WO2011098843A2

WO2011098843A2 - Lactic acid production from starch-based materials by amylolytic lactic acid bacteria

Info

Publication number: WO2011098843A2
Application number: PCT/HR2011/000001
Authority: WO
Inventors: Anita Slavica; Srdan Novak; Antonija Trontel; Božidar ŠANTEK
Original assignee: Sveučilište u Zagrebu
Priority date: 2010-02-10
Filing date: 2011-01-07
Publication date: 2011-08-18
Also published as: WO2011098843A3

Abstract

The Invention pertains to a procedure for producing lactic acid or its salts. The procedure describes a simultaneously conducted saccharification of starch from a starch material and fermentation of sugars thereof into lactic acid by selected bacterium that produce amylolytic enzymes, which eliminates the need for the use of commercial enzymes as normally practiced in prior art.

Description

Lactic acid production from starch-based materials

by amylolytic lactic acid bacteria

Invention description

According to the International Patent Classification, the present Invention belongs to Class C012P7/56 or Subclass C12R.

This Invention pertains to a process for the production of lactic acid or salts thereof. The process describes simultaneous saccharification of starch from starch-based materials and the fermentation of sugars thereof to lactic acid by a selected bacterial strain that produces amylolytic enzymes, which thus makes the hitherto established practice of utilizing commercial enzymes superfluous.

In bioprocesses various species of microorganisms are used to obtain many different products of commercial interest. Bioprocesses are used in the production of rather simple compounds such as alcohols (ethanol, butanol), organic acids (citric, itaconic, gluconic, lactic acids) and amino acids (glutamic acid, lysine), and also in the production of more complex compounds such as antibiotics (penicillin, tetracycline), enzymes, vitamins (riboflavin, B 12, β- carotene) and hormones, so in such processes the bioprocess production successfully competes with the chemical synthesis. The production of lactic acid by sugar fermentation by lactic acid bacteria is a long known process.

The term lactic acid bacteria (LAB) means a specific group of similar non-sporogeneous Gram-positive bacteria that produce lactic acid as a final product of metabolism. Most of these bacteria can ferment only simple sugars (mainly some mono- and di-hexoses) and are unable to ferment starch. The existing processes of lactic acid production from starch as basic material require some extra processes of starch degradation into fermentable sugars. These starch degradation processes precede fermentation and represent a significant item in the overall process costs. Described in prior art are also some attempts to combine the starch degradation process (so-called saccharification) and the fermentation process within a single production phase, i.e., in the same reaction vessel, so these processes are labeled SSF (abbrev. of Simultaneous Saccharification and Fermentation).

The term fermentation means a process of microbial conversion of carbon sources via different metabolic pathways in the cells of various microorganisms to a certain product in anaerobic or microaerophilic conditions, therefore, virtually without oxygen. For an unimpeded course of fermentation it is necessary to supply these microorganisms with all needed nutritive substances. Such substances include a carbon source, as well as the sources of nitrogen, phosphorus, and other biogenic elements, plus the growth factors.

The term carbon source means organic matter that is degraded by microorganisms for metabolic energy acquisition, where the products of degradation are used for the synthesis of cellular material, i.e., for cell growth and reproduction. In the lactic acid fermentation the carbon source are fermentable sugars that the LAB degrade into lactic acid through a series of biochemical reactions serving for metabolic energy acquisition, where lactic acid accumulates in the cell's environment as a product of fermentation.

The term lactic acid means 2-hydroxypropane acid or its salt, a lactate, e.g., calcium lactate or sodium lactate. In the lactic acid production the alkaline or earth alkaline salts of lactic acid are preferred. Lactic acid has one chiral carbon atom and exists in two enantiomeric forms, D- and L-lactic acid. The term 'lactic acid' is used for both enantiomers, as well as for the (racemic) mixture of these two lactic acid enantiomers.

The term nutrient medium means a solution or suspension consisting of a carbon source and different nutritive substances, growth factors and other ingredients needed for the growth and activity of the microorganism that conducts fermentation. The term fermentation medium means a medium after a completed microbial process. A composition of fermentation medium has changed and, among others, it contains a product of fermentation, such as lactic acid or lactate.

The term nutritive substances means specific basic ingredients of media required for the growth and activity of microorganisms, such as the sources of nitrogen, phosphorus, other biogenic elements, mineral substances, etc.

The term growth factors means certain medium ingredients (e g., amino acids, purine and pyrimidine nucleotides, vitamins) which are added to the medium, because the microorganism cannot synthesize them or synthesize them in a quantity insufficient for conducting of specific metabolic reactions. Added growth factors enable or stimulate the growth and metabolic activity of specific microorganisms.

The well known microbial processes are dominated by liquid-phase media. In some fermentations solid or semi-solid state media are used, hence such processes are referred to as semi-solid state fermentations.

The term semi-solid state fermentation (sssf) means the microbial fermentation process of wet semi-solid-gelatinous substrate in a system with very little or without free-circulating water (less than 10% of free-circulating water).

If starch or some other starch-based material (grits and/or flour of maize, wheat, rye, barley, rice etc.) is used in the lactic acid production as a carbon source, it is necessary before fermentation to degrade this complex carbohydrate into fermentable sugars (mostly glucose and/or maltose) that the LAB will then be able to ferment into lactic acid.

The term starch means a mixture of two polymeric carbohydrates composed of glucose units linked by a- 1,4- and a- 1 ,6- glycosidic bonds. The term amylase means polymeric carbohydrate, one of the two parts of starch molecules, which is consisted of glucose chains linked by <x-l,4-glycosidic bonds. The term amylopectin means polymeric carbohydrate, another part of starch molecules, which is consisted of branched glucose chains linked by a- 1,4- and a- 1,6- glycosidic bonds.

The term starch-based material means renewable (plant) material which is used for starch production or which can also be used directly in the preparation of media. The starch-based material used for starch production is first cleaned and/or washed, dipped then milled and mashed. Mashing of milled material includes heating, gelatinization, liquefaction and saccharification of the mash.

The term starch-based material milling means the preparation of starch-based raw material for starch extraction from starch grains. Starch grains have heavily degradable hemicellulose and protein membranes. During milling the granular starch-based materials are soaked in water (wet milling).

The term mashing means the process of mixing of the milled starch- based materials with water at a proper ratio and making the ingredients of the material into a more water-soluble form. Mashing includes mechanical mixing of milled material with water, followed by gelatinization, liquefaction and saccharification of starch and other mash ingredients.

Starch gelatinization or pasting is swelling of starch grains in water at higher temperatures (for different starch-based materials 100-160°C, for pure starch 49-80°C) and converting starch from insoluble into a water-soluble form. Water molecules react with free hydroxyl groups, thus forming the colloidal gel, which in turn absorbs water and a milky opaque opalescent viscous mass is obtained, called gelatinized starch or starch paste. Gelatinized starch reacts with iodine solution and gives blue coloring.

The term liquefaction of water-soluble gelatinized starch comprises further heating with the addition of acid or enzyme, aimed to further degrade starch into maltooligosaccharides (degree of polymerization, DP_n = 2-50) and to reduce the mash viscosity. The term saccharification means degradation of water-soluble gelatinized starch and maltooligosaccharides by acidic or enzymic hydrolysis to fermentable (simple) sugars, mainly glucose and/or maltose (degree of polymerization, DP_n = 1-2).

The term degree of polymerization (DP_n) means the number of glucose units in maltooligosaccharides, e.g., with maltose DP_n is 2, with glucose 1, with maltotriose 3.

The term maltooligosaccharides means starch degradation products consisting of glucose units joined by <x-l,4-glycosidic bonds, with the degree of polymerization (DP_n) ranging from 2 to 50.

The term reducing sugars (RS) and the term dextrose equivalent (DE) mean any titer of free carbonyl groups expressed as glucose (in g L^"1) and serves as a measure of the progress of saccharification.

The term enzymatic hydrolysis means starch and maltooligosaccharide degradation by corresponding amylolytic enzymes or amylases at optimum temperature and pH values of the mash.

The term amylolytic enzymes or amylases comprises three groups of enzymes referred to as α-, β- and γ-amylases. The enzymes are classified according to the type of chemical catalyzing reactions and, based on this classification, are divided into different classes with different numbers attached to them. The enzymes with the initial three numbers EC 3.2.1. belong to class hydrolases (3), subclass glycosylases (3.2) and group glycosydases (3.2.1).

The term a-amylase (EC 3.2.1.1) means the enzyme that catalyses the partial hydrolysis of polysaccharides containing three or more D-glucose units joined by the a- 1,4- glycosidic bond. The trivial names of this enzyme are: glycogenase, diastase, fungal α-amylase or bacterial a-amylase, whereas its systematic name is 1,4-a-D-glucan glucanohydrolase. The term β-amylase (EC 3.2.1.2) means the enzyme that catalyses the hydrolysis of a- 1,4-glycosidic bonds from the non-reducing end of polysaccharide where two glucose subunits are separated yielding in the end a mixture of maltose and higher maltooligosaccharides. The trivial names of this enzyme are: glycogenase, saccaharifying amylase, 1,4-a-D-glucan hydrolase, whereas its systematic name is 4-a-D-glucan maltohydrolase.

The term glucoamylase (or γ-amylase) (EC 3.2.1.3) means the enzyme that catalyses the hydrolysis of a-l,4-D-glycosidic bonds and 1,6-a-D-glycosidic bonds with the non-reducing end of polymers separating one glucose unit each. The trivial names of this enzyme are: amyloglucosidase, γ-amylase, lysosomal a-glucosidase, acidic maltase, exo-l,4-a-glucosidase, glucose amylase, y-l,4-glucan glucohydrolase, 1,4-a-D glucan glucohydrolase, whereas its systematic name is 4-a-D-glucan glucohydrolase.

Some microorganisms produce extracellular amylolytic enzymes which can degrade starch and maltooligosaccharides in the cell's environment into fermentable sugars (glucose, maltose). The amylolytic lactic acid bacteria also belong to this microbial group.

The term amylolytic lactic acid bacteria (ALAB) mean the LAB group that can synthesize the extracellular amylases and degrade the starch substrates into maltose and glucose and then ferment them into lactic acid as the final product of energy metabolism.

The processes of two-step lactic acid production from starch-based materials, where first the enzymic or acidic saccharification and then fermentation are conducted, are long known in prior art, so there is no need to elaborate on them here. The subject of this patent is a special form of simultaneous saccharification and fermentation (SSF) in the lactic acid production from starch-based materials. Some processes belonging to this category have also been described in prior art. For example, the EP 354828 Al patent discloses a lactic acid production process by using various commercial starch-degrading enzymes, as well as various LAB species for fermentation of formed sugars to lactic acid. Added to the medium are various nutritive substances of organic and inorganic origin to improve the LAB growth and activity. The bioprocess referred to in the patent EP 354828 Al involves the incubation of the LAB species Lactobacillus delbrueckii subsp. lactis or the LAB species Lactobacillus rhamnosus in a medium with water-soluble wheat starch to which glucoamylase is added for starch degradation. The publication by Hofvendahl et al. (1999, Appl. Biochem. Biotechnol, 52, 163-169) describes the SSF process conducted by enzymes and by the LAB Lactococcus lactis in a medium with wheat starch. In work by Linko et al. (1996, Enz. Microb. Technol.19, 118-123) also describe the SSF bioprocess conducted with an addition of enzyme for starch degradation and by the LAB L. casei in a medium containing barley starch. The US Pat. No. 2588460 discloses a lactic acid production process conducted by Lactobacillus delbrueckii in a medium with water-soluble maize starch to which glucoamylase is added. The work by Mercier et al. (1992, J. Chem. Technol. Biotechnol. 55, 111-121) describes the SSF process conducted by Lactobacillus amylophlus in a medium containing water-soluble maize starch. The US Patent No. 5464760 and the WO Patent No. 94/13826 disclose lactic acid production processes where a mixed Lactobacillus culture produces D- and L-lactic acid in a medium with potato starch after starch degradation by glucoamylase. Similarly, in work of Tsai et al. (1998, Appl. Biochem. Biotechnol. 70/72, 417-428) describe the SSF lactic acid production process by using Lactobacillus species in a medium with potato starch. All these processes have three characteristics: a) they use more or less purified starch, mostly water-soluble starch as basic material; b) added to the medium are complex nutritive substances of organic and inorganic origin, such as yeast extract, peptone, different salts, etc.; c) added to the medium are the commercial amylo lytic enzymes responsible for simultaneous saccharification and fermentation.

The base of the present Invention is to further improve the SSF process manifested in the following: (a) the use of microorganism L. amylovorus which belongs to the ALAB group that possesses the starch-degrading enzymes, hence no need to add the commercial enzymes in the fermentation preparation phase (as in the two-step process) nor during fermentation (as in SSF); (b) corn grits is directly used instead of more or less purified and processed starch preparations; c) the use of this material can significantly reduce or completely eliminate the addition of simple and complex nutritive substances of organic and inorganic origin; d) the application of grits concentration of 100 g L^"1 leads to the formation of the semi-solid consistency of the medium, so what we have here at work is sssf, semi-solid state fermentation. Various nutritive substances of organic and inorganic origin that satisfy the requirements of microorganism during its growth and lactic acid production may be added to the medium. The nutritive substances of organic origin (e.g. peptone, meat extract, yeast extract, polysorbate) may be added in a concentration up to 40 g L^"1, organic salts (e.g. sodium acetate and ammonium citrate) in a concentration up to 20 g L^"1, whereas the nutritive substances of inorganic origin (e.g. dipotassium hydrogen phosphate, manganese sulfate and magnesium sulfate) are added in a concentration up to 4 g L^'1. As far as we know, no combination of SSF and sssf concerning lactic acid production is known in prior art. A combination of these two processes, SSF and sssf, makes the lactic acid production process much more complex than the traditional processes in liquid media. The SSF/sssf process involves a heterogeneous system consisting of three phases: (a) solid corn grits particles, (b) insoluble starch, and c) soluble starch and other carbohydrates dissolved in the liquid phase. In this process the cells of ALAB L.amylovorus are suspended in the liquid phase, and attached on the solid particles of corn grits and/or starch to which they are bound by molecular forces. The development of such a complex bioprocess where renewable materials of varying consistency and chemical composition are used in a semi-solid heterogeneous multiphase substrate, the physical and chemical properties of which are continuously changing during the bioprocess, requires an exceptional knowledge of microbiological, biochemical and engineering parameters, as well as the right choice and adjustment of analytical methods by which the changes in certain characteristics of the bioprocess are monitored. For that reason, an expert with average training in that area could not conceive and set up the process without extensive research in this direction.

Prominent in prior art are the works by Cheng et al. (1991, J. Midustr. Microbiol. 7, 27- 34), Zhang et al. (1991, Biotechnol. Lett. 13, 733-738) and Xiaodong et al. (1997, Biotechnol. Lett. 19, 841-843) in which the SSF process is conducted by using ALAB Lactobacillus amylovorus in media with water-soluble maize starch and addition of complex nutritive substances of organic and inorganic origin (salts, yeast extract, peptone, etc.). Unlike the present Invention, in the work by Cheng et al. (1991) application of the commercial amylolytic enzymes during the SSF process is described. The other two works describe the processes where starch fermentation is conducted without adding the amylolytic enzymes before and during fermentation. In the work by Xiaodong et al. (1997), the initial starch concentrations in the medium are very low (10 g L^"1) and do not correspond to those in industrial application. In the work by Zhang et al. (1991) high initial starch concentrations (120 g L^"1) are used, which correspond to those applied on industrial scale. The said work essentially differs from this Invention in a number of aspects: a) the mentioned work applies water-soluble starch, whereas this Invention applies corn grits in a high concentration (100 g L^"1); b) as a result, the process according to this Invention is conducted as simultaneous saccharification and fermentation (SSF), and as semi-solid substrate fermentation (sssf), whereas in the mentioned work it is conducted as a simple fermentation in the liquid medium; c) in the mentioned work significant quantities of nutritive substances (salts, yeast extract, etc.) are added to the medium, whereas in this Invention, due to the application of corn grits in place of starch, the process is conducted without or with minimum addition of nutritive substances. The nutritive substances of organic origin (e.g. peptone, meat extract, yeast extract, polysorbate) may be added in a concentration up to 30 g L^"1, or 40 g L^"1, organic salts (e.g. sodium acetate and ammonium citrate) in a concentration up to 15 g L^"1, or 20 g L^"1, whereas the nutritive substances of inorganic origin (e.g. dipotassium hydrogen phosphate, manganese sulfate and magnesium sulfate) are added in a concentration up to 3 g L^"1, or 4 g L^"1. As far as we know, there is no reference that would combine these three essential features of the Invention, which at the same time have a potential of major savings in terms of labor, raw material and energy.

Therefore, the Invention introduces a method of producing lactic acid or its salt from corn grits or other starch-containing materials (cereals and tubers, lignocellulosic/hemicellulosic hydro lisates, alfaalfa, cotton seed hulls, jerusalem arthicoke, corn comb, corn stalks, wheat and other bran, rye and other flours, sweet sorghum, mud residue after pressing of sugar beet, cassava, waste from processing of vegetables and other waste, which are subjected to simultaneous saccharification and fermentation in a semi-solid substrate, i.e. in the combined SSF/sssf process conducted by moderately thermophilic microorganisms of Lactobacillus amylovorus or similar ALAB species and strains of kind producing extracellular amylolytic enzymes that degrade starch in a process that does not require addition of the commercial amylolytic enzymes, where the addition of nutritive substances is substantially reduced and may also be omitted altogether, which makes this process essentially different compared with prior art. This process can also be conducted in conventional bioreactor vessels with mechanical stirrers (e.g., Rushton turbine), but also in a horizontal rotating tubular bioreactor (HRTB) (Santek et al., 1996a, Bioproc. Eng. 14, 195-204; Santek et al., 1996b, Bioproc. Eng. 14, 223- 229; Santek et al., 1998a, Bioproc. Eng. 18, 467-473; Santek et al., 1998b, Bioproc. Eng. 19, 19- 28; Santek et al., 1998c, Bioproc. Eng. 19, 91-102; Santek et al., 2000, Bioproc. Eng. 23, 265- 274; Santek et al., 2004, Kern. Ind. 53 (1), 7-24; Ivancic et al., 2004a, Proc. Biochem. 39(8), 995- 1000; Ivancic et al., 2004b, Bioprocess Biosyst. Eng. 26, 169-175; Slavica et al., 2004, J. Biosci. 29, 169-177; Rezic et al., 2006, Proc. Biochem. 41, 2024-2028; Santek et al., 2006, Chem. Biochem. Eng. Q. 20(4), 389-399; Rezic et al., 2007, World J. Microbiol. Biotechnol. 23 (7), 987-996), which has not been described in prior art.

Upon completed fermentation, lactic acid can be separated from the fermentation medium and additionally purified up to the required degree of purity (e.g. technical, food grade, pharmaceutical purity) using one of the separation and purification methods described in prior art, such as filtration and concentration of the medium after a bioprocess, membrane separation, distillation, vacuum evaporation, extraction (liquid-liquid, backflow), electrodialysis, adsorption, ion exchange, precipitation, crystallization and suchlike, as well as combinations of herein listed methods (patents: US 2008/0261285 Al, Roel, Purac; US005464760A, Tsai et al.; WO 94/113826, Tenlin et al.; US005510526A, Baniel et al.; publications: Narayanan et al., 2004, Electron. J. Biotechnol. 7 (2), 167-179; Demirci et al., 2003, Biotechnol. Bioeng. 83(7), 749-759, Cao et al., 2002, Biochem. Eng. I 11, 189-196; Chen and Ju, 2002, Appl. Microbiol. Biotechnol. 59, 170-174; Schugerl, 2000, Biotechnol. Advances 18, 581-599; Monteagudo and Aldavero, 1999, 1 Chem. Technol. Biotechnol. 74, 627-634; etc.).

It was quite surprising to find that the processes of simultaneous saccharification and fermentation of semi-solid substrate without any or with minimum addition of nutritive substances, conducted according to the present Invention, had yielded high values for product yield coefficient, Y_P/_S (expressed as g of lactic acid per g of consumed starch). Thus in one implementation of the Invention the product yield coefficient is at least 0.80 g g^"1, in a more favorable version of the Invention at least 0.94 g g^"1, and in the most favorable implementation of the Invention at least 0.97 g g^"1. By adjusting the physical parameters, the product yield coefficient can be maintained within a range from 0.93 to 0.95 g g^"1. What has also been noticed is a rapid homofermentative production of lactic acid (qp = 0.40-0.61 h^"1). EXAMPLES - Part 1

Submerged Processes

media, cultivation conditions, and results

[0010] The amylo lytic lactic acid bacterium Lactobacillus amylovorus DSM 20531^T has been cultivated, and maintained on MRS agar and in MRS broth (according to De Man, Rogosa and Sharpe) media with glucose (γο = 20 g L^"1) or water-soluble starch (γ₀ = 10 g L^"1) as the sole and main carbon source. The concentrations of other ingredients of these media were not changed (De Man et al., 1960, J. Appl. Bact. 23, 130-135). The broth and agar MRS media were prepared by dissolving the weighed ingredients, all except Tween 80® and starch, in demineralized water. Prior to sterilization, a corresponding volume of Tween 80® was added to the medium. The sterilization of the media was performed in an autoclave at 121°C over 20 minutes, and the media were cooled to about 40°C before inoculation. The starch solution was sterilized separately and then added to the sterile MRS medium (without a carbon source) in order to achieve the final starch concentration of 10 g L^"1. The MRS broth media thus prepared were then used for preparation of inocula in test tubes without shaking (the total volume of the microbial suspension cultivated at 40°C over = 12 h was 10 mL), and after that were also used for the cultivation of microbial culture in 500 mL Erlenmeyer flasks with shaking in a laboratory shaker (the total volume of the bacterial suspension cultivated at 40°C over ~ 12 h was 400 mL). The corresponding sterile MRS medium (V = 5 L) for the cultivation of bacterial culture and for the production of lactic acid in the laboratory stirred tank bioreactor (Chemap AG, Switzerland) was inoculated with the overnight pregrown culture of L. amylovorus DSM 20531^T (40°C ~ 12 h) from the Erlenmeyer flasks (total volume of the microbial suspension for inoculation was 125 mL).

The medium in the laboratory bioreactor was sterilized indirectly by steam at 121°C over 20 minutes. The cultivation of L. amylovorus DSM 20531^T in the MRS medium with glucose or starch was performed by using the batch procedure at a temperature of 40°C, stirrer rotation speed of 400 min^"1 and pH value of the medium of 5.5 ± 0.2 (c_Nao_H = 10 moL L^"1). During the batch cultivation of L. amylovorus DSM 20531^T in the described conditions the kinetic and stechiometry parameters were attained as shown in Tables 1 and 2. What is important is that in this process high values for product yield coefficient, Y_P/_S (expressed as g of lactic acid g^"1 of consumed starch) were achieved within a range from 0.93 to 0.95 g g^"1, plus rapid homo fermentative lactic acid production (q_P = 0.40-0.61 h^"1; Table 2).

Table 1 - Duration of the growth phases (tGRowm PHASE, lag phase, lag, exponential phase, exp) and values for the specific growth rate (μ), substrate consumption rates (q_s), biomass yield coefficient (Υχ s) and the productivity of microbial biomass (Pr_x) were estimated from experimental data obtained during the batch cultivation of L. amylovorus DSM 20531^T in the laboratory stirred tank bioreactor. microbial growth

^GROWTH PHASE (J¹) μ qs Υχ/s Prx medium lag exp (h-¹) (g L-¹) (h-¹) (g g ¹) (g L-' h-¹)

MRS-glucose

(Yo = 20 g L-¹) 4-5 5 0.41 4.90 0.38 0.25 0.45

MRS-water-soluble

starch 2 6 0.67 4.38 0.29 0.44 0.44

(Yo = 10 g L-¹)

Table 2 - Values for lactic acid formation rate (qp), product yield coefficient (Yp/s), produd/biomass yield coefficient (Yp/x), and the productivity of lactic acid (Pr_p) were estimated from experimental data obtained during the batch cultivation of L. amylovorus DSM 20531^T in the laboratory stirred tank bioreactor. lactic acid production

q^p Xpma Yp/s Ypx

medium (h-¹) (g L^"1) (g g ¹) (g g ') (g L-'h^'1)

MRS-glucose

(Yo = 20 g L-¹) 0.40 18.56 0.93 3.79 1.69

MRS-water-soluble starch

(Yo = 10 g L-¹) 0.61 10.52 0.95 3.40 1.05 EXAMPLES - Part 2

SSF processes

media, cultivation conditions, and results

[0011 ] ALAB L. amylovorus DSM 20531^T is also adapted for cultivation in different starch- based media with a relatively high initial concentration of carbon sources (MRS-water-soluble starch γ₀ = 100 g L^"1; MRS -corn grits γο = 100 g L^"1; and corn grits with minimum addition or without addition of nutritive substances, γο = 100 g L^"1) at different cultivation temperatures (30- 50°C). The media were prepared by weighing the corresponding ingredients, adding Tween 80® and starch or (only) corn grits to demineralized water. When necessary, the ingredients were sterilized together (without separate sterilization of the starch solution) at 121°C in the laboratory bioreactor with continuous mixing (150 min^"1) over 20 minutes (MRS-water-soluble starch and MRS-corn grits) or 30 minutes (corn grits). The laboratory stirred tank bioreactor Biostat Cplus (Sartorius BBI Systems GmbH, Germany; VK = 6 L) was used. The sterile medium in the laboratory bioreactor was inoculated with 2.5% (vol/vol) of overnight pregrown bacterial culture previously inoculated two times in succession into the MRS-medium with water-soluble starch, in the same way as described in the foregoing chapter (chapter [0010]).

The SSF lactic acid production process was first investigated in the MRS-medium with water- soluble starch (γ₀ = 100 g L^"1), then in the MRS-medium with corn grits and afterwards in the corn grits medium without addition of nutritive substances (Table 3).

The initial pH value of the media for cultivation in the laboratory bioreactor was 6.2 ± 0.2 and was not corrected during cultivation until, due to lactic acid production, the pH value was brought down to 5.5 ± 0.2. After reaching these pH values of the medium, the sodium hydroxide solution (cNaOH = 5 moL L^"1) was automatically dosed. The SSF bioprocess was carried out as long as the automatic dosing of the alkali solution was going on, i.e. as long as the L. amylovorus DSM 20531^T was producing lactic acid in these media in the selected conditions. Thus the SSF bioprocess in the MRS medium with corn grits took 160 h, and in the corn grits suspension 216 h. Table 3 - Media composition for inocula cultivation (MRS-water-soluble starch; γ₀ = 100 g L^"1) and for the SSF lactic acid production process (MRS-water-soluble starch, MRS-corn grits and corn grits without addition of nutritive substances; γ₀ = 100 g L^"1) in the laboratory stirred tank bioreactor . ingredient MRS-water-soluble starch MRS-corn grits com grits water-soluble starch 10 or 100 g L^"1 - - corn grits (=70% starch) - 100 g L^"1 100 g L^"1 peptone 10 g L-¹ 10 g L" - meat extract 10 g L^"1 10 g L" - yeast extract 5 g L" 5 g L- -

K₂HP0₄ 2 g L^"' g L- - sodium acetate trihydrate 8.3 g L^"' 8.3 g L-¹ - diammonium citrate 2 g L- 2 g L^"' - magnesium sulfate 0.2 g L^"1 0.2 g L^"1 - manganese sulfate 0.05 g L^"1 0.05 g L^"1 -

Tween 80 1 mL 1 mL - demineralized water 1 L 1 L 1 L

The product yield coefficient, Yp/s (expressed as g of lactic acid g^"1 of consumed starch), amounted to 0.94 (in the MRS-water-soluble starch medium, 0.97 in the MRS-corn grits medium, and 0.80 in the corn grits medium without addition of nutritive substances).

The achieved Yp/s values 0.94 and 0.97 g g^"1 show that with the described process high lactic acid yields can be achieved on starch-based materials without any addition of the commercial amylo lytic enzymes (traditional processes can reach Y_P/_S values of about 0.90 g g^"1), with significant savings in raw material, energy, labor, and investment costs compared to the conventional two-step process. In this regard, it should be noted that an exceptionally high Y_P/S value was reached during the SSF/sssf procedure (Yp/s = 0.80 g g^"1) where the corn grits was the only medium ingredient, something unrecorded in available literature. What should be pointed out here is that the process with corn grits is a combined SSF/sssf process. Furthermore, the highly efficient SSF/sssf process with corn grits was also conducted in the horizontal rotating tubular bioreactor (FJRTB) as described by Santek at al. (Santek et al., 1996a, Bioproc. Eng. 14, 195-204; Santek et al., 1996b, Bioproc. Eng. 14, 223-229; Santek et al., 1998a, Bioproc. Eng. 18, 467-473; Santek et al., 1998b, Bioproc. Eng. 19, 19-28; Santek et al., 1998c, Bioproc. Eng. 19, 91-102; Santek et al., 2000, Bioproc. Eng. 23, 265-274; Santek et al., 2004, Kern. Ind. 53 (1), 7- 24; Ivancic et al., 2004a, Proc. Biochem. 39(8), 995-1000; Ivancic et al., 2004b, Bioprocess Biosyst. Eng. 26, 169-175; Slavica et al., 2004, J. Biosci. 29, 169-177; Rezic et al., 2006, Proc. Biochem. 41, 2024-2028; Santek et al., 2006, Chem. Biochem. Eng. Q. 20(4), 389-399; Rezic et al., 2007, World J. Microbiol. Biotechnol. 23 (7), 987-996). The procedure was conducted at room temperature (20-26°C) and without adjustment of the pH value of the medium, the value of which ranged from 3.8 to 6.2. The SSF/sssf process in the HRTB was conducted in the batch, the fed-batch, the repeated-batch and the continuous mode.

Claims

PATENT CLAIMS

1. The lactic acid production procedure, characterized in that it includes the following steps:

a) simultaneous saccharification and fermentation, specifically simultaneous saccharification and fermentation of semi-solid substrate in the bioreactor by only one species of amylolytic lactic acid bacteria

b) separation of lactic acid from the fermentation medium

c) purification of lactic acid

2. The lactic acid production procedure, according to Claim 1, characterized in that it includes the following steps:

b) separation of lactic acid from the fermentation medium

c) purification of lactic acid

where in step a) no amylolytic enzymes are added and nutritive substances are added in a concentration of 0 g L^"1 to 75 g L^"1.

3. The lactic acid production procedure, according to Claim 2, characterized in that it includes the following steps:

b) separation of lactic acid from the fermentation medium

c) purification of lactic acid

where in step a) neither the amylolytic enzymes nor the inorganic nutritive substances are added, whereas nutritive substances are added in a concentration of up to 60 g L^"1.

4. The lactic acid production procedure, according to Claim 2, characterized in that it includes the following steps:

b) separation of lactic acid from the fermentation medium

c) purification of lactic acid

where in step a) neither the amylolytic enzymes nor the organic substances are added, whereas the inorganic substances are added in a concentration of up to 15 g U¹.

5. The lactic acid production procedure, according to Claim 1 , characterized in that it includes the following steps:

a) simultaneous saccharification and fermentation, specifically simultaneous saccharification and fermentation of semi-solid substrate in the bioreactor by only one kind of amylolytic lactic acid bacteria

b) separation of lactic acid from the fermentation medium

c) purification of lactic acid

where in step a) neither the amylolytic enzymes nor the organic nutritive substances nor the inorganic substances are added.

6. The lactic acid production procedure, according to Claim 1, characterized in that during simultaneous saccharification and fermentation the bacteria of genus Lactobacillus are used as amylolytic lactic acid bacteria.

7. The lactic acid production procedure, according to Claim 6, characterized in that during simultaneous saccharification and fermentation the bacteria of genus Lactobacillus amylovorus are used as amylolytic lactic acid bacteria.

8. The lactic acid production procedure, according to Claim 5, characterized in that during simultaneous saccharification and fermentation the bacterium of genus Lactobacillus amylovorus DSM 20531^T is used as amylo lytic lactic acid bacteria.

9. The lactic acid production procedure, according to Claim 1 , characterized in that Step c) of lactic acid purification is performed by filtration and concentration of the medium after the bioprocess or by separation, or distillation, or vacuum evaporation, or extraction, or electrodialysis, or adsorption, or ion exchange, or precipitation, or crystallization, or a combination of two or more methods listed herein.

10. The lactic acid production procedure, according to any of the Claims 1-5, characterized in that the simultaneous saccharification and fermentation occurs in the same bioreactor.

11. The lactic acid production procedure, according to Claim 10, characterized in that the simultaneous saccharification and fermentation occurs in a standard bioreactor with a stirrer, or a bioreactor with different supports, or a tower bioreactor, or a horizontal tubular bioreactor and other types of bioreactors.

12. The lactic acid production procedure, according to Claim 10, characterized in that the simultaneous saccharification and fermentation occurs in the horizontal rotating tubular bioreactor.

13. The lactic acid production procedure, according to Claim 1, characterized in that it additionally involves cultivation, and maintenance of amylolytic bacteria in media with glucose or water-soluble starch as the only carbon source.

14. The lactic acid production procedure, according to Claims 1-2, characterized in that the product yield coefficient, expressed as g of lactic acid g^"1 of consumed starch, is at least 0.80.

15. The lactic acid production procedure, according to Claims 1-2, characterized in that the product yield coefficient, expressed as g of lactic acid g^"1 of consumed starch, is at least 0.94.

16. The lactic acid production procedure, according to Claims 1-2, characterized in that the product yield coefficient, expressed as g of lactic acid g^"1 of consumed starch, is at least 0.97.

17. The lactic acid production procedure, according to Claims 1-5 and 14-16, characterized in that the semi-solid substrate is composed of any starch-containing material.

18. The lactic acid production procedure, according to Claim 17, characterized in that the semi-solid substrate is composed of corn grits.

19. The lactic acid production procedure, according to Claim 17, characterized in that the semi-solid substrate is composed of starch and MRS-medium ingredients.

20. The lactic acid production procedure, according to Claim 17, characterized in that the semi-solid substrate is composed of corn grits and MRS-medium ingredients.