bromoform

Abstract

A process of formulating brominated C1-C2 alkane in a solid carrier, comprising combining in a reaction vessel an aqueous solution of a water-soluble edible polysaccharide and the brominated C1-C2 alkane to form a semi-solid, paste-like or solid mass, drying and comminuting the mass to give brominated C1-C2 alkane -containing polysaccharide in a powder form. A brominated C1-C2 alkane-rich polysaccharide is also provided by the invention.

Description

[0001] Stable solid compositions with high content of bromoform

[0002] Background of the invention

[0003] In the rumen of ruminant animals such as cows, sheep, deer and goats, metabolic processes occur, in which microbes act on the feed eaten by the animal to break it down into smaller compounds, generating utilizable energy and nutrients for the animal. Methane gas (CH4) is formed as a by-product by such fermentation processes; it is named enteric methane. Enteric methane is released by the animal through eructation. Thus, enteric methane represents a potential loss of feed energy in ruminants. What makes the problem even worse is that the enteric methane that is emitted by ruminant livestock to the atmosphere is a greenhouse gas. Though a relatively shortlived greenhouse gas compared to carbon dioxide, the effect of methane emission is not insignificant, as methane traps heat more effectively than carbon dioxide.

[0004] Suppressing production of enteric methane with anti- methanogenic agents incorporated into feed additives supplied to ruminant livestock is known. A recently published review paper by Honan et al. [Feed additives as a strategic approach to reduce enteric methane production in cattle: modes of action, effectiveness, and safety; Animal Production Science, 2022, 62, 1303-1317] mentions classes of compounds that can act through various mechanisms to reduce methane emission from ruminants, such as halogenated compounds, nitrates, fatty acids, and tannins, to mention a few.

[0005] Potentially useful halogenated compounds include chlorine and / or bromine-substituted C1-C2 hydrocarbons, e.g., chloromethanes and bromomethanes, and mixed chloro / bromo methanes, such as chloroform, carbon tetrachloride, methylene chloride, bromochloromethane, dibromoethane and bromoform. These haloalkanes exist as volatile liquids at room temperature and need to be formulated into solid compositions that can be added to a livestock feed premix.

[0006] For solid, orally administrable delivery systems of liquid haloalkanes to gain commercial acceptance, both initial high content of the haloalkane and good stability against volatilization are important, as these haloalkanes evaporate easily at room temperature. Different formulation approaches were reported, e.g., microencapsulation of liquid haloalkanes to form coacervate, triggered by pH change (US 3,660,562); preparation of inclusion complex of the haloalkane with cyclodextrin in a powder form (WO 96 / 14062 and WO 2023 / 150832), emulsification of the haloalkane or addition to gelatine and solidification in a mold (WO 2023 / 010170), formulation of the haloalkane with the aid of an edible polymer and waxes (WO 2022 / 124914), and a composition comprising an haloalkane and organosulfur (WO 2022 / 136857).

[0007] Amongst the haloalkanes, bromoform is emerging as a potent methanogenic inhibitor. Because the microalga Asparagopsis taxiformis produces bromoform during cultivation, it has been proposed to use this microalga as a feed additive to reduce methane production in ruminant livestock (WO 2020 / 113279, WO 2022 / 237993, WO 2023 / 015042, and AU 2023 / 200792). Synthetically produced bromoform can also be used. Bromoform - a colorless liquid with a boiling point of 150°C - can be prepared by a reaction of acetone with sodium hypobromite; the Br3C-C (0)-CH3 formed undergoes alkaline hydrolysis to give bromoform and sodium acetate. Another approach is to start with chloroform and replace the chlorine atoms with bromine, by the action of molten aluminum bromide (US 1,891,415) or by bubbling gaseous hydrogen bromide through a solution of aluminum chloride in chloroform (US 2,553,518). The invention

[0008] Experimental work reported below shows an efficient method of incorporating C1-C2 bromine-containing alkane, e.g., bromoform or dibromomethane, into edible polysaccharides, e.g., gums, especially gum arabic, in aqueous solutions. Despite the high volatility of brominated C1-C2 alkanes such as bromoform, it is possible to obtain gum powder showing high and stable bromoform holding capacity, e.g., not less than 150 mg bromoform per 1 g of gum, over long storage periods.

[0009] When bromoform is gradually added under stirring to a suitably proportioned aqueous solution, or aqueous / alkanol solution, of a water-soluble edible gum such as gum arabic, then the mixture progressively turns into a homogeneous thick slurry. After the mixture is allowed to stand for some time, a semi-solid, almost non-flowable paste is formed, which, following drying and comminution, affords a powder with high bromoform content. Stability testing of the powder indicates that the bromoform content remains essentially constant with the passage of time, suggesting that the bromoform molecules are tightly trapped within the solid matrix consisting of the edible gum.

[0010] Accordingly, the invention is primarily directed to a process of formulating brominated C1-C2 alkane such as bromoform and dibromomethane in a solid carrier, comprising combining in a reaction vessel an aqueous solution of a water-soluble edible polysaccharide, preferably gum, and brominated C1-C2 alkane (e.g., bromoform) to form a semi-solid, paste-like or solid mass, drying and comminuting the mass to give a brominated Cl- C2 alkane-containing polysaccharide in a powder form. The reaction vessel may further include a lower alkanol.

[0011] A convenient way to incorporate the brominated C1-C2 alkane

[0012] (for example, bromoform and / or dibromomethane) into the gum is by the addition of the brominated C1-C2 alkane to a reaction vessel that was previously charged with an aqueous solution of a water-soluble edible gum. Gums suitable for use in the invention, such as gum arabic, are commercially available in a powder form. Gum arabic powder consists of ~80-82% carbohydrates (a mixture of glycoproteins and polysaccharides, predominantly a mixture of polymers of arabinose and galactose), 11-14% moisture, ~3.6-3.9% ash, 2.0-2.4% proteins and 0.1-0.6% lipids with slight variation among available grades (composition is by weight %). Other properties of gum arabic powders suitable for use in the invention were reported by Abel et al. in Food Research 4 (Suppl. 1) (2020) 107 - 115, e.g., particle size distribution, density (true density of 1.48 g / cm3, bulk density in the range of 0.59-0.79 g / cm3, tapped density in the range of 0.73 to 0.83 g / cm3), flowability (expressed by Hausner ratio in the range of 1.13 to 1.30) and compressibility (Carr index in the range of 11 to 23%). Polysaccharides (non-gum) such as pectin may also be considered for use in the invention.

[0013] The gum, e.g., gum arabic, dissolves in water at room temperature, creating slightly acidic-nearly neutral solutions. The powdered gum can be either suspended in water at room temperature, following which the resulting slurry is heated to a temperature >50°C, e.g., ~ 55 to 65°C, or added to water that was previously heated to >50°C. Under stirring at this elevated temperature range, complete dissolution of the gum is quite fast, giving aqueous solutions with 15:100 to 35:100 gum to water weight ratio. As shown below, this is not the final concentration of the gum in the water, as it is more effective to divide the total amount of the gum into several portions that are supplied incrementally to the reaction vessel, before, during and after the addition of the brominated C1-C2 alkane.

[0014] Usually, the amount of gum in the initial solution is between a fifth and a third of the total amount of the gum needed for the formulation, e.g., between a fifth and a quarter. The weight ratio between the total amount of the gum and water is from 75:100 to 125:100, e.g., from 100:100 to 120:100.

[0015] The brominated C1-C2 alkane, e.g., bromoform, is fed to the aqueous gum solution, usually after the solution is allowed to cool down, so that the process is run at room temperature. Addition of the brominated C1-C2 alkane (e.g., bromoform) takes place in a gradual manner. The brominated C1.-C2 alkane, e.g., brominated methane, which includes bromoform (CHBr3) or dibromomethane (CH2Br2), can be supplied to the reaction vessel in a neat form and preferably alternatively, as a solution with a water-miscible, readily volatile solvent, e.g., a lower alkanol such as ethanol or isopropanol. example, bromoform in a neat form, or a bromoform and ethanol mixture prepared beforehand at a weight mixing ratio of

[0016] 5:1 to 2:1, is slowly added to the aqueous gum arabic solution, e.g., at a feed rate of 3 ml / min to 7 ml / min on a laboratory scale. As the organic component (neat bromoform or bromoform / ethanol solution) is slowly introduced into the aqueous solution under stirring, a homogeneous slurry is formed. The weight ratio between water and the water-miscible organic solvent, e.g., ethanol, when present (the lower alkanol facilitates the homogenization of the mixture) is from 400:100 to 180:100, e.g., from 330:100 to 200:100, e.g., from 250:100 to 210:100 (i.e., predominance of water over ethanol). The weight ratio between bromoform and water ranges from 200:100 to 100:100, e.g., from 160:100 to 140:100. Similar ratios apply for other brominated C1-C2 alkanes, e.g., dibromomethane.

[0017] Next, as the remaining amount of the gum powder is added preferably in an incremental / portionwise manner with stirring, the slurry becomes increasingly thicker. The stirred mixture takes up a large amount of gum arabic when this amount is divided into a few roughly equal portions, with time breaks of not less than 15 minutes between each addition.

[0018] The addition of bromoform, or bromoform / ethanol solution, and the subsequent addition of the gum powder are carried out over a period of time, hereinafter referred to as the 'addition time'. The addition time is not less than 60 minutes. Subsequently, the mixture is stirred, e.g., for 10 to 50 minutes, e.g., 30 minutes.

[0019] After preparation of the mixture has been completed, it is held (unstirred) in a vessel for a period of time hereinafter referred to as the 'hold time'. The hold time is usually not less than 0.5 hour, >1 hour, >2 hours, >3 hours, e.g., not less than 5 hours.

[0020] The components (gum, water, the brominated C1.-C2 alkane such as bromoform, and ethanol) are present in the mixture in carefully selected proportions, aimed at progressively thickening and maintaining the homogeneity of the mixture, which gradually transforms as a whole into a bromoform-rich, paste-like, semisolid / solid material. Under some conditions, however, the mixture may be allowed to break up into two distinct phases, a major phase consisting of a paste and a minor phase consisting of a very thin slurry / solution, in which case the paste is processed in the manner described below.

[0021] It should be noted that an excessive amount of ethanol may lead to separation of the reaction mass into an organic phase (i.e., ethanol and bromoform solution) and an aqueous phase (i.e., the gum solubilized in water). A large excess of water may result in the formation of a non-viscous aqueous solution, with complete, or almost complete, solubilization of the gum, ethanol and bromoform (the latter is sparingly soluble in water). An insufficient excess of bromoform does not destroy the homogeneity of the mixture, but the lower the excess of bromoform, the thinner the slurry, and the less probable the recovery of bromoform-rich gum powder.

[0022] After an appropriate hold time, a very thick, largely homogeneous slurry or paste, containing the brominated C1-C2 alkane (e.g., bromoform or dibromomethane), water and optionally ethanol, is obtained, usually with a paste strength, referring to the content of gum arabic in the paste before the drying step, of e.g., from 23 to 35% by weight, meaning that the remainder, consisting of water, bromoform and optionally ethanol, is from 65 to 77% w / w.

[0023] The drying step to remove water, ethanol and excess bromoform generally involves thermal drying using conventional dryers commonly applied for drying pastes, e.g., vacuum dryers. For example, a useful drying program consists of drying under reduced pressure at a first temperature Tl, 40°C≤Tl<70°C, over at least 10 hours, e.g., 15-20 hours, and then optionally at a second temperature T2, 70°C<T2<95°C, e.g., 80°C<T2<95°C, under reduced pressure over not less than 10 minutes but usually not more than 50 minutes, e.g., about 30 minutes. Stability testing shows that bromoform remaining after the drying at 70°C<T2<95°C is strongly locked in the gum arabic matrix and does not escape even when the powder is kept under conditions of open-air storage.

[0024] Alternatively, the paste is dried at above 100°C, for example, at 120°C under vacuum over a period of 3-5 h until it becomes breakable. The solid material is ground into small particles with a size below about 5 mm. Then the powder is further dried, e.g., at 120°C under vacuum for 2-3 h. After that, the powder is ground and sieved to below 1 mm size. Finally, the powder is heated at 50°C under vacuum over a period of 12 h to complete the drying.

[0025] For example, the paste is placed on trays mounted on shelves in a vacuum dryer, the pressure in the vacuum dryer is decreased, e.g., up to 100 mbar, and the paste is heated to a first temperature 40°CdTl<70°C with a stronger vacuum optionally applied as the heating progresses. The material is ground to reduce particle size to facilitate further removal of the solvents and obtain a powder form of the product which again is exposed to reduced pressure (e.g., up to 30 mbar) at 70°C<T2<95°C, e.g., 80°C<T2<95°C, to give submillimeter gum arabic particles with high brominated C1-C2 alkane (e.g., bromoform) content, e.g., not less than 10%, e.g., not less than 15%, preferably from to 20 to 25% by weight. It should be noted that ~ 30-33 wt.% of bromoform-containing powders may be provided by the invention with heating under reduced pressure at Tl. A portion of the bromoform is liberated if the powder is exposed to the conditions of the second drying step. Bromoform left in the powder after the powder is dried at T2 under reduced pressure is named herein "strongly locked bromoform" and can be as high as 20 to 25 wt.% based on the total weight of the powder.

[0026] Accordingly, another aspect of the invention is therefore a brominated C1-C2 alkane rich-polysaccharide such as bromoformrich polysaccharide, e.g., bromoform-rich gum arabic, usually in a powder form, preferably with a brominated C1-C2 alkane content (e.g., bromoform content or dibromomethane content) of at least 10%, e.g., >15%, >20% based on the total weight of the powder, e.g., the powder of the invention consists of 70 to 90 wt.% gum arabic, e.g., from 75 to 85 wt.%, for example, from 75 to 80 wt.% (typically including a few percentages of tightly bound water remaining after the drying under reduced pressure, e.g., <10 wt.%, e.g., 5-10 wt.%, ~8 wt.% of tightly bound water, which is considered as part of the amount of the gum matrix) and from 10 to 30 wt.% of brominated C1-C2 alkane such as bromoform (e.g., 15 to 25 wt.%, for example, from 20 to 25 wt.%) and remnant ethanol (less than 300 ppm). The concentration of the brominated C1-C2 alkane (e.g., bromoform) in the gum powder of the invention can be quantified by different methods, such as for example by thermal decomposition of a sample by Parr Bomb method in the presence of sodium peroxide and sucrose, or by gas chromatographic determination of bromoform after full dissolution of a gum arabic-bromoform sample in water.

[0027] Infrared (IR) transmittance spectrum of the bromoform-rich gum arabic powder provided by the present invention shows strong absorption peaks characteristic of bromoform (major peaks are at approximately 3000 cm-1, 1150 cm-1and 650 cm-1) and broad absorption bands and peaks assigned to arabic gum (at 3000-3500 cm-1, 2850-2950 c 1600 cm-1and 1000-1100 cm-1).

[0028] The bromoform-containing gum arabic powder is storage stable, showing low vapor pressure, attesting to the fact that bromoform molecules are strongly locked within the gum particles and do not escape easily into the gaseous phase. The vapor pressure measured for bromoform-rich powders of the invention is comparabic (± 15%) to the vapor pressure of commercial gum arabic powder both at T=20°C and / or T=40°C, after the commercial powder, with dso=4O-6O micron, has been dried under vacuum at 50°C for 8-15 hours (gum arabic is fairly hygroscopic and holds moisture even after such drying; this moisture accounts for the vapor pressure of the reference gum arabic). Vapor pressure is measured by freezing a sample under a vacuum of less than 1 mbar at a temperature below the melting point of bromoform, disconnecting the vacuum, gradually heating the sample to a target temperature, and measuring the vapor pressure at the target temperature by a manometer connected to a flask holding the sample.

[0029] We use the term brominated C1-C2 alkane-rich polysaccharide / gum, e.g., bromoform-rich polysaccharide / gum, to describe solid composition consisting of particles made of polysaccharide / gum, with weight ratio polysaccharide / gum to the brominated C1-C2 alkane (e.g., bromoform) ranging from 9:1 to 2:1, e.g., 9:1 to 3:1, preferably from 6:1 to 3:1, more preferably from 5.6:1 to 3:1. Typically, the particles are obtained by causing the polysaccharide / gum to take up excess brominated C1-C2 alkane (e.g., bromoform), and, following drying under the conditions set out above, liberate a portion of the bromoform, still leaving significant bromoform content, in particular strongly locked bromoform, in the solid composition.

[0030] The composition is devoid of synthetic polymers like polyethylene maleic anhydride copolymer and polyethylene imine. The composition of the invention may also be gelatine-free.

[0031] To minimize depletion of the brominated C1-C2 alkane-rich gum arabic owing to escape of the volatile bromoform (or dibromomethane) during storage and shipping, edible coatings can be applied on the particles, or the particles can be formulated in a suitable liquid carrier.

[0032] For example, coating can be applied to bromoform-rich gum arabic particles to provide a hydrophobic wall barrier, to increase hydrophobicity and reduce moisture uptake by the hygroscopic gum arabic, and to minimize escape of the easily volatile bromoform and maintain constant levels of bromoform in the gum arabic matrix. One technique is hot melt coating. For example, after the paste is formed and before the drying step, the paste is heated in the presence of a coating agent (at a concentration of 1 to 30-40% relative to the paste) at a temperature above the melting point of the coating agent, with stirring to uniformly distribute the molten coating agent within the paste. On cooling, the coating agent solidifies to form a hydrophobic film over the gum arabic particles. For example, a coating agent that can be applied in this manner is stearic acid, working at a temperature of 85°C. Addition of microcrystalline cellulose to the paste before drying is another option for creating a coating. Another useful hydrophobic coating consists of a food-grade natural wax such as carnauba wax, which is formulated in a sprayable coating formulation (such as hot tetrahydrofuran solution), to be applied onto the dried bromoform-rich gum arabic powder and create a thin film on the surface of the particles.

[0033] Fluid bed coating is another method that can be used, i.e., the particles consisting of brominated C1-C2 alkane-rich polysaccharide are fluidized with gas while the coating material is injected (through nozzles) either from above ("Top-Spray"), laterally ("Tangential-Spray") or sprayed into the particles bed in the direction of the gas flow ("Bottom- Spray") . As shown in the experimental work reported below, good results were obtained with the latter method, specifically by Wurster coating, namely, using a fluid bed coater with Wurster configuration. The coating material was injected in a molten form into the fluid bed coater and solidified by cooling on the particles (hot melt coating). Alternatively, a coating solution, obtained by dissolving a coating material beforehand in a solvent, was injected into the fluid bed coater, and a coating layer was created upon solvent evaporation (wet coating).

[0034] For example, molten hydrogenated oil (such as hydrogenated rapeseed oil and hydrogenated palm oil) and molten plant- derived wax (such as Carnauba wax) were applied as coating materials on particles of bromoform-rich polysaccharide in a fluid bed coater with the Wurster configuration (the weight ratio of the bromoform-rich polysaccharide to the molten coating material is 65-75 : 35-25, e.g., 70:30). For example, a film forming, cellulose-based, polymer such as ethyl cellulose was dissolved in acetone / isopropanol mixture and the solution was sprayed into a fluid bed coater with the Wurster configuration to coat the particles (the weight ratio of the bromoform-rich polysaccharide to the dissolved coating material is 80-90 : 20-10, e.g., 85:15).

[0035] Accordingly, the invention further relates to a brominated Cl- C2 alkane-rich polysaccharide, which comprises a coating layer. Specifically, coated brominated C1-C2 alkane-rich gum arabic, e.g., coated bromoform-rich gum arabic, is provided by the invention, for example: wax-coated bromoform-rich gum arabic; hydrogenated oil-coated bromoform-rich gum arabic; and cellulose derivative-coated bromoform-rich gum arabic.

[0036] As mentioned above, another approach to increasing the hydrolytic stability of the bromoform-rich gum arabic powder, to prevent premature release of bromoform from the solid formulation, consists of suspending the powder in an edible oil-based carrier. The concentration of the powder in the liquid oil is in a range of 1 to 70% by weight. H-NMR analysis indicates that no bromoform was extracted from the bromoform- GA powder into the oil over a period of four months. Accordingly, another aspect of the invention is a composition comprising an oil-based carrier and the brominated C1-C2 alkane-rich polysaccharide suspended in the carrier, e.g., bromoform-rich gum arabic suspended in oils such as corn oil, sunflower oil, rapeseed oil and soybean oil.

[0037] The bromoform-containing gum powder of the invention can be delivered to ruminant livestock through blending with feed composition or, even better, through addition to premix formulations. Premix formulations consist of feed additives such as vitamins, minerals and other nutritional ingredients and are regularly incorporated into the feeding regime of livestock. Premix formulations may be produced in a pelleted form and added to livestock daily feed as appropriate. The powder of the invention will be mixed with the other constituents of the premix formula in a batch or continuous mixer. The blend will be used as is or fed and processed in a conventional feed pellet machine (available from various manufacturers, such as Anyang Best Complete Machinery Engineering Co., Ltd), pressed out, cut, and discharged to give cylindrically shaped pellets with the appropriate size. An effective bromoform administration dosage is considered to lie in the range from 0.1-1.5 mg / kg animal / day (see, for example, the recently published paper of Eason et al. in NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH, 2023, https: / / doi.org / 10.1080 / 00288233.2023.2248948). Owing to the high bromine content of the powder of the invention (~150-250 mg bromoform per gram powder), an addition rate of just ~0.25 to 2.5 grams / day of the powder to the cattle (cow) diet will deliver the required daily dose of bromoform. For example, a premix can be prepared according to the compositions below, by mixing the ingredients together (MIU: Mill! International Unit; KIU Kilo International Unit): Formulation A

[0038] Ingredient amount

[0039] Vitamin A 12.000 MIU

[0040] Vitamin D 3.120 MIU

[0041] Vitamin E 80.000 KIU

[0042] Antiox 15.000 g

[0043] Manganese 81.800 g

[0044] Zinc 168.800 g

[0045] Iron 18.400 g

[0046] Copper 34.700 g

[0047] Iodine 1.300 g

[0048] Cobalt 0.600 g

[0049] Selenium 0.900 g

[0050] Limestone 233.479 g

[0051] Formulation B (US 2017 / 0000805)

[0052] Ingredient amount (% by weight)

[0053] Vitamin D3 0.2

[0054] Vitamin A 0.05

[0055] Vitamin E 2

[0056] Biotin 0.375

[0057] CuSO40.5

[0058] ZnSO40.281

[0059] CoS040.0185

[0060] Na2SeO30.0035

[0061] KI 0.0055

[0062] MnSO40.484

[0063] CaSO421.08

[0064] MgCl275

[0065] The bromoform-rich gum arabic powder may be added to a premix at a concentration of about 0.1-0.5% by weight. Usually about 2 kg of the premix are added per 100 kg of regular food, e.g., consisting of maize silage (30% by weight), grass silage (20% by weight), cereal grain (25% by weight), long hay (15% by weight), protein meal (8 % by weight) and the premix (2% by weight) . Accordingly, additional aspects of the invention include a method of reducing the emission of enteric methane produced by a ruminant animal, comprising administering to the animal an effective amount of brominated C1-C2 alkane-rich polysaccharide, e.g., bromoform-rich polysaccharide, e.g., bromoform-rich gum such as gum arabic; use of a brominated C1- C2 alkane-rich polysaccharide, e.g., bromoform-rich polysaccharide, e.g., bromoform-rich gum such as gum arabic, as a feed additive in the nutrition of a ruminant animal; and a premix feed formulation, comprising brominated C1-C2 alkane- rich polysaccharide, e.g., bromoform-rich polysaccharide, e.g., bromoform-rich gum such as gum arabic.

[0066] Brief description of the drawings

[0067] Figure 1 is an FTIR spectra of commercial gum arabic, bromoform and bromoform-containing gum arabic of the invention.

[0068] Figure 2 shows an experimental setup of a manometric vapor pressure measurement.

[0069] Examples

[0070] Preparation 1 Synthesis of bromoform

[0071] The reaction took place in a 1000 ml three-necked flask fitted with a mechanical stirrer, a gas inlet tube (dip pipe) and a reflux condenser. The top of the reaction vessel was connected to a water trap (filled with 1.5 L of water) via an intermediate empty flask to prevent back-suction of the water- HBr solution into the reactor.

[0072] To the reactor was added 480 g (4 mol) of chloroform, followed by 26.8 g (0.2 mol) of aluminum chloride. The contents of the flask were heated to 57°C in an oil bath (CHCI3 b.p.=61.7°C). HBr gas from a cylinder was passed via a pressure reducing valve through a calibrated rotameter and was bubbled via a dip-tube into the well-stirred reaction mixture until the full conversion of the chloroform into bromoform had been achieved (monitoring by Gas Chromatography). The reaction mixture was cooled to room temperature, following which 200 ml of water was carefully added to the flask to decompose the catalyst, and the mixture was transferred to a separatory funnel.

[0073] The aqueous layer was separated, and the organic layer was washed with 200 ml of 5% w / w of NaHCO3and then with 200 ml of water. The washed organic phase was distilled under a vacuum of 25-30 mbar to afford the final pure bromoform in a yield of 85% based on chloroform and with a purity exceeding 99% (by GC analysis) with the rest being largely dibromochloromethane.

[0074] Example 1

[0075] Preparation of bromoform-containing gum arabic

[0076] A 0.5 L round-bottom flask, equipped with a mechanical stirrer and a thermometer, was charged with gum arabic (10 g; from Dingli Biodegradable Material Co., Ltd) and water (40 g). The initial slurry became clear after 30 min at 60°C, and the solution was cooled down to 30°C.

[0077] A pre-prepared mixture of bromoform (60 g) and ethanol (20 ml) was then added dropwise, resulting in the formation of a homogeneous slurry. Subsequently, 35 g of gum arabic was added in three, approximately equal, portions at 30-40 min intervals under efficient stirring, to distribute the added gum evenly within the slurry that became increasingly pasty.

[0078] Stirring was stopped and the unstirred thick slurry / paste was held for an additional 15 hours at room temperature to afford a very thick, largely homogeneous slurry / paste containing all the bromoform, water and ethanol.

[0079] Then the paste was heated and kept at 50 °C under vacuum for 2 hours to remove most of the water, ethanol, and the excess of bromoform. The vacuum was gradually increased from about 100 mbar to 15 mbar towards the end. The resulting rubber-like material was taken out of the flask and held in the vacuum oven at 50°C. As the drying proceeded, the material was periodically ground by using a mortar and pestle into smaller particles to enhance the further elimination of the volatiles. The drying at 50°C continued until a constant weight was reached (about 15 hours), resulting in a powder with 32.4 wt.% bromoform content. Then the solid particles were heated further to 90°C under a vacuum of 30 mbar over a period of 30 min. A light-beige free-flowing powder product (59 g) with particle size below 1 mm was obtained, which contained 22.9% bromine (Parr Bomb analysis) that corresponded to 24.1% bromoform. FTIR spectra of bromoform (preparation 1), gum arabic purchased from Dingli Biodegradable Material Co., Ltd, and the powder product are shown in Figure 1. The three spectra are presented as nearly overlaid to facilitate comparison. Shifted molecular bands are not seen. The sharp, strong peak that lies in the fingerprint region at 600-700 cm-1, characteristic of haloalkanes, indicates the inclusion of bromoform in the gum arabic powder.

[0080] Examples 2-3 (invention) and 4 to 7 (comparative) Preparation of bromoform-containing gum arabic

[0081] The procedure of Example 1 was repeated with varying amounts of the constituents as tabulated in Table 1 (the first entry in Table 1 corresponds to Example 1 and was included to facilitate comparison).

[0082] Table 1 In addition, two reference experiments (devoid of bromoform) were performed. In the first reference experiment, 45 g of gum arabic was added to 40 g of water in the same portionwise manner described above. No slurry was observed. Instead, a clear, viscous solution was formed. In the second reference experiment, 45 g of gum arabic was added to 40 g of water in the same portionwise manner described above, but with ethanol (20 ml) present as well. A homogeneous, highly viscous slurry was formed.

[0083] Example 8

[0084] Preparation of bromoform-containing gum arabic

[0085] The procedure of Example 1 was repeated, but the bromoform was added to the reaction vessel in a neat form (instead of a bromoform ethanol solution). A homogeneous, very viscous paste-like slurry was obtained, which, following drying and simultaneous particle size reduction, turned into a powder with a high bromoform content (18.6%, determined by the Parr Bomb analysis).

[0086] Example 9

[0087] Stability testing

[0088] Samples of gum arabic powders were prepared by the procedure of Example 1. Samples collected after the drying at 50°C with 32.4 wt.% bromoform content (labeled GA-CHBr3 / 32.4%) and after the drying at 90°C, with 22.0 wt.% bromoform content (labeled GA-CHBr3 / 22%) were tested to determine their stability against escape and loss of the highly volatile bromoform under different storage conditions. The results are given in parts A and B, respectively.

[0089] A: GA-CHBr3 / 32.4%

[0090] Stability testing was run by placing 5 g of the GA-CHBr3 / 32.4% powder in an airtight 100 ml container. Measurement of the bromoform content after a three-month storage period at ambient temperature indicated the same bromoform level, i.e., the sample retained 100% of its original bromoform loading.

[0091] Stability testing in open air at ambient temperature included measurement of the bromoform content of the GA-CHBr3 / 32.4% powder with the passage of time. A loss of 10% of the initial amount of bromoform occurred fairly rapidly, i.e., within a few days, with the concentration dropping from 32.4% to 29%. But then the bromoform content stabilized and remained constant until the end of the test period, as indicated by measurements that were taken twenty days, sixty days, and ninety days after the beginning of the test. The initial loss of bromoform may be attributed to evaporation of loosely bound bromoform, located in proximity to exterior surfaces of the gum arabic-bromoform particles. However, the bromoform is mostly "trapped" inside the particles and does not evaporate over a long period of time, conferring exceptional stability to the gum arabic-bromoform formulations of the invention.

[0092] Stability Experiments in open air at 50°C were also performed. A loss of about 40% of the initial amount of bromoform occurred fairly rapidly, with the concentration dropping from 32.4% to 19.4% over a period of 17 days. But further, the bromoform content stabilized and remained constant until the end of the test period of 40 days, as indicated by measurements that were taken 31 days and 40 days after the beginning of the test. The initial loss of bromoform may be explained by the reasons set forth above.

[0093] B: GA-CHBr3 / 22%

[0094] The GA-CHBr3 / 22% powder showed no reduction in bromoform content after two months of storage in open air conditions at ambient temperature, indicating that the bromoform remaining after the heating at 90 °C under vacuum of 30 mbar is strongly locked inside the gum arabic matrix. Example 10

[0095] Vapor pressure measurements of bromoform-containing gum arabic and reference / comparative samples

[0096] The experimental set-up used for vapor pressure measurements is shown in Figure 2. A 0.5-liter beaker was placed on an electric hot plate. The beaker was charged with a freezing mixture consisting of crushed ice and sodium chloride. A 50 mL roundbottom flask Fl, placed in the freezing mixture, was used as a sample holder.

[0097] The system was connected to an oil vacuum pump through a bottle trap and a two-way valve VI to attain a vacuum < Imbar, and to a manometer via a T-valve V2 to measure the vacuum inside flask Fl. First, the entire system was evacuated using the oil pump to ensure the complete dryness of the system and to remove any residual gases. After ensuring that the system had no leaks, flask Fl was charged with the test sample weighing 5 g. The flask contents were frozen at -14 °C in the freezing mixture. The temperature inside Fl was measured by a thermocouple positioned at the side of Fl

[0098] When the sample was completely frozen (the freezing point of bromoform is +8 °C), the entire system (including flask Fl) was opened to a vacuum for 10-15 min. The manometer showed < Imbar. The vacuum was then disconnected from the vacuum pump by closing valve VI and turning T-valve V2 in a position that enabled keeping flask Fl and the manometer connected. The 0.5- liter beaker holding the freezing mixture was removed. The electric hot plate was turned on, and the sample was gradually heated to the desired temperature using a water heating bath. The temperature and pressure were monitored until equilibrium, where the pressure was stable for at least 5 min. The compositions of the samples, temperatures measured by the thermocouple, and vapor pressure recorded by the manometer are tabulated in Table 2. The tests were performed in duplicate.

[0099] Table 2

[0100] Daniel R. Stull, Vapor pressure of pure substances, Industrial &

[0101] Engineering Chemistry, vol. 39, Number 4, pp.517-550, 1947. The results tabulated above show that bromoform does not contribute to the vapor pressure produced by a powder sample of the invention, indicating that the bromoform is fully and strongly 'locked' inside the GA particles, attesting to the safety of the formulation.

[0102] Example 11

[0103] Preparation of dibromomethane-containing gum arabic

[0104] A 0.25 L round-bottom flask, equipped with a mechanical stirrer and a thermometer, was charged with gum arabic (10 g; from Dingli Biodegradable Material Co., Ltd) and water (40 g). The initial slurry became clear after 30 min at 60 °C, and the solution was cooled down to ambient temperature.

[0105] A pre-prepared mixture of dibromomethane (20 ml, 49.8 g) and ethanol (20 ml) was then added dropwise, resulting in the formation of a homogeneous slurry. Subsequently, 35 g gum arabic was added in three, approximately equal, portions at 15 min intervals under efficient stirring, to distribute the added gum evenly within the slurry that became increasingly pasty.

[0106] Stirring was stopped, and the thick paste was poured onto a stainless plate while the thickness of the layer on the plate was about 1 cm. After about 24 h in the hood, the originally viscous liquid paste turned into a hard rubber-like material.

[0107] Subsequently, the paste was kept at 50 °C under vacuum for 4 hours to remove most of the water, ethanol, and the excess of dibromomethane. The vacuum was gradually increased from about 100 mbar to 15 mbar towards the end. The resulting solid, crispy material was taken out of the oven and ground by using a mortar and pestle into smaller particles to enhance the further elimination of the volatiles. The drying at 50 °C under vacuum continued for an additional 12 h, resulting in a light beige powder (55 g) with 20 wt.% dibromomethane content.

[0108] Examples 12A-12D

[0109] Coating of bromoform-containing gum arabic

[0110] All coatings were performed in a fluid bed lab coater equipped with a Wurster configuration using the bottom spray method.

[0111] 12A: Bromoform-containing gum arabic powder of Example 1 was coated with molten hydrogenated rapeseed oil (heated to 150°C), sprayed while the powder was maintained at 35-40°C. The coated particles collected consisted of 70% by weight of bromoform-containing gum arabic powder and 30% by weight of rapeseed oil.

[0112] 12B: Bromoform-containing gum arabic of Example 1 was coated with molten Carnauba wax (heated to 150°C), sprayed while the powder was maintained at 35-40°C. The coated particles collected consisted of 70% by weight of bromoform-containing gum arabic powder and 30% by weight of Carnauba wax.

[0113] 12C: Bromoform-containing gum arabic powder of Example 1 was coated with molten hydrogenated palm oil (heated to 150°C), sprayed while the powder was maintained at 35-40°C. The coated particles collected consisted of 70% by weight of bromoformcontaining gum arabic powder and 30% by weight of palm oil.

[0114] 12D: Bromoform-containing gum arabic powder of Example 1 was coated with a coating solution prepared beforehand by dissolving ethyl cellulose (N4 grade) in a mixture of acetone and isopropanol. The powder was maintained at 40-43°C. The organic solvents evaporated completely, affording coated particles consisting of 85% by weight of bromoform-containing gum arabic powder and 15% by weight of ethyl cellulose.

Claims

Claims1) A process of formulating brominated C1-C2 alkane in a solid carrier, comprising combining in a reaction vessel an aqueous solution of a water-soluble edible polysaccharide and the brominated C1-C2 alkane to form a semi-solid, paste-like or solid mass, drying and comminuting the mass to give brominated C1-C2 alkane-containing polysaccharide in a powder form.2) The process according to claim 1, wherein the polysaccharide is a gum .3) The process according to claim 1 or 2, wherein the reaction vessel further comprises a lower alkanol.4) The process according to claim 2 or 3, comprising dissolving gum arabic in water to form a solution, adding the brominated C1-C2 alkane and optionally ethanol to the solution, introducing further amounts of gum arabic in a portionwise or incremental manner, whereby a progressively thickening slurry is formed, and holding the slurry for a sufficient time to obtain a semi-solid, paste-like or solid mass.5) The process according to any one of claims 1 to 4, wherein the brominated C1-C2 alkane is selected from bromoform and dibromomethane.6) The process according to claim 5, wherein the brominated Cl-C2 alkane is bromoform.7) The process according to any one of the preceding claims, wherein the weight ratio between the total amount of the gum added and water is from 75:100 to 125:100, the weight ratio between the brominated C1-C2 alkane and water is from 200:100 to 100:100, and the weight ratio between water and ethanol, when present, is from 400:100 to 180:100.8) The process according to claim 7, wherein the weight ratio between the total amount of the gum added and water is from 100:100 to 120:100, the weight ratio between the brominated Cl- C2 alkane and water is from 160:100 to 140:100, and the weight ratio between water and ethanol, when present, is from 250:100 to 210:100.9) The process according to any one of the preceding claims, wherein a paste is obtained, having a content of gum arabic before the drying step in the range from 23 to 35% by weight, with the reminder consisting of water, the brominated C1-C2 alkane and optionally ethanol.10) The process according to any one of the preceding claims, wherein the brominated C1-C2 alkane is bromoform, comprising drying the paste under reduced pressure at a first temperature in the range of 40°CdTl<70°C to substantially remove water, ethanol when present, and excess bromoform.11) The process according to claimat a second temperature in the range of 70°CdT2<95°C under reduced pressure to remove further amounts of water and ethanol when present, and releasable bromoform.12) The process according to any one of the preceding claims, wherein the brominated C1-C2 alkane is bromoform, and the bromoform-containing polysaccharide consists of gum arabic powder with not less than 15% by weight bromoform, calculated based on the total weight of the powder.13) A brominated C1-C2 alkane-rich polysaccharide.14) The brominated C1-C2 alkane-rich polysaccharide of claim 13, consisting of gum arabic in a powder form, with >15% of brominated C1-C2 alkane based on the total weight of the powder.15) The brominated C1-C2 alkane-rich polysaccharide according to claim 14, which is bromoform-rich gum arabic or dibromomethane- rich gum arabic.16) The bromoform-rich gum arabic powder according to claim 15, consisting of from 70 to 80 wt.% of gum arabic, including water held by the gum arabic, and from 20 to 30 wt.% bromoform.17) The bromoform-rich gum arabic powder according to any one of claims 13 to 16, showing vapor pressure comparabic to the vapor pressure of a bromoform-free commercial gum arabic powder at T=20°C and / or T=40°C, after the bromoform-free commercial powder, with dso=4O-6O micron, has been dried under vacuum of 15 mbar at 50°C for 15 hours.18) The brominated C1-C2 alkane-rich polysaccharide according to any one of claims 13 to 17, comprising a coating layer.19) Coated brominated C1-C2 alkane-rich polysaccharide according to claim 18, which is selected from the group consisting of: wax-coated bromoform-rich polysaccharide; hydrogenated oil-coated bromoform-rich polysaccharide; and cellulose derivative-coated bromoform-rich polysaccharide.20) Coated bromoform-rich gum arabic according to claim 19.21) A composition comprising an oil-based carrier and the brominated C1-C2 alkane-rich polysaccharide according to any one of claims 13 to 19 suspended in the carrier.wherein the brominated C1-C2 alkane-rich polysaccharide is bromoform-rich gum arabic.23) Use of a brominated C1-C2 alkane-rich polysaccharide according to any one of claims 13 to 19 as a feed additive in the diet of a ruminant animal.24) The use according to claim 23, wherein the brominated C1-C2 alkane-rich polysaccharide is bromoform-rich gum arabic.25) Premix feed formulation, comprising, or prepared from, brominated C1-C2 alkane-rich polysaccharide according to any one of claims 13 to 19.26) Premix feed formulation according to claim 25, wherein the brominated C1-C2 alkane-rich polysaccharide is bromoform-rich gum arabic.

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