DE2818668C3 - Microbiological medium - Google Patents

Description

OH
RC NR ₁

25th

where R and Ri = hydrogen, alkyl or aryl mean and m ° n under alkyl and aryl both non-substituted groups and groups, which are substituted with halogen or nitrogen, understands, was brought into contact.

35

The subject of the invention is the microbiological medium mentioned in the claim.

Liquid media for the growth of microorganisms in submerged culture fermentation are mostly sterilized with high pressure steam before they are inoculated with the desired product-producing organism. High pressure steam is an effective sterilizing agent for fermentation media; however, its use is associated with many disadvantages. One of the most obvious drawbacks is the cost of the energy required to heat and cool large reaction vessels and large volumes of fermentation media. which are nowadays commonly used for Fermentalionsverfahren, is required to, <> a time of high energy costs and limited energy resources is considerable economic disadvantage of steam sterilization.

In addition, high-pressure water steam makes the use of the use of inexpensive materials y, for the devices, such as fiberglass, impossible, and instead makes use of relatively expensive materials, such as stainless steel and steel with a high carbon content requires addition, a relatively long period of time necessary in order _{to effectively sterilize large volumes b} o of Mikrobert media, which has an unfavorable effect on the economy of the process. In addition, steam sterilization often leads to a loss of nutritional values in the microbial media as a result of excessive chemical and physical changes in the nutrient components, which leads to a reduction in the production of desired products and thus to a decrease in the production of nutrients Effectiveness of the overall process leads.

As an alternative to steam sterilization, the Biocidal chemical treatment of fermentation media known beta-propiolactone and ethylene oxide are for example, have been described as effective chemical sterilants; see »The Chemical Sterilization of Liquid Media with Beta-Propiolactone and Ethylene Oxide ", Toplan et al, in" Journal of Biochemical and Microbiological Technology and Engineering ", Volume III, No. 3, pages 311 to 323 (1961). Another chemical antimicrobial agent used in fermentation processes is formaldehyde (U.S. Patent 2,991,231), Furacilin (U.S. Patent 32 02 587) and antibiotics such as penicillin and streptomycin (US Pat. No. 3,494,832). With the exception of ethylene oxide, however, have aTL-chemical properties Means that have been used for the production of microbiological media are only limited biocidal effectiveness, d. h “They work specifically against or prevent certain classes of microorganisms merely the growth of certain contaminating organisms and are therefore only of limited value. Although ethylene oxide is a broad spectrum microbiocide with which one can obtain high levels of microbial purity is due to its use in commercial processes Safety concerns excluded. The high degree of flammability of ethylene oxide has the effect that it forms explosive mixtures with air in very low concentrations. Hence, its use in Fermentation processes are restricted to specially designed autoclaves, which operate under the strictest safety precautions must be operated, and therefore the use of ethylene oxide in processes in the hardly practical on an industrial scale.

The effectiveness of glutaraldehyde as a biocide is described in detail in the literature. In US Pat. Nos. 30 16 328, 36 97 222 and 39 12 450, for example, methods for sterilizing contaminated objects, such as surgical and dental appliances, are disclosed. described with glutaraldehyde solutions. However, it is precisely its strong effectiveness as a biocide that makes glutaraldehyde unsuitable as a chemical sterilizing agent for fermentation medicine. Glutaraldehyde has a biocidal activity against practically all types of microorganisms, including those organisms which are inoculated into the microbiological medium in order to produce the desired products by means of Kiiliurfermentation. In contrast to the sterilization with high pressure steam, ako does not decrease the biocidal effectiveness of (iltilaral dehydration after the liquid medium and the related support device have been sterilized. ils been considered as sterilizing agents for fermentation.itKinssysteine in common ancestors

The object of the invention is to create a sterile microbiological medium using olutaraldehyde as a sterilizing agent, the biocidal effectiveness of glutaraldehyde being switched off after the sterilization is complete. The culture medium is initially brought into contact with glutaraldehyde in a sufficient concentration to obtain the desired degree of microbiological purity.

ren the pH of the medium to a value of more than 6.5 set; then the medium with one or more nitrogen-containing compounds from the Group ammonia, primary amines, secondary amines, heterocyclic amines, ureas and amides (cf. Claim) brought into contact to inactivate glutaraldehyde and its biocidal effects turn off. The pH of the medium is above 6.5 during this inactivation stage held. The medium can then be inoculated with the desired organism in order to start the fermentation to initiate.

The term "sterilization" used here means reducing the level of microbial contamination to very low levels and is not im here used in the strictest sense, in which it means the absolute elimination of all impurities.

The present invention is therefore a microbiological medium produced by sterilization one to be used in microbe fermentation 2η the microbiological medium, characterized in that it has been obtained by

a) the medium with glutaraldehyde until reaching the desired level of microbiological Purity has been brought into contact,

b) the pH of the sterilized medium obtained in step a) was adjusted to above 6.5 and

c) the medium obtained in step b) with a medium sufficient to inactivate the glutaraldehyde Amount of ammonia, primary amines, secuno The amines, heterocyclic amines, urea compounds and the armies of the following Structure:

OH

RCN R ₁

where R and Ri = hydrogen. Mean alkyl or aryl and alkyl and aryl are understood to mean both unsubstituted groups and groups which are substituted by halogen or nitrogen.
Appropriately, alkyl groups with I to 6, better 4 ·; I used up to 4 carbon atoms.

The necessary operating conditions in the sterilization, such as the concentration of glutaraldehyde in the culture medium, the pH of the medium and the duration of the treatment, are determined by the desired degree of microbial purity for a particular fermentation process. At a pH of less than about h. ">, A glutaraldehyde concentration of less than about 0.5% by weight, based on the lermentalion nutrient solution, is generally sufficient 5 is held, usually results in a two-hour treatment at a Glular.ildchyd Konzentralion from about 0.05 wt ^u / o of the medium the desired degree of purity smikröbischer; wherein: a GlütaiaidchydkönzefH Iration from about 0.2 percent ₁ = ⁰ The duration of the treatment is generally about 30 minutes with the same temperature and the same temperature. The temperature of the liquid medium during the treatment can be between about 15 and 75 ° C ; a temperature of about 25 to 30 * C is favorable. the pH'Werl the medium kanri between about 2 and 10, but advantageously it is less than about 6.5, more preferably between about 5 and 6. at a pH of about 6.5 ammonium salts, which are often as a substrate component n are present in fermentation media, chemically react with glutaraldehyde, which means that significantly higher concentrations of glutaraldehyde are necessary to sterilize the medium. This reaction is of course also undesirable in that it removes from the medium nutrients intended to promote the fermentation reaction. Therefore, the sterilization of media containing ammonium salts, such as ammonium sulfate and ammonium phosphate, is expediently carried out at pH values below about 6.5 in order to prevent the reaction of glutaraldehyde with the ammonium compounds.

Nitrogen-containing compounds which, according to the invention, are suitable for chemically inactivating glutaraldehyde are, are z. B .:

Ammonia, methylamine, ethylamine,

Propylamine. Butylamine, tert-butylamine,

Nonylamine, octadecylamine, dimethylamine,

Diethylamine, dipropylamine,

Diisopropylamine, cyclohexylamine,

Piperazine, morpholine, urea,

Methyl urea, ethyl urea,

Dimethyl urine, formamide, g

Methylformamide, ethylformamide,

N-methylformamide, N-ethylformamide,

N-propylformamide, benzamide,

N-phenylformamide and the like

Ammonia is preferred in terms of availability and price.

According to the invention, the nitrogen-containing compounds can either be added directly to the medium become to react with free glutaraldehyde; however, if necessary, they can also be used with a pH values above 63 can be formed in situ from a coordinatively covalent ammonium compound. So can e.g. B. ammonium hydroxide and / or ethylammonium hydroxide are added to the medium where They turn into reactive at a pH above 63 nitrogen-containing compounds, namely ammonia or ethylamine. being transformed. According to the invention suitable k <· - 'dinatively covalent ammonium compounds are z. B .:

Ammonium hydroxide. Ammonium chloride,

Ammonium sulfate. Ammonium phosphate,

Ammonium acetate. Ammonium propionate,

Methylammonium hydroxide.

Ethylammonium hydroxide.

Methylammonium acetate.

Methacrylic sulfate.

Dimethylammonium hydroxide.

Diethylammonium acetai.

Dipropylammonium hydroxide and the like.
The nitrogen-containing compounds or, if appropriate, the coordinatively covalent ammonium compounds can be added to the medium in any suitable manner. If the compound is a gas. like ammonia, methylamine or dimethylamine, it can be blown directly into the liquid medium; Liquids such as ammonium hydroxide ₍ ethylamine, diethylamine U _1, etc., can be added volumetrically or flat weight directly to the medium. Solid compounds such as urea, ammonium phosphate and ammonium phosphate, are expediently filled with a solvent such as water, thinner and the

obtained solution is before addition to the medium mil |

Steam or with a chemical sterilant or sterilized by filtration.

As mentioned earlier, ammonium compounds such as ammonium phosphate and ammonium sulfate are used in commonly used as a nutrient in certain fermentation media. If the pH is this Medium is above about 6 5, these compounds can be converted into nitrogen-containing compounds which react chemically with glutaraldehyde. According to a favorable embodiment of the invention if an excess of a coordinatively covalent ammonium compound is added to the medium during the initial preparation, which exceeds the amount required as a substrate component for the fermentation broth admitted. The excess amount is expediently the etv / a stoichiometric amount of Anr with glutaraldehyde is required. The inactivation of giutaraldehyde can then be recorded by simply adjusting the pH of the medium to above 6.5 to prevent the formation of nitrogenous To favor compound from the coordinatively covalent ammonium compound. The pH of the The medium is then kept above 6.5 until the reaction with glutaraldehyde is complete.

Inactivation of glutaraldehyde can be carried out at any suitable temperature, although a temperature of about 20-30C is preferred. The pH of the medium can vary from about 6.6 to 14 jn; however, it is best between about 7 and 9. The reaction time generally varies between about 1/2 hour and 4 hours; it depends on the pH of the medium and on the effectiveness of the nitrogen-containing compound or the coordinatively γ-, covalent ammonium compound. Ammonium salts with groups of very weak acids, such as ammonium carbonate, generally require less time to inactivate glutaraldehyde than ammonium salts with groups of strong acids, such as ammonium sulfate. The ₄ n reactivity of some typical ammonium compounds is therefore decreasing in the following order:

Ammonium hydroxide. Ammonium carbonate.
Ammonium acetate. Ammonium phosphate. 4i

Ammonium chloride and ammonium sulfate.
Normally, for a fermentation medium at 25 ° C that is kept at a pH of about 7.5, a reaction time of about 1 to ¹ 1/2 hours is necessary for inactivation when a coordinatively covalent ammonium compound, such as ammonium hydroxide. is used.

In order to bring about complete inactivation of the glutaraldehyde, the amount of nitrogen-containing compounds in the medium should be approximately stoichiomic-Irish equivalent to the amount of glutaraldehyde in the medium. In particular, the number of reactive "NH" bonds should correspond to the number of available aldehyde groups in giutaraldehyde; per Mon! Glutaraldehyde are two aldehyde ω groups present ^{- For example, 2 1} mol "ammonia, which has three reactive" ά-Βίη 'fertilizers per mole, are required to react stoichiometrically with 1 mol of glutaraldehyde. Likewise, 1 mol ethylamine (which is 2 has reactive "NH" bonds per mole) or 2 JvIoI diethylamine (which has 1 reactive "NH" bond per mole) per mole of giularaldehyde.

If necessary, an excess of the troublesome amount can also be used.

example 1

The following experiment was carried out to address the sterilization of glutaraldehyde generation media for penicillin-G silver culture fermentation and to get a comparison with steam sterilization.

Three fermentation devices with a capacity of 14 l each, which are equipped with an automatic pH adjustment device, a dissolved oxygen probe, an automatic glucose delivery system, and a automatic foam control system (feed on demand) were all equipped with the the following growth medium:

component Amount, g Glucose 8:00 pm (NH ₄ I ₂ SO ₄ 120.00 KH ₂ PO ₄ 24.0G CaSO ₄ · 2H ₂ O 66.00 Na ₂ SO ₄ 4.00 MgSO ₄ • 7H ₂ O 5.60 FeSO ₄ 0.45 MnCl ₂ 0.40 ZnSO ₄ 0.40 CaCl 0.4 (1 CuSO ₄ 0.06 water 8000.00

The automatic glucose delivery system of each unit was primed with an aqueous solution containing 25% by weight Containing glucose and 2.08% by weight phenylacetic acid. these solutions were for 30 minutes at 121'C and sterilize with steam under a steam pressure of 2.08 ata L

The automatic foam regulating addition units were each with 100 g of a fermentation foam regulating agent charged, which throughout the attempts to combat foaming was used.

Fermentation preparation No. 1 and its contents were sterilized with steam for 45 minutes at 121 ° C and under a pressure of 2.08 ata. Of the The pH value after sterilization was 5.2 and was increased by adding a sodium hydroxide solution (5.0 n) 6.8 set. It took about 5 hours. • irr. the fermentation device and the contents 121 ° C to maintain the temperature for the prescribed time and then the Device for inoculation to cool to 24 ° Cao.

Fermentation device No. 2 was not sterilized and the pH of the medium was 5.6. This pH was maintained for 1 hour and then increased with a sodium hydroxide solution (5, Q n) 6.8 set. The temperature of the medium was 24 ° C

32 g of a 25.2% by weight aqueous glutaraldehyde solution was added to the contents of fermenter # 3, resulting in a glutaraldehyde content of about 0 ₍ 1% by weight based on total charge. The temperature of the medium was 24 ° G immediately after the addition

the pH of the medium was 5.5 and this value was maintained for 1 hour. After that it was the pH of the medium was adjusted to 7.0 using a sodium hydroxide solution (5.0 nj. During the following 80 minutes, the pH of the medium was automatically adjusted by the pli adjustment device set and held at a value of 6.8. It should be noted that - while part of the (NfU ^ SOj in solution is converted into ammonia, which then rengiert with the Glutarnldchycl - the pH value of the medium tends to decrease due to the presence of sulfal ions in the solution. It is therefore the Regular addition of an alkaline material, such as sodium hydroxide, is necessary to keep the medium up to keep a constant pH. As soon as the • inactivation of glutaraldehyde is complete. is the pl I-word of the medium is constant.

vvii ι miiuili:

were each with 200 ecm of an inoculation medium, which under Using a particular strain of Penicillium chrysogenum had been bred, vaccinated and 48 Incubated for hours at 25 "C in shake flasks with a capacity of 500 ecm. The wet volume of the My / els in the inoculum was after centrifugation at 5000 rpm, for a period of 15 minutes 16%.

The speed of each inoculated fermenter was set at 360 rpm and the air flow rate was set at 4000 cc / min. set. The temperature in each device was maintained at 24.0 ± 0.5 ° C throughout the fermentation. The saturation dissolved oxygen level in each device was 8.8 mgO ₂ / liter at an indicated value of 100%.

At 24 hours after inoculation, the dissolved oxygen content of the nutrient solution was in each Fermentation device as follows: No. 1-97%: No. 2- 10%; No. 3 - 95%. A microscopic one Examination using a simple staining procedure revealed that Ferments Nos. 1 and No. 3 predominantly the organism Penicillium chrysogenum with practically no trace of other contaminants Contained organisms. In contrast, ferment No. 2 contained only traces of Penicillium chrysogenum and also considerable amounts of contaminants consisting of bacilli and cocci bacteria. As a result of the strong presence of contaminating organisms in fermentation device No. 2 it became necessary to stop this fermentation after 30 hours.

The fermentations in devices # 1 and # 3 were continued for 196 hours and were facilitated by adding a sterile glucose-phenylacetic acid solution to each device. After 196 Hours of microscopic examination of the nutrient solutions revealed that both cultures were essentially off Penicillium chrysogenum passed with no visible evidence of contamination.

The penicillin G content of the nutrient solutions was measured by the method of Bethel and Bond (J. Anal. Chem-Volume 86, pages 448 to 457, 1961) and was as follows: 865 units / ml in fermenter # 1 and 858 units / ml in fermenter # 3.

Example 2

The following experiment was performed to test the sterilization of a Cephaiosporin wCft fermentation medium with glutaraldehyde to show and around a

Comparison with water vaporization n.

Three Fcrmentatioiisvofrichliingch with 14 l capacity were all with the following Ccphaiosporin Fermentaliohsniedium loaded:

component Crowd, {j Beet molasses 240.0 Animal rest .120.0 proteins Corn steep water 40.0 D, L-methionine 40.0 Methyl oleate 160.0 CaCO, 12.0 NII ₁ CO ₁ 7.0 Silicone angled foam U, 160 middle water 8000.00

Formation Device No. I and its Contents were sterilized with steam for 45 minutes at 12PC and under a pressure of 1.05 aiii. That Sterilizing the system and lowering the temperature to 29 "Ci .1111 vaccination took about 6 hours.

The pH of the medium in the fermentation device No. 2 was set to 5.5 and the temperature was held at 29 "C for 1 hour. The pH was then adjusted to 6.8 using an ammonium hydroxide solution (30% w / w).

To the contents of fermentation device No. 3, 64 g of 25% (wt.%) Aqueous was added Glutaraldehyde solution was added and the temperature was maintained at 29 ° C. The pH of the medium was 5.5 after the addition of glutaraldehyde. and this value was maintained for 1 hour. Thereafter the pH was adjusted by adding an ammonium hydroxide solution set to 6.8 and held there for 1 hour.

The fermentation devices No. 1, 2 and 3 were then each with 500 ecm of an inoculation medium, the 48 Grown for hours using a specific strain of Cephalosporium acremonium was inoculated and fermentation was carried out at 29 ° C using a stirring speed of 300 rpm, and an air flow rate of 4000 cc / min. carried out.

After a fermentation of 96 hours it was found that the fermentation devices No. 1 (sterilized with steam) and No. 3 (treated with glutaraldehyde) each more than 500 units / ml Contained cephalosporin "C". Fermentation device # 2 (non-sterilized) did not contain any Trace of cephalosporin "C" and microscopic examination showed it was at least 5 overgrown with various types of contaminating organisms.

Example 3

The following experiment was carried out to test the sterilization with glutaraldehyde of production media to demonstrate that used in the production of ethyl alcohol through anaerobic fermentation and to make a comparison with steam sterilization.

Two anaerobic exercise devices with 4 liters each Capacity were each charged with 2000 cem of distilled water and 50C / g Minncsoia beet molasses.

FerinenialiofmOrrichlung No. I was (5 min long at 121 ° C and under a steam pressure of 1.05 atm in an autoclave with steam treated. The pH of the medium in the fermentation (ionizing device was then adjusted to 4.9 by adding lactic acid.

Fermcntaliönsvorrichtung No. 2 was 5 g of a 25% strength (% by weight) aqueous glutaraldehyde solution was added, the pH being 5.6 and the temperature Was 25 ° C. After 20 minutes, the pH value was increased by adding about 8 ecm of a 30% (% by weight) Ammonium hydroxide solution adjusted to 7.0 and held at this level for 1 hour. After that it was the pH value adjusted to 4.9 with the help of lactic acid.

The fermentation devices No. I and 2 were each inoculated with 2 g of Saccharomyccs ccrevisiae, and the Fermentation was carried out at 25 ° C.

The ethyl alcohol content of the nutrient solutions was determined by gas chromatography, the received the following results:

Time Ethanol, vol .-% (± 0.1%) Fermentation reservoir Fermentation SId. No. I. vorr. No. 2 0 0 0 12th U 1.4 24 4.6 4.8 48 6.0 5.8

component

Amount, g

Yeast extract 24

Malt extract 24

Peptone 40

Glucose 160

Distilled water 8000

I)

2 (1

SO

35

Example 4

This example aims at sterilization with glutaraldehyde of production media used in the manufacture of xanthan gum show and a Create a comparison to steam sterilization. This example also shows what the effect of simply adjusting the pH of the culture medium following the Sterilization raised to above 6.5, however, failed to inactivate the medium a nitrogenous compound add the glutaraldehyde (fermentation device no. 2).

Three fermentation devices, each with 141 casks. capacity were all charged with the following xanthan gum production medium:

The fermentation device No. I was ^{sterilized for 20 minutes at 121 °} C. and under a pressure of 1.05 atmospheres mil water vapor. The pH of the medium was adjusted to 7.0 by adding sodium hydroxide.

Fermentation batch # 2 was made at 29 ° C sterilized by adding 30 g of a 25% strength (% by weight) aqueous glutaraldehyde solution, the pH was adjusted to 5.5. The pH was held at 5.5 for 1 hour. After that he was through Addition of a 5N sodium hydroxide solution adjusted to 7.2 and at this value for 2 hours held.

Fermentation device no. 3 was sterilized at 29 "C by adding 32 g of a 25% (% by weight) glutaraldehyde solution, the pH being adjusted to 5.5. The pH was adjusted to J for 1 hour ₁ J gciiiiiicii.Dtii'iUL'n wind cf diii'ün additions of about 8 ecm of a 30% (wt .-%) ammonium hydroxide solution to 7.0 and held at this value for 2 hours.

All three fertnentationsvorrichtungen were then with 1000 ecm of an inoculation medium, which pure Xanthamonas campestris cultures, inoculated, and fermentation was carried out at 29 ° C, at a Air flow rate of 4000 cc / min. and at a stirring speed of 300 rpm.

Immediately after inoculation, the dissolved oxygen level in the nutrient solution decreased in each Fermentation device down to about 92% saturation. what on the growth of Xanthamonas campestris indicates. The dissolved oxygen content in the fermentation device was within 15 minutes No. 2 again 100%; this suggested that the biocidal properties of glutaraldehyde were still effective were, because the medium did not contain any ammonium compound ioder nitrogen-containing compound had been added to inactivate the glutaraldehyde. Of the Dissolved oxygen levels in Fermenter # 2 remained the same for the entire 48 hours Fermentation to 100% and no change in viscosity was observed.

The content of dissolved oxygen in the fermentation devices Nos. I and 3 continued to decline and reached a level of saturation within 12 hours of 0. The stirring speed was set to 500 rpm in these fermentation devices, to maintain a dissolved oxygen level of about 20 to .30%. The viscosity of the nutrient solutions in fermenter # 1 and # 3 continued to gain weight over the next 48 hours, and increased At the end of the fermentation the solutions were practically too viscous to flow.

A microscopic examination of the extremely viscous flowable media in the fermentation device No. 1 and No. 3 showed that the cultures were predominantly the organism Xanthamonas campestris contained. Microscopic examination of the liquid in fermentation device # 2 revealed that only a very small amount of this organism was present.

Claims (1)

Claim: Microbiological medium made by sterilizing one during microbe fermentation microbiological medium to be used, characterized in that it is obtained has been by a) the medium with glutaraldehyde until achieved the desired degree of microbiological purity has been brought into contact, b) the pH of the sterilized medium obtained in step a) is adjusted to above 6.5 was and c) the medium obtained in step b) with a ts Inactivation of glutaraldehyde sufficient amount of ammonia, primary amines, secondary amines, heterocyclic amines, urea compounds and amides of the following Structure: