CS242635B1 - A method of preparing a fermentation broth for oefaloaporin C biosynthesis - Google Patents

Abstract

The invention describes aerobic fermentation with the strain Acremonium chrysogenum on a suitably digested soil before etherification. The required suitable ratio of dissolved carbon and nitrogen is achieved by adding 2-8% by weight of waste mycelium of Acremonium chrysogenum based on 100% of the fermentation soil to the fermentation soil before etherification, isolated from a previous completed fermentation of cephalosporin C by the production strain Acremonium chrysogenum. This mycelium contains proteolytic and amylolytic enzymes and at the same time own organic sources of carbon and nitrogen from the cell mass and from the residues of the captured unconsumed fermentation soil. After adding such waste mycelium to the fermentation soil, after 60 minutes of mixing at 50°C, hydrolytic processes are intensified, which increases the soluble nitrogen in the soil and reduces the degree of polymerization of carbohydrates. Ultimately, this leads to an increase in the yield of cephalosporin C production.

Description

(54) Method for the preparation of fermentation broth for biosynthesis of oefaloaporin C

The invention describes an aerobic fermentation with a strain of Acremonium chrysogenum on a suitable digested soil prior to eterilization. The desired appropriate dissolved carbon to nitrogen ratio is achieved by adding 2-8% by weight to the fermentation broth before eterilization. Acremonium chrysogenum waste myelium based on 100% of the regeneration soil isolated from the previously completed fermentation of cephalosporin C by the production strain Acremonium chrysogenum. This mycelium contains proteolytic and amylolytic enzymes and, at the same time, actually organic sources of carbon and nitrogen from the cell mass and from the residues of the unused fermentation fall. After the addition of such waste myelium to the fermentation broth, after 60 minutes of stirring at 50 ° C, the hydrolytic processes are intensified, thereby increasing the soluble nitrogen in the soil and reducing the polymerization degree of carbohydrates. Ultimately, this leads to an increase in the yield of cephalosporin C production.

242 635

1242 635

The present invention describes a process for the preparation of a fermentation pod for the biosynthesis of cephalosporin C (CFS-C) by fermentation with the strain Acremonium chrysogenum.

Cephalosporin C is produced by submerged aerobic fermentation using a suitable culture, in practice, mostly with strains of Acremonium chrysogenum and suitable sterile nutrient sources. substrates and fats and their hydrolysates, which also act as defoamers. As sources of organic nitrogen are used peanut, soybean, bone, fish meal, corn extract, cotton seed extract, various yeast cell hydrolyzates and others. According to the enzymatic metabolism of sulfur in the production strains, either inorganic sulfur such as (NH4) / gSQ ^ or organic compounds, of which DL-methionine has proven to be the best, can be used as the sulfur source.

These patents are protected by U.S. Pat. No. 3,816,257, issued May 11, 1974, and by U.S. Pat. No. 3,082,155, issued October 19, 1963 and U.S. Pat. U.S. Pat.

239 j21 of 22.2.1973 and MS. AO No. 198,425 dated 8 August 1932.

Since it is known that the formation of cephalosporin and its by-products can be controlled by a suitable ratio of dissolved carbon and nitrogen, the fermentation broths are sometimes cleaved to the desired level before sterilization by a hydrolytic procedure. For this purpose, enzyme preparations are used which catalyze such hydrolysis and / or hydrolysis. more drastic chemical routes using acids or bases.

Our proposed method of preparation of the fermentation broth for the cephalosporin biosynthesis is characterized by using waste mycelium Acremonium chrysogenum isolated from previous production as a substrate having proteolytic and amylolytic enzymes and at the same time actually organic sources of carbon and nitrogen from the cell mass and from the retained fermentation broth. E.g. with the addition of 2.5 wt. % wet filter cake containing 30 wt. dry matter after

242 635 production of cephalosporin C for the finished fermentation broth, the soluble amino nitrogen increased by 3% at 60 ° C and stirred for 60 minutes, and the polysaccharides experienced a polymerization step of 3%. · Ultimately, this resulted in an increase in cephalosporin production activity of 19%. %. A calmer advantage of such a method of preparation is that the wastes that are laborious to dispose of are partially reduced.

The examples given above merely illustrate the principle of the invention, but do not limit its validity.

Example no

Replacement of 0.5% by weight. % of the solid components of the fermentation broth e. waste mycelia in the pdde for cephalosporin C biosynthesis by fermentation with the strain Acremonium chrysogenum. As fermentation platform, the following composition was used: corn starch 4, 13% w / w, peanut agar 5.85% w / w, mycelium waste 0.5% w / w, glucose 1% w / w, sucrose 0, 48) wt.%, CaCO ^ 0.97 (wt.%, Methyl oleate 2, ≥4 wt.%, DL-methionine 1.65) wt.%, PH after sterilization 7.1. The idda was maintained at 30 ° C (60 ° C) for 60 minutes after mixing all components, while the weight loss resulting from the evaporation of the soil was compensated by the addition of water.

This stage was compared in a laboratory fermentation experiment with a control stage designated as production stage XIII of the following composition: maize starch 4.5% by weight, peanut flour 6)% by weight, glucose 1.23% by weight, sucrose 0.5 % by weight, CaCO-1)% by weight, methyl oleate 2, s)% by weight, DL-methionine 1,?

Both steps were sterilized at 12 ° C for 30 minutes in an autoclave and inoculated with 1% v / v inoculum of Acremonium chrysogenum CCM-F-644 prepared 43-72 h by culturing the production strain on the inoculation stage designated as Inoculation stage V. sucrose 1.5% by weight, ammonium acetate 0.3% by weight, DL-methionine 0.1% by weight, maize extract Ο, βφ by weight, methyl oleate Ο, φ by weight. followed by 48-72 h cultivation on the production stage XIII.

Fermentation was performed on a rotary shaker at 3 Hz · 40 mm eccentric at 27.3 * 0 in 500 ml beakers filled with 40 ml fermentation flask. Eermentation lasted 168 hours. At the end of fermentation, activity in the test and control flasks was determined by the Claridge spectrophotometric method in three parallel flasks. The stick values were averaged and the mean value was expressed in vyjád relative to the control.

Results:

check trial podium

500 µg / ml δ 50 µg / ml

-3Example δ.2

Substitution of mass. solids of fermentation broth waste mycelia in the cephalosporin C biosynthesis soil by fermentation with the strain Acremoniua chrysogenum. Composition of the experimental soil: corn starch 4.1 wt.%, Peanut flour 5.1 wt.%, Mycelium ^ wt.%, Glucose 1.15 # wt., Sucrose 0.4 # wt. 9% by weight, methyl oleate 2, 4% by weight; DL-methionine 1; Conditions for preparation of test soil, preparation of inoculum, fermentation and evaluation of the experiment as in Example 1.

Results control 8 500 µg / ml test platform 8 680 yug / ml,

Example of phenot

Replacement of 2% solids of the fermentation broth with 2% / trash mycelium in the cephalosporin C biosynthesis broth by fermentation with strain Acremonium chrysogenum. The composition of the test pod as in Example 2, but after mixing all the components, the test pod was briefly heated to 90 ° C and then stirred for 30 minutes with gradual cooling at ambient temperature of 20 ° C. Inoculum preparation, fermentation and experiment evaluation as in Example 1.

Results: control of 8,500 µg / µL of experimental conditions 8,620 yug / ml.

Example 4.

2% wt. solids of fermentation broth with 2 wt. waste nycellia in the cephalosporin C biosynthesis lead

Composition of the experimental soil: corn starch 4.1% by weight, peanut flour 5.1% by weight, mycelium waste 2% by weight, glucose 1.15% by weight, sucrose 0.45% by weight, CaCOCO 0.95 1.5 wt.%, rapeseed oil unrefined 1.5 wt.%, soybean oil 0.07 wt.%, DL-methionine 1.5 wt. This pod was compared with a β pod labeled as production pod XIII modified with the following composition: corn starch 4.5 # wt., Peanut flour 6 # wt., Glucose 1.25 # wt., Sucrose 0.5 # wt., CaCO 3. 1 # wt., Unrefined rapeseed oil 1.7 # wt., Soybean oil 0.8 # wt., DL-methionine 1.7 # wt.

Both lines were inoculated with 10 # vol inoculum of Acremonium chrysogenum CCM-F-644 prepared 48-72 h by culturing the production strain on an inoculation broth consisting of: sucrose 1.5 # weight, ammonium acetate 0.5 # weight, DL-methionine 0.1% w / w, maize leach 0.88% w / w, rapeseed oil unrefined 0.8% w / w, followed by 48-72 hours cultivation on a composition consisting of: corn starch 4.5% w / w, ara-41

242 635 Aid flour 6% by weight, glucose 1.25% by weight, sucrose 0.5% by weight, CaCO3 1% by weight, rapeseed oil unrefined 2.5% by weight, DL-methionine 1.7% by weight .

In addition, the conditions of soil preparation, seed material, fermentation and evaluation of the experiment as in Example No. 1 are as follows.

Results: control 8,000 µg / ml experimental soil 8040 µg / ml.

Example δ.5

Substitution of 3% wt. % solids with 3 wt. wasteful mycelia in pdde for cephalosporin C bioeynesis

Composition of experimental soil: maize extract 8% by weight, corn starch 2.9% by weight, dextrin 5.5% by weight, glucose 0.46% by weight, sucrose 1.38% by weight, CaCO ₃ 1% by weight. , / NH _4/2 S0 _4, 1.29 wt.%, MgS0 ₄ 0.27 wt.%, KH ₂ P0 ₄ 0.45 wt.%, soybean oil 2.3% vol.

This pdda was compared to the control pdda labeled as pdda production IIIa of the following composition: maize leach 8.7 wt%, corn starch 3.2 wt%, dextrin 6 wt%, glucose 0.5 wt%, sucrose 1, 5% by weight, MgSO ₄ 0.3% by weight, KH ₂ PO ₄ 0.5% by weight, soybean oil 2.5% by volume

Both soils were inoculated with 10% by volume inoculum of Acremonium chrysogenum CCM-F-750 prepared for 72 h by culturing the production strain on an inoculum seed with the composition: sucrose 2% by weight, ammonium acetate 0.5% by weight, DL-methionine 0.1% % by weight, corn liquor 0.88% by weight, methyl oleate 0.3% by weight

Moreover, the conditions for preparation of the soil, seed material, fermentation and evaluation of the experiment as in Example No. 1 are used.

Results: control 9050 µg / ml experimental pdda 9140 µg / ml.

Claims (1)

SUBJECT MATTER 7 f 242 635 Method for the preparation of the fermentation broth for the biosynthesis of cephalosporin C by fermentation with the strain Acremonium chrysogenum characterized by. The method according to claim 1, wherein the mycellium Acremonium chrysogenum with a dry matter content of 30% by weight is used as a supplementary source of nutrients, proteolytic and amylolytic enzymes. obtained after filtration of the fermentation broth from the previous cephalosporin C fermentation in an amount of 2 - 4 wt. based on 10% of the fermentation broth.