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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus

Definitions

• the present invention pertains to a method for the production of lactic acid or a salt thereof wherein starch is subjected to a process of simultaneous saccharification and fermentation .
• Fermentation processes are exploited for the manufacture of a vast number of products of considerable commercial interest and which are difficult to produce synthetically. Fermentation is used in industry to produce simple compounds among which alcohols, such as ethanol and butanol; organic acids, such as citric acid, itaconic acid, (R) - or (S) -lactic acid and gluconic acid; etones; amino acids, such as glutamic acid and lysine; but also more complex compounds as antibiotics, such as penicillin and tetracyclm; enzymes; vitamins, such as riboflavin, vitamin B12 and beta- carotene; and hormones. Also in the brewing, wine, dairy, leather and tobacco industry fermentation processes are used.
• liquefied starch means starch that has been subjected to the process of liquefaction, which means acid, enzymic, or thermomechanical fluidification of starch leading to a dissolved or soluble form.
• malto-oligosaccharides means maltosaccharides composed of ⁇ - 1, 4-glucosidic linkages of anhydroglucose units having a degree of polymerization (DP n ) of 2 - 50 (n) such as maltose, maltotriose, rnaltotetraose, or maltopentaose and the like.
• degree of polymerization or DP n means the number of anhydroglucopyranose units in a given saccharide .
• Maltose having two ⁇ -1, 4-glucos ⁇ d ⁇ c linked glucose residues may be referred to as DP 2 ; maltotriose, having three ⁇ -1, -glucos ⁇ d ⁇ c linked glucose residues may be referred to as DP 3 and so on.
• maltodextrin means a starch hydrolysis product having a DE of less than 20.
• DE extrose eguivalent
• D-glucose on a dry basis means the composition based on the absence of water. For instance, a product containing 25 wt .
• lactic acid means 2-hydroxy- propiomc acid in either its free acid or salt form.
• the salt form of lactate is specifically referred to as lactate-salt, e.g. as either the calcium salt of lactic acid or calcium lactate. Alkali and earth alkali metal salts of lactic acid are preferred. Lactic acid contains a chiral carbon atom, and for that reason can exist as (R) and (S) enant omer.
• lactic acid contains a chiral carbon atom, and for that reason can exist as (R) and (S) enant omer.
• lactic acid as used m this application includes the pure (R) and (S) isomers, and mixtures thereof including the racemic mixture.
• enantiomeric purity for an excess (S)- ⁇ somer means
• Enantiomeric purity 100 % x ⁇ ( (S) -isomer) / ( (R) - somer ⁇ (S) -isomer) ⁇
• saccha ⁇ fication means the acid- or enzymic-hydrolysis of starch or liquefied starch or maltodextrines or maltooligosaccharides that ultimately results in the production of D-glucose or maltose or small maltooligosaccharides or mixtures thereof.
• ⁇ -amylase means an enzyme belonging to the functional-class with the EC number 3.2.1.1 and the trivial name glycogenase, diastase, fungal cc-amylase, or bacterial ⁇ —amylase, and the systematic name 1, - ⁇ -D-glucan glucanohydrolase .
• ⁇ -Amylases partially depolymenze polysaccharides containing three or more 1,4- ⁇ -lmked D-glucose units by endohydrolysis of 1, 4- ⁇ -D-glucos ⁇ d ⁇ c linkages.
• the term "glucoamylase” means an enzyme belonging to the functional-class with the EC number 3.2.1.3 and the trivial name glucoamylase, ⁇ -amylase, lysosomal ⁇ -glucosidase, acid maltase, exo-1,4- ⁇ -glucosidase, glucozyme, AMG, or GAM, and the systematic name 1,4- ⁇ -D-glucan glucohydrolase.
• Glucoamylases hydrolyze terminal 1,4- lmked ⁇ —D-glucose residues successively from the non-reducing ends of the chains with release of ⁇ -D-glucose, and they hydrolyze the ⁇ - 1,6 linkages.
• the term "pullulanase” means an enzyme belonging to the functional-class with the EC number 3.2.1.41 and the trivial name limit-dextrmase, debranchmg enzyme, or amylopectm 6-glucano- hydrolase, and the systematic name ⁇ -dextr ⁇ n-6-glucanohydrolase or pullulan 6-glucanohydrolase . Pullulana ⁇ es debranched starch by hydrolysmg the ⁇ -1, 6 linkages.
• starch means any purified or crude starch or liquefied starch, or any starch-or liquefied starch-containing material.
• Wheat, corn, rye, and potato starch are examples of starches that can usefully be applied m the present invention .
• SSF saccha ⁇ fication and fermentation
• Fermentation of starch or liquefied starch to lactic acid is known in the art, for example from EP 354828 Al, wherein a process for producing lactic acid is disclosed, which includes incubating Lactobacillus delbrueckii subsp lactis or Lactobacillus rhamnosus in fermentation broth containing liquefied wheat starch and a preparation of a glucoamylase to produce a lactate-containmg fermentation broth.
• Hofvendahl et al m Appl . Biochem. B otechnol., 52: 163-169 (1999) describe an SSF-system with Lactococcus lactis in fermentation broth-containing wheat starch. Linko et al in Enz .
• Microb Technol., 19: 118-123 (1996) describe an SSF-system with Lactobacillus casei in fermentation broth-containing barley starch.
• US 2588460 discloses a process to produce lactic ac d which includes incubating Lactobacillus delbrueckii fermentation broth- containing liquefied corn starch and a preparation of a glucoamylase to produce a lactate-containmg fermentation broth.
• Mercier et al J. Che , Technol. Biotechnol . , 55: 111-121 (1992) describe an SSF- system with Lactobacillus amylophilus in fermentation broth- contaimng liquefied corn starch Cheng et al m J. Indust.
• Biotechnol , 70/72: 417-428 describe an SSF-system with a Lactobacillus species in fermentation broth-contammg potato starch.
• the term "fermentation broth” refers to both media m the form originally provided to microorganisms as a source of nutrients, growth factors and carbohydrates and media produced after some or all of the originally provided nutrients, growth factors, or carbohydrates have been consumed and fermentation products including lactic acid have been excreted into the media by the microorganisms
• microorganisms such as Lactobacillus species, Lactococcus species, Streptococcus species, Enterococcus species, and Sporolactobacillus species are producers of lactic ac d
• certain properties make these organisms less suitable for the industrial manufacture of lactic acid, including the fact that they have a growth temperature in the range of 30-50° C, with an optimum at 30- 37° C, which makes it more difficult to avoid infections m industrial scale fermentation systems, which compromise the enantiomeric purity of the lactic acid during the fermentation than when higher temperatures can be used. It is one of the objects of this invention to provide a method, which is devoid of this disadvantage of the known SSF processes .
• the growth-temperature range of these microorganisms is not congruent with the application temperature range of glucoamylase and pullulanase, which is in the range of 50 - 70° C and of ⁇ -amylase, which is in the range 80 - 105° C.
• a lactic fermentation with the microorganisms operated as SSF at a temperature which is optimal for these microorganism is sub-optimal for the activity of the enzyme.
• thermophilic nutritionally less demanding lactic acid-producing bacteria are more favorable.
• the invention therefore pertains to a method for the production of lactic acid or a salt thereof wherein starch is subjected to a process of simultaneous saccha ⁇ fication and fermentation, the method comprising saccharifying starch in a medium comprising at least a glucoamylase and simultaneously fermenting the starch using a microorganism, and optionally isolating lactic acid from the medium, characterized that a moderately thermophilic lactic acid-producing microorganism is used.
• moderately thermophilic Bacillus species for the manufacture of lactic acid from simple sugars such as glucose and sucrose is known in the art.
• the term "moderately thermophilic” means bacterial strains, which are capable of growing at temperatures between 30 - 65°C, with an optimum between 40 - 60° C, more preferably between 50 - 60° C.
• US 5002881 and DE 4000942 disclose processes to produce lactic acid which include incubating strains of Bacillus coagulans at 48 - 54° C in a simple fermentation medium containing either glucose or sucrose as carbohydrate to produce a lactate containing fermentation broth.
• DE 4000942 Olsen in Kemisk, 25: 125-130 (1944) and Payot et al m Enzyme Microb.
• Technol. - 24: 191-199 (1999) describe a lactic fermentation at 50 - 60° C without sterile conditions with Bacillus coagulans (i.e. Lactobacillus cereale) in fermentation medium with sucrose as carbohydrate to produce a lactate-containing broth.
• Bacillus coagulans i.e. Lactobacillus cereale
• US 6022537 discloses a moderately thermophilic amylolytic organism, Bacillus thermoamylovorans, which is useful for producing lactic acid.
• enantiomerically pure lactic acid or a salt thereof can now easily be prepared.
• enantiomerically pure means that the enantiomeric purity is at least 95%, preferably at least 98%, and more preferably at least 99%.
• Bacillus coagulans and Bacillus thermoamylovorans are excellent lactate producers, they also have certain properties which at first sight would render these organisms less suitable candidates for the manufacture of enantiomerically pure lactic acid from starch or liquefied starch.
• Bacillus thermoamylovorans is heterolactic, which means that a fraction of the carbohydrate substrate is fermented to products other than lactic acid (ethanol, acetic acid, and formic acid) , thereby lowering the product yield (mol lactic acid per mol carbohydrate) and rendering the product lactic acid more difficult to purify.
• Environm. Microbiol., 61: 656-659 (1995); and Internat. J. System. Bacteriol., 45: 9-16 (1995) disclose that the lactate yield on glucose is typically in the order of 88 - 92% (mol lactic acid per mol glucose) . Furthermore, it was also disclosed that the enantiomeric purity of the (S) -lactic acid was 98%. Bacillus coagulans is homolactic, which means that the carbohydrate is fermented to lactic acid only. Furthermore DE 4000942 discloses that the enantiomeric purity of the lactic acid is close to 100%. Bacillus coagulans on the other hand ferments starch or liquefied starch or maltodextrines or maltooligosaccharides poorly.
• Bacillus coagulans and Bacillus thermoamylovorans have a relatively high pH optimum (6.0 - 7 0) for lactic ac d production, which is substantially higher in pH units than the preferred application (working) pH range of glucoamylase (3.5 - 5) .
• the application pH range of glucoamylase is thus not congruent with that of the microorganism.
• a lactic fermentation with the moderately thermophilic Bacillus coagulans and Bacillus thermoamylovorans operated as SSF at a pH that is optimal for the microorganism reduces the activity of the glucoamylase. This can be compensated by adding more glucoamylase, but this will add to the costs.
• glucoamylase can give a reversed reaction to produce ⁇ -1, 4, ⁇ —1, 6 and ⁇ —1 , 3 linkages from glucose producing sugars, such as maltose, isomaltose and nigerose, which are poorly or not fermentable by moderately thermophilic lactic acid bacteria (e g Reilly (1985) : Enzymic degradation of starch in: Starch Conversion Technology (van Beynum and Roels ed) , Marcel Dekker Inc, New York, pp 101-142)
• SSF moderately thermophilic lactic acid bacteria prevents the accumulation of free glucose in fermentation broth thereby preventing or reducing the rate of reversion reactions and thereby preventing the accumulation of non- fermentable sugars such as isomaltose and nigerose.
• the starch is saccharified, fermented, and optionally liquefied in a mixture of glucoamylase and at least one of pullulanase and ⁇ -amylase.
• the instant process results m that substantially less residual sugars are obtained compared with conventional fermentation processes, making direct isolation of lactic acid possible.
• the present invention thus provides n an advantage of an easy and inexpensive work-up procedure, not longer necessitating a complex extraction step to separate the lactic acid from residual sugars.
• An additional advantage of the instant process is the possibility of using crude starch, since the low residual sugar amounts not longer require the use of purified starch as starting product. Usually residual sugar contents less than 5 g/1, preferably less than 2 g/1 are easily obtainable with the process of the invention.
• saccharification is performed by glucoamylase or a mixture of glucoamylase and ⁇ -amylase and/or pullulanase.
• Glucoamylase (EC 3.2.1.3, 1, - ⁇ -D-glucan glucohydrolase) cleaves primarily ⁇ -1, 4-glucosidic linkages at the non-reducing ends of both starch and the fragments left from ⁇ -amylase hydrolysis.
• Glucoamylase is an exohydrolase and attacks di-, oligo- and polysaccharides-conta mg glucose predominantly bound together by ⁇ -1, 4-glucosidic linkages, though ⁇ -1, 6-glucosidic linkages and ⁇ -1, 3-glucos ⁇ d ⁇ c linkages are attacked at lower rates.
• This is of considerable industrial interest, as starch-hydrolysates after ⁇ -amylase treatment contain ⁇ -1, 6-glucosidic linkages that must be cleaved if acceptable glucose yields are to be obtained. Since the activity of glucoamylase towards ⁇ -1, 6-glucosidic linkages is low some commercially available glucoamylase preparations contain pullulanase.
• Pullulanase is a so-called debranchmg enzyme, which cleaves the ⁇ -1, 6-glucosidic linkages present in starch and liquefied starch.
• enzyme-containing glucoamylase as ma n component as well as combination preparations containing glucoamylase and pullulanase appeared suitable for the process according to the present invention.
• the application pH range for glucoamylase and pullulanase was found to be optimal between 3.5 and 5.
• the application temperature range was found optimal between 55 - 70 °C.
• the effect of calcium lactate and pH on the activity and stability of the enzyme was found not to become progressively worse as a result of the accumulation of fermentation product.
• AMGTM E (ex Novo Nordisk), a glucoamylase with a high debranch ng activity, DextrozymeTM E (containing glucoamylase and pullulanase) (ex NOVO Nordisk) , and OptimaxTM 7525 HP (containing glucoamylase and pullulanase) (ex Genencor) were also tested and again no large difference was observed between these preparations.
• DextrozymeTM E performed slightly less than AMGTM E and OptimaxTM 7525 HP regarding overall fermentation performance.
• AMGTM E and OptimaxTM 7525 HP performed almost identical and both enzyme preparations were studied n detail (see Experimental) .
• the fermentation is performed by a moderately thermophilic lactic acid producing microorganism such as microorganisms derived from a strain of Bacillus coagulans, Bacillus thermoamylovorans, Bacillus smithn, Geobacillus stearothermophilus, or from mixtures thereof. These microorganisms are capable of growing at temperatures between 30 - 65° C. Thus these microorganism are better adjusted to work in the optimal working temperatures of the enzymes and the process according to the invention is usually conducted at a temperature between 30 - 70 °C.
• the method of the invention is usually performed at pH 3 - 8.5, preferably at pH 5 - 6, more preferably at pH 5.35 - 5.80, most preferably at pH 5 50 - 5.60.
• thermophilic lactic ac d- producmg microorganism it is an advantage to adapt the thermophilic lactic ac d- producmg microorganism to the pH range as used in the process.
• the pH strongly influences the activity.
• the enzymes become progressively more active at low pH values.
• the common pH ranges m which moderately thermophilic lactic acid-produc g microorganism perform optimal is between 6.0 and 7. Therefore, it is preferred to use moderately thermophilic lactic acid-producing microorganism, which has been adapted to have its maximum performance at low pH (between 5 and 6, preferably between 5.35 - 5.80, most preferably between 5.35 - 5.80).
• the technique of adapting the microorganism is known in the art.
• Adaptation or acclimation of moderately thermophilic lactic acid-producmg bacteria to improve the performance at pH 5.35 - 5.8 was accomplished by carrying out 40 - 50 serial transfers in fermentation medium at pH 5.6.
• the simultaneous saccha ⁇ fication and fermentation may be performed on a starch slurry or on any other starch-conta mg composition. Said starch may be liquefied or not. If a non-liquefled starch slurry or any other starch containing composition is used, the saccha ⁇ fication and fermentation may be combined with liquefaction and also an ⁇ -amylase may be present n the fermentation medium.
• a full saccharification step may last up to 72 hours. However, it is possible to do a pre-saccharification of typically 40 - 90 minutes and then complete saccharification during fermentation. Said pre-saccharification step may be conducted at a temperature above 50° C, just prior to the fermentation.
• the most widely used process is a simultaneous saccharification and fermentation (SSF) process where there is no holding stage for the saccharification, meaning that fermenting organism and enzyme (s) is (are) added together.
• SSF simultaneous saccharification and fermentation
• the moderately thermophilic lactic acid producing microorganism and enzyme (s) may also be added simultaneously.
• the process according to the invention may also be performed by a pre- saccharification followed by a simultaneous saccharification and fermentation wherein an additional portion of the enzymes is added with the addition of the moderately thermophilic lactic acid- producmg microorganism.
• a starch slurry or slurry of a suitable starch-contammg material or liquefied starch is fed into a fermenter.
• the microbial inoculum and nutrients are also fed into the fermenter.
• a suitable base such as calcium hydroxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, magnesium oxide, ammonia, ammonium hydroxide or a suitable carbonate such as calcium carbonate, sodium carbonate, sodium hydrogencarbonate, potassium carbonate, potassium hydrogencarbonate, magnesium carbonate, ammonium carbonate may be added for pH control.
• gelatinized starch or starch containing material is broken down (hydrolyzed) to maltodextrines with an average DE between 10 and 30.
• gelat ization means the process that transforms starch granules into starch paste
• the hydrolysis may be carried out by acid treatment or enzymatically by ⁇ -amylase treatment.
• the ⁇ - amylase derived from a microorganism or a plant.
• Preferred a- amylases are of fungal or bacterial origin. A definition of ⁇ - amylase is given above.
• the enzymatic liquefaction process may be carried out at a pH between 5 and 6, preferably between 5.35 and 5.80, and more preferably between 5.50 and 5.60.
• enzymatic liquefaction may advantageously be combined with either the pre- saccharification or the simultaneous saccharification and fermentation or with both.
• Acid hydrolysis although its use is not wide spread, may also be used.
• the raw material may be milled
• a side stream from starch processing such as co- called B-starch may also be used.
• Liquefaction is known in the art and needs no further elucidation here. As indicated-above, the liquefaction may also take place in combination with the saccharification and fermentation
• the hot slurry is heated to between 60 - 95° C, preferably 80 - 85° C, and at least an ⁇ -amylase is added; 2) the slurry is jet-cooked at a temperature between 95- 140° C, preferably 105 - 125° C to complete gelanitization of the slurry; 3) the slurry is cooled to 60 - 95° C and more ⁇ -amylase is added to finalize hydrolysis.
• the liquefaction process may be carried out at pH 5 - 6, in particular at a pH between 5.35 and 5.80, most preferably at pH 5.50 - 5.60.
• lactic acid After combined saccharification and fermentation the formed lactic acid is optionally isolated from the fermentation medium and purified when necessary.
• Conventional purification/isolation methods for lactic acid are distillation, extraction, electrodialysis, adsorption, ion-exchange, crystallization and the like, and combinations of the above-mentioned purification/isolation methods. Distillation is the most commonly used technique. However, as explained above, with the process according to the invention the formation of non-fermentable sugars is reduced. Therefore, isolation or purification steps may be less complicated and sometimes even redundant. Further details on how to carry out distillation, and other techniques for recovering of lactic acid are well known to the skilled person. The invention is further illustrated by the following experiments, which are included without restricting the invention thereto.
• the culture Bacillus coagulans that was adapted to pH 5.65 was cultivated in a 3-liter glass jacketed stirred reactor equipped with temperature and pH control (Applikon, Schiedam, The Netherlands) .
• the culture was routinely maintained, by transferring every 24 hours 180 - 200 ml of an actively fermenting culture to a fermenter containing a freshly prepared batch of maltodextr e- medium (see Table 1) .
• Fermentations were performed with pure maltodextrines as well as liquefied starch from the Cargill Refinery in Blair (Nebraska, USA) and glucose syrup also from the Cargill Refinery in Blair (Nebraska, USA) (see Table 1) .
• the temperature of the fermenters was controlled at 54 - 56° C by means of a circulating water bath.
• the pH set point was a compromise between the optimum pH of the enzyme and the optimum for Bacillus coagulans that was adapted to pH 5.65.
• the fermentation was started at pH 6.0 and as a result of the formation of lactic acid allowed to drop. Upon reaching pH 5.55 - 5.75 the pH was controlled by the automatic addition of calcium hydroxide (250 g/1) . After 24 hours fermentation the culture received a second portion of AMG ETM (Novozymes) . The fermentation was allowed to proceed until the demand for calcium hydroxide ceased. This was the case after 30 - 40 hours incubation. Results of SSF experiments
• the benchmark for the SSF fermentation was set by the fermentation with glucose syrup (DE 98) as substrate.
• the SSF study was started with a maltodextrine preparation from Roquette Freres (GlucidexTM 12, DE 11-14) and successfully finished.
• Two glucoamylase preparations were extensively tested: AMG ETM from Novozymes (glucoamylase with high debranching activity) and OptimaxTM 7525 HP from Genencor (glucoamylase/pullulanase) .
• a typical SSF fermentation on pure maltodextrines was free of residual sugars within 2 days.
• the mam residual sugars present were glucose and isomaltose, however, at a consistently lower concentration than m comparable cultures that received DE 98 glucose syrup as substrate.

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