Method for preparing granules containing guanidinoacetic acid

Abstract

The present application relates to a method for preparing a granule comprising guanidinoacetic acid, the method comprising the steps of: a) providing a fermentation broth comprising guanidinoacetic acid and biomass; b) reducing the water content in the fermentation broth provided in step a) to obtain a concentrated fermentation broth; and c) subjecting the concentrated fermentation broth of step b) to wet granulation.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing guanidinoacetic acid-containing granules, to specific guanidinoacetic acid-containing granules, and to a method for supplementing animal feed with said guanidinoacetic acid-containing granules.

[0002] Guanyl compounds, i.e., compounds containing a guanidine group, are widely used as energy supplements, antidiabetics, anti-inflammatory drugs, antihistamines, and antihypertensive drugs. Many of these guanyl compounds are synthesized by amidinotransfer reactions, in which the amidino group of arginine is transferred to various amidino group acceptors, such as guanidinoacetic acid, 3-guanidinopropionic acid, 4-guanidinobutyric acid, 2-guanidinoethanol, hydroxyguanidine, and homoarginine, among others. Biocatalytic amidinotransfer reactions using arginine as a substrate are usually reversible and inhibited by the by-product ornithine. Biosynthesis of some guanyl compounds remains challenging because they are inhibited by ornithine and require the expensive substrate arginine.

[0003] Guanidinoacetic acid (GAA), which acts as a direct precursor of creatine, has recently attracted renewed interest as a nutritional additive due to its creatine-recovering effect and its high stability in aqueous solution. Furthermore, it is widely used in the pharmaceutical industry and as a feed additive in poultry farming. The European Feed Safety Authority (2009) concluded that GAA does not possess mutagenic or genotoxic properties and poses no risk to the environment. Given the widespread use of GAA, there is a large industrial demand for it.

[0004] In 1861, GAA was first prepared by chemically reacting cyanamide with glycine. Currently, GAA is primarily synthesized chemically by reacting glycine or sodium glycinate with a guanylating agent, such as O-alkylisourea or cyanamide. Purification of GAA is cumbersome because the final product is contaminated with the initial guanylating agent or toxic substances, such as iminodiacetic acid or methyliminodiacetic acid, and the production method is not environmentally friendly. Therefore, biotechnological GAA production from renewable raw materials is highly desirable and promising.

[0005] GAA can be synthesized from arginine and glycine by arginine:glycine amidinotransferase (AGAT, EC:2.1.4.1) in some vertebrates, but only a few prokaryotes, such as cyanobacteria, can produce GAA through specialized metabolic synthesis. AGAT catalyzes the reversible transfer of the amidino group from arginine (donor) to the amino group of glycine (acceptor), producing GAA and ornithine. To produce one mole of GAA, one mole of arginine is required, resulting in the production of one mole of ornithine.

[0006] Downstream processing for chemically produced guanidinoacetic acid includes crystallization, mechanical dehydration including washing of the resulting crystals containing guanidinoacetic acid, e.g., GAA, drying the crystals, granulating the dried crystals with a granulating (adhesive) agent, and drying the granules to obtain a free-flowing, low-dusting product.

[0007] This rather complex downstream process can be simplified if guanidinoacetic acid, GAA, is produced in a fermentation process, in principle the broth from said fermentation process can be fed directly to the granulation.

[0008] For example, WO 2022 / 243116 A1 discloses a method for the fermentative production of guanidinoacetic acid (GAA), comprising culturing a suitable microorganism in a suitable medium under suitable conditions and allowing GAA to accumulate in the medium to form a GAA-containing fermentation broth. This document also discloses that the method can further comprise frying and / or granulating the GAA-containing fermentation broth.

[0009] German Patent No. 1031366 A1 discloses that the granulation of a feed additive containing amino acids and / or vitamins, and optionally components of a fermentation broth, is carried out in a circulating fluidized bed. This document also discloses a granular feed additive containing 40-100% by weight of L-amino acids and up to 20% by weight of fermentation broth components and / or biomass formed during fermentation.

[0010] German Patent No. 102007034102 A1 discloses glycocyanamine-containing moldings, in particular granules and extrudates, which are abrasion-resistant and flowable, and a method for producing the same. The moldings have a flowability of 350 to 850 kg / m. 3 It has a bulk density of 1000 kJ / g, a particle size of 32 to 2750 μm, and a glycocyanin content of 55 to 99.9 wt. % based on the total weight, making it particularly suitable as a feed additive.

[0011] However, such fermentation broths tend to settle if not stirred, resulting in inefficient granulation. These effects are thought to be due to the low dry matter content and low viscosity typically found in fermentation broths. For example, GAA fermentation broths have a dry matter content of less than 10% by mass, and the crystals contained therein have a narrow particle size distribution. For example, GAA crystals in the fermentation broth have a particle size distribution of d10.3 = 4.62 μm; d50.3 = 10.50 μm; and d90.3 = 136.6 μm. Specifically, prolonged operation of the granulator due to the high water content in the fermentation broth has been observed to result in low dry matter gain within the granulator. To make matters worse, this low dry matter gain is compounded by interparticle attrition, resulting in a particle size reduction from an initial d50.3 of 343 μm to, for example, a d50.3 of 206 μm.

[0012] Therefore, there remains a need for a method for preparing granules containing guanidinoacetic acid GAA that overcomes the above problems. Such a method should enable the production of granules containing guanidinoacetic acid GAA without the use of binders.

[0013] It has been found that the above problems can be solved by reducing the water content in the fermentation broth to obtain a concentrated fermentation broth before subjecting it to wet granulation.

[0014] Therefore, one object of the present invention is a method for preparing a granule comprising guanidinoacetic acid, the method comprising: a) providing a fermentation broth comprising guanidinoacetic acid and biomass; b) reducing the water content in the fermentation broth provided in step a) to obtain a concentrated fermentation broth; and c) subjecting the concentrated fermentation broth of step b) to wet granulation.

[0015] The method according to the present invention is not only simpler and more cost-effective than the standard process, but also sustainable because it does not produce any waste. Rather, all materials from the fermentation process are included in the final product, except for the water extracted in step b) and any steam from the wet granulation in step c). Thus, the product obtained from the method according to the present invention contains additional biomass that is not only consumable for animals but also provides additional nutritional value. In comparison, the standard process for preparing granules containing guanidinoacetic acid begins with chemically produced guanidinoacetic acid and involves additional downstream processes, including crystallization, mechanical dehydration and washing, drying, mechanical granulation facilitated by a granulating agent, and then further drying.

[0016] In one embodiment of the method according to the invention, the fermentation broth is concentrated in step b) to a dry matter content of at least 10% by weight.

[0017] Preferably, the fermentation broth is concentrated in step b) to a dry matter content of at least 15, 20, 25, 30 or 35% by weight.

[0018] In a preferred embodiment of the method according to the invention, the fermentation broth is concentrated in step b) to a dry matter content of 40-60% by weight.

[0019] Preferably, the fermentation broth provided in step a) and / or the fermentation broth obtained in step b) has a biomass content in the range of 1 to 10% by weight, or 1 to 5% by weight, based on the total weight of the fermentation broth or concentrated fermentation broth, respectively.

[0020] In the context of the present invention, the term wet granulation, as known to those skilled in the art, refers to the formation of granules by subjecting a liquid formulation containing suspended or dissolved solids to an agitation system, for example, under the influence of an impeller (in a high-shear granulator), a screw (in a twin-screw granulator), or air (in a fluidized-bed granulator). The agitation that occurs within the system and the wetting of the ingredients in the formulation cause agglomeration of primary powder particles to produce wet granules. The granulation fluid (fluid) comprises a solvent or carrier material, which must be volatile so that it can be removed by drying and nontoxic depending on the intended use. Typical liquids used in wet granulation include water, ethanol, and isopropanol, either alone or in combination, and the liquid solution can be either aqueous or solvent-based. Aqueous solutions have the advantage of being safer to handle than other solvents. Fermentation broth, being an aqueous system / formulation, fulfills these advantages.

[0021] In principle, the wet granulation of the method according to the present invention is not subject to any restrictions regarding a specific wet granulation technique. Thus, the wet granulation can be high shear granulation, twin screw granulation, or fluidized bed granulator. However, fluidized bed granulation has several advantages over other wet granulation techniques. These advantages are, among others, as follows: Fluid bed granulator is a one-unit system, which makes fluid bed granulation a relatively simple method, thus saving labor costs, transportation losses, and time. This technology uses airflow to evaporate the liquid, thus avoiding a waste stream. Heat transfer in a fluid bed granulator is 2 to 6 times greater than that produced by a tray dryer. The method can be automated once the parameters are optimized. Drying occurs evenly and the method prevents staining.

[0022] These aspects contribute to making the method according to the invention simpler and more cost-effective.

[0023] In another embodiment of the process according to the invention, the wet granulation in step c) is fluidized bed granulation.

[0024] The water and dissolved molecules (or solids) contained in the fermentation broth in step a) can form bonds between particles already present in the fermentation broth that are strong enough to stick them together. Particle bonding using a liquid is a combination of capillary and adhesive forces until a more permanent solid bond is established. However, when the granules obtained in this way dry, they may collapse. Therefore, water may not be strong enough to create and maintain the bond. The fermentation broth used in the method according to the present invention contains biomass, which can hold or attract guanidinoacetic acid particles to form granules. Therefore, the biomass in the fermentation broth can already act as a granulating or binding agent. However, if the biomass does not provide this function at all or to the required extent, it is beneficial to add a granulating agent during the method according to the present invention.

[0025] In one embodiment of the process according to the invention, prior to step c), a granulating agent is added to the concentrated fermentation broth obtained in step b).

[0026] In principle, any binder or binding agent can be used as a granulating agent that holds or draws other materials together to form an overall mechanical or chemical cohesive structure through adhesion or cohesion. However, granulating agents used to prepare granules containing guanidinoacetic acid should not be toxic or have any inherent effects on animals. Furthermore, any potential incompatibility with guanidinoacetic acid must be excluded. The use of organic binders as granulating agents in the method according to the present invention offers the advantage that they are typically non-toxic and compatible with guanidinoacetic acid.

[0027] In a preferred embodiment of the process according to the invention, the granulating agent is an organic binder.

[0028] Organic binders, such as starch, cellulose ethers, cellulose esters, polyvinyl alcohol, and / or mixtures thereof, hold or draw together solid materials to form an overall cohesive mechanical and chemical structure through adhesion or cohesion. Additionally, they are also non-toxic and compatible with guanidinoacetic acid.

[0029] In another preferred embodiment of the process according to the invention, the granulating agent is starch, cellulose ether, cellulose ester, polyvinyl alcohol, and / or mixtures thereof.

[0030] It is beneficial to keep the amount of granulating agent in the prepared granules as low as possible.Therefore, the concentration of guanidinoacetic acid and the concentration of any additional mass with additional nutritional value are not reduced more than necessary.In the method according to the present invention, only up to 5% by weight, preferably 1-5% by weight or 2-4% by weight of granulating agent is required based on the weight of dry binder relative to the dry matter in the fermentation broth.

[0031] In yet another embodiment of the process according to the invention, the amount of granulating agent is at most 5% by weight, based on the weight of dry binder with respect to the dry substances in the fermentation broth.

[0032] Preferably, the amount of granulating agent is in the range of 1 to 5% by weight, or 2 to 4% by weight, based on the weight of dry binder with respect to the dry matter in the fermentation broth.

[0033] Fluidized-bed spray granulation can be defined as a particle formation process in which a solid-containing liquid is converted into a non-dusty granular solid in one step by drying. Essentially, hot air is used to fluidize a bed of already formed particles (seed particles) of the same or different dissolved components. In the context of the present invention, the liquid being processed is a solution or suspension, which is continuously sprayed onto or into the bed from a space above the bed surface by an atomizing nozzle. The high-temperature fluidization evaporates the solvent, leaving the dissolved material on the surface of the seed particles. If the solids dissolved in the sprayed liquid and the particles in the fluidized bed are made of the same material, the process is called layered granulation. If different solids are sprayed, the particles are coated, forming a distinct layer on the seed particles. If the sprayed droplets impinge on the fluidized particles (seed particles) and do not rebound, they spread on the particle surface, eventually drying, leaving behind solids that form a layer around the particles. Thus, layer-by-layer growth of the particles is achieved. The resulting particles are called granules or granules. The process according to the present invention is preferably layered granulation. Thus, the seed particles fed to the fluid bed granulator also comprise or consist of guanidinoacetic acid.

[0034] In a further embodiment of the process according to the invention, the wet granulation in step c) is an agglomeration granulation.

[0035] In the context of the present invention, guanidinoacetic acid containing seed particles with a d50.3 of 300-400 μm has been successfully used to prepare guanidinoacetic acid containing particles with particle size distributions of d10.3 of 220-720 μm, d50.3 of 300-1100 μm, and d90.3 of 420-1700 μm. In particular, the seed particles have a particle size distribution of d10.3 of 150-250 μm, d50.3 of 300-400 μm, and d90.3 of 475-575 μm.

[0036] Preferably, seed particles with a d50.3 of 300 to 400 μm are used in step c) of the process according to the invention.

[0037] The examples of the present invention show that granules comprising guanidinoacetic acid and biomass and having particle size distributions d10.3 of 220-720 μm, d50.3 of 300-1100 μm, and d90.3 of 420-1700 μm have improved or at least beneficial particle properties, such as good flowability, and / or improved particle quality, such as high bulk density.

[0038] Therefore, another object of the present invention is a granule comprising guanidinoacetic acid and biomass, the granule having a particle size distribution d10.3 between 220 and 720 μm, d50.3 between 300 and 1100 μm, and d90.3 between 420 and 1700 μm.

[0039] In principle, the granules according to the present invention are not subject to any limitations regarding the method for their preparation, as long as the method in question allows for the production of granules with the required particle size distribution. Nevertheless, it is preferred that the granules containing guanidinoacetic acid are obtained by the method according to the present invention. This is because the method according to the present invention, including the wet granulation of step c), further improves the quality aspects of the product, such as particle size or immediate properties, by adhering the existing solids to larger granules. In addition to being simpler, more cost-effective, and more sustainable, the method according to the present invention, including a specific granulation step, also leads to improved product quality aspects of the guanidinoacetic acid-containing particles, such as an increased particle size distribution with a d10.3 of 220-720 μm, a d50.3 of 300-1100 μm, and a d90.3 of 420-1700 μm, as well as improved particle properties, such as good flowability.

[0040] The examples of the present invention show that the use of a granulating agent, such as a binder, is not essential to provide granules containing guanidinoacetic acid. Nevertheless, the additional presence of a granulating agent, such as a binder, can be beneficial in some cases to hold the granules together, especially under difficult or extreme conditions, such as under the influence of pressure, such as mixing.

[0041] In one embodiment, the granulate according to the present invention further comprises a granulating agent.

[0042] Preferably, the granulating agent is an organic binder, in particular starch, cellulose ether, cellulose ester, polyvinyl alcohol, and / or mixtures thereof.

[0043] In one embodiment, the granules according to the invention have the following particle size distribution: d10.3 of 300-720 μm, d50.3 of 450-1100 μm, and d90.3 of 700-1700 μm, preferably a particle size distribution: d10.3 of 300-720 μm, d50.3 of 600-1100 μm, and d90.3 of 800-1700 μm.

[0044] Guanidinoacetic acid is widely used as an energy supplement, antidiabetic, anti-inflammatory, antihistamine and antihypertensive drug.

[0045] A further object of the present invention is a method for supplementing animal feed, said feed being supplemented with granules comprising guanidinoacetic acid according to the invention and / or granules comprising guanidinoacetic acid obtained by the method according to the invention.

[0046] Working Example: I. Example 1 of the Present Invention A fermentation broth with a biomass content of 45 g / kg was prepared. The fluidized bed granulator used consisted of an electric heater, which heated gas (air, nitrogen, CO2, or a combination thereof) to a high temperature (180°C) and then flowed through a distribution tray to fluidize seed particles (d50.3: 343 μm). A spray nozzle supplied by a pump was used to atomize the fermentation broth into fine droplets, which dried on the seed particles, forming a solid layer on the surface or on the seed particles, or acted as a binder to combine the seed particles into larger aggregates or a combination thereof. The fluidization gas was passed through a filter before being released into the environment.

[0047] Broths with less than 10% dry matter by mass, containing biomass and crystals with a particle size distribution of d10.3: 4.62 μm; d50.3 = 10.50 μm; and d90.3 = 136.6 μm, tended to settle without stirring. This can cause problems in broth handling and granulation because particles settle and low dry matter content makes granulation inefficient. Specifically, high water content resulted in less dry matter gain in the granulator, leading to longer granulator runs. Longer run times led to increased interparticle attrition, resulting in a decrease in particle size, e.g., from an initial d50.3 value of 343 μm to 206 μm.

[0048] Therefore, the broth was evaporated by vacuum evaporation at 50–100 mbara until the dry matter content was up to 50% by mass. In addition to the benefit of reducing the water in the broth, the increase in viscosity also resulted in a more homogeneous liquid due to a decrease in the settling velocity and therefore a decrease in the tendency of suspended particles to settle.

[0049] The following fluidized bed granulations were carried out using various granulating agents, such as corn starch, CMC (carboxymethylcellulose), and PVA (polyvinyl alcohol), in the range of 2-4% by weight (based on the weight of dry binder relative to the dry substance in the broth). An increase in particle size distribution (PSD) could be achieved with all additives and at all concentrations used.

[0050] Because the granulation experiments using a granulating agent resulted in an increase in particle size distribution, additional granulation experiments were also performed without a granulating agent. Surprisingly, a significant increase in particle size distribution was also observed when the evaporated fermentation broth was subjected to granulation using additional granulating agent. [Table 1]

[0051] II. Example 2 of the Present Invention To verify these results and to investigate the influence of the initial biomass concentration, a second experiment was carried out without the inclusion of any additional granulating agent or any similar. Here, a fermentation broth was used at 15 g of biomass per kg of broth, which was evaporated to a dry matter content of approximately 50% by mass. In this run, the results of the previous experiment could be repeated, and in particular, a significant increase in the particle size distribution was again found. Using a significantly smaller starting material, the effect of growth on the particle size distribution was highlighted without reaching the limits of the experimental equipment for fluidizing the material. [Table 2]

[0052] III. Liquidity Determination The flowability of the particles was determined using five different funnels, each with a different orifice diameter. The first funnel had a 2.5 mm orifice diameter, the smallest orifice diameter. Product that passed through this orifice was graded with a flowability value of 1. Next were the second, third, and fourth funnels with orifice diameters of 5 mm, 8 mm, and 12 mm. Product that passed through the orifice of the second funnel was graded with a flowability value of 2, product that passed through the orifice of the third funnel was graded with a flowability value of 3, and product that passed through the orifice of the fourth funnel was graded with a flowability value of 4. The fifth and final funnel had an 18 mm orifice diameter; product that passed through this orifice was graded with a flowability value of 5. All product that did not pass through the orifice of the fifth funnel was graded with a flowability value of 6. A flowability value of 5 or higher indicates that the product in question may not be suitable for automated, high-output feed mills. Table 3 below summarizes the liquidity determination results.

[0053] IV. Determining the angle of repose The flow properties of powder materials are determined by measuring the height of the cone formed. For the experiment, a sieve with a mesh size that allowed particles to pass unimpeded was fixed 60 mm from the top of a metal cylinder. The material to be evaluated was gently scraped through the sieve until a geometrically uniform cone was formed at the top of the metal cylinder. The angle of repose was calculated using the following formula:

number

number

number

[0054] V. Bulk density (ρ b ) and tamped apparent density (ρ t ) determination Bulk density is the mass of a specified volume of powder or granular material. Tamped apparent density indicates the change in volume after tamping the mass obtained in bulk density measurement. A standardized test apparatus was used, consisting of a metal tripod, a funnel with a defined tulip-shaped outlet, and a rotating slide. The test apparatus was placed on top of a 250 mL glass cylinder. After filling the metal funnel with the test material, the rotating slide was opened and the test material was allowed to flow evenly until 20 mL of the test material was filled into the cylinder. The bulk density was then calculated using the following formula:

number

number

[0055] The tamped apparent density was determined in accordance with DIN 53194 using a tamping device, for example a JEL ST 2 from Fa. Engelsmann AG, Ludwigshafen. The tamped apparent density was calculated according to the following formula:

number

[0056] VI. Determination of the Carr Index The Carr index is a measure of the compressibility of a powder and is calculated by the following formula:

number

number

number

[0057] The results of the determination of flowability, angle of repose, bulk density, tamped apparent density, and Carr index are summarized in Table 3. [Table 3]

[0058] A comparison of the data in Table 3 shows that the products obtained by the method according to the invention, Examples 1 and 2, are of the same or at least comparable quality to the comparative materials, namely the commercial products GuanAmino® (Evonik) or CreAmino® (Alzchem Group). However, the comparative materials were prepared by chemical processes, and the material thus obtained was further processed in highly complex processes. In comparison, the products of Examples 1 and 2 were obtained by a significantly simplified method according to the invention. The effect of higher water uptake, likely due to hygroscopic biomass in the product, can be addressed.

[0059] VII. Determination of particle size distribution Particle size distribution (PSD) is measured by laser diffraction. This method uses the effect that particles scatter light at specific angles depending on the size of the particle. Smaller particles scatter light at wider angles, larger particles at narrower angles. Therefore, specific patterns of light can be detected depending on the overall PSD of the measured sample.

[0060] Here, PSD was measured using a laser diffraction particle size analyzer LA 950 (Horiba). The measured samples were suspended in isopropanol. Before measurement, the samples were sonicated for 60 or 120 seconds. Alternatively, the samples were measured without sonication. It was found that samples sonicated for 60 and 120 seconds yielded comparable results within the accuracy of the measurement, although samples measured without sonication yielded higher values. Therefore, the values ​​measured after 60 seconds of sonication are referred to here.

[0061] VIII. Overview Comprehensive experimental investigations have shown that it is possible to produce granules containing guanidinoacetic acid (GAA) from fermentation broth containing guanidinoacetic acid by evaporation and granulation, particularly fluidized-bed granulation. The standard process begins with a chemically produced product, which undergoes complex downstream processes including crystallization, mechanical dewatering and washing, drying, mechanical granulation facilitated by a granulating agent, and drying again. In comparison, the method of the present invention is much simpler. Specifically, it is simpler, more cost-effective, and more sustainable than the standard process. A further advantage of the method of the present invention is that all materials derived from the fermentation process are included in the final product, resulting in no waste products other than water from evaporation and water vapor from wet granulation, e.g., fluidized-bed granulation. Furthermore, all residues from fermentation have potential nutritional value and can be consumed by animals. Finally, the granules obtained from the method of the present invention are of the same, or at least similar, quality to those from the standard process.

Claims

1. 1. A method for preparing a particulate material comprising guanidinoacetic acid, comprising: a) providing a fermentation broth comprising guanidinoacetic acid and biomass; b) reducing the water content in the fermentation broth provided in step a) to obtain a concentrated fermentation broth; and c) subjecting the concentrated fermentation broth of step b) to wet granulation.

2. 2. The method of claim 1, wherein the fermentation broth is concentrated in step b) to a dry matter content of at least 10% by weight.

3. 3. The method according to claim 1 or 2, wherein the fermentation broth is concentrated in step b) to a dry matter content of 40 to 60% by weight.

4. 4. The method according to any one of claims 1 to 3, wherein the wet granulation in step c) is fluidized bed granulation.

5. 5. The method according to any one of claims 1 to 4, wherein a granulating agent is added to the concentrated fermentation broth obtained in step b) before step c).

6. The method of claim 5, wherein the granulating agent is an organic binder.

7. 7. The method of claim 5 or 6, wherein the granulating agent is starch, cellulose ether, cellulose ester, polyvinyl alcohol, and / or mixtures thereof.

8. 8. The method according to any one of claims 5 to 7, wherein the amount of granulating agent is up to 5% by weight, based on the weight of the dry binder with respect to the dry matter in the fermentation broth.

9. 9. The method according to any one of claims 1 to 8, wherein the wet granulation of step c) is an agglomeration granulation.

10. A granule comprising guanidinoacetic acid and biomass, the granule having a particle size distribution of d10.3 between 220 and 720 μm, d50.3 between 300 and 1100 μm, and d90.3 between 420 and 1700 μm.

11. The granule of claim 10 further comprising a granulating agent.

12. 12. A granule according to claim 10 or 11, having a particle size distribution d10.3 of 300 to 720 μm, d50.3 of 450 to 1100 μm, and d90.3 of 700 to 1700 μm.

13. 13. A method for supplementing animal feed, wherein the feed is supplemented with the guanidinoacetic acid containing granules according to any one of claims 10 to 12 and / or the guanidinoacetic acid containing granules obtained by the method according to any one of claims 1 to 9.

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