CS203545B1 - Method of preparing fermentative molasses medium for the citric fermentation - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention relates to a process for the intensive production of citric acid in fermentation of molasses while suppressing the production of gluconic acid.

In industrial strains of Aspergillus niger, besides the high productivity of citric acid, the property that it produces no or negligible amounts of secondary acids, in particular oxalic acid and gluconic acid, is also appreciated. By-acids are generated at the expense of the main product and make it difficult to isolate from the fermented solution and purify it. Oxalic acid is more common than gluconic acid. Study of the occurrence of oxalic acid during lemon fermentation has shown that the tendency to produce more or less (or no) oxalic acid is due both to the specificity of the Aspergillus niger strain used and to the specific molasses that can induce oxalic acid production in individual strains. or to a lesser extent (Leopold J. and Zd. Valtr, Zentrabl. Bakteriol. II. Abt. 113, 24, 1960). As is well known, a generally neutral and especially alkaline reaction promotes the formation of oxalic acid. The same applies to the formation of gluconic acid, but less is known about the conditions of its formation during lemon fermentation. In principle, the same applies to oxalic acid, since the tendency to produce gluconic acid is both a property of the Aspergillus niger strain used (Leopold J., Die Nahrung 3,987, 1959) and, secondly, the formation of gluconic acid can be induced by the molasses used. demonstrate the following observations. While the presence of oxalic acid during lemon fermentation can be suppressed by adjusting the substrate to a certain ratio of alkali and phosphate and maintaining a pH of about 2.0 throughout the process (U.S. Pat. No. 3,335,067, 1967, Process for producing citric acid. Miles). Laboratories), a similar intervention for the elimination of gluconic acid is not yet known. Some bacteria (Klebsiella aerogenes) produce gluconic acid by dehydrogenation and subsequent dephosphorylation of glucoso-6-phosphate (DM Neijssel, DW Tempest, Arch. Microbiol. 105, 183, 1975), while glucose is the substrate of Aspergillus niger glucosooxyase. It consists of two identical subunits and serves as a coenzyme flavinadenine nucleotide (Harubito T., N. Osamu, O. Kazuji, J. Biochem. - Tokyo, 78, 835, 1976). Meyrhofa (Franke W., Zentralbl. Bakteriol, II. Abt. 191, 194, 1963), making it worse

Λ supplied tricarboxylic cycle and reduced production of citric acid. A variety of sugars, especially glucose and some organic acids, induce the formation of glucose oxydase (Franke W., L. Nochek. K. Acye, Arch. Microbiol. 51, 323, 1965). In addition to the work that sought to suppress the production of foreign acids, efforts to intensify the production of the main product, i.e. citric acid, by adding some substances to the fermentation medium continued. Years ago, the addition of arsenic, resp. sodium cyanide (Chughtal TD and T. Walker, Biochem. J. 48, 524, 1951; 51, 484, 1954), potassium sulfate (Leopold J., Z. Valtr, US Pat. Nos. 92291 - 1960 and 97292 - 1960), alcohols (Quadar MA et al., Pakistan J. Biochem. 1, 43, 1988) as well as apple cider and malt flower (Ilczuk Z., Zentralbl. Bakteriol. II. Abt. 593, 1972). 1970 (Krčmář S., J. Tachmyna, J. Tomovičová), the yield of citric acid can be increased by adding ethylenediaminetetraacetate to the fermentation molasses medium.

A study of molasses produced from varieties of sugar beet grown in the subtropical region around the Mediterranean found that when fermented to citric acid, the production strain of Aspergillus niger, which always produces only citric acid without side acids on domestic molasses citric acid also has a considerable amount of gluconic acid. Thus, it is an example where molasses induces the production of gluconic acid in the production strain. In addition, the molasses have the particularity that the yield of citric acid at the optimum dose of potassium ferrocyanide (i.e. the dose at which optimum yield of citric acid is obtained without further intervention in the fermentation solution) is low and dependent on the initial pH of the fermentation medium. affects the production of gluconic acid. By way of illustration, Table 1 shows the results of fermentation experiments of one species of molasses from said region. While the A. niger strain used here yields high yields of citric acid (70-80%) on domestic molasses and produces no foreign acids, the optimum yield of citric acid on the foreign molasses is very low and gluconic acid production is unusually high. The sensitive dependence of the formation of both acids on the original pH of the fermentation medium is striking. At pH 7.10, the formation of citric acid is maximal, the formation of gluconic acid is minimal; the more we deviate from the optimum pH, the more the ratio of both acids changes to the detriment of citric acid. Table 2 shows the yields of both acids for the other 4 molasses of the same type (underscored J numbers; maximum yields of citric acid are slightly higher, yields of gluconic acid are similar to those of the sample of Table 1 and show a great dependence on In all these experiments, the ratio of the two acids is such that it is no longer possible to speak of lemon fermentation but of mixed fermentation of citric acid and gluconic acid, which is worthless for industrial purposes, and such molasses would not be operationally viable with acceptable economic results.

T a b u 1 k a 1

Influence of fermentation medium pH on citric acid yields (in% on sugar used at optimum dose of potassium ferrocyanide Yields in% pH of citric acid gluconic acid

7.25 26.8 25.6

7.10 34.7 12.7

6.90 27.7 14.5

6.80 20.9 20.2

6.55 15.1 24.5

These drawbacks are overcome by the invention, which is based on the action of disodium ethylenediaminetetraacetate (chelatone 3, EDTA) on the fermentation medium. The fermented molasses solution is cooked with the optimum dose of potassium ferrocyanide and, after topping up and cooling with water, EDTA is dissolved in it at 50 to 60 ° C or below. According to the invention, the optimum pH of the fermentation medium (6.0 to 7.2) as well as the optimum dose of EDTA, about 0.001 mol / l, must be tested in advance according to the invention. The effect of ethylenediaminetetraacetate on yields of citric acid and gluconic acid in several species of said molasses (I to IV) is evident from the results of the experiments shown in Table 2. The optimum EDTA concentration here was 0.001 mol / l medium; by its addition, the pH of the medium decreased slightly.

0 3 5 4 5

Effect of ethylenediaminetetraacetate (EDTA) on yield of citric acid and gluconic acid (in% on sugar used) at different pH of fermentation medium in different molasses [I-IV]

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For each molasses species tested, the yields of citric acid and gluconic acid are sensitive to the pH of the fermentation medium. Optimal but low yields of citric acid (from 36.6 to 41.7 o / o) are accompanied by yields of gluconic acid from 7.5 to 3.13%. The EDTA additive according to the invention will increase the yield of citric acid very much at the upper pH of each molasses species, namely from 36.6% to 58.4% for molasses I, from 41.8% to 56.1% for molasses II, III from 41.7 to 60.6% and from molasses IV from 40.2 to 51.8%, ie by 11 to 29 O / o. At the same time, according to the invention, the occurrence of gluconic acid in molasses I and II is completely suppressed, and in molasses IV its yield is reduced to 1.3%; although for molasses III the reduction in gluconic acid yields was not as effective (from 27% to 19%), the yield of citric acid increased from 35% to 60%. In order to maximize the stimulation of citric acid production and at the same time to most effectively suppress the production of gluconic acid, the optimum pH of the fermentation medium and the optimum dose of ethylenediaminetetraacetate should be tested for each type of molasses used. Thus, by applying the invention, molasses that would not be processable can be fermented with acceptable to good yields of citric acid and technologically harmless amounts of gluconic acid.

Already cited MS. No. 135387, the addition of ethylenediaminetetraacetate to the fermentation medium is recommended to increase the yield of citric acid. However, there are some fundamental differences between this patent and the present invention. According to the cited patent, an inlase substrate is prepared with an amount of potassium ferrocyanide 20 to 40% lower than the optimum dose and at the earliest after 30 min. 0.0005 to 0.001 moles of ethylenediaminetetraacetate per 1 L are added at a temperature above 60 ° C, thereby increasing the yield of citric acid by about 20%. In contrast to this process, in the present invention, the substrate is cooked with the entire optimal dose of potassium ferrocyanide and ethylenediaminetetraacetate is added to the finished substrate at a temperature of 60 ° C and below. In the invention, a large pH effect is observed on the change in yield of citric acid and gluconic acid in the range of 6.0 to 7.2, while the Cs. patent

No. 135387 is satisfied with the mention of adjusting the pH to 7.0 to 7.2 (see Example 1 of the cited patent). Thus, the present invention differs from the quoted natent at essential points.

Example 1

In 660 parts of water, dissolve 800 parts of molasses, 0.46 parts of phosphoric acid (wt. 1.25), 2.0 parts of sulfuric acid (wt. 1.565) and 3.2 with stirring and heating to 80 ° C. parts of potassium ferrocyanide. The solution (containing about 32% sugar) is brought to a boil for 3/4 hours and then cold, non-toxic water is allowed to reach its final volume (sugar concentration is 15%). The temperature of the resulting solution is about 55 ° C, pH 6.65. Add 0.001 mol of disodium ethylenediaminetetraacetate to 1 L, bringing the pH of the solution to 6.50. After fermentation for 8 to 9 days, 60.6% citric acid (in% of sugar used) and only traces of gluconic acid are formed. Without the addition of ethylenediaminetetraacetate, yields of citric acid are 36.0% and gluconic acid 18.7%, calculated in% of the sugar used.

Example 2

With another molasses, a fermentation solution is prepared in the same manner as in Example 1, except that 1.8 parts of sulfuric acid and 2.9 parts of potassium ferrocyanide are used; The pH of the final solution is 6.95. Upon addition of 0.0013 mol of disodium ethylenediaminetetraacetate to a 1 L substrate, the pH drops to 6.79. The yield of citric acid in the fermented solution is 58.7% (calculated on the sugar used), the gluconic acid was not detectable. In a control experiment without the addition of ethylenediaminetetraacetate, yields of citric acid were 32.4% and gluconic acid were 27.1% (calculated on the sugar used).

Claims (1)

Process for preparing fermented molasses medium for lemon fermentation using potassium ferrocyanide and disodium ethylenediaminetetraacetate, characterized in that the molasses solution is boiled with an optimum dose of potassium ferrocyanide, i.e. a dose at which an optimum acid yield is obtained. OF THE INVENTION without further interfering with the fermentation solution of the molasses, the disodium ethylenediaminetetraacetate at a concentration of 0.0005 to 0.002 mol per liter of fermentation medium is added to the cooled solution at a temperature of 40 to 60 ° C and the pH of the fermentation medium is adjusted to 6, 0 to 7.2.