EP1957659A1 - Fermentative production of organic compounds - Google Patents

Abstract

The invention relates to a method for producing at least one organic compound comprising at least 3 C-atoms or at least 2 C-atoms and at least 1 N-atom by fermentation. Said method comprises the following steps: i) a starch source is ground in order to obtain a grinding material which contains at least one part of the non-starch containing solid components of the starch source; ii) the grinding material is suspended in an aqueous liquid in an amount such that the dry mass content in the suspension is at least 45 wt. %, iii) the starch component is hydrolysed in the grinding material by liquefying and optionally, subsequently, sweetening, in order to obtain an aqueous medium M, which contains the hydrolysed starch components and at least one part of the non-starch containing solid components of the starch source, is obtained; and iv) the aqueous substance M obtained in step iii) is used for fermentation in order to cultivate a micro-organism which is capable of over-producing the organic compound; the suspension obtained in step ii) is heated in step iii) by introducing vapour into the suspension, in order to obtain temperatures greater than the pasting temperature of the starch obtained in the grinding materials.

Description

Fermentative production of organic compounds

description

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. The range of microbial metabolites produced in this way, d. H. organic compounds, this includes z. B. low molecular weight volatile compounds such as ethanol, non-volatile metabolites such as amino acids, vitamins and carotenoids and a variety of other substances.

For such generally known microbial fermentation processes, different carbon sources are used, depending on the different process conditions. These range from pure sucrose via beet and cane molasses, so-called "high test molasses", to glucose from starch hydrolysates, and for the biotechnological production of L-lysine, acetic acid and ethanol are also cited as large-scale co-substrates (Pfefferle et al., Biotechnogical Manufacture of Lysines, Advances in Biochemical Engineering / Biotechnology, Vol. 79 (2003), 59-1 12).

Based on the carbon sources mentioned, various methods and procedures for the sugar-based, fermentative production of microbial metabolites are established. Using the example of L-lysine, these are described, for example, by Pfefferle et al. (supra) with regard to strain development, process development and large-scale production.

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. For this purpose, the starch from a natural source of starch such as potatoes, cassava, cereals, z. As 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.

In addition to the use of such pre-purified starch sources, the use of non-purified starch sources for the production of carbon sources for the fermentative production of microbial metabolites has also been described. Typically, the 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. B. the purification of liquefied and saccharified starch solutions (JP 57159500), or provide methods that should allow the production of fermentation media from renewable resources (EP 1205557).

By contrast, non-purified starch sources are widely used in the fermentative production of bioethanol. Here, 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. In this case, 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. In "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.

Since the desired product is obtained by distillation in the fermentative production of bioethanol, the use of starch sources from the dry-milling process in non-prepurified form is not a serious problem. When applying a dry-milling process for the production of other microbial metabolites is However, the introduced via the sugar solution in the fermentation solids flow problematic, since this can have a negative effect on the fermentation, z. B. with regard to the oxygen transfer rate or the oxygen demand of the microorganisms used (cf .. see Mersmann, A. et al .: Selection and Design of African Bioreactors, Chem. Eng. Technol. 13 (1990), 357-370) as well as the subsequent work-up can not aggravate significantly.

In addition, 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.

In the fermentative production of organic metabolites other than ethanol, it is in principle disadvantageous to supply the sugar-containing media with low sugar concentrations to the fermentation, since this leads to a disproportionate dilution of the fermentation broth and consequently reduces the achievable final concentration of the desired products, which on the one hand increases costs causing their recovery from the fermentation medium and decreases the space-time yield. These considerations are particularly relevant in the case when a starch hydrolyzate prepared for high volume bioethanol production, which conventionally has low sugar or glucose concentrations of up to about 30 or 33% by weight, partially undergoes a minor volumetric minor fermentation to produce others Chemicals to be supplied.

Because of the difficulties and limitations presented, dry-milling processes, such as are widely used for bioethanol production, have hitherto remained of no significant economic importance in the fermentative production of other microbial metabolites than ethanol.

Attempts to transfer the dry-milling concept and the principal advantages associated with this method to the large-scale production of microbial

Metabolites have hitherto only been described using cassava as a source of starch. Thus, 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 μm. 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.

It was therefore an object of the present invention to provide a further process for the production of organic compounds by fermentation, which requires no or at least no complete prior separation of the non-starchy solid constituents contained in the starch source. In particular, the process should allow for continuous hydrolysis of the starch component of the starch source. In addition, it should be characterized by easy handling of the media used and their ease of use in fermentation. In particular, the process should allow the use of cereals as a source of starch.

It has surprisingly been found that a fermentative process for preparing organic compounds can be carried out efficiently despite the inherently high solids input, if the sugar required for fermentation is provided in the form of an aqueous medium which is obtainable by

i) grinding a starch source to obtain a millbase containing at least one

Contains part of the non-starchy solid components of the starch source;

ii) suspending the millbase in an aqueous liquid in an amount such that a dry matter content in the suspension of at least 45% by weight results,

iii) 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:

iii) 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 a portion of the non-starchy solid constituents of the starch source; and

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;

wherein in step iii) the suspension obtained in step ii) by introducing

Steam is heated in the suspension to temperatures above the gelatinization temperature of the starch contained in the millbase.

Despite the high dry matter content in the suspension used for the hydrolysis, the hydrolysis can be carried out without difficulty in the manner according to the invention, and correspondingly high concentrations of metabolizable sugars are obtained. It surprisingly does not matter whether the sugar is present after hydrolysis in the form of mono- or disaccharides or in the form of oligosaccharides (= dextrins). The high content of solid, non-starchy components of the starch source in the resulting medium surprisingly does not interfere with the fermentation. In addition, by the method according to the invention viscosity problems, as they can occur during liquefaction of the starch source at higher concentrations of ground material, largely avoided. Due to the associated high dry matter content high concentration of metabolizable sugars in the fermentation medium, 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. Of course, the medium M obtainable according to the invention is suitable as a sugar source in the batch phase of the fermentation. The terms "starch content" and "starch component" are used interchangeably here and below.

With respect to the aqueous medium M obtained in step iii), the terms "aqueous medium", "liquid medium" and "aqueous sugar-containing liquid" are used synonymously.

The term "liquefaction" as used herein means the hydrolytic degradation of starch into oligosaccharides, especially dextrins.

The terms "saccharification" or "saccharification" here and in the following mean the hydrolysis of dextrins to monosaccharides, in particular to monosaccharides such as glucose. By a "saccharifying enzyme" is thus understood an enzyme which hydrolyzes dextrins to monosaccharides.

The term "dextrin" is understood here and below as meaning 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.

The terms "content of glucose equivalents" and "sugar concentration" refers to the total concentration of mono-, di- and oligosaccharides in the medium potentially available for fermentation. The term "glucose equivalents" also includes the metabolizable sugars or sugar units other than glucose.

The terms "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. Preferably, the starch source is selected from cereals and especially among corn, rye, triticale and wheat grains. In principle, 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.

For the preparation of the sugar-containing liquid medium in step i) the respective starch source with or without the addition of liquid, for. As water, ground, preferably without the addition of liquid. It can also be a dry grinding combined with a subsequent wet grinding.

For dry grinding, hammer mills, rotor mills or roll mills are typically used; For wet grinding, agitating mixers, stirred ball mills, circulation mills, disk mills, annular chamber mills, vibrating mills or planetary mills are suitable. In principle, 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.

By grinding, a suitable for the subsequent process steps grain size is set. It has proved to be advantageous if the grinding stock obtained during grinding, in particular in dry grinding, in step i) flour particles, d. H. particulate components having a particle size in the range of 100 to 630 microns in a proportion of 30 to 100 wt .-%, preferably 40 to 95 wt .-% and particularly preferably 50 to 90 wt .-%. Preferably, the millbase obtained contains 50 wt .-% of flour particles having a particle size of more than 100 microns. As a rule, 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. For cereal flours, 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. In the case of a low degree of milling, 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.

According to the invention, 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. Based on the starchy constituents of the millbase (and thus on the amount of metabolizable sugar in the medium M), 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 .-%.

Subsequently, 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. This process is often referred to as mashing.

In general, 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.

In principle, it is possible to use 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.

To carry out the hydrolysis, 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. Preferably, the vapor is introduced with overpressure into the suspension. Accordingly, 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. By introducing the vapor, the suspension heats up instantly to temperatures above 90 ° C, ie to temperatures above the gelatinization temperature.

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. By injecting the steam with overpressure, the suspension is not only heated but also mechanical energy is introduced into the system, which promotes a further division of Mahlgutpartikel, causes a particularly uniform energy input, and thus a particularly uniform gelatinization of the granular starch particles in the millbase Episode has. Typically, these devices have a tubular geometry. Preferably, 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. In this nozzle, a needle or arranged on a longitudinally displaceable rod cone is arranged. Needle or cone forms a gap with the cone of the nozzle. By moving the needle or the rod in the longitudinal direction, the size of the gap and thus the cross-sectional area of the nozzle opening can be adjusted in a simple manner, whereby one can regulate the speed of the steam input in a simple manner.

Typically, 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.

In continuous reaction, the steam-treated suspension is usually subsequently transferred to a post-reaction zone in order to continue the gelling of the starch components. 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. In one embodiment, 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. In another embodiment of the invention, the post-reaction zone has a tubular geometry.

After leaving the post-reaction zone, the suspension is usually relaxed and then performs a liquefaction. Preferably, 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. As a rule, 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. As a rule, the liquefaction in step ii) takes place in the presence of at least one starch-liquefying enzyme, which is generally selected from α-amylases. Other active and stable starch-liquefying enzymes under the reaction conditions can also be used.

In principle, all starch-liquefying enzymes, in particular α-amylases (enzyme class EC 3.2.1.1) can be used to liquefy the starch content in the millbase, for example α-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 α-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 α-amylases for liquefaction.

Advantageously, the amounts of starch-liquefying enzyme, in particular α-amylase, are chosen so that rapid and complete degradation of the starch to oligosaccharides is achieved. The total amount of starch-liquefying enzyme, in particular α-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 α-amylase (resp. the starch-liquefying enzyme used) can be introduced into the reaction vessel or added during the liquefaction step.

For an optimal effect of the α-amylase (or of the starch-liquefying enzyme used), 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.

To stabilize the enzymes used, if appropriate, 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 α-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.

For complete degradation of the starch to dextrins, 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. Optionally, one or more additional aliquots of α-amylase, z. B. in the range of 0.001 to 0.5 wt .-% and preferably 0.002 to 0.2 wt .-%, based on the total amount of the starch source used, are added to the reaction mixture.

In a preferred embodiment of the invention, 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. In this way, an aqueous starch hydrolyzate is obtained, 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. For this, the aqueous product obtained after liquefaction is treated with an enzyme causing the saccharification, typically a glucoamylase, under the conditions customary for this purpose.

For the saccharification of the dextrins (ie oligosaccharides) it is possible in principle to use all glucoamylases (enzyme class EC 3.2.1.3), in particular 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.

In general, 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. Preferably, the aqueous liquefaction product will first be adjusted to these temperatures and then mixed with the enzyme which causes the saccharification. Advantageously, prior to the addition of 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. After addition of the saccharifying enzyme, 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.

Since a ground stock is used for the preparation of the aqueous hydrolyzate which contains substantially all constituents of the starch source or at least in addition to the starch also a part of the solid non-starchy constituents (ie, there is no complete separation of the non-starchy solid constituents of the starch source) , 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. 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. 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.

To increase the overall sugar yield or to obtain free amino acids, further 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.

In another embodiment of the invention, one will make no or only partial saccharification before fermentation. The saccharification then takes place at least partly during the fermentation, ie in situ. For example, one can proceed in such a way that one part of the amount contained in the liquid medium Dextrins, e.g. B. in the range of 10 to 90 wt .-% and in particular in the range of 20 to 80 wt .-%, based on the total weight of the dextrins (or the original starch), saccharified and the resulting sugar-containing medium used in the fermentation. 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. In the latter case, it is particularly advantageous that during fermentation the speed of saccharification, in particular of glucose release, is automatically adjusted to the requirements of the microorganisms on the one hand by the amount of biomass and on the other hand by the expression level of the strain's own saccharifying enzymes.

Advantages of in situ saccharification are on the one hand reduced investment costs, on the other hand, a higher glucose concentration in the batch (batch) may optionally be presented by a delayed release of glucose, without any inhibition or metabolism change of the microorganisms used. In E. coli leads to a high glucose concentration 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. Accordingly, 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.

Depending on the process procedure, 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. As 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. Typically, 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. For this purpose, 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.

In general, 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. For this purpose, 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. For this purpose, the entire medium M of lysis or killing is supplied. This can, for. B. thermally or chemically. However, in the context of the method according to the invention, it has not proved necessary to carry out a sterilization step as described hereinbefore fermentation, but rather it has proven advantageous to perform no such sterilization step. Accordingly, 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. For this purpose, 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.

For example, 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 Nährmedienbestandteile 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. Here, the sugar contained in the fermentation medium is metabolized and the desired metabolite is formed (so-called batch process or batch phase).

In the fed-batch mode, after the batch phase, z. B. if the

Total sugar concentration has fallen below a certain value, 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

vi) adding the medium M to the fermentation medium F, in which the hydrolyzed starch components contained in the medium M, d. H. the sugars by which the organic compound overproducing microorganisms are metabolized to form the organic compound.

In step v), for example, a conventional sugar-containing medium, usually a glucose solution, can be adjusted to 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. In this case, 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. in the range of> 0 to 5 wt .-%, calculated as glucose equivalents and based on the total weight of the fermentation medium F is. 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. In this case, the sugar introduced in the fermentation medium F is metabolized and the desired metabolite is formed.

By adding according to step vi) of the aqueous medium M to the fermentation medium F, the fermentation process is maintained and the metabolite overproduced by the microorganism accumulates in the fermentation broth. Here, the volume ratio of fed medium M to the present and containing the microorganisms batch medium (fermentation medium F) 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. In particular, via the feed rate of the sugar-containing liquid medium, the sugar content in the fermentation broth can be regulated. As a rule, 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.

In a preferred embodiment, the fermentation medium F in step v) (ie here the batch medium) 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. For this purpose, 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.

Preferably, one will proceed so that 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.

It is understood that according to the invention in the batch phase and / or in the fed batch phase, 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. In one embodiment of the invention, 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.

By the method according to the invention, 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).

However, it is also possible to use the 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.

The term 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. Gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid; Diols having preferably 3 to 8 C atoms, for. For example, propanediol and butanediol; polyhydric (also referred to as higher) alcohols with 3 or more, eg. B. 3, 4, 5 or 6 OH groups, for. Glycerol, sorbitol, mannitol, xylitol and arabinitol; long-chain (also referred to as longer-chain) alcohols having at least 4 carbon atoms, eg. B. with 4 to 22 carbon atoms, z. B. butanol; Carbohydrates, eg. Hyaluronic acid and trehalose; aromatic compounds, e.g. Aromatic amines, vanillin and indigo; Vitamins and provitamins, eg. As ascorbic acid, vitamin B ₆ , vitamin B ₁₂ and riboflavin, cofactors and so-called nutraceuticals; Proteins, e.g. 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.

The term "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.

In particular, 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.

It is obvious to a person skilled in the art that the compounds produced in this way by fermentation are in each case obtained in the enantiomeric form produced by the microorganisms used (if different enantiomers exist). So you get z. B. of the amino acids usually the respective L-enantiomer.

The 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.

Table A:

In preferred embodiments of the invention, 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.

Further preferred embodiments of the invention relate to the use of the sugar-containing liquid medium obtainable according to the invention in a fermentative production of

- 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.

In a further particularly preferred embodiment, 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. As well as 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. To carry out the fermentation, 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.

In a preferred embodiment, 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

3-hydroxybutyric acid;

Ketones with 3 to 10 carbon atoms, such as acetone;

Alkanols having 4 to 10 C atoms, such as butanol; and alkandiols with 3 to 8

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 delbrückii, Lactobacillus leichmannii, Propionibacterium arabinosum, Propionibacterium schermanii, Propionibacterium freudenreichii, Clostridium propionicum, Clostridium formicoaceticum, Clostridium acetobutylicum, Rhizopus arrhizus and Rhizopus oryzae.

In a preferred embodiment, the microorganism used in the fermentation is a strain of the genus Corynebacterium, in particular a strain of Corynebacterium glutamicum. In particular, it is a strain of the genus Corynebacterium, especially of the Corynebacterium glutamicum, which overproduces an amino acid, especially lysine, methionine or glutamate.

In a further preferred embodiment, the microorganism used in the fermentation is a strain of the genus Escherichia, in particular a strain of Escherichia coli. In particular, it is a strain of the genus Escherichia, especially of Escherichia coli, which overproduces an amino acid, especially lysine, methionine or threonine.

In a specific preferred embodiment, the metabolite produced by the microorganisms in the fermentation is lysine. To carry out the fermentation 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. In principle, both a continuous and a batch (batch or fed-batch) mode of operation into consideration, preferred is the fed-batch mode of operation. In a further particularly preferred embodiment, the metabolic product produced by the microorganisms in the fermentation is methionine. To carry out the fermentation, analogous conditions and procedures as described for other carbon sources can be used here, eg. In WO 03/087386 and WO 03/100072.

In a further particularly preferred embodiment, the metabolic product produced by the microorganisms in the fermentation is tartaric acid. To carry out the fermentation analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In WO 01/021772.

In a further particularly preferred embodiment, the metabolite produced by the microorganisms in the fermentation is 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.

In a further particularly preferred embodiment, the metabolic product produced by the microorganisms in the fermentation is fumaric acid. To carry out the fermentation, 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.

In a further particularly preferred embodiment, the metabolite produced by the microorganisms in the fermentation is succinic acid. To carry out the fermentation analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In Int. J. Syst. Bacteriol. 26, 498-504 (1976); EP 249773 (1987), invent .: Lemme et al. Datta; US 5504004 (1996), invent .: Guettler, Jain et al. Soni; Arch. Microbiol. 167, 332-342 (1997); Guettler MV, Rumler D, Jain MK., Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. Int J Syst Bacteriol. 1999 Jan; 49 Pt 1: 207-16; US 5,723,322, US 5,573,931, US 5,521,075, WO 99/06532, US 5,869,301 or US 5,770,435.

In a further particularly preferred embodiment, the metabolic product produced by the microorganisms in the fermentation is a phytase. To carry out the fermentation here analogous conditions and Procedures described for other carbon sources, e.g. In WO 98/55599.

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. As fibers and unused sugar, and unused buffer and nutrient salts contains. 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.

Before isolation or depletion of a microbial metabolite or separation of the volatile constituents of the fermentation broth, 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

WO 2005/116228 (PCT / EP2005 / 005728) of the Applicant, to which reference is made for further details.

The isolation or depletion of the metabolite from the fermentation broth, d. H. 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. Both for the isolation of recyclable materials and for the separation of solids, ie 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).

To isolate 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. Alternatively, the desired product can also be isolated directly from the fermentation broth, z. Example by using chromatographic methods or extraction method. As a chromatographic method, in particular ion exchange chromatography should be mentioned, in which the desired product can be selectively isolated on the chromatography column. In this case, the separation of the solids from the remaining fermentation broth is advantageously carried out z. B. by decantation, evaporation and / or drying.

In the case of volatile or oily compounds, a control of the maximum temperatures during workup, in particular during drying, is generally required. Advantageously, 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

(PCT / EP2005 / 005728) of the applicant. Examples of compounds which can be advantageously prepared in this way are γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, furthermore propionic acid, lactic acid, propanediol, butanol and acetone. For the purposes of the present invention, 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. By far, it means that after removal of the volatile constituents, a solid or at least semi-solid residue remains, which can optionally be converted by the addition of solids in a solid product. As a rule, this means removing the volatile constituents up to a residual moisture content of not more than 30% by weight, frequently not more than 20% by weight and in particular not more than 15% by weight. In general, 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. In general, at least one, in particular the final stage for obtaining the metabolite in solid form will comprise a drying step.

In the one-step procedure, 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.

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. In one or more subsequent stages, 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. Thus, upon removal of the fermentation broth volatiles, 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. Accordingly, but also - preferential example, small amounts of the desired nonvolatile microbial metabolite, usually a maximum of 20 wt .-%, z. B. 0.1 to 20 wt .-%, preferably not more than 10, in particular not more than 5 wt .-%, more preferably at most 2.5 wt .-% and most preferably at most 1 wt .-%, based to the total dry weight of the metabolite, removing the volatile constituents of the fermentation broth together with them. In a very particularly preferred embodiment, 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.

If desired, prior to removing the volatiles, a part, e.g. B. 5 to 80 wt .-% and in particular 30 to 70 wt .-%, the non-starchy solid constituents are separated from the fermentation broth, z. B. by centrifugation or filtration. Optionally, such pre-separation will be performed to remove coarser solid particles containing no or low levels of nonvolatile microbial metabolite. For prefiltration, known in the art, conventional methods, eg. B. using coarse mesh screens, nets, perforated plates, or the like. Optionally, a separation of coarse solid particles can also take place in a centrifugal separator. The apparatuses used here, such as decanters, centrifuges, sedanizers and separators, are likewise known to the person skilled in the art. In this way, a solid or semisolid, for. Pasty residue containing the nonvolatile metabolite and nonvolatile, usually solid non-starchy constituents of the starch source, or at least large portions thereof, often at least 90% by weight or the total amount of solid non-starchy ingredients.

By adding formulation auxiliaries such as carrier and coating materials, binders and other additives, 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.

The commonly used 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. For further details, reference is made to PCT / EP2006 / 066057 (earlier application DE 10 2005 042 541.0).

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.

The addition of 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.

Thus, 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. The addition of formulation auxiliaries during drying can, for. B. play a role when a metabolic product containing solution or suspension is sprayed. In particular after drying, 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. 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. In the context of the product formulation or the workup after completion of fermentation applicable methods known to those skilled in the art, apparatuses, auxiliaries or general and specific embodiments are further described in EP 1038 527, EP 0648 076, EP 835613, EP 0219 276, EP 0394 022, EP 0547 422, EP 1088 486, WO 98/55599, EP 0758 018 and WO 92/12645.

In a first variant of this embodiment (II), 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.

In a second variant of this embodiment (II), the volatile constituents are removed by evaporation. The evaporation can be done in a conventional manner. Examples of 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.

In a preferred embodiment, the devolatilization of the fermentation broth comprises a spray drying process or a fluidized bed drying process, including fluid bed granulation. For this purpose, 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

vii) the medium M obtained in step iii) containing non-starch-containing solid component of the starch source, a subset of not more than 50 wt .-%, z. B. in the range of 5 to 45 wt .-%, based on the total weight, and the remaining amount of a fermentation for the preparation of a first metabolite (A), z. A nonvolatile metabolite (A) in solid form or a volatile metabolite (A); and

viii) this subset, optionally after prior complete or partial separation of the non-starchy solid constituents of the starch source, a fermentation for producing a second metabolite (B), which is identical or different from the metabolite product (A).

In the case of separation of the non-starchy solid constituents according to vii), 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).

This procedure makes it possible to use in the separate fermentation according to vii) microorganisms for which certain minimum requirements must be satisfied, eg. B. with respect to the oxygen transfer rate. For such microorganisms used in the separate fermentation according to viii) come z. B. Bacillus species, preferably Bacillus subtilis into consideration. 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. Accordingly, 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. For example, the same amino acid to be used as a food additive. Nosäure prepared using the according to viii) solids depleted fermentation broth. By completely or partially separating the non-starchy solid constituents, the cleaning effort in the workup of the metabolite, the scope of which requires a higher purity, for. As a food additive, can be reduced.

In a further preferred embodiment of this procedure, z. B. proceed as follows. It implements a preferably large-volume fermentation for the production of metabolites A, z. As of 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. According to vii), 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 Metwechselpro - 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.

If the microbial metabolite (A) prepared in the large-volume fermentation is ethanol, then 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.

In a preferred embodiment of the procedure described above, the metabolite B produced by the microorganisms in the fermentation is 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.

To carry out this variant of the process, a preferably large-volume fermentation is implemented for the production of metabolic products A, z. Of amino acids such as lysine, methionine, glutamate or, of citric acid or of etha- nol, as described above. According to vii) a part of the medium M obtained after step iii) is removed and according to viii) by conventional methods, for. As centrifugation or filtration, completely or partially freed from the solids. 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. After lysis of the cell mass, 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. Subsequently, the riboflavin is dried, preferably by means of spray and fluidized bed dryers. Alternatively, the riboflavin-containing fermentation mixture produced in accordance with viii) can be worked up according to analogous conditions and procedures, such as, for. As described in EP 1048668 and EP 730034. After pasteurization, 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.

In a further preferred embodiment of this procedure, the metabolic product B produced by the microorganisms in the fermentation is pantothenic acid. To carry out the fermentation analogous conditions and procedures can be applied here, as have been described for other carbon sources, eg. In WO 01/021772.

To carry out this variant of the method can, for. B. as described above for riboflavin. 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. In this case, 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.

The 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).

After drying and / or formulation or formulation, 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 following examples are given to illustrate certain aspects of the present invention, but are not to be construed as limiting in any way.

Examples

I. Grinding the starch source

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.

Table I

II. Enzymatic starch liquefaction and saccharification

11.1) liquefaction in the jet cooker For the continuous liquefaction of a dry-milled cornmeal, two stirred tanks of 250 L volume are installed, from which the cornmeal, mashed in water, is fed to the jet cooker, alternately at hourly intervals. A typical approach for these boilers is by submitting 1 17 kg of water to which an α-amylase, e.g. B. Termamyl SC, in a concentration of 0.10 wt .-% (based on the amount of flour) is added. Subsequently, 133 kg maize flour are added and mixed in at approx. 45 ° C in several steps. After setting a Ca ²⁺ concentration of 50 ppm, z. B. by addition of CaCl ₂ , the pH is adjusted in a range between 5.6 and 5.8. After addition of all components, 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). In a tube reactor downstream of the jet cooker with a holding time of 5 minutes, part of the gelatinized starch is degraded to dextrins (first liquefaction). Subsequently, the temperature of the reaction mixture is reduced by flashing to 90 ° C, with about 5 kg / h of steam go off. At 90 ° C, 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.

11.1) saccharification

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.

IN THE. Strain ATCC 13032 lysC ^fbr

In some of the following examples, 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.

tribe

It became the modified wild-type with feedback-deregulated aspartokinase

ATCC13032 lysC ^fbr used.

Production of the inoculum

Cells were incubated at 30 ° C overnight after streaking on sterile CM + CaAc agar (composition: see Table 1, 20 min at 121 ° C). The cells were then scraped off the plates and resuspended in saline. 25 ml of the medium (see Table 2) in 250 ml Erlenmeyer flasks with two baffles were each inoculated with such an amount of the cell suspension thus prepared that the optical density reached a value of OD ₆ io = 0.5 at 610 nm.

Table 1: Composition of the CM + CaAc agar plates

Production of the fermentation broth

The composition of the flask medium is shown in Table 2. The experiment was carried out in triplicate.

Table 2: Piston media

^* adjusted with dilute aqueous NaOH solution

After inoculation, the flasks were incubated at 30 ° C for 48 hours and agitated (200 rpm) in a humidified shaker cabinet. After the termination of the fermentation, 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).

Example 2:

Liquefied and saccharified corn flour hydrolyzate prepared according to Regulation II was used in shake flask experiments using Aspergillus niger.

tribe An Aspergillus niger phytase production strain with 6 copies of the phyA gene from Aspergillus ficuum under the control of the glaA promoter was generated analogously to the preparation of NP505-7 described in detail in WO 98/46772. The control used was a strain with 3 modified glaA ampicons (analogous to ISO505) but without integrated phyA expression cassettes.

Production of the inoculum

20 ml of the preculture medium (see Table 3) in 100 ml Erlenmeyer flasks with a chicane are in each case inoculated with 100 μl of a freezing culture and incubated for 24 h at 34 ° C. with agitation (170 rpm) in a humidified shaker cabinet.

Table 3: Composition of the preculture medium

^* adjusted with dilute sulfuric acid

50 ml of the main culture medium (see Table 4) in 250 ml Erlenmeyer flasks with a baffle are each inoculated with 5 ml preculture

Production of the fermentation broth

The composition of the flask medium is shown in Table 4. From each sample, two flasks were set up.

Table 4: Flask media

Adjust with dilute sulfuric acid

After inoculation, 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). 10 μl of the enzyme solution were mixed with 140 μl of 6.49 mM phytate solution in 250 mM sodium acetate buffer, pH 5.5 (Phytate: dodecasodium salt of phytic acid). After one hour of incubation at 37 ° C, the reaction was stopped by adding the same volume (150 μl) of trichloroacetic acid. An aliquot of this mixture (20 μl) was transferred to 280 μl of a solution containing 0.32 NH ₂ SO ₄ , 0.27% by weight ammonium molybdate, and 1.08% by weight ascorbic acid. This was followed by a 25-minute incubation at 50.degree. The absorbance of the blue colored solution was measured at 820 nm.

Claims

claims

1. 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 the following steps:

i) grinding a starch source to obtain a millbase containing at least a portion of the non-starchy solid components of the starch source;

ii) suspending the millbase in an aqueous liquid in an amount such that a dry matter content in the suspension of at least 45% by weight results,

iii) 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 a portion of the non-starchy solid constituents of the starch source; and

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;

wherein, in step iii), the suspension obtained in step ii) is heated by introducing steam into the suspension at temperatures above the tabletting temperature of the starch contained in the millbase.

2. A process according to claim 1, wherein the heating is carried out with steam in a jet cooker.

3. The method according to any one of the preceding claims, wherein cooling the heated suspension of the ground material by flash evaporation to temperatures below the gelatinization temperature and then the liquefaction of the starch ke in the presence of a starch-liquefying enzyme.

4. The method according to any one of claims 1 or 2, wherein the suspension is added at least one strength-liquefying enzyme before heating.

A method according to any preceding claim, wherein the hydrolysis of the starch comprises a saccharification step.

6. Method according to one of the preceding claims, additionally comprising the following steps:

v) culturing the microorganism capable of overproduction of the organic compound in an aqueous, metabolizable sugar-containing fermentation medium F; and

vi) adding the aqueous medium M to the fermentation medium F, wherein the hydrolyzed starch constituents 10 contained in the aqueous medium M are metabolized by the microorganisms to form the organic compound.

7. The method according to claim 6, wherein the fermentation medium F in step v) substantially the aqueous medium M, which is used for overproduction of organic

15 compound competent microorganisms, nutrient salts, conventional excipients and

Water for dilution includes.

8. The method according to any one of the preceding claims, characterized in that the millbase used in step ii) at least 20% of the starch source

20 contained, solid, non-starchy components of the starch source.

9. The method according to any one of the preceding claims, characterized in that one uses grain grains as the starch source.

10. The method according to any one of the preceding claims, wherein the starch-liquefying enzyme is an α-amylase.

1 1. A method according to any one of the preceding claims, characterized in that the organic compound prepared under optionally hydroxyl groups

30 carrying mono-, di- and tricarboxylic acids having 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, long-chain alcohols having at least 4 C atoms, carbohydrates, aromatic

Compounds, vitamins, provitamins, cofactors, nutraceuticals, proteins, carotenoids, ketones of 3 to 10 carbon atoms, lactones, biopolymers and cyclodextrins are selected.

12. The method according to any one of the preceding claims, wherein the microorganism used for fermentation 40 is selected from natural or recombinant microorganisms containing at least one of the following metabolites overproduce: 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 -Atomen, alkanediols having 3 to 8 carbon atoms and polyhydroxyalkanoates.

The method of claim 12, wherein the microorganism is selected from those that overproduce one or more amino acids.

14. The method of claim 12, wherein the microorganism is selected from those that overproduce one or more aliphatic mono- and dicarboxylic acids having 3 to 10 carbon atoms.

The method of claim 12, wherein the microorganism is selected from those that overproduce one or more enzymes.

The method of claim 15, wherein the microorganism is selected from those that overproduce a phytase.

17. The method according to any one of the preceding claims, wherein the microorganisms are selected from the genera Corynebacterium, Bacillus, Ashbya, Escherichia, Aspergillus, Alcaligenes, Actinobacillus, Anaerobiospirillum, Lactobacillus, Propionibacterium, Clostridium and Rhizopus.

18. A method according to claim 17, wherein the microorganism is selected from strains of the genus Corynebacterium.

19. The method according to any one of the preceding claims, wherein depleted at least one microbial metabolite from the fermentation broth or i solves and then largely removed the volatile components of the fermentation broth, to obtain a solid or semi-solid protein composition.

A process according to any one of claims 1 to 18, wherein the volatile constituents of the fermentation broth are at least partially removed without prior isolation or depletion of a non-volatile microbial metabolite, and optionally without prior separation of solid constituents to obtain a solid formulation of a nonvolatile microbial metabolite ,