MXPA00006216A - Aqueous lysine-containing animal feed supplements and process for the production thereor - Google Patents

Abstract

Animal feed supplement derived from a fermentation broth is a liquid with a pH below 7 containing:(a) L-lysine and/or its salts;(b) 0-100%of the broth's biomass;and (c) at least a major part of the broth's other dissolved and suspended components. An Independent claim is also included for a process for producing a solution or suspension containing lysine and/or its salts, comprising:(a) producing a fermentation broth containing L-lysine;(b) optionally removing at least part of the biomass;(c) optionally adding L-lysine or a solution containing L-lysine;(d) adjusting the broth to a pH below 7;and (e) optionally concentrating the broth;where steps (b)-(e) can be performed in any order.

Description

AQUEOUS SUPPLEMENTS FOR FORKS CONTAINING LYSINE AND PROCEDURE FOR PREPARATION FIELD OF THE INVENTION The invention relates to aqueous supplements for forages containing L-lysine, which optionally contain microorganisms and these preferably inactivated by fermentation, and to a process for the preparation of that form of preparation. BACKGROUND OF THE INVENTION The essential amino acid L-lysine is widely used as a supplement for forages. It is known that L-lysine is produced by fermentation of strains of coryneform bacteria, especially with Corynebacterium glutamicum. Due to the great importance, we work continuously to improve these procedures. of preparation. The improvements of the process can consist of technical measures of fermentation such as agitation and oxygen feed, or the composition of nutritive media such as sugar concentration during fermentation, or post-processing to the form of a product, by means of ion exchange chromatography or crystallization. According to the state of the technician, three different groups of product forms are known.

REF .: 120833 contain L-lysine, which are prepared from fermentation broths containing lysine. The most known group are the products in the form of powder or crystalline purified or pure L-lysine, which is typically in the form of a salt such as L-lysine monohydrochloride. Another group of solid product forms, such as that described in EP-B-0533039, also contains -L-lysine, the ingredients used during the preparation by fermentation and eventually the inactivated biomass of the microorganisms used. Products in the form of powder, crystalline or also granulated have clear disadvantages on handling and use. The influences of the climate, such as high humidity, have a disadvantageous influence on the quality of the product, since due to the possible formation of lumps and impair the storage capacity and dosage of the product in use. The preparation of products containing L-lysine in powder form can lead to the development of unwanted powder. The third group of products containing L-lysine includes concentrated aqueous alkaline solutions, and thus overcomes the aforementioned problems (EP-B-0534865). Correspondingly to the different product forms, the most diverse procedures for the preparation of products containing L-lysine from fermentation broths containing L-lysine are known. For the preparation of solid pure L-lysine, two essentially different processes are currently known. Lysine can be obtained as monohydrochloride (Lys-HCl) by crystallization of the corresponding fermentation broths, after the inactivated biomass has been separated by suitable methods. The purification of the filtrate before a subsequent concentration is generally carried out by means of chromatography-ion exchange through several steps. For this first the fermentation broths are acidified separated from the biomass by means of hydrochloric acid. { HCl) or sulfuric acid (H2S04), to facilitate the adsorption of lisxin in the exchange resins. In addition to the L-lysine prepared by fermentation, other non-specific, different cations, which are found in the fermentation broths, are linked. In general, several interchangeable columns are sequentially connected to obtain a pure product. The adsorbed lysine is subsequently eluted preferentially with an ammoniacal solution and the ion intermingling column is regenerated. The resulting lysine solution is then concentrated and after neutralization with hydrochloric acid, lysine HCl is obtained in crystalline form. Another method allows obtaining lysine in the form of a crystalline salt after purification with activated charcoal (SU-183581). Here the fermentation broths containing lysine are inactivated by means of a standard procedure with moist heat and separated by means of filtering the biomass. After the acidification of the filtrate at a pH of 5, 4-5% of activated carbon is added under constant stirring at 50-55 ° C., to separate unwanted impurities from the filtrate and to avoid the coloration of the crystallisate. In a subsequent filtration, the active carbon is separated and then the dissolved sulphate is precipitated with the addition of calcium hydroxide in the form of calcium sulfate. This is filtered, the ammonia fraction is separated in a rotary evaporator under vacuum and the solution is extracted until recrystallization occurs under cooling. The disadvantage of the two processing methods consists in the many individual steps of the process and the costly purification procedures by means of ion exchange chromatography. By means of the elimination of annoying salts or the use of different means of elution, additional waste streams are presented, which must be purified or disposed of in an expensive manner.

These disadvantages are overcome according to EP-B-0533039 because the entire fermentation load eventually including the biomass is concentrated and spray-dried at a high temperature. In US-A 5,990,350 a process for the preparation of a granulate containing free biomass L-lysine is described. In this process, the biomass is first separated from the fermentation broth by means of ultrafiltration and discarded. The filtrate obtained in this way is concentrated by means of repeated evaporation of the aqueous action. The concentrate obtained in this way is dried by means of spray granulation in the flowing layer to form a granulate and is processed. EP-B-0534865 discloses a process for the preparation of basic aqueous solutions containing L-lysine from fermentation broths. In the process described therein, the bismase is separated from the fermentation broth and discarded. By means of a base such as sodium, potassium or ammonium hydroxide, a pH value between 9 and 11 is adjusted. The mineral constituents (inorganic salts) after concentration and cooling are separated from the broths by means of crystallization. and, either they are used as fertilizer or they are discarded (deposed). Task of the Invention The task of the invention consists in presenting new forms of preparation of aqueous L-lysine, useful as forage supplements, and their salts. It is also the task of the invention to present a method that is more economical and more productive than the currently known methods. DESCRIPTION OF THE INVENTION The essential amino acid lysine is used to a large extent as a supplement for forages. The microorganisms of the genus Corynebacterium are characterized by the ability to produce in relatively short times, high concentrations of L-lysine "in the corresponding fermentation medium.The production processes are carried out in general as feeding procedures (Fedbatch). -Line prepared by fermentation is mainly processed at this time in the form of a crystalline product, powder or granulate The object of the invention is a forage supplement based on fermentation broths, characterized in that a) it contains L-lysine and / or its salts, b) the biomass formed during fermentation in an amount of 0 to 100%, preferably 50 to 100% and especially 90 to 100%, c) at least a significant fraction of the other dissolved and suspended ingredients of the fermentation broths, d) is in liquid form, optionally in the form of suspension and e) has a pH <(less than) 7. The forage supplement has a lysine content (as a lysine base) of 10 to 35% by weight, preferably 15 to 35% by weight, especially preferred 18 to 35% by weight, and very especially preferred 21 to 34% by weight, in proportion to the amount total of the supplement. The pH is < (minor a) 7, preferably 2 to 6.5, especially preferred 2.5 to 6 and very especially preferred 2.5 to 5. The ration of the total dry mass of the supplement is from 10 to 55% by weight, preferably 20 to 55% by weight , especially preferred 35 to 55% by weight and most especially preferred 45 to 54% by weight. The dry mass also contains, in addition to lysine, the biomass of the producing microorganisms, the inorganic and organic constituents of the fermentation broths and the by-products formed during fermentation, as long as they are not removed by a suitable method such as separation. or filtration. If the biomass is left completely or partially in the supplement, then it contains the protein of the microorganism in a maximum concentration of 4% by weight. The supplement contains as inorganic parts, among others calcium, magnesium, phosphorus in the form of phosphate and sulfur in the form of sulphate and as organic constituents among other vitamins, as well as, for example, biotin and thiamine or sugar, such as, for example, isomaltose. To organic by-products formed in small quantities during fermentation, belong the L-amino acids selected from the group of L-alanine, - L-asparagine, L-glutamine, L-methionine, L-threonine and L-valine. In addition to these are organic acids which carry one to three carbsyl groups, such as, for example, lactic acid, acetic acid and alic acid. Finally also belong to these sugars such as trehalose. These compounds are eventually desired when they improve the value of the supplement. Another object of the invention is a process for the preparation of aqueous solutions / suspensions - of lysine and / or its salts which is characterized in that a) a broth containing L-lysine is prepared by means of fermentation, b) optionally by When the addition of a substance containing L-lysine is adjusted to a desired concentration of lysine, c) 'the pH value of the solution / suspension is adjusted to <7, and d) the broths thus obtained are optionally concentrated under vacuum, the sequence of steps b), c) and d) being modified. Another object of the invention is finally a process for the preparation of aqueous solutions / suspensions of lysine and / or its salts, which is characterized in that a) a broth containing L-lysine is prepared by means of fermentation, b) the value pH of the broths thus obtained conforms to < (minor a) 7, c) optionally the biomass solution / suspension is completely or partially separated, d) optionally by means of the addition of a compound containing L-lysine, the desired concentration of lysine is adjusted, e) and the broths thus obtained are optionally concentrated under vacuum, the sequence of steps b), c), d) and e) being modified. LJ-isine-producing mutant coryneform bacteria are expressly described in the state of the art as for example in US-A-4, 657, 860. These strains can be grown continuously or discontinuously in batch processes or in feeding procedure (fed batch) or repeated fed batch procedure in order to produce L-amino acids. A summary of known cultivation methods can be found in the text by Chmiel (Bioprozestechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the text by Storhas (Bioreaktoren und periphere Einrichtungen (Vie eg Verlag, Braunschweig / iesbaden, 1994)). The culture medium used must suitably be sufficient for the requirements of the strains in question. Sugar sources and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, melase, starches - and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil can be used as carbon sources. fatty acids such as palmitic acid, stearinic acid and linoleic acid, alcohols such as glycerin and ethanol and organic acids such as acetic acid. These substances can be used individually or in mixtures. Nitrogen-containing organic compounds such as peptone, yeast extract, meat extract, malt extract, corn steep water, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, phosphate can be used as nitrogen source. of ammonium, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used alone or as mixtures. Phosphoric acid, potassium dihydrophosphate or dipotassium hydrophosphate or the corresponding sodium-containing salts can be used as phosphorus sources. The culture medium must also contain salts of metals such as magnesium sulfate or iron, which are necessary for growth. Finally, growth promoters can be used as amino acids • and vitamins in addition to the substances mentioned. Suitable precursors can be added to the culture medium. The additives mentioned can be added to the culture in the form of a single charge or in a suitable form added during cultivation. To control the pH of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acidic compounds such as phosphoric acid or sulfuric acid are suitably used. To control foaming, anti-foaming agents such as polyglycol fatty acid or silicone oils can be used. To maintain the stability of the plasmids, selective substances, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or gaseous mixtures containing oxygen such as air are introduced into the culture and mixed by means of suitable agitation systems or gaseous streams. The culture temperature is typically between 25 ° C and 37 ° C. The culture is continued until a maximum amount of L-lysine has been formed. This objective is normally reached in the course of 10 to 160 hours. Examples of suitable fermentation media can be found, for example, in patent documents EP-B-0 532 8678, US-A-5, 840, 551 and US-5, 990, 350. The analysis of L-lysine can be carried out automatically based on anion exchange chromatography with the subsequent derivation of ninhydrin as described by Spackman et al. (Apalytical Chemistry, 30, (1958) 1190), or by reverse phase CLAP, as described by Lipdrsth et al. (Analytical Chemistry (1979) 51: 1167-1174). The fermentation broths used preferably have a content of L-lysine > (greater than) 60 g / L (as a lysine base) with a fraction of unmetabolized sugar < (less than) 5.0 g / L. With a total dry mass fraction of > 10% by weight is the fraction of the dry biomass preferably between 1 to 4% by weight. The content of fermentation by-products and vitamins (amino acids, organic acids) is here < (less than) 2% by weight. In the process according to the invention, the biomass present in the first fermentation broths is generally inactivated or killed by means of for example a heat treatment. Eventually inactivation may also be omitted. The pH of the fermentation broths is then adjusted by means of an inorganic acid such as, for example, sulfuric acid, hydrochloric acid or phosphoric acid, an organic acid such as, for example, citric acid, acetic acid or formic acid or a mixture of different acids , up to a pH < (minor a) 7, preferably 2 a &5.5, especially preferred 2.5 to 6 and most especially 2.5 to 5. Eventually before or after the acidification of the biomass is completely or partially separated with generally known methods of separation or filtration . A separation of the mineral constituent parts in general is not required. The acid solution or suspension containing L-lysine is then concentrated by known methods (such as with a rotary evaporator, thin-film evaporator or precipitated film evaporator) preferably vacuum, until a product is obtained liquid with a lysine content (as L-lysine base) of 10 to 35% by weight, preferably 15 to 35% by weight, especially preferred 20 to 35% by weight, and very especially preferred 21 to 34% by weight , up to a total dry mass content of 10 to 55% by weight, preferably 20 to 55% by weight, especially preferred 35 to 55% by weight, and very especially preferred 45 to 54% by weight. This generally produces that the broths are extracted in such a way that mineral constituents (inorganic salts) of the fermentation broths are preferably not precipitated and that the lysine is dissolved. Optionally, the desired concentration of -L-lysine in the product can be adjusted by the addition of a substance containing L-lysine during a desired step of the process. The solution / suspension obtained in this way has an acidic pH value, can be transported easily, dosed without problems, is microbially stable and better storable than an alkaline solution. The acidification can also be carried out after or during the concentration. Anions that function as sulfate, chloride, phosphate, citrate etc. they can already be fed before the fermentation of the medium in the form of commercial salts. The suspension concept considers that the preferred inactivated microorganisms are preferably not dissolved in the product according to the invention. Examples: The present invention will be clarified in detail with the help of the following exemplary embodiments. Example l. Preparation of fermentation broths containing L-lysine. Fermentation processes for the preparation of broths containing lysine that can be prepared according to the invention have been described expressly in different previous patents (EP-B 0 532 867 and US-A ,840,551). Example 2. Preparation of a biomass-free product. According to example 1, 20kg-of a fermentation broth with a content of about 9. 5% by weight (as a lysine base). The biomass present - in the fermentation broth, was first inactivated by means of a heat treatment for 30 min. at 80 ° C. The inactivated biomass was then separated by means of centrifugation for 20 min. at 4000rpm (Biofugue-Stratos laboratory centrifuge, Heraeus, Dusseldorf Germany). 1. 0 liter of the residue clarified in this way was adjusted to a Ph value of. about 4 by means of stepwise addition of concentrated sulfuric acid. In the centrifugation the liquid content was subsequently reduced to approximately 50% of the dry content in a rotary vacuum evaporator (rotary laboratory evaporator Büchi Rotavapor RE-120, Büchi-Labortechnik GmbH, Constance, Germany). The product-liquid obtained in this way had an L-lysine content of 31.5% by weight (as a lysine base) and a Ph value of 4. The total dry mass was 49.3% by weight. Example 3. Preparation of a product containing biomass. According to example 1, 20kg of a fermentation broth with a content of approximately 9.5% by weight (as a lysine base) was prepared. The biomass present in the fermentation broth was first inactivated by means of a heat treatment for 30 min. at 80 ° C. The ipactivated biomass was separated, then by means of centrifugation for 20 min. at 4000Rpm (Biofugue-stratss laboratory centrifuge, heraeus, Dusseldorf Germany). 1.0 liter of the rinse in this manner was adjusted to a Ph value of approximately 4 by the stepwise addition of concentrated sulfuric acid. In the centrifugation, the liquid content was subsequently reduced to approximately 50% of the dry content in a vacuum rotary evaporator (rotary laboratory evaporator Büchi Rotavapor RE-120, Bühi-Labortechnik GmbH, Constance, Germany). The liquid product obtained in this way had an L-lysine content of 21.8% by weight (as a lysine base) and a Ph value of 4, the total dry mass, was 50.7% by weight. After storage for 16 months at 20 ° C, no significant change in the content could be determined. It is noted that in relation to this date, the best method known by the applicant to bring the said invention into practice is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following: - -

Claims (6)

REIVIWPICATIONS
1. l.- Forage supplement to fermentation broth base, characterized in that a) it contains L-lysine and / or its salts, b) the biomass formed during fermentation in an amount of 0 to 100%, preferably 50 to 100 % and especially from 90 to 100%, c) at least a significant fraction of the other ingredients dissolved and suspended from the fermentation broths, d) is in liquid form, optionally in the form of a suspension and e) has a pH < (minor a) 7.
2. 2.- Solutions / suspensions containing L-lysine according to claim 1 with an L-lysine content of 10 to 35% by weight, based on the total amount of the solution / suspension.
3. 3. Solutions / suspensions containing L-lysine according to claim 1 with a pH value of 2-6.5.
4. 4. Process for the preparation of solutions / suspensions containing lysine and / or its salts, characterized in that, a) a broth containing L-lysine is prepared by means of fermentation, b) optionally by means of the addition of a substance containing L-lysine a desired concentration of lysine is adjusted, c) the pH value of the solution / suspension is adjusted to < 7, and d) the brines thus obtained are optionally concentrated under vacuum, the sequence of steps b), c) and d) being modified.
5. 5. Process for the preparation of an additive for forage containing lysine and / or its salts, which is characterized in that a) a broth containing L-lysine is prepared by means of fermentation, b) the pH value of the broths thus obtained conforms to < (minor a) 7, c) optionally the biomass solution / suspension is completely or partially separated, d) optionally by means of the addition of a compound containing L-lysine, the desired concentration of lysine is adjusted, e) and the broths thus obtained are optionally concentrated under vacuum, the sequence of steps b), c), d) and e) being modified.
6. 6. - Use of solutions / suspensions containing L-lysine and / or its salts according to claim 1, as supplements for forages.