阅读说明：本技术 支链酮酸(bcka)的混合物和制备这样的混合物的方法 (Mixtures of branched ketoacids (BCKA) and methods of making such mixtures ) 是由 C·贾雷基 D·费特 于 2019-07-26 设计创作，主要内容包括：本发明涉及制备两种或更多种支链酮酸的均匀混合物的方法,其中在第一步骤中,将两种或更多种游离酮酸混合,和在第二步骤中,将混合的酮酸与一种或多种碱土金属盐共结晶,并涉及含有支链酮酸混合物的食物、食品补充剂或药物产品,其用于支持肌肉结构、提高肌肉性能和改善总体健康状况,同时通过与摄入相应氨基酸相比减少的氮供应和改善的体内氮代谢而减轻了氮解毒代谢。(The present invention relates to a method for preparing a homogeneous mixture of two or more branched-chain keto acids, wherein in a first step two or more free keto acids are mixed and in a second step the mixed keto acids are co-crystallized with one or more alkaline earth metal salts, and to a food, food supplement or pharmaceutical product containing a mixture of branched-chain keto acids for supporting muscle structure, increasing muscle performance and improving overall health, while reducing nitrogen detoxification metabolism by a reduced nitrogen supply and an improved nitrogen metabolism in vivo compared to the intake of the corresponding amino acids.)

1. A process for preparing a homogeneous mixture of two or more keto acids, wherein

In a first step, two or more free keto acids are mixed, and

in a second step, the mixed keto acid is co-crystallized with one or more alkaline earth metal salts.

2. The process according to claim 1, wherein the alkaline earth metal is selected from magnesium and calcium, preferably calcium.

3. The process according to any one of the preceding claims, wherein the alkaline earth metal salt is selected from the group consisting of calcium carbonate, calcium hydroxide, calcium acetate, calcium chloride, calcium oxide, magnesium hydroxide and magnesium acetate.

4. The method according to any one of the preceding claims, wherein the keto acid is selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine, ketophenylalanine and hydroxymethionine, preferably from the group consisting of ketoleucine, ketovaline and ketoisoleucine.

5. The method according to any one of the preceding claims, wherein the free keto acid is added to the water or mother liquor solution to which a stoichiometric amount of the alkaline earth metal salt has been added in a desired molar ratio.

6. The method according to any one of the preceding claims, wherein the free keto acid is added as an aqueous solution or as a solution in an organic solvent, preferably selected from the group consisting of methyl isobutyl ketone, acetone and tert-butyl methyl ether.

7. The method according to any one of the preceding claims, wherein the mixed ketoacid is purified prior to the co-crystallization step using one of the following methods: steam distillation, solvent extraction, ion exchange chromatography or crude crystallization with alkaline earth metal salts.

8. The method according to claim 7, wherein the mixed ketoacid is purified using a combination of steam distillation or solvent extraction prior to the co-crystallization step.

9. Mixture of at least two keto acids obtainable by the method according to any of the preceding claims, characterized in that the mixture contains mixed alkaline earth metal salts and has a uniform particle size distribution and a uniform crystal form.

10. Mixture according to claim 9, characterized in that the particle size is not more than 400 μm, preferably not more than 300 μm, more preferably not more than 200 μm or most preferably not more than 150 μm.

11. Mixture according to one of claims 9 or 10, characterized in that the mixture contains a mixed calcium or magnesium salt of ketoleucine and ketoisoleucine.

12. Mixture according to one of claims 9 or 10, characterized in that the mixture contains a calcium or magnesium salt of ketoleucine and ketovaline in admixture.

13. Mixture according to one of claims 9 or 10, characterized in that the mixture contains a calcium or magnesium salt mixture of ketoisoleucine and ketovaline.

14. Mixture according to one of claims 9 or 10, characterized in that the mixture contains a mixed calcium or magnesium salt of ketoleucine, ketoisoleucine and ketovaline in a rough ratio of 2:1:1.

15. Mixture according to one of claims 9 or 10, characterized in that the mixture contains a calcium or magnesium salt mixture of ketoleucine, ketoisoleucine, ketovaline, hydroxymethionine and ketophenylalanine.

16. Food, food supplement or pharmaceutical product containing a mixture according to any one of claims 9 to 15.

17. Use of a mixture according to any one of claims 9 to 15 for the preparation of a food, food supplement or pharmaceutical product.

Technical Field

The present invention relates to a method for preparing a homogeneous mixture of two or more branched-chain keto acids, wherein in a first step two or more free keto acids are mixed, and in a second step the mixed keto acids are co-crystallized with one or more alkaline earth metal salts. The invention also relates to food, food supplements and pharmaceutical products containing a mixture of branched chain keto acids for supporting muscle structure, enhancing muscle performance and improving overall health while mitigating nitrogen detoxification metabolism by reduced nitrogen supply compared to ingestion of the corresponding amino acids and by improved nitrogen metabolism in the body.

Background

Lack of physical movement is a risk factor that may lead to a decrease in physical performance and thus a reduction in quality of life. To prevent the decrease of physical ability and to reestablish physical ability, physical exercise is indispensable in which a series of cellular processes such as muscle damage and muscle breakdown, muscle regeneration, muscle hypertrophy and muscle fiber transformation occur. In the cellular processes, energy and protein metabolism play a decisive role. The supply of amino acids therefore plays a decisive role for the metabolic processes that take place in muscle tissue. In particular, the branched-chain amino acids valine, leucine and isoleucine are essential substrates and important regulators in protein biosynthesis and are the main nitrogen sources for glutamine and alanine synthesis in skeletal muscle. In addition, alanine is also an important precursor of gluconeogenesis, and glutamine acts as a nitrogen transporter between organs.

The average required amount of protein is about 660mg/kg body weight, which may however be significantly increased by physical exercise. The protein demand can generally be met by a balanced diet, which is however not easily achieved. Physical exercise, due to increased protein degradation and decreased protein synthesis, leads to a change in the demand for nutrients and, in addition, to a changed metabolic location result, which is caused, for example, by the effect of physical exercise on the hormonal system, and finally, also lacks knowledge about a proper diet with increasing physical load, especially in relation to age, so that malnutrition can occur rapidly.

For these reasons, it appears logical to use food supplements in individuals experiencing physical stress. In this regard, studies have been conducted with different results, which are related to the effect of creatine supplementation on the efficacy of subjects. Furthermore, it is known that muscle regeneration can be promoted by a high carbohydrate supply.

In the past, the use of Branched Chain Amino Acids (BCAAs) as dietary substitutes has also been extensively studied, but there are no clear results. Although in one study it was reported that mental and physical performance was improved by supplementation with BCAA (Blomstrand, E. et al, Eur. J. appl. Physiol. Occup Physiol 63:83-88,1991), in another study no effect on physical performance was found (van HG, Raayakers, Saris, Wagenmakers, J. Physiol 486(Pt3), 789-.

The alpha-keto acids of the branched-chain amino acids likewise play an important role in the metabolism of amino acids, in particular in skeletal muscle and liver. One third of muscle protein consists of branched chain amino acids, which cannot be formed by the body, but must be ingested with food. In muscle, especially in the case of physical exertion, proteins are continuously synthesized and broken down, with the amino acid breakdown accompanied by transfer of the amino group to the carrier to form the corresponding a-keto acid. The keto acid obtained can then be further enzymatically oxidized to produce energy. The carrier is transported to the liver where toxic ammonia is released, which must be converted to urea and excreted via the kidneys.

The use of alpha-keto acids derived from branched-chain amino acids for pharmaceutical purposes has long been known. For example, in particular, alpha-ketoisocaproate (ketoleucine) can be used to reduce protein breakdown in muscle and to reduce urea formation resulting from protein breakdown after muscle activity (US 4,677,121). The use of ketoleucine in malnutrition, muscular dystrophy or uremia, and other diseases that are secondary consequences of protein breakdown in muscle is also described in this document. In this case, the administration of ketoleucine is by intravenous injection. Furthermore, it has been proposed to administer alpha-keto acids of leucine, isoleucine and valine to patients who must maintain a protein-reduced diet, for example due to renal failure (US 4,100,161). The role of alpha-keto acids in protein metabolism for various medical indications is also described in Walser, M.et al, Kidney International, Vol.38 (1990), p.595-604.

In contrast, in the field of functional foods, branched-chain amino acids are used directly to support muscle growth, for example in athletes (Shimomura, Y., et al, American Society for Nutrition). The use of alpha-keto acids of leucine, isoleucine and valine for improving muscle performance and also for supporting muscle recovery after fatigue is described in US 6,100,287, in which salts formed from the corresponding anionic keto acids with cationic amino acids, such as arginine or lysine, as counterions are used. However, polyamines are also formed as a result, which are known to cause apoptosis (programmed cell death). Excretion of polyamine breakdown products is via the kidneys, which are thus subject to further stress.

WO 2008/122613 describes food supplements containing alpha-keto analogs of branched chain amino acids for supporting muscle structure, increasing muscle performance and improving overall health, while reducing nitrogen detoxification metabolism by reduced nitrogen supply and improved nitrogen metabolism in vivo compared to ingestion of the corresponding amino acids. In particular, food supplements having a combination of α -ketoisocaproate and α -ketoisovalerate or α -ketoβ -methylvalerate or a combination of α -ketoisovalerate and α -ketoβ -methylvalerate or a combination of all three α -keto acids or their salts are disclosed. Furthermore, specific ratios of different α -keto analogs are preferred.

It is known from US 2011/0257236a1 and US 4,677,121 that two combinations of the branched chain amino acids L-leucine, L-isoleucine and L-valine and their respective keto acids in a ratio of about 2:1:1 are able to inhibit muscle damage during strenuous exercise. In addition, it is well established that the keto acids leucine, isoleucine, valine and hydroxymethionine are used in medicaments for maintaining protein levels in the case of chronic kidney disease (described in US 4,100,160 and US 4,100,161). Despite the reported methods and advantages of the combined processing of branched-chain L-amino acids, up to now, the processing of individual branched keto acids and hydroxymethionine as calcium salts has been reported.

However, when the branched ketonates are prepared separately and mixed at a later stage, the branched ketonates have different particle size distributions and their crystal forms are also different. In particular, the calcium salt of ketoisoleucine has very large crystals. As a result, it is difficult to prepare a homogeneous mixture of the branched ketonates and the product must be milled to provide a homogeneous mixture suitable for use in a health care product. In order to provide a homogeneous mixture with a uniform particle size distribution, a dry blending process may for example be applied, wherein several components with different particle sizes need to be weighed, premixed, ground and mixed again to ensure homogeneity. As a first step, the appropriate amount of individually released amino acids is weighed for the pre-mixing step, according to the desired composition in the mixture. For pre-mixing, the mixture needs to be transferred to and mixed in a dryer to obtain a homogeneous mixture. In a subsequent step, the mixture needs to be milled to obtain a suitable uniform particle size distribution. In the final mixing step, the mixture needs to be transferred to a dryer to obtain a homogeneous amino acid mixture.

Starting from the prior art, there is a need for food supplements with a combination of branched chain keto acids that promote post-exercise health, increase muscle synthesis and muscle efficiency and permanently reduce metabolic nitrogen burden. More specifically, there is a need for a homogeneous mixture composed of branched keto acids in a predetermined ratio, and a simplified method of preparing a homogeneous mixture having a uniform particle distribution, the mixture containing a ketone analog and a ketone group and a hydroxyl component of an essential amino acid.

Disclosure of Invention

The problem is solved by providing a method for preparing a homogeneous (homogenes) mixture of two or more keto acids, wherein

-in a first step, mixing two or more branched keto acids, and

-in a second step, co-crystallizing the mixed keto acid with one or more alkaline earth metal salts.

It has surprisingly been found that after co-crystallization of two or more keto acids, homogeneity of the product is achieved without additional processing. The obtained mixture has a uniform particle size distribution and a uniform (homogenes) crystal form.

In a preferred embodiment, the alkaline earth metal is selected from magnesium and calcium, preferably calcium.

It is particularly preferred that the alkaline earth metal salt is selected from calcium carbonate, calcium hydroxide, calcium acetate, calcium chloride, calcium oxide, magnesium hydroxide and magnesium acetate.

In an alternative embodiment of the invention, the keto acid used in the method according to the invention is selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine, ketophenylalanine and hydroxymethionine. In a preferred embodiment, the keto acid is selected from the group consisting of ketoleucine, ketovaline, and ketoisoleucine.

In one embodiment of the invention, the free keto acid is added in the desired molar ratio to water or to a mother liquor solution to which a stoichiometric amount of the alkaline earth metal salt has been added.

In this embodiment, the aqueous phase is preferably saturated with product by recycling the mother liquor. This recycling of the mother liquor has an unexpected effect-due to the optimal saturation of the mother liquor, it was found that the target composition could be achieved without adjusting the composition to compensate for the varying solubility. The free acid is added in the desired ratio to the mother liquor solution to which the stoichiometric amount of calcium salt has been added.

In an alternative embodiment, the free keto acid is added as an aqueous solution or as a solution in an organic solvent, preferably selected from the group consisting of methyl isobutyl ketone, acetone and tert-butyl methyl ether.

In a preferred configuration of the invention, the mixed ketoacid is purified using one of the following methods prior to the co-crystallization step: steam distillation, solvent extraction, ion exchange chromatography or crude crystallization with alkaline earth metal salts.

The ketoacids may be prepared via chemical synthesis or via a fermentation process. Purification is usually achieved by solvent extraction, or by steam distillation followed by salt formation.

In a further preferred configuration of the invention, the mixed ketoacid is purified using a combination of steam distillation to provide an aqueous solution of the purified ketoacid in a similar ratio to the input. Taking into account the difference in boiling points: the results were surprising for the ketovaline free acid (70-80 ℃ at 5 mbar), the ketoleucine free acid (about 100 ℃ at 5 mbar) and the ketoisoleucine free acid (about 115 ℃ at 5 mbar).

Another subject of the present invention is a mixture of at least two keto acids obtainable by the process as described above, wherein the mixture comprises mixed alkaline earth metal salts and has a uniform particle size distribution and a uniform crystal form.

In a preferred embodiment of the invention, the particle size in the mixture is not more than 400 μm, preferably not more than 300 μm, more preferably not more than 200 μm or most preferably not more than 150 μm.

In a particularly preferred configuration, the mixture contains a calcium or magnesium salt of ketoleucine and ketoisoleucine in admixture.

In a particularly preferred arrangement, the mixture contains a calcium or magnesium salt mixture of ketoleucine and ketovaline.

In a particularly preferred arrangement, the mixture contains a calcium or magnesium salt mixture of ketoisoleucine and ketovaline.

In a particularly preferred arrangement, the mixture contains a calcium or magnesium salt blend of ketoleucine, ketoisoleucine and ketovaline in a ratio of approximately 2:1:1.

In a particularly preferred arrangement, the mixture contains a calcium or magnesium salt mixture of ketoleucine, ketoisoleucine, ketovaline, hydroxymethionine and ketophenylalanine.

The invention also relates to a food, food supplement or pharmaceutical product containing a mixture of keto acids according to the above embodiments.

In addition, additional nitrogen-free additives may be added to the food supplement. Those which may be particularly emphasized are energy-donating compounds which are preferably selected from carbohydrates, such as glucose, but also additives which promote the regeneration process, such as vitamins, in particular vitamin a, vitamin B1, B2, B6 and B12, vitamin C, vitamin D, vitamin E, vitamin K, pantothenic acid, nicotinic acid, folic acid, biotin, choline and inositol. In addition, antioxidants such as beta-carotene, potassium citrate, citric acid, lactic acid, tocopherol, sodium ascorbate or potassium ascorbate or ascorbic acid may also be present in the food supplement. Minerals and trace elements selected from the group consisting of sodium, potassium, magnesium, calcium, iron, zinc, manganese, copper, selenium, chromium, phosphorus and iodine may also be used as additives. In this case, the additives are added in amounts conventional in the food field.

Preferred food supplements may contain, for example (in each case the preferred daily dose):

10-500mg of sodium is added,

10-500mg of potassium, in the form of potassium,

50-500mg of calcium, in particular calcium,

10-300mg of magnesium, in particular magnesium,

1-20mg of zinc, and the zinc,

5-50mg of iron (Fe),

0.1-1mg of iodine,

5-100 mug of selenium,

5-100 mug of chromium,

up to 100mg of vitamin B1,

up to 100mg of vitamin B2,

up to 100mg of vitamin B6,

up to 200mg of vitamin B12,

up to 5g of vitamin C, and,

up to 500mg of vitamin E,

up to 300mg of pantothenic acid,

up to 1g of nicotinic acid, with a total amount of niacin,

up to 10mg of folic acid,

up to 1mg biotin.

Further additives which may be considered as additives are saturated or unsaturated fatty acids, in particular C6-C22 fatty acids. In addition, fats and oils selected from sunflower oil, sesame oil, rapeseed oil, palm oil, castor oil, coconut oil, safflower oil, soybean oil, lard, tallow and fish oil may be used. In addition, preservatives, food dyes, sweeteners, taste enhancers and/or aroma substances may also be present in the food supplement in conventional amounts known to the person skilled in the art. In particular, taste masking substances are considered as additives, since, for example, free α -keto acids may taste acidic or their salts may taste unpleasant. If the amount of additive used is relatively large, a nitrogen-free additive may be used in this case. However, particularly preferred food supplements do not contain nitrogen-containing additives.

The claimed food supplement can be used, for example, in the form of a powder, tablet, mini-tablet, pill, granule, sachet, capsule or in the form of a solution or suspension. In tablet form, the α -keto acid or salt thereof is preferably formulated in the food supplement in about 30 to 80% by volume, preferably with nitrogen-free additives, in particular carbohydrates, fats and oils, and, if appropriate, amino acids, such as leucine, isoleucine and valine, which may be present in the food supplement in an amount of about 70 to 20% by volume.

For example, the capsule may be filled with: the composition of the invention or the coated granules in the form of coated pellets, wherein "coating" means coating with at least a coating layer. In another embodiment, a capsule that is itself coated with a coating may be filled with: coated or uncoated pellets, powders, or coated or uncoated granules.

If it is desired to administer the food supplement directly in powder or tablet form, it may be advantageous to add conventional carriers. Suitable carriers are, for example, linear or (hyper) branched polyesters, polyethers, polyglycerols, polyglycolides, polylactides, polylactide-co-glycolides, polytartrates and polysaccharides or polyethylene oxide based dendrimers, polyether dendrimers, coated PAMAM dendrimers, for example polylactide-co-glycolide coatings or polyaryl ethers.

The tablets, pills or capsules may additionally be provided with a coating, for example to allow the food supplement to be released first in the intestinal tract. In this case, the following encapsulating materials are preferably used: carboxymethyleneCellulose, cellulose nitrate, polyvinyl alcohol, shellac, carrageenan, alginates, gelatin, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose acetate succinate (HPMC-AS), cellulose acetate trimellitate, ethylcellulose, polyglycerol, polyesters or copolymers based on methacrylic acid and methacrylic acid/acrylic acid esters or their derivatives (for example)。

Conversely, if a solution or suspension of the food supplement is administered, it may be useful to add an emulsifier or colloid in order to be able to absorb all the desired components as well as possible in an aqueous system. Suitable additives are, for example, polyvinyl alcohols, glycerol esters of edible fatty acids, their esters of acetic, citric, lactic or tartaric acid, polyoxyethylene stearate, carbohydrate esters of edible fatty acids, propylene glycol esters, glycerol esters or sorbitan esters, or sodium lauryl sulfate.

The invention further relates to a food product (functional food) comprising the claimed food supplement. These may be, for example, beverages or bars (bars) particularly suitable for consumption of the food supplement.

In this case, the food product may be mixed with the claimed food supplement during their preparation or a formulation of the food supplement may be subsequently added to the food product, for example in the form of a powder or tablet. Mention may be made here, for example, of effervescent tablets or powders dissolved in mineral water.

The described food supplement or food product can in principle be used throughout the day, but is recommended in particular during or after physical exertion. Physical exercise can result in muscle adaptation, which includes muscle damage, muscle hypertrophy, and muscle transformation. In this case, the training unit is considered to be a combination of the training phase and the regeneration phase. A poor design of the training unit may lead to e.g. an overtraining syndrome, which is manifested as a prolonged fatigue and a reduced physical performance. This overtraining syndrome is often caused or exacerbated by malnutrition.

In said aspect, the food supplement of the invention is particularly directed to athletes, in this case both recreational and top athletes, including power athletes, and those interested in health and fitness. It is also particularly advantageous to use food supplements by elderly persons who are known to often have limited nitrogen economy and limited nitrogen excretion capacity.

The invention further relates to the following uses of the claimed food supplement: for the preparation of products which can be taken orally, such as functional foods, in particular beverages, gels, creams, bouillons, energy bars and the like, as well as tablets, powders, which can be provided, for example, in sachets, bags, tubes, and for supporting muscle building, the efficacy of the muscular system, for protecting the muscular system against cell damage under stress, for improving the overall health, overall physical performance and for supporting muscle regeneration after physical stress, while alleviating metabolism with respect to nitrogen detoxification.

The invention also covers food products for medical purposes, such as food products, beverages, supplements, special food products for medical purposes or pharmaceutical products.

Thus, the invention also encompasses the use of a mixture according to the above embodiments for the preparation of a food, food supplement or pharmaceutical product.

Detailed Description

Examples

The starting material for the process is an aqueous sodium salt solution of a keto acid prepared via a chemical or fermentation process. Table 1 lists the preferred concentrations of the starting solutions.

Table 1: preferred concentrations of the starting solution of the sodium salt of a single keto acid.

The ketoacids may be prepared via previously reported chemical or fermentation processes. Purification can be achieved by steam distillation, solvent extraction or crude precipitation from the fermentation solution, followed by salt formation.

An example of a process flow is depicted in FIG. 1, where FIG. 1 shows co-distillation and co-crystallization of the calcium salt of ketoleucine: ketovaline: ketoisoleucine at approximately 2:1:1. As shown in fig. 1, the branched-chain keto acids Ketoleucine (KIC), Ketovaline (KIV), and Ketoisoleucine (KMV) were combined and acidified at about pH 1. With addition of water, a combined steam distillation is carried out, followed by Ca (OAc)₂And (4) salifying. The resulting product is concentrated and co-crystallized and can be isolated in the last step.

Example 1: purification of Branched Chain Keto Acids (BCKA) using combined steam distillation

A sodium salt solution of ketoleucine (331g of a 6.9% by weight solution containing 22.8g of the sodium salt), ketoisoleucine (61g of an 18.8% by weight solution containing 11.4g of the sodium salt) and ketovaline (144g of an 8.6% by weight solution containing 12.4g of the sodium salt) was added to an acid-resistant container at the concentrations indicated above to give a ratio based on active substance of 2:1: 1.2. The amount of ketovaline described in this example is suitable for use in the process of using fresh liquid for salification (see examples 5-7 for description). When recycled mother liquor is used, the ketovaline fraction can be reduced to 1.

About 165g of 50 wt% sulfuric acid was added to adjust the pH of the solution to pH <1 with stirring at ambient temperature. After adjusting the pH, heat and vacuum are applied to effect distillation at 60-80 deg.C/200-.

Additional water was added and the distillation was carried out under vacuum of 270-300 mbar at a maximum of 80 ℃ until about 42g/g of keto acid was distilled off. The distillate contained about 2.2 wt.% BCKA.

It has been found that the three branched-chain keto acids can be distilled simultaneously, thereby providing an aqueous solution of the purified keto acid in a similar ratio to the input (see FIG. 2, which shows the area% composition of the free keto acid during the combined water vapor distillation).

Analysis of the base solution (residue) showed that the recovery of the keto acid was almost quantitative. Furthermore, it was found that the efficiency of the combined distillation in terms of water vapour consumption is overall better when compared to the distillation of the individual keto acids. This is particularly true for ketoisoleucine, as shown in figure 2.

	Total weight of	Keto acid content	g water/g keto acid
				Keto acid mixtures	529.8g	12.0g	44.2
Ketoclaine	534.0g	12.0g	44.5
				Kevalonic acid	425.6g	12.0g	35.5
Ketone isoleucine	638.3g	12.0g	53.2

Table 2: efficiency of distillation of free keto acids, expressed as grams/water per gram of free acid

Example 2: purification of branched-chain keto acids using solvent extraction

The following keto acids were added to water as their respective free acids, sodium or calcium salts in the following molar ratios to provide an approximately 5% (weight/volume) solution: ketovaline ketoleucine ketoisoleucine was about 1:2: 1. The solution was adjusted to pH <1 with aqueous hydrochloric acid at ambient temperature and the acidic solution was then extracted with methyl isobutyl ketone (MIBK). The MIBK solution containing the free keto acid was heated to 60 ℃, followed by the addition of calcium carbonate solids to adjust the pH to >3 (target: 3-5, slightly more than in the given examples). The biphasic mixture was heated to 80 ℃ to allow the layers to settle and then separated. The lower aqueous layer (containing product) was further extracted with MIBK at 80 ℃ to remove impurities and then vacuum distilled to remove residual MIBK and partially concentrate the batch, cooling the aqueous layer to ambient temperature to crystallize the product. The solid was isolated by filtration, washed with water and then dried to dryness under vacuum at maximum 75 ℃.

Example 3: purification of branched-chain keto acids by precipitation from fermentation solutions

An approximately 5% aqueous solution of the ketoleucine fermentation solution was acidified with aqueous hydrochloric acid to a pH of approximately 2. Calcium carbonate solids were added at ambient temperature until the pH was >3 (target: about 3-5). The precipitated solid was isolated by filtration, washed with water and then dried to dryness under vacuum at maximum 75 ℃.

Example 4 (comparative): preparation of calcium salts of individual branched keto acids

The process of crystallizing (or more precisely salifying) the free keto acid with a calcium salt (usually calcium carbonate, calcium hydroxide or calcium acetate) is well established. The free keto acid may be added as an aqueous solution (if sufficiently diluted) or as a solution in one or more organic solvents such as, but not limited to, methyl isobutyl ketone, acetone, t-butyl methyl ether.

When the three ketonates are prepared separately, they have different particle size distributions and their crystal forms are also completely different. FIG. 3 shows a photomicrograph of a single branched calcium ketonate salt (magnification: 100 times, scale bar 200 μm); a) ketovaline Ca salt; b) ca salts of ketoleucine; c) ca salt of ketoisoleucine. In particular, the calcium salt of ketoisoleucine has particularly large crystals. The corresponding Particle Size Distribution (PSD) of the individual branched calcium ketonates is shown in fig. 4 and clearly shows the varying particle size distribution of the calcium salts of ketovaline, ketoleucine and ketoisoleucine; a) ketovaline Ca salt; b) ca salts of ketoleucine; c) ketone isoleucine Ca salt-x axis represents particle size in μm, and y axis represents% of particles. The red line depicts the sum of the particles.

As a result, it is difficult to prepare a homogeneous mixture of the ketonic acid salts, and the product must be milled to obtain a mixture suitable for use in healthcare or any other application.

Example 5: preparation of a mixture of calcium salts (with calcium acetate)

In the case of co-crystallization, the composition of the starting free keto acid must take into account the variability of the solubility of the calcium salt (compare table 3, which shows the solubility in water at 20 ℃, in g/L) in order to achieve the desired ratio in the finished product:

table 3: water solubility of keto acids and alpha-hydroxymethionine (in g/L at 20 ℃ C.)

In general, in order to maximize the yield of the crystallization process, recycle of the mother liquor is often employed. In the case of co-crystallization, this recycling of the mother liquor has an unexpected effect — due to the optimal saturation of the mother liquor, it was found that the target composition can be achieved without adjusting the composition to compensate for the varying solubility as described above, the free acid can simply be added in the desired ratio to the mother liquor solution to which the stoichiometric amount of calcium salt has been added. This is illustrated in figure 5 for the branched ketoacids ketovaline, ketoisoleucine and ketoleucine.

Combined, acidifying and steam distillation

In this case, a sodium salt solution of ketoleucine (331g of a 6.9% by weight solution containing 22.8g of the sodium salt), ketoisoleucine (61g of an 18.8% by weight solution containing 11.4g of the sodium salt) and ketovaline (144g of an 8.6% by weight solution containing 12.4g of the sodium salt) was added to an acid-resistant container at the concentrations specified in Table 1 to give a ratio based on active substance of 2:1: 1.2. The amount of ketovaline described in this example is suitable for use in the case of fresh solutions for salification (see examples 5-7 for description). When recycled mother liquor is used, the ketovaline fraction can be reduced to 1. At ambient temperature and with stirring, about 165g of 50 wt% sulfuric acid was added to adjust the pH of the solution to pH < 1. After the pH adjustment, heat and vacuum were applied to achieve distillation at 60-80 deg.C/200-300 mbar. Additional water was added and the distillation was carried out under vacuum of 270-300 mbar at a maximum of 80 ℃ until about 42g/g of keto acid had distilled off. The distillate contained about 2.2 wt.% BCKA.

Salt formation and separation

To a solution of 7.7 grams (about 0.5 molar equivalents) of calcium acetate in 23.3 grams of water (about 25% solution) was added 550 grams of a free keto acid distillate containing about 12 grams of branched chain keto acid. After checking the pH, an additional amount of 25% calcium acetate solution was added to reach a pH of 3-4. Once the pH was adjusted, the batch was heated to 75-80 ℃ and distilled under 250-350 mbar vacuum to concentrate as described in the following steps.

First batch with fresh liquid

The batch was concentrated to about 15 wt% at 75-80 ℃ and then cooled to about 20-25 ℃ to crystallize. After stirring for about 1 hour at about 20-25 ℃, the product was isolated by filtration and dried. The product yield was 11.8g, corresponding to 78% (reference starting material). The mother liquor was retained for subsequent batches.

Subsequent batches with recycled mother liquor

To 78g of the mother liquor was added 15g (0.5 molar equivalent) of calcium acetate. To this calcium acetate/mother liquor solution, 550g of a free keto acid distillate was added, containing ketoleucine in a ratio of 2:1: ketoisoleucine: about 12g branched ketoacids of ketovaline. The expected pH is 3-4. The batch is concentrated to about 11-13% by weight at 75-80 ℃ and then cooled to about 20-25 ℃ to crystallize. After stirring for about 1 hour at about 20-25 ℃, the product was isolated by filtration and dried. The product yields were 12.2-14g, corresponding to 80-94%. The mother liquor was retained for subsequent batches.

On the other hand, the product prepared by co-crystallization is homogeneous, as shown in fig. 6, and has a more uniform particle size distribution (as shown in fig. 7), which makes it suitable for applications without further processing. FIG. 6 shows a micrograph of the co-crystallized branched calcium ketonate (2:1:1 ketoleucine: ketoisoleucine: ketovaline) (magnification: 100 times, scale bar 200 μm). FIG. 7 shows the Particle Size Distribution (PSD) -x-axis shows the particle size in μm and y-axis shows the% of particles for the co-crystallized branched calcium ketonate salt (2:1:1 ketoleucine: ketoisoleucine: ketovaline). The red line depicts the sum of the particles.

The effect of uniform particle size distribution can be further seen when comparing the particle size distribution of a mixture of BCKA co-crystallized according to the present invention with a mixture of BCKA without any co-crystallization or co-processing.

As shown in fig. 8a), a uniform particle size distribution can be measured for the co-crystallized branched ketonate. However, when the same branched ketoacid salt was mixed without any co-crystallization or co-processing, three different peaks of the three ketoacids could be detected, wherein the ketoisoleucine had much larger particles than the ketoleucine and the ketovaline which showed the smallest particle size, as shown in fig. 8 b). FIG. 8 shows the Particle Size Distribution (PSD) of a mixture of a) cocrystallized branched ketoacid calcium salt (2:1:1 ketoleucine: ketoisoleucine: ketovaline) and b) branched ketoacid calcium salt (2:1:1 ketoleucine: ketoisoleucine: ketovaline) -the x-axis shows the particle size in μm and the y-axis shows the volume% of the particles.

Example 6: preparation of calcium salt mixture (with calcium carbonate)

Branched ketoacids were added to the BCKA mother liquor (about 5% (weight/volume)) in the following molar ratio: ketovaline ketoleucine ketoisoleucine 1.1:2: 1. The mixture was heated to 60 ℃ and then about 0.5 molar equivalent of calcium carbonate was added in portions. The suspension was heated to >75 ℃ and after stirring for a period of time, the reaction mixture was cooled to ambient temperature. The solid was isolated by filtration, washed with water, and dried.

Example 7: preparation of calcium salt mixture (with calcium hydroxide)

A 5-10% solution (which may be single phase or biphasic) comprising 2 equivalents of a ketoacid mixture consisting of 1.1:2:1 ketovaline: ketoleucine: ketoisoleucine in water (or stock solution) is prepared and the solution is heated to about 50-70 ℃. At this temperature, about 1 equivalent of calcium hydroxide was added. Once the addition was complete, the contents were heated to 75-90 ℃ to dissolve the solids, and then the solution was cooled to ambient temperature. The resulting solid was isolated by filtration and washed with water. The solid may be dried under vacuum at up to 60 ℃. The yields achieved are from 40 to 80%. Higher yields are achieved when recycled mother liquor is used.

Example 8: preparation of calcium salt mixtures

The following calcium salts were prepared using methods analogous to those described in example 2 (for the formation of the free acid) and examples 4 to 7 for salt formation/isolation:

ketobenzalanine/alpha-hydroxymethionine

Ketobenzalanine/Kevalnine

alpha-Hydroxymethionine/Ketone isoleucine

Example 9: preparation of magnesium salt mixture (with magnesium acetate)

To an approximately 5-10% solution of approximately 1 equivalent of magnesium acetate tetrahydrate in water at 50-75 deg.C was added 30-60% of a solution of methyl isobutyl ketone (MIBK) of a ketoacid mixture consisting of 1.1:2:1 of ketovaline ketoleucine ketoisoleucine (BCKA). The biphasic solution is heated to 75-85 ℃ and the phases are separated at this temperature. The aqueous solution was concentrated under vacuum to a minimum volume at a maximum of 80 ℃, then after addition of n-butanol, concentrated again to remove residual water of n-butanol (by azeotropic distillation) to afford a solid. The solid may be dried under vacuum at up to 70 ℃. The yields achieved are about 80-90%. The characteristics of the different magnesium salts of branched keto acids are shown in table 4.

Table 4: summary of magnesium salts of branched keto acids: single ketonate and branched ketonate mixtures (2:1:1 ketoleucine: ketoisoleucine: ketovaline)

The following mixtures were also prepared:

a mixed magnesium salt of ketoleucine and ketoisoleucine,

a mixed magnesium salt of ketoleucine and ketovaline, and

a mixed magnesium salt of ketoisoleucine and ketovaline.

Example 10: preparation of magnesium salt mixture (with magnesium hydroxide)

A 5-10% solution (which may be single-phase or two-phase) comprising 2 equivalents of the keto acid in water (or mother liquor) is prepared and the solution is heated to about 50-70 ℃. At this temperature, about 1 equivalent of magnesium hydroxide was added. Once the addition was complete, the contents were concentrated under vacuum at 75 ℃ until a solid was obtained. The solid was dried. The yield achieved was > 90%.

Example 11: preparation of mixed calcium/magnesium salt mixtures in different proportions

A 5-10% solution (which may be single-phase or two-phase) comprising 2 equivalents of the keto acid in water (or mother liquor) is prepared and the solution is heated to about 50-70 ℃. At this temperature, a total of about 1 equivalent of magnesium hydroxide/calcium is added in a ratio of 2:1, 1:1 or 1: 2. Once the addition was complete, the contents were concentrated under vacuum at 75 ℃ until a solid was obtained. The solid was dried. The yield achieved was > 90%. The characteristics of the mixed calcium/magnesium salt with branched-chain keto acids in different mixture ratios are shown in table 5.

Table 5: summary of Mixed calcium/magnesium salts of branched-chain keto acids with different mixture ratios