DD226590A1 - MEANS FOR PHAGENHEMMUNG IN MICROBIAL PRODUCTION PROCESSES - Google Patents

Abstract

The invention relates to agents for the inhibition of DNA and RNA phages in microbial production processes and thus for the stabilization of biotechnological processes. The task was to find chemical agents that inactivate and / or inhibit their proliferation due to their chemical constitution after addition to production formulations, so that phage-related process disturbances are prevented. The object is achieved by using agents which contain differently substituted piperidinoacetanilides or compounds derived therefrom. The compositions according to the invention are added to the fermentation compositions directly or in dissolved form. They may also contain solubilizers and / or other formulation auxiliaries. The compounds of the invention act against various DNA and RNA phages in different hosts and are particularly suitable because of their broad spectrum of activity for securing phagengefaehrdeter production processes.

Description

Title of the invention;

Means for phage inhibition in microbial production processes

Field of application of the invention

The invention relates to the stabilization of production in phage-prone biotechnological processes, insofar as they are operated using or gaining prokaryotic organisms (bacteria, cyanobacteria).

Characteristic of the known technical solutions

Measures to reduce or suspend phage risk in microbiological-biotechnological processes, which are increasingly being used primarily for material production, substance transformation and biomass production, are of considerable importance. Phage infections and their negative effects in bacterial production processes can be avoided with certainty by aseptic ("sterile") fermentation. Because of the considerable cost involved, such a method can only be used for products that are tolerable at a high price (eg antibiotics). In addition, phagesein fractures are also in sterile procedures, z. As by technical defects or lysogenic bacterial strains containing phages in the form of prophages in the bacterial cells themselves, possible.

Another way to reduce phage risk is to recover and utilize bacterial mutants that are resistant to one or more phages. The limits of this method are

that there is no resistance to all phages of a bacterial strain,

- that the resistant mutants are mostly no Hocbleistungsstamme,

- and that the pbagen in turn can mutate again and thus break the resistance of the host organisms

... (z BS Seto, T. Osawa and acid Yamamoto, Agr Biol Ghem · 22, 261-266, 1968; T. Oki, and A. O _z aki, Agr Biol · CBEM 22, 320 -.. 328, 1968).

Analogous to the virus chemotherapy or Propfaylaxe in other organisms, the direct control of phages has been proposed by means of suitable chemicals for microorganisms. However, this has so far also led only to partial successes. Examples of this type are the use of calcium-binding or chelating substances, reducing chemicals, base analogues, amino acids, antibiotics or other compounds against phages, which are harmful factors in the microbial production of acetone and butanol, amino acids, nucleic acids, antibiotics, dairy products, biomass u * a. 1978, pp. 16 and 26, for further examples: N. Kaahima et al., Agr. Biol. Chem. 4Ό, 41-47, 1976, H. Lipavska et al , Folia microbiol., 22., 60-63, 1978). The general application of such agents is contrary to the fact that the substances in the phage-effective concentration can also inhibit the bacterial proliferation of the production strain itself. Another disadvantage is the phage specificity of the agents. The compounds listed each acted against only one or a few biotecbnologisch important phages. For example, the indole specified as Pfaageninhibitor for Bacillus subtilis (T. Ukita and K. Kusai, Jap Pat, No. 32 - 1097, 1957) has no influence on the propagation of PZ phages in a production strain of this bacterial species according to own investigations. Also, the now-developed phage inhibitors with a broader spectrum of efficacy (B. Kiesel et al., DD-IAP Ή Ή? -, 1981;

) are not applicable in all potentially possible production strains and are not effective against the totality of all phages. The cbemoprophylaxis or therapy of phagal infections in microbial production processes to be used independently or as part of integrated measures therefore requires the widest possible range of different pabin inhibitors.

Object of the invention

It is the object of the invention to reduce the phage risk present in microbial production processes and the associated production losses or decreases by the use of novel chemical phage inhibitors, which differ from the previously known in their spectrum of action, especially by against both deoxyribonucleic acid ( DUA) - phage as well as against ribonucleic acid (RUA) -Pbagen of many host organisms are effective, so that at the same time Wirttsverträglicbkeit a broader application in biotechnology as a standalone measure as well as in the context of integrated antiphagal regimes is possible.

Explanation of the essence of the invention

The invention has for its object to use chemical agents that inhibit the phage proliferation due to their chemical constitution without affecting the host organisms (production strains), thereby enabling antiphagous prophylaxis or therapy and stabilize the yields of microbial growth and synthesis processes. The chemical agents should have a broad spectrum of action against different representatives of both the DITA al3 and the RHA phages and thus effectively protect a pbagengefäed production strain from various harmful phages and possibly also their mutants. In addition, corresponding inhibitors should also be usable for the protection of different microbial production processes.

The object is achieved according to the invention that are used for phage control in microbiological-technical production processes means, the compounds of the general formulas

(I)? 1H-CH-CO-HH-Ar

(ID

(CH ₀ ) 'H-CH-CO-HH-Ar

(III)

(IV)

(CH ₂ ) H-CH-CO-HH-Het

ΠH-CH-CO-HH-Het

(V) H-CH-CO-HH-Ar

(VI) H-CH-CO-HH-Ar

X "

(VII) Y

H-CH-CO-HH-Het

(VIII) H-CH-CO-HH-Het

(IX) Z

H-CH-CO-HH-Ar

(X) ^R 1 H-CH-CO-HH-Ar

(XI) Z

H-CH-CO-HH-Het

Uli)

-CH-CO-HH-Heΐ

^XY

contain, where Y = 0, S; Z = -IiH-, -UR ₃ or -II (R ₄ , R ₅ ); X = complementary anion; R ₁ = -H or alkyl radicals; R ₂ = alkyl substituents; R ₃ = -H or alkyl radicals or acyl, suidrophyl, phosphoryl or phosphonyl radicals; R., Rc = identical or different alkyl radicals. Ar may be the benzene ring or differently substituted aryl radicals and Het may be substituted and unsubstituted hetaryl radicals. η = 4.5 and 6; ρ = 1, 2, in the case of R. = -H, the optical antipodes or the racemates can be used · The compound types (I), (III), (V), (VII), (IX) and (XI ) can be used as free bases or in salt form.

The compounds can be used together with solubilizers and / or other formulation auxiliaries. It has proven to be advantageous if the active substance content accounts for 50 to 95 % of the composition.

The compounds of the general formulas (I) to (XII) according to the invention exhibit antipag- onal effects in different Pbagen / prokaryotic systems, eg. In the DHA phage systems

PZ-A / Bacillus subtilis,

PZ-B / Bacillus subtilis

χ /, Escherichia coli C 600

LPP-1 / Plectonema boryanum

and the RNA-Pbagen systems

M 12 / Eschericbia coli W 1665 F ⁺ and Qβ / Escberichia coli AB 301.

In order to eliminate the harmful effect of Pbagen in liquid cultures (fermentation broth) of bacterial production strains (stagnation or decrease in cell concentration), the means are added in such amounts to the culture media that an active substance concentration of 10 to 10. "^ G / ml results.

embodiments

To mark the antiphagal properties of the compounds according to the invention, the plaque inhibition test with agar cultures in the variant of the diffusion perforated plate test was used. In the selection of the test systems as different phages were considered: virulent or temperate, belonging to different morphological groups, Uukleinsäuretyp DITA or RKA, necessary for Pfaagenvermehrung host organisms grampositive or gram negative, their diet beterotropb or autotrophic. At the same time, bacteriophages that had appeared as harmful factors in biotechnological processes were also included.

Essentially, the following DKA phage / host systems were used:

PZ-A / Bacillus subtilis

Phage virulent ("pseudotemperent"), morphological group C, host gram positive, heterotrophic; production strain

PZ-B / Bacillus subtilis

Phage virulent, morphological group A, host gram positive, heterotrophic; production strain

X / Escherichia coli G 600

Pbage temperate, morphological group B, host gram negative, heterotrophic

LPP-1 / Plectonema boryanum

Phage virulent, morphological group C host gram negative, photoautotrophic; cyanobacterium

In addition, the following RSA phage / host systems were included in the investigations:

M 12 / Escherichia coli W 1665 F ⁺

Pbage virulent, morphological group E, serological group I, host gram negative, heterotrophic

Qβ / Escherichia coli AB 301

Phage virulent, morphological group E, serological group III,

Host gram-negative, heterotrophic ·

The plaque inhibition test was performed as an agar diffusion hole test. To prepare the Doppelscbicbtplatten the still liquid Deckschichtagar was inoculated at 48 ⁰ C with phages and host organisms and poured onto the pre-dried at 37 ⁰ C for 30 min base layer "holes were punched into the culture media after their solidification holes of 10 mm diameter and 0.05 ml of a 0.1 molar, in some cases, 1% solution of the test substances filled. After one hour of diffusion time at room temperature, the plates were incubated (PZ-Pbagen at 28 ⁰ C, Λ, M 12 and Qß at 37 ⁰ C and LPP-1 at 25 ⁰ C and

HOo Ix). As a result of the diffusion of the test compounds in the agar substrate, a concentration gradient is formed in the radial direction in which effective phage inhibition can be recognized as a non-plaque zone or zone with fewer and / or smaller plaques. The evaluation was carried out by measuring the radial widths of these Plaquehemmzonen. All approaches consisted of three to five parallels and were usually repeated several times. The experiments carried out and evaluated in the manner described show that the compounds according to the invention in the different test systems cause considerable plaque inhibition zones (ie regions with inhibition of phage propagation) and thus act antiphagally against very different DNA phages and also RITA phages. Examples 1 to 3 show some results with DUA phages and in example series 4 those with RUA phages.

The examples given show that many of the substituted piperidinoacetanilides of the invention, e.g. As piperidinoacetanilide hydrobromide and 4-i * luor-piperidinoacetanilid-bydrochlorid, are effective against numerous phages. It is apparent from the sometimes considerable width of the zones of inhibition that the antiphagical activity of these compounds extends over a larger concentration range. This fulfills important preconditions for use in stabilizing microbiological-biotechnological production processes.

Example series 1

Antiphagous activity of inventive compounds against PZ-A and PZ-B bacteriophages in the hole test with Bacillus subtilis

connection

Piperidinoacetanilid-bydrobromid

3-PlUor-piperidinoacetanilide-bydrocbloride 4-fluoro-piperidinoacetanedihydrate hydrochloride 3-gblor-piperidinoacetanilide-bydrocbloride 3-chloro-piperidinoacetanilide-hydrobromide 3-trifluorometbyl-piperiacetoacetanilide-hydro-

chloride

3-Styptyl-piperidinoacetanilide-bydrochloride 4-Ethoxy-piperidinoacetanilazole hydrochloride 3-Nitro-piperidinoacetanilide-bydrocbloride 4-Hitro-piperidinoacetanilide-hydrocfaloride 2-Cblor- (IT-metbyl) -piperidinoacetanilide; iodide 4-Metbyl- (l-inetbyl ) -piperidinoacetanilide-iodide Morpbolino-acetanilide-bydrobromide 4-Bromo-piperidinoacetanilide 4-Metbyl- (N-metbyl) -morpboliniuiD-acetanilide-

iodide piperidino-acet-2-aminobenzaldehyde

-GH-C O-KH-f OH-

Plaquebemm- 5 Off in mm z one (radial strain PZ-B PZ-A 3.0 6.3 3.0 3.7 5.7 5.3 2.7 3.0 7.0 10.0 8.7 6.7 3.7 2.7 0 3.0 3.3 2.7 5.7 3.7 1.7 1.7 5.7 3.0 2.0 0 0 3.7 0 6.7 1.7 4.7

1.3

Example series 2

Antiphagous activity of compounds according to the invention against Λ bacteriophages in the hole test with Escherichia coli

connection

3-Chloropiperidinoacetanilide hydrobromide 4-iodo-piperidinoacetanilide hydrochloride 3-Metbyl-piperidinoacetanilide-bydrochloride 2-Methoxy-piperidinoacetanilide-hydrocoboride 3-Metboxy-piperidinoacetanilide-bydrochloride 3-Etboxy-piperidinoacetanilide-bydrocbloride

Plaquebemmzone

(radial dimension in mm)

4.7 1.7 1.3 1.0 2.3 1.0

Example series 3

Antiphagous activity of compounds according to the invention against LPP-1 cyanopbagen in the hole test with Plectonema boryanum

connection

Piperidinoacetanilide hydrochloride piperidinoacetanilide hydrobromide 2-fluoro-piperidinoacetanilide-bydrochloride 4-i'-fluoro-piperidinoacetanilide hydrochloride 3-iodo-piperidinoacetanilide hydrochloride 4-iodo-piperidinoacetanilide hydrochloride 2-trifluoromethyl-piperidinoacetanilide

hydrochloride

3-Metbyl-piperidinoacetanilide-bydrochloride 3-Methyl-piperidinoacetanilide-hydrobromide 2-Methoxy-piperidinoacetanilide-bydrochloride 3-Metboxy-piperidinoacetanilide hydrochloride 4-Methoxy-piperidinoacetanilide-bydrocbloride 3-Ethoxy-piperidinoacetanilide hydrochloride 4-Etboxy-piperidinoacetanilide-bydrochloride 3 Nitro-piperidinoacetanilide-byrocrocoride 2-Gblor - (! 5-methyl) -piperidiniuin-acetanilide

iodide

Plaquehemmzone less and / or smaller plaques (radial dimension in mm) 0 plaque-free 12.0 7.3 3.0 6.3 6.0 18.7 0 16.0 0 9.0 4.0 12.0 0 6.7 0 22.0 0 20.7 5.0 9.7 0 20.0 4.7 16.7 5.0 17.0 0 19.7 0 14.3 5.7

Example 4

Antipagal activity of inventive compounds against M12 and Qß bacteriophages in the hole test with Escherichia coli

Connection plaque inhibition zone

(radial dimension in mm)

M12

Piperidinoacetanilide hydrocoboride Piperidinoacetanilide hydrobromide

2-fluoro-piperidinoacetanilide-bydrocbloride 3-i'-fluoro-piperidinoacetanilide hydrochloride 4-fluoro-piperidinoacetanilide-hydrocoboride 3-trifluorometbyl-piperidinoacetanilide-hydro-

cblorid

3-Metbyl-piperidinoacetanilide-bydrobromide 3-Metboxy-piperidinoacetanilide-bydrocbloride 4-Uitro-piperidinoacetanilide-bydrochloride Piperidinoacet- (4-pyridino) amide 4-Bromo-piperidinoacetanilide

4.0 4.0 4.5 4.0 3.0 3.0 2.5 2.5 3.0 2.5 5.0 4.0 2.5 2.5 3.0 3.0 4.0 3.0 3.0 3.5 3.5 2.0 2.5 0

CH-

Claims (4)

Erfindungsansprücfae 1. Agent for bacteriophage or _# Cyanopbagenbekämpfung in microbiological production processes, characterized in that antiphagal agents of the general formulas (I) to (XII) (ID (CH ₀ ) "H-CH-CO-NH-Ar (III) (CH ₂ ) _n U-CH-CO-HH-Het (IV) (CH ₂ ) N-CH-CO-HH-Het (V) Y (VI) N-CH-CO-im-Ar IT-CH-C 0-in-Ar X " (VII) Y U-CH-CO-NH-Het (VIII) H-CH-CO-HH-Het ^R 1 (IX) Z N-CH-CO-UH-Ar (X) Z is N-CH-CO-SH-Ar (XI) Z N-CH-CO-NH-Het 2. Composition according to item 1, characterized in that the antiperspirant active ingredients are present individually or in any combination with each other. 2 m S ~ ( ^GE 2 ^ m \ ^R 1 (XII) Z N-CH-CO-NH-Het where Y = O, S; Z = -MH, -HH-R- or -Η "R ₄₁ R ₅ ); X = complementary anion; R = -H or alkyl radicals; Rp = alkyl substituents; R 1, = -H or alkyl radicals or acyl radicals; R.sup.5, R.sup.5 = identical or different alkyl radicals, Ar represents the benzene ring or differently substituted aryl radicals and Het represents substituted or unsubstituted hetaryl radicals, m = 2, η s 4, 5 and 6, ρ = Can be 1, 2, are included together with conventional excipients and carriers · 3. Means according to item 1 and 2, characterized in that the V / irkst off concentration is on average between 1 and 99 % . 4 · Means according to items 1 to 3, characterized in that Solubilizers and / or formulation auxiliaries are included.