DD149875A3 - PROCESS FOR MICROBIAL PRODUCTION OF A MIXTURE OF CITRIC ACID AND ISOZITRONIC ACID - Google Patents

Abstract

1. A process for the microbial production of a mixture of citric acid and isocitric acid by yeasts on different carbon sources, characterized in that the nutrient solution in the growth phase just enough of a nitrogen source is added to obtain a biomass concentration 5 to 15 g / l, and that in the subsequent production phase in known per se conditions for a citric acid accumulation (acidic pH, trapping of the acid, presence of effectors, good ventilation, etc.). 2. The method according to claim 1, characterized in that both phases, the growth and the production phase, are performed as spatially separated process stages.

Description

170 314 -

The invention relates to a process for the preparation of a mixture of citric and isocitric acids by yeasts with carbon sources such as n-alkanes, sugars or short-chain aliphatic oxygen-containing compounds.

The accumulation of the citric acids by yeasts is "known to occur after completion of the ϊ / achstuinsphase, which is indicated by a maximum Hefebiomassekonzentration and depletion of the nitrogen source.Generally, the procedure is that .a citric acid-forming strain, mostly belonging to the genus Candida, is pulled out in a seed culture and inoculation with a fermentation solution containing, in addition to customary salts and possible supplines, a suitable source of carbon in concentrations corresponding to the amount of citric acid mixture to be obtained and mostly containing calcium carbonate for deadening the acids, numerous inventions take into account the influence of 'on the Synthesis involved enzymes by certain chemical agents and describe the addition of heavy metal ions and other effectors, thereby increasing the acid excretion or promoted the formation of citric acid at the expense of isocitric acid In particular, strains of the species Candida lipolytica are described as particularly effective in the yeasts of the genus Candida. Also, the breeding of high-performance strains by mutation is known. Also, species such as Candida zeylanoides are listed, which are particularly suitable for a citric acid production. The amounts of excreted acids are given in very different ways. They may, in the most favorable case, be 200 g / l citric plus isocitric acids, although angages η are the most frequent between 50 and 100 g / l. ,

170 314 - ^ -

A disadvantage of the known method is that a high biomass concentration is generally desired, which is not desirable in every case, because a high biomass concentration required a correspondingly high amount of starting materials, stirring and aeration energy and a.j3 cleaning operations

The purpose of the invention is the preparation of a mixture of citric acid and isocitric acid in high yield with reduced use of starting material.

The invention is therefore based on the object to modify the proportions of nutrients.

According to the invention, the production of the citric acid is carried out in such a way that the actual production phase is carried out by imitation by the nitrogen source. This means that just as much of a nitrogen source is added to the medium as an undisturbed propagation of the yeast compound high Zitroneη acid formation optimal amount is needed. Surprisingly, this amount is at low concentration values. At values between 5 and 7 g of yeast dry matter, the maximum of the citric acid accumulation has already been reached, which is maintained over a wide range up to about 15 g / l

Specifically working »vie follows: yeast, preferably of the genus Candida is grown under aseptic and aerobic conditions in a medium that contains just as much of a nitrogen source, a biomass ¹ volume between 5th to 15 g / l (as dry matter) to As a reference value for the calculation of the amount of nitrogen source - usually an ammonium salt - serves the relationship .:

Targeted amount of biomass

N source (calculated to N) in g / l = in g / 1

12.5

assuming a yeast protein content of 50 % .

The other components of the medium - and LiLneralsalze \ depending on the need of the yeast strain also Supp- ι line (such "B autolysate.) Are in a known Bey contraction, parked on the amount of nitrogen source submitted. The carbon source and the calcic acid carbonate (as another substance to trap the acids): may, already at the beginning of the cultivation in full height or else the part exclusively to the citric acid; Synthesis diont, be added after completion of the growth phase. The amount of calcium carbonate is expediently to be such that the pH value does not become less than 3 until the end of the process. After completion of the growth phase, the process is continued under aerobic and aseptic conditions for about 3-5 days.

It is perfectly within the meaning of the invention, if for a Zi-. troneηSäurenproduktion cultured yeast strain is cultivated in a separate propagation stage and the actual citric acid synthesis takes place in a second or in several stages, the individual stages spatially separated, can be performed in their own fermentors *

A separation according to the invention in at least two phases allows the addition of such substances to the phase of citric acid excretion, which influence these or reduce the proportion of isocitric acid in favor of the proportion of citric acid and vice versa. Such substances are for. Ferric, lead, zinc and manganese ions, fluoroacetate or citrate; Methanol and certain vitamins such as thiamine or vitamin-containing substrates, such as yeast autolysate · *

The following examples illustrate the method according to the invention; ren::

'. Example Λ . ·

In a Hührfermentor of 1 1 volume 550 ml of a "nutrient solution of the following composition are sterilized:

"0.4 i> NH ₄ Cl

0.1 % KH ₂ PO _{4 :} ·; ν- 0.05 ίο MgSO ₄ ... 7 H ₂ O

0.5 %. Yeast extract (70 #)

0.5 % CaCO ₃

20 ml of Para, ffinöl- (η-paraffins ^c -jo ~

The fermentor is then inoculated with 40 ml of a well-agitated, 18-hour shake culture of Candida lipolytica. "The culture broth nutrient solution had the same composition as above. * Air is vented at 50 l / h and the temperature is raised to 30 + 10 ⁰ C held * After 40 h, the biomass synthesis is terminated, the Fermentation solution contains 1.5 % yeast dry

The fermentation solution is transferred under aseptic conditions Id to a sterilized fermenter of the same type as above, in which a pH - value of 4.5 to 5 is kept constant by means of a regulating device with the aid of constantly slurried 10% lime milk. In addition, it is aerated with 60 1-Laft / h. After 10 hours aseptically add 100 ml paraffia oil (η-paraffins C, q - C ₂ q) »After another 80 hours the fermentation is over · The" volume of the fermentation solution is 305 ml "

The yield is - calculated to the initial volume of 600 ml - 31 g / l citric and 60 g / l isocitric acid ·

Example 2 .

1 l of a nutrient solution, which is filled up with tap water, eathält

1.5 ml paraffin oil (n ~ araffine C ^ ₀ - C ₂₀ )

0.6 g of N (as NH.Cl)

0.5 g KH ₂ PO ₄

0.2 g of MgSO ₄ .7H ₂ O

0.02 g CaCl ₂

2.5 g yeast extract (70 % dry matter)

In each case 100 ml of this medium are filled with 10 ml 500 ml shake flasks, sterilized and inoculated with 10 ml of a cocaine solution of Candida lipolytioa. Shake at 30 ° C. for 30 hours.

β

Thereafter, the η content of the fermentation solution is Ό, Ό08 g / l and the / biomass concentration 7.5 g / l · Aseptically added to each flask 8 g CaCO-, 15 ml paraffin oil and the flasks are shaken for 4 days at 30 ⁰ C. , The yield is 50 g / l citric acid and 55 g / l isocitric acid ·

Example 3 Γ. , , , '.': \ '.': "' ' .Λ-. , -.. '.'.. '' 'Will be used the same medium and the same Verfahrensdurchführüng as in Example 2 »only the paraffin oil is sugar (20 g / l glucose for the Wachstuinsphase and 50 g / l for the production phase) The yield of citric acid (isocitric acid was not determined) is 35 g / l · ·: ·,;

Example 4 '/ ^r -.''' - ·

0.8. g / l. N (as

20 v ^{ml of} " ^v " η-paraffins (

.0.5 g £ H2 ^P0 4

0.2 g MgSO _A ".7 H ₂ O

H-

0.02 g. CuSO ₄

_: 0.01 g of ZnCl ₂

0.02 'g CoSO ₄ .7H ₂ O,

0.02 g MnSO ₄ .4H ₂ O

"0.02 g of CaCl ₂

are filled with tap water to 1 · 1, "zehü; Stokes elkolber ¹⁾ 'by 500 mL; volume: filled, sterilized, inoculated with 10 ml of an inoculum of Candida guilliermondii and for 40 hours at" per 100 ml in '30 ⁰ C shaken , Thereafter, the N-,. Content of the fermentation solution 0.02 g / l and the biomass concentration 10 / g / l .. To each. Shake corpus added under aseptic conditions 1 ml of 10% yeast autolysate solution and 5 g CaCO 2. .

Shake vigorously at 30 ^° C. for 4 sec. The yield is 10 g / l citric and 25 g / l IsiDzitron acid.

Claims (2)

170 314 - ⁶ - Patent to spriiche; ~~~ "_._...-- .._: ... - 1 · Method for micromixing Preparation of a mixture. of citric acid and isocitric acid by yeasts on different carbon sources, characterized in that the nutrient solution in the growth phase contains just enough of a nitrogen source to obtain a biomass concentration of 15 to 15 g / l and in the subsequent production phase in per se "known" conditions for a lemons acid accumulation (acidic pH, trapping of the acid, presence of effectors} good ventilation and the like.). 2. The method according to claim 1, characterized in that "both phases, the Y / achstuius- and the production phase _{s are performed} as spatially separated process stages.