Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/14—Glutamic acid; Glutamine

Definitions

• the present invention relates to the fermentative preparation of organic compounds having at least 3 C atoms or having at least 2 C atoms and at least 1 N atom using a sugar-containing medium which comprises at least a portion of the non-starchy solid components of the starch source for culturing of microorganisms.
• Sugar-containing liquid media are a basic nutrient source for many fermentation processes; the sugars contained in the media are metabolized by the microorganisms used to obtain organic products of value.
• starch An important source of carbon for the microorganism-mediated fermentative production of microbial metabolites is starch. This must first be liquefied and saccharified in upstream reaction steps before it can be used as a carbon source in a fermentation.
• starch from a natural source of starch such as potatoes, cassava, cereals, z.
• a natural source of starch such as potatoes, cassava, cereals, z.
• wheat, corn, barley, rye, triticale or rice usually obtained in pre-purified form and then enzymatically liquefied and saccharified, and then in the actual Fer- mentation for the production of the desired metabolic products.
• non-purified starch sources for the production of carbon sources for the fermentative production of microbial metabolites.
• starch sources are first crushed by grinding.
• the millbase is then subjected to liquefaction and saccharification. Since, in addition to starch, this millbase naturally also contains a number of non-starch-containing constituents which can adversely influence the fermentation, these constituents are usually separated off before the fermentation.
• the removal can be carried out either directly after grinding (WO 02/077252, JP 2001-072701, JP 56-169594, CN 12181 11), after liquefaction (WO 02/077252, CN 1 173541) or following the saccharification (CN 1266102; Beukema et al .: Production of fermentation syrups by enzymatic hydrolysis of potatoes; potatoe saccharification to give culture medium (Conference Abstract), Symp. Biotechnol. Res Neth., (1983) 6, NL8302229). In all variants, however, a largely pure starch hydrolyzate is used in the fermentation.
• More recent processes for the fermentative production of organic compounds comprise in particular a purification of the starch sources prior to fermentation, for.
• non-purified starch sources are widely used in the fermentative production of bioethanol.
• the starch sources usually whole cereal grains, first dry milled and then hydrolyzed the starch component of the starch source under the action of enzymes.
• the hydrolysis both discontinuously, z. B. in stirred tanks, as well as continuously, for. B. To be performed in jet cookers.
• Corresponding process descriptions can be found z.
• the Alcohol Textbook - A reference for the beverage, fuel and industrial alcohol industries Jaques et al. (Ed.), Nottingham Univ. Press 1995, ISBN 1-8977676-735, Chapter 2, pp. 7 to 23, and in McAloon et al., “Determining the cost of producing ethanol from starch and lignocellulosic feedstocks," NREL / TP-580-28989, National Renewable Energy Laboratory, October 2000.
• the viscosity of the suspension can reach a critical value as a result of the introduction of solids already in the preparation of the starchy suspension, whereby z. B. a suspension with more than 30 wt .-% maize flour in water is no longer homogeneously miscible (Industrial Enzymology, 2nd ed., T. Godfrey, S. West, 1996). This limits the glucose concentration in the conventional procedure. This is not relevant in view of the fermentative production of bioethanol, since higher concentrations due to the toxicity of the product for the yeasts used for fermentation could not be implemented in any case meaningful.
• JP 2001/275693 describes a process for the fermentative production of amino acids, in which the starch source used is peeled cassava nodules which have been ground dry. For carrying out the process, however, it is necessary to set a particle size of the ground material of ⁇ 150 ⁇ m. In the filtration used for this purpose, parts of the grinding stock used, including non-starchy constituents, separated before liquefaction / saccharification of the starch contained and subsequent fermentation. Moderate sugar concentrations are achieved in this process. A similar process is described in JP 2001/309751 for the preparation of an amino acid-containing feed additive.
• Increased sugar concentrations in the liquid medium used for fermentation can be achieved by using a method of saccharification largely containing the solid, non-starchy components of the starch source by the method described in WO 2005/116228 (PCT / EP2005 / 005728) of the Applicant , A separation of the solid, non-starch-containing components contained in the starch source before fermentation has surprisingly proved to be unnecessary.
• a similar process using starch sources selected from cereal grains is described in the applicant's PCT / EP2006 / 066057 (earlier patent application DE 10 2005 042 541.0). For a continuous supply of sugary media with high sugar concentration, however, this method is relatively expensive.
• the process should allow for continuous hydrolysis of the starch component of the starch source.
• it should be characterized by easy handling of the media used and their ease of use in fermentation.
• the process should allow the use of cereals as a source of starch.
• hydrolysis of the starch constituent in the millbase by liquefaction and optionally subsequent saccharification to obtain an aqueous medium M containing the hydrolyzed starch constituents of the starch source and at least containing part of the non-starchy solid constituents of the starch source, wherein the hydrolysis comprises heating the suspension of the millbase by introducing water vapor into the suspension at temperatures above the pasting temperature of the starch contained in the millbase.
• the invention thus provides a process for preparing at least one organic compound having at least 3 C atoms or having at least 2 C atoms and at least 1 N atom by fermentation, comprising, in addition to steps i) and ii), the following steps:
• step iv) using the aqueous medium M obtained in step iii) in a fermentation to cultivate a microorganism which is capable of overproducing the organic compound;
• step iii) the suspension obtained in step ii) by introducing
• the high content of solid, non-starchy components of the starch source in the resulting medium surprisingly does not interfere with the fermentation.
• viscosity problems as they can occur during liquefaction of the starch source at higher concentrations of ground material, largely avoided.
• the medium can be used in a particularly advantageous manner in the feeding phase of the fermentation, whereby an undesirable dilution is largely avoided or at least significantly reduced.
• the medium M obtainable according to the invention is suitable as a sugar source in the batch phase of the fermentation.
• starch content and “starch component” are used interchangeably here and below.
• aqueous medium M obtained in step iii the terms “aqueous medium”, “liquid medium” and “aqueous sugar-containing liquid” are used synonymously.
• reaction means the hydrolytic degradation of starch into oligosaccharides, especially dextrins.
• sacharification or “saccharification” here and in the following mean the hydrolysis of dextrins to monosaccharides, in particular to monosaccharides such as glucose.
• sacharifying enzyme an enzyme which hydrolyzes dextrins to monosaccharides.
• oligosaccharides obtained by hydrolytic degradation of starch which as a rule consist of 3 to 18, in particular 6 to 12 monosaccharide units, in particular of glucose units.
• glucose equivalents and “sugar concentration” refers to the total concentration of mono-, di- and oligosaccharides in the medium potentially available for fermentation.
• glucose equivalents also includes the metabolizable sugars or sugar units other than glucose.
• overproducing and overproduction are used herein and hereafter with respect to a microorganism to refer to its property of producing one or more of its metabolites in an amount in excess of the amount needed to replicate the microorganism, whereby it comes to the enrichment in the fermentation medium, wherein the enrichment can take place extra- or intracellularly.
• Suitable starch sources for grinding are, above all, dry grain crops or seeds which, when dried, have at least 40% by weight and preferably at least 50% by weight of starch. These are found in many of today's large-scale crops such as corn, wheat, oats, barley, rye, triticale, rice and sugar beets, potatoes, cassava and various types of millet, z. Sorghum and MiIo.
• the starch source is selected from cereals and especially among corn, rye, triticale and wheat grains.
• the method according to the invention can also be used with analogue starch sources perform such as a mixture of starchy corn kernels or seeds.
• the respective starch source with or without the addition of liquid, for.
• liquid for.
• water ground, preferably without the addition of liquid. It can also be a dry grinding combined with a subsequent wet grinding.
• hammer mills, rotor mills or roll mills are typically used;
• agitating mixers, stirred ball mills, circulation mills, disk mills, annular chamber mills, vibrating mills or planetary mills are suitable.
• other mills come into consideration.
• the amount of liquid required for wet milling can be determined by the skilled person in routine experiments. Usually it is adjusted so that the content of dry matter in the range of 10 to 20 wt .-% is.
• the millbase obtained contains 50 wt .-% of flour particles having a particle size of more than 100 microns.
• at least 95% by weight of the ground flour particles have a particle size of less than 2 mm.
• the measurement of the grain size is carried out by sieve analysis using a vibration analyzer.
• a small grain size is basically advantageous for achieving a high product yield. Too small a particle size, however, can lead to problems, in particular due to lump formation / agglomeration, during mashing of the ground material during liquefaction or during work-up, eg. As in the drying of the solids after the fermentation step, lead.
• Flours are usually characterized by the degree of milling or by the type of flour, and these correlate with one another in such a way that the index of flour type increases as the degree of pulverization increases.
• the Ausmahlungsgrad corresponds to the amount by weight of the recovered flour, based on 100 parts by weight of the millbase used. While grinding first pure, finest flour, z. B. from the interior of the grain, is obtained in further milling, so with increasing Ausmahlungsgrad, the proportion of Rohfaser- and shell content in the flour, the starch content is lower.
• the Ausmahlungsgrad is therefore also reflected in the so-called flour type, which as a number for classification of flours, in particular of cereal flours, and based on the ash content of the flour (so-called ash scale).
• the flour type or the type number indicates the amount of ash (minerals) in mg, which remains when burning 100 g flour powder substance.
• a higher type number means a higher degree of milling since the kernel of the cereal grain contains about 0.4% by weight, while the shell contains about 5% ash by weight.
• the cereal flours are therefore predominantly composed of the comminuted flour body, ie the starch constituent of the cereal grains; if the degree of milling is higher, the cereal flours also contain the crushed, protein-containing aleurone layer of the cereal grains; in the case of meal, the constituents of the proteinaceous and fat-containing seedling as well as the raw fiber and ash-containing seed shells.
• Flours having a high degree of milling or a high type number are generally preferred for the purposes according to the invention. If grain is used as the source of starch, then preferably the whole unpeeled grains are ground and processed further, if appropriate after prior mechanical separation of germ and husks.
• the millbase used contains at least one part, preferably at least 20% by weight, in particular at least 50% by weight, especially at least 90% by weight and especially not less than 99% by weight of those contained in the ground cereal grains starchy solid ingredients, according to the degree of milling.
• the proportion of non-starchy solid constituents millbase is preferably at least 10 wt .-% and in particular at least 15 wt .-%, z. B. between 15 and 75% by weight and especially between 20 and 60 wt .-%.
• step ii) the millbase in step ii) with an aqueous liquid, for. B. fresh water, recycled process water, z. B. from a subsequent fermentation, or mixed with a mixture of these liquids, to obtain an aqueous suspension.
• an aqueous liquid for. B. fresh water, recycled process water, z. B. from a subsequent fermentation, or mixed with a mixture of these liquids.
• This process is often referred to as mashing.
• such an amount of the starch source or of the millbase will be mixed with the aqueous liquid that the resulting suspension has a dry matter content of at least 45% by weight, frequently at least 50% by weight, in particular at least 55% by weight. %, especially at least 60 wt .-%, z. B. 45 to 80 wt .-%, preferably 50 to 75 wt .-%, in particular 55 to 70 wt .-% and especially 60 to 70% by weight.
• the aqueous liquid used for suspending the solid millbase to a slightly elevated temperature, for. B. in the range of 40 to 70 ° C, pre-tempering. It is preferred that the temperature of the liquid be as high as is selected, that the resulting suspension has a temperature below the vitrification temperature, preferably at least 5 K below the gelatinization temperature of the starch. Preferably, the temperature of the suspension will not exceed 60 ° C, especially 55 ° C.
• Suspending particulate millbase in the aqueous liquor may be both batch and continuous in conventional equipment, such as intermittently in agitator mixers or in continuously operated mixing equipment for solids mixing with liquids, for example in mixers downstream of the rotor stator Principle work.
• the aqueous suspension containing the millbase is first heated by introducing steam into a temperature above the gelatinization temperature of the starch contained in the starch source or the millbase.
• the temperature required for the respective starch is known to the person skilled in the art (see the cited "The Alcohol Textbook - A reference for the beverage, fuel and industrial alcohol industries", Chapter 2, p. 11) or can be determined by him in routine experiments Typically, the mixture is heated to a temperature which is at least 10 K and in particular at least 20 K, for example 10 to 100 K, in particular 20 to 80 K, above the respective gelatinization temperature, in particular the suspension is heated to temperatures in the region of 90 up to 150 ° C, and especially in the range of 100 to 140 ° C.
• the water vapor used for heating is typically superheated steam having a temperature of at least 105 ° C, especially at least 110 ° C, e.g. B. 1 has 10 to 210 ° C.
• the vapor is introduced with overpressure into the suspension.
• the steam preferably has a pressure of at least 1.5 bar, e.g. B. 1, 5 to 16 bar, in particular 2 to 12 bar.
• the introduction of water vapor into the suspension is generally carried out by introducing the vapor into the suspension at overpressure, preferably at an overpressure of 1 to 10 or 11 bar, in particular 1.5 to 5 bar, and preferably at high speed.
• overpressure preferably at an overpressure of 1 to 10 or 11 bar, in particular 1.5 to 5 bar, and preferably at high speed.
• the heating with steam is preferably carried out in a continuously operating device, into which the suspension is fed continuously with a specific delivery pressure, which results from the viscosity of the suspension, the conveying speed and the geometry of the device, and in which feed-in sens the suspension the hot steam with positive pressure, based on the delivery pressure, fed via a controllable nozzle.
• a continuously operating device into which the suspension is fed continuously with a specific delivery pressure, which results from the viscosity of the suspension, the conveying speed and the geometry of the device, and in which feed-in sens the suspension the hot steam with positive pressure, based on the delivery pressure, fed via a controllable nozzle.
• the vapor is introduced in the direction of the longitudinal axis of the tubular device.
• the supply of the suspension is usually at an angle of at least 45 ° or perpendicular thereto.
• the controllable nozzle typically has a conical geometry that tapers in the flow direction of the vapor.
• a needle or arranged on a longitudinally displaceable rod cone is arranged. Needle or cone forms a gap with the cone of the nozzle.
• these devices also include a mixing tube into which the suspension is transported after steaming and discharged from the device.
• This mixing tube is usually arranged in the direction of the steam inlet and perpendicular to the feed.
• the mixing tube typically forms a gap with the nozzle through which the suspension is transported. Through this gap additional shear forces act on the suspension during transport and thus increase the mechanical energy input into the suspension.
• the mixing tube can be arranged to be displaceable in the longitudinal direction. By moving the mixing tube can be adjusted in a simple manner, the size of the gap opening and thus the pressure drop in the device.
• Such devices are known under the name jet cookers from the prior art, for example, the device shown in "The Alcohol Textbook", Chapter 2, loc cit, Figure 13 and commercially available, for example under the name HYDROHEATER® Fa. Hydro Thermal Corp. Waukesha WI, USA.
• the steam-treated suspension is usually subsequently transferred to a post-reaction zone in order to continue the gelling of the starch components.
• a post-reaction zone In the post-reaction zone, there is typically overpressure, typically an absolute pressure in the range of 2 to 8 bar.
• the temperatures in the post-reaction zone are typically in the range of 90 to 150 ° C.
• the residence time in this post-reaction zone may be in the range from 1 minute to 4 hours, depending on the temperature of the suspension.
• the post-reaction onszonen typically have a tubular or columnar geometry.
• the post-reaction zone has the geometry of a vertically arranged column.
• the suspension is here after leaving the device for steam treatment one applied in the upper part of the column and removed in the lower part.
• the post-reaction zone has a tubular geometry.
• the suspension After leaving the post-reaction zone, the suspension is usually relaxed and then performs a liquefaction.
• the relaxation is carried out as flash evaporation in order to cool the suspension, preferably to temperatures below 100 ° C., in particular below 85 ° C.
• a liquefaction of the starch thus digested takes place in a separate reaction vessel. The liquefaction can be carried out in the manner described above.
• the liquefaction can be carried out in the usual way.
• the liquefaction in step ii) takes place in the presence of at least one starch-liquefying enzyme, which is generally selected from ⁇ -amylases.
• starch-liquefying enzyme which is generally selected from ⁇ -amylases.
• Other active and stable starch-liquefying enzymes under the reaction conditions can also be used.
• starch-liquefying enzymes in particular ⁇ -amylases (enzyme class EC 3.2.1.1) can be used to liquefy the starch content in the millbase, for example ⁇ -amylases obtained from Bacillus lichenformis or Bacillus stae rothermophilus and especially those which liquefy used by dry-milling processes in the production of bioethanol.
• Preferred enzymes are temperature stable, d. H. they also do not lose their enzymatic activity when heated to temperatures above the gelatinization temperature.
• the ⁇ -amylases suitable for liquefaction are also commercially available, for example from Novozymes under the name Termomyl 120 L, type L; or from Genencor under the name Spezyme. It is also possible to use a combination of different ⁇ -amylases for liquefaction.
• the amounts of starch-liquefying enzyme are chosen so that rapid and complete degradation of the starch to oligosaccharides is achieved.
• the total amount of starch-liquefying enzyme, in particular ⁇ -amylase is usually in the range from 0.002 to 3.0% by weight, preferably from 0.01 to 1.5% by weight and more preferably from 0.02 to 0, 5 wt .-%, based on the total amount of the starch source used.
• the ⁇ -amylase (resp. the starch-liquefying enzyme used) can be introduced into the reaction vessel or added during the liquefaction step.
• step ii) is preferably at least temporarily at a pH in the pH optimum of the liquefying enzyme, frequently at a pH in the weakly acidic range, preferably between 4.0 and 7.0, more preferably between 5.0 to 6.5, usually before or at the beginning of step ii) the pH adjustment is made;
• This pH is preferably controlled during liquefaction and optionally adjusted.
• the adjustment of the pH is preferably carried out with dilute mineral acids such as H 2 SO 4 or H 3 PO 4 or with dilute alkali solutions such as NaOH or KOH.
• the concentration of Ca 2+ ions, z. B. with CaCl 2 to an enzyme-specific optimal value. Suitable concentration levels can be determined by one skilled in the art in routine experimentation. If z. As Termamyl used as ⁇ -amylase, it is advantageous to have a Ca 2+ concentration of z. B. 10 to 100 ppm, preferably 20 to 80 ppm and more preferably about 30 to 70 ppm in the liquid medium, wherein the indication ppm is by weight and g / 1000 kg means.
• the reaction mixture is kept at the set temperature until the starch detection with iodine or optionally another test for the detection of starch negative or at least substantially negative.
• At least a portion or the total amount, usually at least 50%, in particular at least 80%, of the total or total amount of starch-liquefying enzyme is added to the suspension of the millbase in the aqueous liquid prior to heating with steam , In this way, the liquefaction already takes place during heating to temperatures above the gelatinization temperature. The heating with water vapor and the post-reaction phase are carried out accordingly. A subsequent liquefaction in a separate reaction vessel can be omitted. Preferably, however, such liquefaction will be carried out to complete the degradation of the starch into dextrins.
• an aqueous starch hydrolyzate which contains the liquefied starch portion from the millbase, typically dextrins and optionally further oligosaccharides and mono- or disaccharides, and the non-starchy constituents of the millbase, in particular the solid, non-starchy constituents of the liquefaction agent used Grind contains.
• This hydrolyzate can be fed as an aqueous medium M directly to a fermentation to produce the organic compound. Often, however, one will still submit to saccharification.
• the saccharification can be carried out analogously to the known saccharification methods of the prior art.
• the saccharification can be carried out continuously or discontinuously.
• the liquefied medium is this typically completely saccharified in a special saccharification tank before it z. B. is fed to a subsequent fermentation step.
• the aqueous product obtained after liquefaction is treated with an enzyme causing the saccharification, typically a glucoamylase, under the conditions customary for this purpose.
• glucoamylases enzyme class EC 3.2.1.3
• glucoamylases which have been obtained from Aspergilus and especially those which are used for saccharification of materials obtained by dry-milling processes used in the production of bioethanol.
• the glucoamylases suitable for saccharification are also commercially available, for example from Novozymes under the name Dextrozyme GA; or from Genencor under the name Optidex.
• a combination of different glucoamylases can also be used.
• the saccharifying enzyme is usually added to the dextrin-containing hydrolyzate obtained after liquefaction in an amount of 0.001 to 5.0% by weight, preferably 0.005 to 3.0% by weight, and more preferably 0.01 to 1.0% by weight. -%, based on the total amount of starch source used added.
• the saccharification takes place at temperatures in the range of the temperature optimum of the saccharifying enzyme or slightly below, z. At 50 to 70 ° C, preferably at 60 to 65 ° C.
• the aqueous liquefaction product will first be adjusted to these temperatures and then mixed with the enzyme which causes the saccharification.
• the saccharifying enzyme e.g. As the glucoamylase, the pH of the aqueous hydrolyzate to a value in the optimal range of action of the enzyme used, preferably in the range between 3.5 and 6.0; more preferably between 4.0 and 5.5, and most preferably between 4.0 and 5.0.
• the dextrin-containing suspension is preferably for a period of, for. B. 2 to 72 hours or longer, if necessary, in particular 5 to 48 hours kept at the set temperature, wherein the dextrins are saccharified to monosaccharides.
• the progress of saccharification may be by methods known to those skilled in the art, e.g. As HPLC, enzyme assays or glucose test strips, are followed. Saccharification is complete when the concentration of monosaccharides no longer increases or decreases significantly.
• the aqueous hydrolyzate obtained after liquefaction and optionally saccharification also comprises some or all of the non-starch-containing solid constituents of the starch source. This often requires the entry of a non-negligible proportion of phytate, z. B. from the grain crop.
• phytases In order to avoid the resulting inhibiting effect, it is advantageous to add one or more phytases to the hydrolyzate before it is fed to a fermentation step.
• the addition of the phytase can be done before, during or after liquefaction, provided that it has the required heat stability. It can be used any phytases, as far as their activity is limited under the reaction conditions in each case at most insignificant. Preference is given to phytases with a temperature stability (T50)> 50 ° C and particularly preferably> 60 ° C.
• T50 temperature stability
• the amount of phytase is usually 1 to 10,000 units / kg of starch source, and more preferably 10 to 4,000 units / kg of starch source.
• enzymes for example pullulanases, cellulases, hemicellulases, glucanases, xylanases, glucosidases or proteases, may be added to the liquor or during the saccharification.
• the addition of these enzymes can positively influence the viscosity, i. H. (eg by cleavage of long-chain (also referred to as longer-chain) glucans and / or of (arabino) xylans), the release of metabolizable glucosides and the release of (residual) starch cause.
• the use of proteases has analogous positive effects, with additional amino acids as growth factors for the fermentation can be released.
• the saccharification then takes place at least partly during the fermentation, ie in situ.
• Further saccharification can then take place in situ in the fermentation medium.
• the saccharification can furthermore be carried out directly in the fermenter with the elimination of a separate saccharification tank.
• In situ saccharification may be carried out with the addition of saccharifying enzymes as described above, or in the absence of such enzymes, since many microorganisms themselves are capable of metabolizing oligosaccharides.
• the dextrins are either taken up as such by the microorganism and metabolized or, after previous saccharification by strain-own saccharifying enzymes, eg. B. strain-specific glucoamylases, hydrolyzed and then metabolized.
• strain-own saccharifying enzymes eg. B. strain-specific glucoamylases
• a higher glucose concentration in the batch may optionally be presented by a delayed release of glucose, without any inhibition or metabolism change of the microorganisms used.
• E. coli leads to a high glucose concentration z.
• z For example, to form organic acids (acetate), while Saccharomyces cerevisae in this case z.
• B. switches to fermentation, although in aerated fermenters sufficient oxygen is present (Crabtree effect).
• a delayed release of glucose is adjustable by controlling the glucoamylase concentration. As a result, the aforementioned effects can be suppressed and it can be submitted to more substrate, so that the resulting from the supplied feed stream dilution can be reduced.
• the aqueous hydrolyzate obtained after liquefaction and optionally carried out saccharification, ie the medium M typically has a dry matter content of at least 45 wt .-%, often at least 50 wt .-%, in particular at least 55 wt .-%, especially at least 60 wt. %, z. B. 45 to 80 wt .-%, preferably 50 to 75 wt .-%, in particular 55 to 70 wt .-% us specifically 60 to 70 wt .-% to.
• the aqueous medium M obtained after hydrolysis generally has a sugar concentration, calculated as glucose equivalents of at least 35% by weight, frequently at least 40% by weight, in particular at least 45% by weight, especially at least 50% by weight. %, z. B. 35 to 70 wt .-%, in particular 40 to 65 wt .-%, in particular 45 to 60 wt .-% and especially 50 to 60 wt .-% to. based on the total weight of the medium M.
• the glucose equivalents contained in the medium M obtained are present in the form of monosaccharides or oligosaccharides, in particular dextrins.
• Main ingredient are typically monosaccharides such as hexoses and pentoses, z.
• glucose fructose, mannose, galactose, sorbose, xylose, arabinose and ribose, in particular glucose or oligosaccharides of these monosaccharides.
• the proportion of monosaccharides other than glucose in the free form or as constituents of the oligosaccharides in the medium M can vary depending on the starch source used and the non-starch-containing constituents contained therein and can be modified by the process, for example by digestion of cellulose components by addition of cellulases.
• the proportion of glucose, in free or bound form, among the glucose equivalents of the medium M is in the range from 50 to 99% by weight, in particular from 75 to 97% by weight and especially from 80 to 95% by weight. %, based on the total amount of glucose equivalents.
• the aqueous medium M obtained in step iii) is used according to the invention in step iv) for the fermentative preparation of the desired organic compound.
• the medium M is fed to a fermentation, where it serves for culturing the microorganisms used in the fermentation.
• the respective organic compound is obtained here as a volatile or nonvolatile microbial metabolite.
• the dextrin-containing medium M to fermentation temperature usually in the range of 32 to 37 ° C, cool before feeding it to the fermentation.
• the aqueous dextrin-containing medium M can optionally be sterilized before fermentation, wherein the microorganisms are usually killed by thermal or chemical methods.
• the aqueous medium M is usually heated to temperatures above 80 ° C.
• the killing or lysis of the cells can take place immediately before the fermentation.
• the entire medium M of lysis or killing is supplied. This can, for. B. thermally or chemically.
• a preferred embodiment of the invention relates to a process in which the medium M obtained in step iii) is fed directly, ie without prior sterilization, to the fermentation. Fermentation involves the metabolism of the sugars contained in the medium. If the sugars contained in the medium are in the form of oligosaccharides, especially in the form of dextrins, they are either taken up as such by the microorganism or after previous saccharification by added or strain-own saccharifying enzymes, in particular glucoamylases, and metabolised , If no saccharifying enzymes are added and the sugars contained in the medium are present in the form of oligosaccharides, especially dextrins, saccharification of the liquefied starch components takes place in parallel to the metabolization of the sugar, in particular the monosaccharide glucose, by the microorganisms.
• the fermentation can be carried out in the usual manner known to the person skilled in the art.
• the desired microorganism will usually be cultured in the liquid medium obtained by the method described here.
• the fermentation process can be operated both batchwise and semicontinuously (fed-batch mode, including fed-batch with intermediate harvesting), with the semi-continuous mode being preferred.
• the medium M obtained by the process of the invention or a conventional sugar source i. H. metabolizable mono-, di- and / or oligosaccharides or media containing metabolizable mono-, di- and / or oligosaccharides, optionally after dilution with water and addition of conventional media components such as buffers, nutrient salts, nitrogen sources such as ammonium sulfate, urea, etc., complex Nährmedienzierer containing amino acids, such as yeast extracts, peptones, CSL and the like, inoculate with the desired microorganism and multiply under fermentation conditions until the microorganism concentration reaches the steady state desired for the fermentation.
• the sugar contained in the fermentation medium is metabolized and the desired metabolite is formed (so-called batch process or batch phase).
• the medium M is added continuously or discontinuously to the fermentation medium.
• a typical embodiment of the method according to the invention is the fed-batch mode, which comprises the following steps: v) cultivating the microorganism capable of overproducing the organic compound in an aqueous fermentation medium F; and
• a conventional sugar-containing medium usually a glucose solution
• a suitable sugar concentration by dilution with an aqueous liquid, in particular water, and the media components customary for fermentation, such as buffers, nutrient salts, nitrogen sources, such as ammonium sulfate, Urea, etc., complex nutrient media ingredients containing amino acids such as yeast extracts, peptones, CSL, and the like.
• the ratio of amount of sugar to liquid will usually be selected preferably such that the total concentration of monosaccharides in the fermentation medium F less than 6 wt .-%, z. B.
• the sugar-containing batch medium thus prepared is inoculated with the desired microorganism and the microorganism in the batch medium (fermentation medium F) is increased under fermentation conditions until the concentration of microorganisms reaches a desired stationary state for the fermentation.
• the sugar introduced in the fermentation medium F is metabolized and the desired metabolite is formed.
• the volume ratio of fed medium M to the present and containing the microorganisms batch medium is generally in the range of about 1:10 to 10: 1 and preferably about 1: 5 to 5: 1 and especially in the range of 1: 1 to 5: 1.
• the sugar content in the fermentation broth can be regulated.
• the feed rate will be adjusted such that the monosaccharide content in the fermentation broth is in the range from> 0% by weight to about 5% by weight, and in particular does not exceed a value of 3% by weight.
• the fermentation medium F in step v) essentially comprises the medium M, the microorganisms capable of overproduction of the organic compound, nutrient salts, customary Excipients such as bases or buffers and optionally water for dilution.
• the medium M is optionally diluted to the desired sugar concentration, z. B. in the range of 0.1 to 10 wt .-%, calculated as glucose equivalents and based on the total weight of the medium M, and this directly for preparing the fermentation medium F (batch medium) use.
• the sugar content of the dextrin-containing medium used to maintain the fermentation according to step vi), is usually higher, z. B. in the o. G. Areas to minimize the dilution of the fermentation medium F.
• an aqueous medium M with a higher sugar concentration, z. B. at least 40 wt .-%, especially at least 45 wt .-% and especially at least 50 wt .-%, calculated as glucose equivalents and based on the total weight of the aqueous medium M produces.
• This medium M is then used on the one hand according to step v) after dilution with water to prepare the batch medium (fermentation medium F) and on the other hand according to step vi) for addition to the fermentation medium F.
• the main amount preferably at least 60 wt .-%, in particular at least 70 wt .-% of the used in the fermentation to be metabolized sugar from the medium M.
• a part e.g. B. 1 to 50 wt .-%, in particular 5 to 40 wt .-% and especially 10 to 30 wt .-% of the used in the fermentation to be metabolized sugar from conventional sugar sources.
• the conventional sugar sources include mono- and disaccharides such as glucose and sucrose, but also media containing metabolizable mono-, di- and / or oligosaccharides in a concentration of at least 50% by weight and which are substantially free of water insoluble solids, for example glucose syrups, sucrose syrups, thick juices, maltose syrup, dextrin syrups, but also waste products of sugar production (molasses), in particular molasses from beet sugar production but also molasses from cane sugar production.
• mono- and disaccharides such as glucose and sucrose, but also media containing metabolizable mono-, di- and / or oligosaccharides in a concentration of at least 50% by weight and which are substantially free of water insoluble solids, for example glucose syrups, sucrose syrups, thick juices, maltose syrup, dextrin syrups, but also waste products of sugar production (molasses), in particular molasses from beet sugar production but also molasses from
• volatile and non-volatile, in particular non-volatile, microbial metabolites having at least 3 C atoms or having at least 2 C atoms and 1 N atom can be produced by fermentation.
• Non-volatile products are understood as meaning those compounds which can not be obtained from the fermentation broth by distillation by distillation. These compounds generally have a boiling point above the boiling point of water, often above 150 ° C and especially above 200 ° C. Normal pressure on. They are usually compounds that are solid under normal conditions (298 K, 101, 3 kPa).
• aqueous medium M in a fermentation for the production of nonvolatile microbial metabolites which have a melting point below the boiling point of water or / and an oily consistency at atmospheric pressure.
• nonvolatile microbial metabolites herein includes, in particular, organic, optionally 1 or more, e.g. B. 1, 2, 3 or 4 hydroxyl-bearing mono-, di- and tricarboxylic acids having preferably 3 to 10 carbon atoms, for. Tartaric, itaconic, succinic, propionic, lactic, 3-hydroxypropionic, fumaric, maleic, 2,5-furandicarboxylic, glutaric, levulinic, gluconic, aconitic and diaminopimelic acids, citric acid; proteinogenic and non-proteinogenic amino acids, e.g.
• Lysine glutamate, methionine, phenylalanine, aspartic acid, tryptophan and threonine; Purine and pyrimidine bases; Nucleosides and nucleotides, e.g. Nicotinamide adenine dinucleotide (NAD) and adenosine 5'-monophosphate (AMP); lipids; saturated and unsaturated fatty acids having preferably 10 to 22 carbon atoms, e.g.
• propanediol and butanediol polyhydric (also referred to as higher) alcohols with 3 or more, eg. B. 3, 4, 5 or 6 OH groups, for.
• Enzymes such as amylases, pectinases, acidic, hybrid or neutral cells, esterases such as lipases, pancreases, proteases, xylanases and oxidoreductases such as laccase, catalase and peroxidase, glucanases, phytases; Carotenoids, z. Lycopin, beta-carotene, astaxanthin, zeaxanthin and canthaxanthin; Ketones having preferably 3 to 10 carbon atoms and optionally 1 or more hydroxyl groups, for. Acetone and acetoin; Lactones, e.g.
• Gamma-butyrolactone, cyclodextrins, biopolymers e.g. B. polyhydroxyacetate, polyester, z. Polylactide, polysaccharides, polyisoprenoids, polyamides; as well as precursors and derivatives of the aforementioned compounds.
• Other compounds of interest as nonvolatile microbial metabolites are described by Gutcho in Chemicals by Fermentation, Noyes Data Corporation (1973), ISBN: 0818805086.
• cofactor includes non-proteinaceous compounds that are necessary for the occurrence of normal enzyme activity. These compounds can be organic or be inorganic; the cofactor molecules of the invention are preferably organic. Examples of such molecules are NAD and nicotinamide adenine dinucleotide phosphate (NADP); The precursor of these cofactors is niacin.
• the term "nutraceutical” includes food additives that are beneficial to the health of plants and animals, especially humans. Examples of such molecules are vitamins, antioxidants and certain lipids, e.g. B. polyunsaturated fatty acids.
• the metabolites produced under enzymes amino acids, vitamins, disaccharides, aliphatic mono- and dicarboxylic acids having 3 to 10 carbon atoms, aliphatic hydroxycarboxylic acids having 3 to 10 carbon atoms, ketones having 3 to 10 carbon atoms, alkanols having 4 to 10 C atoms and alkanediols having 3 to 10 and in particular 3 to 8 carbon atoms selected.
• microorganisms used in the fermentation depend in a manner known per se on the respective microbial metabolites, as explained in detail below. They may be of natural origin or genetically modified. Examples of suitable microorganisms and fermentation processes are, for. In table A, for example.
• the organic compound prepared is optionally mono-, di- and tricarboxylic acids bearing 3 to 10 carbon atoms, proteinogenic and non-proteinogenic amino acids, purine bases, pyrimidine bases; Nucleosides, nucleotides, lipids; saturated and unsaturated fatty acids; Diols having 4 to 10 C atoms, polyhydric alcohols having 3 or more hydroxyl groups, longer-chain alcohols having at least 4 C atoms, carbohydrates, aromatic compounds, vitamins, provitamins, cofactors, nutraceuticals, proteins, carotenoids, ketones of 3 to 10 C atoms, lactones, biopolymers and cyclodextrins selected.
• a first preferred embodiment of the invention relates to the use of the sugar-containing liquid medium obtainable according to the invention in a fermentative production of enzymes such as phytases, xylanases or glucanases.
• a second preferred embodiment of the invention relates to the use of the sugar-containing liquid medium obtainable according to the invention in a fermentative production of amino acids such as lysine, methionine, threonine and glutamate
• a further preferred embodiment of the invention relates to the use of the sugar-containing liquid medium obtainable according to the invention in a fermentative preparation of vitamins such as pantothenic acid and riboflavin, precursors and secondary products thereof.
• Mono-, di- and tricarboxylic acids in particular aliphatic mono- and dicarboxylic acids having 3 to 10 carbon atoms such as propionic acid, fumaric acid and succinic acid, aliphatic hydroxycarboxylic acids having 3 to 10 carbon atoms such as lactic acid; longer-chain alkanols as mentioned above, in particular alkanols having 4 to 10 C atoms, such as butanol;
• Diols as mentioned above, in particular alkanediols having 3 to 10 and in particular 3 to 8 C atoms, such as propanediol;
• Ketones as mentioned above in particular ketones having 3 to 10 carbon atoms, such as acetone; and - carbohydrates as mentioned above, in particular disaccharides such as trehalose.
• the metabolic product produced in the fermentation by the microorganisms is polyhydroxyalkanoates such as poly-3-hydroxybutyrate and copolyester with other organic hydroxycarboxylic acids such as 3-hydroxyvaleric acid, 4-hydroxybutyric acid and others which are described in US Pat Steinbüchel (loc. Cit.) Are described, for.
• long-chain (also referred to as longer-chain) hydroxycarboxylic acids such as 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxytetradecanoic acid, and mixtures thereof.
• analogous conditions and procedures can be used here, as have been described for other carbon sources, eg. See, for example, S. Y. Lee, Plastic Bacteria Progress and Prospects for Polyhydroxyalkanoate Production in Bacteria, Tibtech, Vol. 14, (1996), pp. 431-438.
• the microorganisms used in the fermentation are therefore selected from natural or recombinant microorganisms which overproduce at least one of the following metabolic products:
• Enzymes such as phytase, xylanase or glucanase; Amino acids such as lysine, threonine or methionine; - vitamins such as pantothenic acid and riboflavin; Precursors and / or derivatives thereof; Disaccharides such as trehalose; aliphatic mono- and dicarboxylic acids having 3 to 10 C atoms, such as propionic acid, fumaric acid and succinic acid; aliphatic hydroxycarboxylic acids having 3 to 10 C atoms such as lactic acid; Polyhydroxyalkanoates such as poly-3-hydroxybutyrate and copolyester of
• C atoms such as propanediol.
• Suitable microorganisms are usually selected from the genera Corynebacterium, Bacillus, Ashbya, Escherichia, Aspergillus, Alcaligenes, Actinobacillus, Anaerobiospirillum, Lactobacillus, Propionibacterium, Rhizopus and Clostridium, in particular from strains of Corynebacterium glutamicum, Bacillus subtilis, Ashbya gossypii, Escherichia coli, Aspergillus niger or Alcaligenes latus, Anaerobiospirillum succiniproducens, Actinobacillus succinogenes, Lactobacillus delbschreibii, Lactobacillus leichmannii, Propionibacterium arabinosum, Propionibacterium schermanii, Propionibacterium freudenreichii, Clostridium propionicum, Clostridium formicoaceticum, Clostridium acetobutylicum
• the microorganism used in the fermentation is a strain of the genus Corynebacterium, in particular a strain of Corynebacterium glutamicum.
• it is a strain of the genus Corynebacterium, especially of the Corynebacterium glutamicum, which overproduces an amino acid, especially lysine, methionine or glutamate.
• the microorganism used in the fermentation is a strain of the genus Escherichia, in particular a strain of Escherichia coli.
• it is a strain of the genus Escherichia, especially of Escherichia coli, which overproduces an amino acid, especially lysine, methionine or threonine.
• the metabolite produced by the microorganisms in the fermentation is lysine.
• analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In Pfefferle et al., Supra, and U.S. 3,708,395.
• the fed-batch mode of operation preferred is the fed-batch mode of operation.
• the metabolic product produced by the microorganisms in the fermentation is methionine.
• analogous conditions and procedures as described for other carbon sources can be used here, eg. In WO 03/087386 and WO 03/100072.
• the metabolic product produced by the microorganisms in the fermentation is tartaric acid.
• analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In WO 01/021772.
• the metabolite produced by the microorganisms in the fermentation is riboflavin.
• riboflavin To carry out the fermentation analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In WO 01/011052, DE 19840709, WO 98/29539, EP 1186664 and Fujioka, K .: New biotechnology for riboflavin (vitamin B2) and character of this riboflavin. Fragrance Journal (2003), 31 (3), 44-48.
• the metabolic product produced by the microorganisms in the fermentation is fumaric acid.
• analogous conditions and procedures as described for other carbon sources can be used here, eg. In Rhodes et al., Production of Fumaric Acid in 20-L Fermentors, Applied Microbiology, 1962, 10 (1), 9-15.
• the metabolite produced by the microorganisms in the fermentation is succinic acid.
• succinic acid a compound that produces succinic acid.
• the metabolic product produced by the microorganisms in the fermentation is a phytase.
• the metabolic product produced by the microorganisms in the fermentation is a phytase.
• the fermentation results in a fermentation broth, in addition to the desired microbial metabolite essentially the biomass produced during the fermentation, the non-metabolised components of the liquefied starch solution and in particular the non-starchy solid components of the starch source, such as.
• This liquid medium is also referred to in the present application as a fermentation broth, wherein the fermentation broth also comprises the added dextrin-containing medium (1), in which a partially or incomplete fermentative conversion of the sugars contained therein, d. H. partial or incomplete microbial metabolism of the usable sugars (eg mono- and disaccharides) has taken place.
• a sterilization step is optionally carried out in the manner described above.
• a specific embodiment (I) of the invention relates to a process in which at least one microbial metabolite is depleted or isolated from the fermentation broth. The volatiles of the fermentation broth are then substantially removed to yield a solid or semi-solid protein composition. A more detailed description of how to carry out such a process and the resulting protein composition is the subject of
• the organic compound having at least 3 C atoms or at least 2 C atoms and at least one N-atom (hereinafter also desired product) is generally carried out in such a way that at least one metabolic product depleted or isolated from the fermentation broth that the content this metabolic product in the remaining fermentation broth at most 20 wt .-%, in particular at most 10 wt .-%, especially at most 5 wt .-% and especially at most 2.5 wt .-%, each based on the total weight of the remaining fermentation broth is ,
• the isolation or depletion of the microbial metabolite from the fermentation broth can be done in one or more steps.
• An essential step here is the separation of the solid constituents from the fermentation broth. This can be carried out either before or after the isolation of the desired product.
• solid-liquid phase separation conventional methods are known in the art, which also include steps for coarse and fine cleaning of recyclable materials and for packaging (eg described in Belter , P.A., Bioseparations: Downstream Processing for Biotechnology, John Wiley & Sons (1988), and Ullmann's Encyclopedia of Industrial Chemistry, 5th Edition on CD-ROM, Wiley-VCH).
• the product of value it is advantageously possible to proceed by first removing the solid constituents from the fermentation broth, e.g. B. by centrifugation or filtration, and then isolated the desired product from the liquid phase, for. Example by crystallization, precipitation, adsorption or distillation.
• the desired product can also be isolated directly from the fermentation broth, z.
• a chromatographic method in particular ion exchange chromatography should be mentioned, in which the desired product can be selectively isolated on the chromatography column.
• the separation of the solids from the remaining fermentation broth is advantageously carried out z. B. by decantation, evaporation and / or drying.
• these compounds can also be prepared by formulating them in quasi-solid form (pseudo-solid form) on adsorbents. Suitable adsorbents for this purpose are, for. In WO 2005/116228
• Examples of compounds which can be advantageously prepared in this way are ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, furthermore propionic acid, lactic acid, propanediol, butanol and acetone.
• these compounds in pseudo-free formulation are also understood to be nonvolatile microbial metabolites in solid form.
• a further specific embodiment (II) relates to a process in which the volatile constituents of the fermentation broth are substantially removed without prior isolation or removal of a nonvolatile microbial metabolite, and optionally without prior separation of solid constituents, resulting in a solid formulation of a nonvolatile microbial Metabolism product receives.
• a more detailed description of the implementation of such a method can be found in the PCT / EP2006 / 066057 (earlier patent application DE 10 2005 042 541.0) of the applicant.
• the volatile constituents of the fermentation broth are advantageously up to a residual moisture content in the range of 0.2 to 30 wt .-%, preferably 1 to 20 wt .-%, particularly preferably 2 to 15 wt .-% and most preferably 5 to 15 wt .-%, based on the determined after drying total weight of the solid components, remove from the fermentation broth.
• the residual moisture content can be determined by conventional methods known in the art, for. Example by means of thermogravimetry (Hemminger et al., Methods of thermal analysis, Springer Verlag, Berlin, Heidelberg, 1989).
• Recovery of the nonvolatile metabolite (s) in solid form from the fermentation broth may be accomplished in one, two or more stages, especially in a one or two step procedure.
• at least one, in particular the final stage for obtaining the metabolite in solid form will comprise a drying step.
• the volatile constituents of the fermentation broth if appropriate after the aforesaid preliminary separation, are removed until the desired residual moisture content has been reached.
• the fermentation broth In the two- or multi-step procedure, the fermentation broth, first concentrate, z. Example by means of (micro-, ultra-) filtration or thermally by evaporation of a portion of the volatiles.
• the proportion of volatile constituents removed in this step is generally from 10 to 80% by weight and in particular from 20 to 70% by weight, based on the total weight of the volatile constituents of the fermentation broth.
• the residual volatiles of the fermentation broth are removed until the desired residual moisture content is achieved.
• the removal of the volatile constituents of the liquid medium takes place according to this embodiment (II) essentially without a prior depletion or even isolation of the desired product.
• the non-volatile metabolic product is substantially not removed along with the volatiles of the liquid medium, but remains with at least a portion, usually the majority and, in particular, the total of other solid components from the fermentation broth in the residue thus obtained.
• small amounts of the desired nonvolatile microbial metabolite usually a maximum of 20 wt .-%, z. B.
• the desired non-volatile microbial metabolite remains at least 90% by weight, in particular at least 95% by weight, especially 99% by weight and especially about 100% by weight, based in each case on the Total dry weight of the metabolic product, as a solid in admixture with the part obtained after removal of the volatiles or the whole of the solid components of the fermentation medium.
• the non-starchy solid constituents are separated from the fermentation broth, z. B. by centrifugation or filtration.
• such pre-separation will be performed to remove coarser solid particles containing no or low levels of nonvolatile microbial metabolite.
• conventional methods eg. B. using coarse mesh screens, nets, perforated plates, or the like.
• a separation of coarse solid particles can also take place in a centrifugal separator.
• the properties of the dried metabolite present together with the solid components of the fermentation can be targeted with respect to various parameters such as active ingredient content, particle size, particle shape, tendency to dust, hygroscopicity, stability, in particular bearings Stability, color, odor, flow behavior, tendency to agglomerate, electrostatic charging, light and temperature sensitivity, mechanical stability and redispersibility are packaged in a manner known per se.
• formulation auxiliaries such as carrier and coating materials, binders and other additives
• formulation auxiliaries include, for. As binders, support materials, powdering / flow aids, also color pigments, biocides, dispersants, antifoams, viscosity regulators, acids, alkalis, antioxidants dants, enzyme stabilizers, enzyme inhibitors, adsorbates, fats, fatty acids, oils or mixtures thereof.
• Such formulation auxiliaries are advantageously used in particular when using formulation and drying processes such as spray drying, fluid bed drying and freeze drying as drying aids.
• formulation and drying processes such as spray drying, fluid bed drying and freeze drying as drying aids.
• the proportion of the aforementioned additives and optionally other additives such as coating materials can vary greatly depending on the specific requirements of the respective metabolite and depending on the properties of the additives used and z. B. in the range of 0.1 to 80 wt .-% and in particular in the range of 1 to 30 wt .-%, each based on the total weight of the finished formulated product or mixture.
• formulation auxiliaries may take place before, during or after the work-up of the fermentation broth (also referred to as product formulation or solid design) and in particular during the drying.
• An addition of formulation auxiliaries prior to working up the fermentation broth or the metabolic product may be particularly advantageous in order to improve the processability of the substances or products to be processed.
• the formulation auxiliaries may be added both to the metabolic product obtained in solid form and to a solution or suspension containing it, e.g. B. after completion of fermentation directly to the fermentation broth or to a solution or suspension obtained in the course of the work-up before the final drying step.
• the adjuvants z. B. are mixed in a suspension of the microbial metabolite; Such a suspension can also be added to a carrier material, for. B. by spraying or mixing.
• a carrier material for. B. by spraying or mixing.
• formulation auxiliaries during drying can, for. B. play a role when a metabolic product containing solution or suspension is sprayed.
• addition of formulation auxiliaries z. B. when applying coatings or coatings / coating layers on dried particles. Both after drying and after a possible coating step further aids can be added to the product.
• the removal of the volatiles from the fermentation broth is carried out in a manner known per se by conventional methods for the separation of solid phases from liquid phases, including filtration processes and processes for volatilization of volatile constituents of the liquid phases. Such methods, which may also include steps for the coarse cleaning of recyclables and steps for packaging, be z.
• filtration processes and processes for volatilization of volatile constituents of the liquid phases Such methods, which may also include steps for the coarse cleaning of recyclables and steps for packaging, be z.
• the non-volatile microbial metabolite if it is present dissolved in the liquid phase, is converted from the liquid phase to the solid phase, eg. B. by crystallization or precipitation. Subsequently, a separation of the non-volatile solid components including the metabolite, z. B. by centrifugation, decantation or filtration. Similarly, one can also separate oily metabolites, wherein the respective oily fermentation products by the addition of adsorbents, eg. As silica, silica gels, clay, clay and activated carbon, converted into a solid form.
• adsorbents eg. As silica, silica gels, clay, clay and activated carbon
• the volatile constituents are removed by evaporation.
• the evaporation can be done in a conventional manner.
• suitable methods for volatilization of volatile components are spray drying, fluidized bed drying or agglomeration, freeze drying, current and contact dryers and extrusion drying.
• a combination of the aforementioned methods with molding methods such as extrusion, pelletizing or prilling can be made. In these latter methods, preferably partially or substantially predried metabolic product-containing mixtures are used.
• the devolatilization of the fermentation broth comprises a spray drying process or a fluidized bed drying process, including fluid bed granulation.
• the fermentation broth optionally after a preliminary separation to remove coarse solid particles containing no or only small amounts of nonvolatile microbial metabolite, fed to one or more spray or fluidized bed drying apparatus.
• the transport or the supply of solids-loaded fermentation broth is advantageously carried out by means of conventional transport devices for solids-containing liquids, eg. As pumps such as eccentric screw pumps (eg., The Fa. Delasco PCM) or high-pressure pumps (eg., The company LEWA Herbert Ott GmbH). Carrying out a fermentation using the sugar-containing liquid medium according to the invention can also be carried out in such a way that
• the solids content of the remaining portion of the medium M is preferably not more than 50% by weight, in particular not more than 30% by weight, more preferably not more than 10% by weight and very particularly preferably at most 5% by weight. In particular, in this case, it is preferable to separate all the solids before fermentation to produce the second metabolite (B).
• the compounds produced by such microorganisms in the separate fermentation are in particular among vitamins, cofactors and nutraceuticals, purine and pyrimidine bases, nucleosides and nucleotides, lipids, saturated and unsaturated fatty acids, aromatic compounds, proteins, carotenoids, especially with vitamins, Cofactors and nutraceuticals, proteins and carotenoids, and especially selected from riboflavin and calcium pantothenate.
• a preferred embodiment of this procedure relates to the parallel production of the same metabolites (A) and (B) in two separate fermentations. This is particularly advantageous if different purity requirements are to be set for different applications of the same metabolic product.
• the first metabolite (A), z. As a feed additive to be used amino acid, for. As lysine, methionine, threonine or glutamate, using the solids-containing fermentation broth and the same second metabolite (B), z.
• the same amino acid to be used as a food additive Noklare prepared using the according to viii) solids depleted fermentation broth.
• z. B. proceed as follows. It implements a preferably large-volume fermentation for the production of metabolites A, z.
• amino acids such as lysine, methionine, glutamate or threonine, citric acid or ethanol, eg. B. according to the methods described in WO 2005/116228 (PCT / EP2005 / 005728) or PCT / EP2006 / 066057 (earlier application DE 10 2005 042 541.0) or according to the methods known for the fermentative production of bioethanol.
• part of the medium M obtained after step iii) is removed.
• the part removed according to vii) can be prepared according to viii) by conventional methods, e.g. As centrifugation or filtration, completely or partially freed from the solids, depending on the requirements of the fermentation for the production of B.
• the thus obtained, optionally completely or partially freed from the solids medium M is according to viii) a fermentation for the production of a Met Touchpro - Duct B fed.
• a stream of solids separated off according to viii) is advantageously returned to the stream of medium M of the large-volume fermentation.
• the medium M produced after step iii) has concentrations of mono-, di- or oligosaccharides, as in the fermentative ethanol production (bioethanol) are common, for. B. in the range of 25 to 33 wt .-%.
• the carrying out of a separation of solids according to step viii) also depends here on the requirements of the fermentation for the production of the respective metabolite B.
• the metabolite B produced by the microorganisms in the fermentation is riboflavin.
• analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In WO 01/011052, DE 19840709, WO 98/29539, EP 1186664 and Fujioka, K .: New biotechnology for riboflavin (vitamin B2) and character of this riboflavin. Fragrance Journal (2003), 31 (3), 44-48.
• a preferably large-volume fermentation is implemented for the production of metabolic products A, z.
• amino acids such as lysine, methionine, glutamate or, of citric acid or of etha- nol, as described above.
• a part of the medium M obtained after step iii) is removed and according to viii) by conventional methods, for.
• the medium M obtained therefrom, which is essentially completely or partially freed from the solids is fed to a fermentation according to viii) for the production of the metabolite B, in this case riboflavin.
• the solid stream separated according to viii) is advantageously returned to the stream of medium M of the large-volume fermentation.
• the riboflavin-containing fermentation broth produced in this manner according to viii) can be worked up by analogous conditions and procedures as have been described for other carbon sources, e.g. In DE 4037441, EP 464582, EP 438767 and DE 3819745.
• the separation of the crystalline present riboflavin is preferably carried out by decantation. Other types of solids separation, e.g. As filtration, are also possible.
• the riboflavin is dried, preferably by means of spray and fluidized bed dryers.
• the riboflavin-containing fermentation mixture produced in accordance with viii) can be worked up according to analogous conditions and procedures, such as, for.
• the fermentation broth is centrifuged here and the residual solids-containing fraction is treated with a mineral acid.
• the riboflavin formed is filtered from the aqueous-acidic medium, optionally washed and then dried.
• the metabolic product B produced by the microorganisms in the fermentation is pantothenic acid.
• analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In WO 01/021772.
• the medium M pre-purified according to vii), preferably substantially freed from the solids, is fed to a fermentation according to viii) for the production of pantothenic acid.
• the reduced viscosity compared to the solid-containing liquid medium is particularly advantageous.
• the separated solids stream is preferably returned to the stream of the sugar-containing liquid medium of the large-volume fermentation.
• pantothenic acid-containing fermentation broth prepared according to viii) can be worked up by analogous conditions and procedures as described for other carbon sources, e.g. In EP 1050219 and WO 01/83799. After pasteurizing the entire fermentation broth, the remaining solids z. B. separated by centrifugation or filtration. The clear-running of the festival Material separation is partially evaporated, optionally mixed with calcium chloride and dried, in particular spray-dried.
• the separated solids can be obtained in the context of the parallel-operated, large-volume fermentation process together with the respective desired microbial metabolite (A).
• whole or ground cereal grains preferably corn, wheat, barley, millet, triticale and / or rye may be added to the product formulation or protein composition.
• the millables used in the following were produced as follows. Whole corn kernels were completely ground using a rotor mill. Using different Schlägerwerke, grinding tracks or sieve installations three different subtleties were achieved. A sieve analysis of the material to be ground using a laboratory vibrating sieve (vibratory analysis machine: Retsch Vibrotronic type VE1, sieving time 5 min, amplitude: 1.5 mm) gave the results listed in Table I.
• the pH is adjusted in a range between 5.6 and 5.8.
• the maize meal suspension is intensively mixed by stirring until use in the jet cooker. This suspension is then fed to the jet cooker at 250 kg / h at a pressure of 5 bar.
• the heating of the maize meal suspension beyond the gelatinization temperature to 105 ° C. is effected by parallel feeding of 25 kg / h of steam (7.5 bar).
• part of the gelatinized starch is degraded to dextrins (first liquefaction).
• the temperature of the reaction mixture is reduced by flashing to 90 ° C, with about 5 kg / h of steam go off.
• the second liquefaction is then carried out in a further tubular reactor over a period of 100 min to completely decompose the starch to dextrins.
• the resulting reaction mixture is then cooled to the saccharification temperature of 61 ° C. by reflashing under a water loss of about 14 kg / h.
• Part of the reaction mixture obtained from 11.1) was saccharified in a random test. For this purpose, about 1000 g of the reaction mixture were transferred to a stirred tank and kept at 61 ° C. with constant stirring. Stirring was continued throughout the experiment. After adjusting the pH to 4.3 with H 2 SO 4 , 17.9 g (15.2 ml) of Dextrozyme GA (Novozymes A / S) was added. The temperature was maintained for about 3 hours, with the progress of the reaction monitored by HPLC. At the end, the glucose concentration was 420 g / kg.
• a modified strain of Corynebacterium glutamicum was used, which was described under the name ATCC13032 lysC fbr in WO 05/059144.
• example 1 Liquefied and saccharified corn flour hydrolyzate prepared according to protocol Il was used in shake flask experiments using Corynebacterium glutamicum.
• the composition of the flask medium is shown in Table 2. The experiment was carried out in triplicate.
• the flasks were incubated at 30 ° C for 48 hours and agitated (200 rpm) in a humidified shaker cabinet.
• the content of glucose and lysine was determined by HPLC.
• HPLC analyzes were performed on Agilent 1100 Series LC equipment.
• the amino acid concentration was determined by high pressure liquid chromatography on an Agilent 1100 Series LC System HPLC. A pre-column derivatization with orthophthalaldehyde allows the quantification of the amino acids formed, the separation of the amino acid mixture takes place on a Hypersil AA column (Agilent).
• the composition of the flask medium is shown in Table 4. From each sample, two flasks were set up.
• the flasks were incubated at 34 ° C for 6 days and agitated (170 rpm) in a humidified shaker cabinet. After termination of the fermentation, the phytase activity with phytic acid as substrate and at a suitable phytase activity level (standard: 0.6 U / ml) in 250 mM acetic acid / sodium acetate / Tween 20 (0.1% by weight), pH 5, 5 buffers determined.
• the assay has been standardized for use in microtiter plates (MTP).

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