SK278555B6 - Method of producing l-lactic acid - Google Patents

Abstract

The production of L-lactic acid is based on that principles, that the micro-organism related to the family called Rhizopus is cultivated with the use of the sub-mersous procedure, while the micro-organism strain closed to the Rhizopus arrhizus CCM 8109 is suitable for these purposes and the above mentioned cultivation is provided at the temperature of 28 up to 48 Celsius degrees, in the environment with pH value of 3,0 up to 7,0 containing 6 up to 25 weight percent of saccharide substrate, however an increase and a decrease of the temperature and pH value closed to the fermentation medium can be provided based on the step by step principle.

Description

Technical field

The invention relates to a process for the production of L-lactic acid.

BACKGROUND OF THE INVENTION

Lactic acid (i.e., 2-hydroxypropanoic acid) is produced by a wide variety of microorganisms. For industrial production, mainly bacteria of the genus Lactobacillus are used, which in addition to the L (+) - form can also form the D (-) and DL-form of this acid. The advantage of bacterial fermentation is the anaerobic mode of the process and the associated simplicity of the cultivation equipment. The disadvantage of a bacterial producer lies in the need for a complex, nutrient-rich production medium, as well as in the production of side metabolites (organic acids, polyols, etc.) [Vickroy, T. B .: In Comprehensive Biotechnology, Vol. 3, p. 761-776, Pergamon Press 1985]

Of the filamentous fungi, the lactic acid producers are mainly Rhizopus, arrhizus and oryzae. Producers are less demanding on nutritional factors and, moreover, show a high stereospecificity (they produce exclusively the L (+) - form of lactic acid). This fact is very important in the fermentation preparation of the lactic acid calcium salt, because with the use of lactobacilli, due to minimal aeration and minimal mixing, calcium carbonate neutralization is problematic and significantly prolongs the fermentation time, affects yields, residual carbohydrate levels and the like.

SUMMARY OF THE INVENTION

It is an object of the process for preparing L-lactic acid according to the invention that a filamentous fungus of the genus Rhizopus is submerged in an aqueous nutrient medium at a temperature of 28-48 ° C, a pH of 3.5 to 7.0 with a content of 8-25% by weight. a saccharide substrate. Rhizopus arrhizus or Rhizopus oryzae species, preferably Rhizopus arrhizus R-41 (CCM 8109) mutants showing high selectivity and conversion rate, can be used as production microorganisms. It is advantageous to ferment at a higher content of the saccharide substrate such that the temperature and the pH of the fermentation medium are gradually increased during fermentation. This regime ensures the preparation of higher concentrations of L-lactic acid while avoiding the possible precipitation of the resulting salts of L-lactic acid, especially calcium. Moreover, it makes it possible to prepare a mixture of free L-lactic acid and its corresponding salt, which substantially improves the efficiency of the isolation of pure L-lactic acid and of the entire process.

During cultivation, the pH is maintained by the addition of alkaline reacting solutions, in particular NaOH, KOH, NH ₄ OH, CaCO ₃ , Ca (OH) _2, preferably calcium ions, which allow easy preparation of free L-lactic acid from fermented medium after addition of sulfuric acid; separation of the resulting calcium sulfate. L-lactic acid will be present both in the culture medium and in the form of the corresponding salts, which also fall within the term "L-lactic acid".

The fermentation medium is vigorously stirred and aerated preferably 0.2-1.0 volume of air per volume of fermentation medium. To inoculate the production fermenter, it is preferable to use a vegetative inoculum which is obtained by 18 to 28-hour submerged culture of the producer spores and is metered into the production fermenter in an amount of 5 to 25% of the total volume.

The fermentation medium contains a carbohydrate substrate in an amount of 6-25 wt. Carbohydrate substrates are understood to be glucose, acidic and enzymatic hydrolysates of starch, sucrose, intermediates and wastes in the production of sucrose, in particular affinades, raffinates, light and heavy juices, clergy and other substrates containing free glucose, respectively. sucrose.

Fermentation is carried out until the substrate is depleted.

E.g. in 48 hours 15% glucose solutions, respectively. sucrose to L-lactic acid with a yield of 90-95% of the theoretical value (from 100 g of glucose and sucrose, respectively, 75 g and 78 g of L-lactic acid, respectively) are formed. The content of other organic acids shall not exceed 0,5% by weight. Some variations in reaction times and yields should be considered depending on the equipment used and the culture conditions.

L-lactic acid can be prepared from the fermented medium after separation of the biomass by treatment with mineral acid or other conventional industrial processes (cation exchangers, reverse osmosis, etc.).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

An inoculation medium containing in 1 ^dm3 of tap water 15 g of glucose, 10 g of molasses, 20 g corn starch 30, 4.0 g (NH ^ SO ,,, 1.6 g KH ₂ PO _4, 0.25 g MgSO ₄ .7H ₂ O, 0.066 g ZnSO ₄ .7H ₂ O, 0.01 g Fe ₂ (SO ₄ ) ₃ .9H ₂ O and 3.0 g corn steep liquor.

0.1 dm ³ inoculum medium in 0.5 dm ³ culture flasks were inoculated with spores of Rhizopus arrhizus CCM 8109 producing micro-organism35 (spore concentration ranging from 1-3.10 ⁶ / ml medium), which were prepared by 14-week culture on solid culture medium. . After cultivation on a rotary shaker at 30 ° C for 24 hours, a submerged culture of production micro-organism 40 is used to inoculate 4.5 dm ³ production medium in a 7 dm ³ fermenter at 7% v / v. Production medium contains in 1 dm ³ tap water: 120 g glucose, 2.0 g (NH4) 2SO4, 0.3 g KH2PO4, 0.4 g MgSO4.7H2O, 0.01 g Fe2 (SO4) 3.9H2O, 0.044 g 45 ZnSO4.7H2O, 0.5 g corn steep liquor. The fermentation itself is performed at 28 ° C, aeration of 3 dm ³ / min, stirring 420 rpm. and overpressure 0,0ΙΌ, 03 MPa. The pH of the fermentation broth is maintained at 5.550 -6.0 by the addition of CaCO3 or Ca (OH) ₂ . After reaching the L-lactic acid concentration g / l, the temperature in the fermenter is gradually raised (2 ° C for each 5 g / l of L-lactic acid produced) and the pH decreases (for every 5 g / l of L-lactic acid produced) by 0.25). After 42 hours of cultivation, the fermentation medium contains 86 g / L of L-lactic acid, i.e. 95.5% of the theoretical value. The residual glucose concentration is less than 0.1 g / L.

Example 2

An enzyme cornstarch hydrolyzate containing 98% D-glucose is prepared. Inoculum preparation, media composition and fermentation conditions are analogous to Example 1 except that the pH of the medium is maintained by the addition of 20% NH ₄ OH. The fermentation time and yields are analogous to Example 1.

SK 278555-6

Example 3

4.2 dm ³ inoculum medium is prepared in a 7 dm ³ fermentation vessel and inoculated with a spore inoculum analogously to Example 1. The composition of the inoculum medium is analogous to Example 1 except that an analogous amount of sucrose is used instead of glucose. After 24 hours cultivation (temperature 30 ° C, aeration of 2 dm ³ / min, stirring 300 rpm, overpressure 0.02 MPa), the submerged culture thus prepared is used to inoculate the production fermenter in an amount of 7% by volume. The production medium contains sucrose at a concentration of 180 g / l instead of glucose. The fermentation itself is carried out in a 20 dm ³ fermentation vessel with a 14 dm ³ fermentation medium. The fermentation is initially carried out at a temperature of 33 ° C, aeration of 8 l / min, stirring at 400 rpm. and overpressure of 0.02 MPa. The pH is maintained by the addition of CaCO ₃ or Ca (OH) ₂ in the range of 5.5-6.0. After reaching a L-lactic acid concentration of 70 g / l, it is increased for every 10 g / l of acid, the temperature is 2 ° C and the pH is lowered by 0.3 pH unit. At a residual sucrose concentration of 0.2 g / l, 20 yields of lactic acid in the range of 92-96% of the theoretical value are achieved.

Industrial applicability

The invention is useful in the production of L-lactic acid or its salts, which are widely used in the food, pharmaceutical, chemical and other fields. 50

Claims (2)

Process for the production of L-lactic acid or its salts, characterized in that a microorganism of the genus Rhizopus, preferably the strain Rhizopus arrhizus CCM 8109, is submerged in an aqueous nutrient medium at a temperature of 28-48 ° C, a pH of 3.0 to 7 % With a content of 6 to 25 wt. a saccharide substrate. Production method according to claim 1, characterized in that during the cultivation the temperature is gradually raised and the pH of the fermentation medium is lowered.