DD282471A5 - METHOD FOR CULTIVATING MICROORGANISMS - Google Patents

Abstract

The invention relates to a method for the cultivation of microorganisms. Field of application is the pharmaceutical industry. According to the invention, 0.5 g / l to 5 g / l of the derivatives of the polyvinyl alcohols according to Fig. I or the alkylcelluloses according to Fig. II or mixtures of both polymeric substances are added to the culture medium. The application is preferably carried out in the cultivation of Bordetella pertussis {cultivation; microorganisms; Pharmaceutical Industry; Culture medium; polyvinyl alcohol; alkylcelluloses; Bordetella pertussis}

Description

For this 2 pages drawings

Field of application of the invention

The invention relates to a method for the cultivation of microorganisms and for the production of products which are formed in the aerobic cultivation of microorganisms. Application is the pharmaceutical industry (antibiotic and vaccine production)

Characteristic of the known state of the art

Known are methods in which you a partial significant increase in cell yield or product formation is achieved ^r ch addition of non-essential materials to conventional, suitable for the cultivation of microorganisms culture media.

Thus it was found that additions of specifically substituted cyclodextrins !! (2,6-di-O-methylated β-cyclodextrins) have a diverse spectrum of activity in the cultivation of microorganisms (yield increase of lankacidin antibiotics, (A.SUZUKI et al., EP 91781-1983), stimulation of the formation of Pertussis antigens, [A.! MAIZUMI et al., J Clin. Microbiol., 17, 5 (1983)], growth stimulation of bifidobacteria, [C. YOSHIKUMI et al., DE patent 3205018)). These so-specific substituted cyclodextrins are relatively expensive due to their complex chemosynthesis. This limits their broad industrial application.

Furthermore, insoluble polymeric anion exchangers are known which can similarly bind growth-inhibiting substances (E. ROWATT, J. Gen. Microbiol., 17,297-326 [1957]). Because of their insolubility in the culture medium, however, these exchange resins substantially complicate the process control in cultivations in industrial bioreactors.

Known are attempts to increase the pertussis antigen yield by addition of soluble polyvinyl alcohols to a nutrient medium according to STAINER & SCHÖLTE (G.GREENSPAN, US Pat. No. 4,551,249).

Furthermore, surfactant-containing ethylene oxide-propylene oxide adducts and non-surfactant polyethylene glycols or mixtures of both substance classes were used to improve mass transfer or to change the surface tension in fermentative microwave protein recovery (SCP), with increases in biomass concentration being detectable by up to 20% (K.TRIEMS et al., DD Patent 201459; GA LUEBBERT et al., DD Patent 153495).

Object of the invention

The aim of the invention is to use in the cultivation of microorganisms cheap and non-toxic substances which promote the growth of the microorganisms and the formation of desired metabolic products.

Explanation of the essence of the invention

There were found water-soluble, polymeric compounds that be! Addition to the culture medium and subsequent cultivator of microorganisms cause a significant increase in cell yield and product formation.

The substances to be used in an inventive manner are derivatives of the polyvinyl alcohols (FIG. 1) as well as alkylated derivatives of cellulose (FIG. II). In the formula image according to FIG. 1, Ri denotes either an alkyl radical having 1-16 C atoms, or an acyl or an acetal radical, wherein 5-20% of the hydrogen of the hydroxyl groups of a polymer molecule is substituted by R, radicals. The molecular weight of the derivatives of polyvinyl alcohols according to Fig. I comprises a range between 5000 and 20,000.

In the alkyl derivatives of the cellulose of Figure Il, R ₂ , Ra and R _{4 are} either methyl groups, with between 27-32% of the hydrogen of the hydroxy groups being replaced by CH ₃ radicals, or R ₂ and R ₄ are methyl groups and R ₃ is a hydroxypropyl rust, wherein between 50% and 93% of all alkyl substituents are represented by methyl groups, or P ₂ and R j are methyl groups. Ind R ₄ is a hydroxybutyl radical, the proportion of which in the total mass of the cellulose derivatives being 2-5% by weight.

According to the invention, 0.5 g / l to 5 g / l of the substances according to FIGS. 1 and 2 are added to the culture medium.

In the case of the cultivation of Bordetella pertussis, an increase of de. Cell yield up to 80% and the yield of haemagglutinierer.den antigens (filamentous hemagglutinin FHA and pertussis toxin PT) reaches 3-30 times. The advantages of the method compared to the prior art are that the substances of FIG. I and FIG. Il are much cheaper than dis specifically substituted cyclodextrins and that, in contrast to the solid Anionenaustauscherha.zen that the process control in the fermentor through Obstruct the constipation of the leaching systems, are soluble in the culture medium.

Compared with the method described by GREENSPAN using unsubstituted polyvinyl alcohol with up to 144 hours of culture time and maximum antigen yield after 8 to 72 hours, very high antigen yields can already be achieved after 12 to 25 hours with the method according to the invention.

Ausführungsbelsplole

The method according to the invention is described below on the basis of the aerobic cultivation of Bordetella pertussis in a 1-I-Erlenmeyer flask.

The surprising effect of the substances added to the nutrient medium with regard to cell density and antigen yield will be explained by way of example on 3 representatives of the substance classes according to FIG. 1 and FIG.

For this purpose, an Erlenmeyer flask with 100 ml of culture medium (composition see Appendix) with the addition of 0.5-3.0 g / l of a derivative of the substance classes according to FIG. Il filled under sterile conditions and inoculated with Bordetella pertussis germs (TOHAMA Phase I). The starting density is 5 × 10 ⁹ -8 × 10 ⁹ germs / ml. The culture is shaken for 12-25 hours at 35-37 ⁰ C with a frequency of 150-17C min " ^1. Subsequently one determines the cell yield and the antigen content.

The anti-clade content is determined by the hemaggluination micromethod of SATO (Y. SATO et al., Inf. Immunity, 7 (6), 992-X99 (1973)).

The results of these embodiments are summarized in Table 1.

Table 1 Haemagglutination titer (HA titer) and cell yields after cultivation of Bordetella pof-jssis phase 1-l Erlenmeyer flask

in the

Nährmedienzusatz amount Kult. duration density HA titer (G / l) (Hrs.) (• 10 ⁹ germs / ml) without _ 23-25 70-120 128-256 Wofatit SBW (Chem. Kombinat Bitterfeld) 10-14 23-25 112-125 256-512 Polyvinyl alcohol (55/02) 1 24 150 1024 Polyvinyl acetate (Hoechst AG, FRG) 0.5 12 102-105 2 048 Trade name: Mowiol 18/88 0.5 15.5 123-126 2 048 see Fig. I. 0.5 21 158-161 4 096 0.5 25 193-195 1 024-2 048 1.5 12 109-113 > 4 096 1.5 15.5 136-137 > 4096 1.5 21 172-182 > 4 096 1.5 25 209-217 > 4096 3.0 12 103-109 4096 3.0 15.5 133-138 4096 3.0 21 170-173 > 4096 3.0 25 202-204 > 4096 Methylcellulose (DOW Chemical, USA) 1.5 18 123-127 > 4 096 Trade name: MethocelA4c 1.5 23 148-149 > 4 096 see Fig. Il Hydroxypropyl methylcellulose 1.5 18 121-124 > 4096 Trade name: Methocel K100 1.5 23 137-145 > 4096 see Fig. Il

attachment

Composition of the culture medium:

One liter of the culture medium used for cultivating Bordetella pertussis germs by the method according to the invention contains, in addition to the additives described:

casein hydrolyzate 1.3-1.5 g of amino nitrogen Glutamic acid (Na salt) 5g NaCl 2.4 g FeSCV 7H ₂ O 1 mg MgSO ₄ -7H ₂ O 10mg CuSO ₄ -5H ₂ O 0.25 mg cysteine hydrochloride 30 mg soluble starch 2g Hefedialysat 75g niacin 5mg nicotinamide 5 mg Water (AB-DDR) ad 1000 ml

Claims (7)

1. A method for the cultivation of microorganisms, characterized in that in a liquid nutrient medium, which carbon and organic nitrogen sources and inorganic salts and water-soluble, polymeric substances according to FIG. I and FIG. II, which are added before the beginning or during cultivation, contains, under microbial conditions, a microorganism strain is cultivated. 2. The method according to claim 1, characterized in that it is the derivatives of the polyvinyl alcohols according to Fig.I in the water-soluble, polymeric substances in which the hydrogen atoms of the hydroxy groups are substituted by alkyl, acyl or Ac jtalreste in such a way in that it affects between 5% and 20% of all hydroxyl groups of a molecule, the molecular weight of the derivatives thus formed being between 5,000 and 20,000. 3. The method according to claim 1, characterized in that it is the derivatives of cellulose according to the water-soluble, polymeric substances of FIG. Il, in which the hydrogen atoms of the hydroxy groups are substituted by methyl, hydroxypropyl or hydroxybutyl in such a way that of which between 27% and 32% of all hydroxy groups of a molecule are affected. 4. The method according to claims 1-3, characterized in that the water-soluble, polymeric substances according to FIG. I and FIG. Il both individually, as well as in the form of mixtures of the substances are added to the nutrient medium, that the total amount of these substances in the Nutrient medium between 0.5g / l and 5g / l amounts. 5. The method according to Ansprüchon 1-4, characterized in that the addition of the water-soluble polymeric substances in the course of cultivation optionally occurring foam is controlled by the addition of chemically acting antifoams or by mechanical Schaumzerstörer or by combination of both agents. 6. The method according to claims 1-5, characterized in that its application in the cultivation of the microorganism Bordetella pertussis with the aim of obtaining an effective against whooping cough vaccine based on a Vollkeim ^ akzine or on the basis of isolated and highly purified antigens of Bordetella pertussis takes place. 7. Process according to claims 1-5, characterized in that its application in the cultivation of the microorganisms Bordetella parapertussis and Bordetella bronchiseptica takes place.