DD260288A1 - METHOD FOR PRODUCING A MICROBIAL NEEDLE MEDIUM - Google Patents

Abstract

The invention relates to a process for the production of microbial nutrient media for the cultivation of microorganisms. The task of developing new, simplified nutrient media with improved properties for the growth of microorganisms is achieved by replacing the complex organic nitrogen sources previously used in microbial nutrient media, such as peptones, meat extracts, meat broth, casein, casein hydrolyzate, with the sulfuric acid hydrolyzate of Biomehl or its precursor biosludge concentrate is used or in combination with various carbon and energy sources without further addition of mineral salts and trace elements results in new nutrient media.

Description

Field of application of the invention

Microbial culture media are used to cultivate microorganisms of various taxonomic assignments. Culturing of the microorganisms is for the purpose of general characterization, biochemical and genetic studies, and for the recovery of microbial biomass and its constituents. Microbial nutrient media can therefore be used in all microbiological laboratories and manufactured in the relevant production facilities for ready-made medicines.

Characteristic of the known state of the art

In addition to assimilable carbon and energy sources, microorganisms require nitrogen sources and various mineral salts, trace elements, vitamins and growth substances to grow. Certain microorganisms require an organic nitrogen source which already contains a large number of the building blocks necessary for the growth of the microorganism cell in prefabricated composition. The nutrient media hitherto used in microbiology for general characterization and for other purposes contain the organic nitrogen source in the form of extracts of high molecular weight protein compounds such as meat extracts, yeast extracts, meat water or meat broth or enzymatically or with mineral acids digested proteins in the form of peptones of various origin such as blood, meat , Fisch and Casein (Handbuch Nährböden, Merck, 1980; Microbiology Handbook, Becton Dickinson GmbH, Heidelberg; Difco Manual). Partially contain these nutrient media and total hydrolysates of certain proteins such. As Casein, so-called Casamino acids (Difco Manual) or hydrolyzates of casein and other natural protein products obtained by sulfuric acid hydrolysis (DEOS 3212673A1).

The disadvantage of such nutrient media is that hydrolysates are used, which are made of valuable starting materials such. As casein or soy protein were obtained. In addition, these nutrient media must be added in addition certain mineral salts such as phosphate compounds and trace elements. Another disadvantage is that the previously used complex organic nitrogen sources, in particular peptones, tarnish the nutrient media and thus complicate certain evaluations.

Object of the invention

The object of the invention is to produce novel, improved nutrient media for microbiological work, which do not have the disadvantages mentioned.

Explanation of the essence of the invention

The invention is based on the object by using a low-cost, readily available, novel complex nitrogen source previously used for microbial nutrient media organic nitrogen sources such as peptones of various origins, extracts of meat and other biological materials such. B. to replace yeast and beyond by

-2- For example Ü88

Combination of this novel complex nitrogen source with suitable carbon and energy sources without further additions of mineral salts to produce novel, simplified nutrient media with improved properties for the growth of various microorganisms.

According to the invention, this object is achieved in that instead of previously obtained in microwave nutrient media enzymatically or by acid digestion of various protein sources (peptones), meat, fish and yeast extracts or acid Caseinhydrolysate the hydrolyzate prepared by sulfuric acid hydrolysis and by neutralization with ammonia water or biomass which is taken from the liquid manure fermentation as underflow and concentrated by separation referred to as biosludge concentrate precursor, or this biomehl or Bioschlammkonzentrathydrolysat in combination with carbon and energy sources such as alcohols, carboxylic acids mono-, di-, oligo- and polysaccharides, without further Addition of mineral salts, but with the addition of vitamins and growth substances for the production of novel, simplified nutrient media with improved properties for the growth of microorganisms.

The process according to the use of biomehl or. Bio-sludge concentrate hydrolyzate instead of previously used complex nitrogen sources or their combination with various carbon and energy sources is basically carried out as follows:

The complex nitrogen sources used in most microwave nutrient media are partially or totally replaced by the addition of biomass or biosludge concentrate hydrolysates prepared by sulfuric acid pressure hydrolysis and subsequent neutralization with ammonia water. Furthermore, biomehl or Bioschlammkonzentrathydrolysate be used advantageously in combination with favorable for the growth of microorganisms carbon and energy sources without further additions of mineral salts and trace elements such as phosphorus, magnesium, iron, manganese, cobalt or nickel salts. As carbon and energy sources while alcohols, mono-, dicarboxylic acids, oils, hydroxy, di- and tricarboxylic acids, aldehydes and ketones, mono-, di-, oligo- and polysaccharides, phenols, aromatic alcohols, and heterocyclic compounds are used. Depending on the requirement of the microorganism to be grown is an addition of vitamins such as vitamin B ₁ , B ₂ , B ₆ , B ₁₂ or various growth substances such. As folic acid or ferrioxamine.

The amounts of biomass or biosludge concentrate hydrolyzate used correspond to an a-NH ₂ -N content of 10 μg / ml " ¹ to 1000 μg / ml" ¹ , but preferably 50 μg / ml " ¹ to 500 μg / ml" ¹ . The novel, simplified in terms of their composition, largely optically clear nutrient media have improved properties for the growth of microorganisms.

embodiment

Embodiments of the invention will be explained, but without limiting it.

example 1

In microwave nutrient media, the previously used complex, organic nitrogen sources are partially or completely replaced by the hydrolyzate of biomehl, the so-changed nutrient media usually sterilized by autoclaving and poured into sterilized Petri dishes. After cooling, inoculation with the microorganisms indicated in Table 1 is carried out by means of a loop. After 3 or 6 days of incubation at 30 ⁰ C, the growth is evaluated. As a control serve with the same microorganisms inoculated unchanged nutrient media, a)

dextrose 10, Og-I - ¹ M79 medium ad. 1.01 1-1 Peptone (from blood) 10, Og-I - ¹ (control) Γ ¹ Al 53 medium Casamino acids (Difco) 1 Og-I -1 Γ ¹ (control) -leextract (Bramsch) 2.0 g · I -1 20.0ml · Γ ¹ 1-1 NaCl 6.0 g · I -1 1-1 Agar Agar 15, Og-I -1 1-1 Distilled water | -1 Variant I 50.0ml · 1-1 Instead of Casamino acids (Difco) 3,0g- 1-1 Biomedical hydrolyzate (BMH) 15.0g - 15.0g-Γ ¹ variants 0,1g- ad. 1.01 Instead of Casamino acids (Difco) 0,5g- and peptone (from blood) 0,5g- Biomedical hydrolyzate (BMH) 1,0g- 25.0ml · Γ ¹ sucrose 5,0g- dextrin 1,0ml- urea NaCl KH ₂ PO ₄ yeast extract Peptone (from blood) FeSO ₄ (1% solution) Agar Agar Distilled water Variant I Instead of peptone (from blood) Biomehlhydrolysat Varianteil Instead of peptone (from blood),

KH ₂ PO ₄ and FeSO ₄ 25.0ml · Γ ¹ Biomehlhydroiysat 40.0g · Γ ¹ Sabouraud medium c) maltose 10.0g-Γ ¹ (control) peptone 15, Og-1 " ¹ Agar Agar ad. 1.01 Distilled water Variant I Instead of peptone 50.0ml · Γ ¹ Biomehlhydroiysat

Table 1

Growth of Penicillium chrysogenum, Penicillium camembertii (after 3 days) and Streptomyces hygroscopicus (after 6 days) on nutrient media variants with biomehlhydroiysate as a complex, organic nitrogen source. The original, unaltered nutrient media serve as control.

Microorganism a) M79 medium '___ b) AL53 medium c) SabouraudM.

Control VaM VaV. II control Var.l Var.ll control Var.l

P. chrysogenum

P.camembatii ++ + + + + + + +

S. hygroscopicus +++ +++ +

+: weak growth ++: moderate growth + + +: good growth + +++: very good growth

Example 2

The use of the complex, organic nitrogen source Biomehlhydroiysat carried out as described in Example 1, as well as the preparation of nutrient media in sterile Petri dishes with the change but that for growth testing except P. chrysogenum and P.camenbertii also Escherichia coli, Bacillus amylofaciens and Bacillus globigii (evaluation of Growth after 24 hours), a)

Casamino acids (Difco) 1,5g -ml "'CAY medium glycerin 20.0ml I " ¹ (control) yeast extract 2,5g · Γ ¹ NaCl 1.0g-r ¹ KH ₂ PO ₄ 1, Og-T ¹ MgSO ₄ .7H ₂ O 0.1 gr ¹ CaCO ₃ 0.005g · Γ ¹ Agar Agar 15.0 g Γ ¹ Distilled water ad. 1.01 Variant I Instead of Casamino acids (Difco) Biomehlhydroiysat 4, OmI-I " ¹ Varianteil Instead of Casamino acids (Difco) Biomehlhydroiysat 10.0ml -Γ ¹ Variant III Instead of Casamino acids (Difco) KH ₂ PO ₄ and MgSO ₄ .7H ₂ O Biomehlhydroiysat 15, OmI-T ¹ Yeast extract (Difco) 4.0 g · Γ ¹ yeast extract agar 53 malt extract 10.0g · Γ ¹ (control) dextrose 4.0g · Γ ¹ Agar Agar 20.0g · Γ ¹ Distilled water ad. 1.01 Variant I Instead of yeast extract (Difco) Biomehlhydroiysat 8, OmI-I " ¹

Table 2

Growth of Penicillium chrysogenum, Penicillium camembertii, Escherichia coli B, Bacillus amylofaciens and Bacillus globigii on nutrient media variants with biomehlhydroiysate as a complex, organic nitrogen source. The original, unaltered nutrient media serve as control.

Microorganism CAY medium

Control var.

Var.ll

Var.l

Yeast Extract Medium 53 Control Var.l

P.chrysogenum P.camembertii E.coliB B.amylofaciens B.globigii

+: weak growth ++: moderate growth +++: good growth ++++: very good growth

Example 3

The nutrient media a) to h) listed below are combined according to the given formulations, prepared by adding the agar and boil-up, and the pH is adjusted to pH 6.8 ± 0.2 with ¹ Ao η NaOH and in steep-flow bottles of 200 each ml bottled. After sterilization in an autoclave for 25 minutes at 120 ^° C., the nutrient media can be poured into previously sterilized petri dishes immediately or later after being re-liquefied in a steam pot or in a boiling water bath. After solidification of the nutrient medium, the inoculation with cell material of the microorganisms specified in Table 3 is carried out by streaking using Impföse on the Nährmedienoberfläche.

Table 3 shows the results of the growth of various microorganisms on the nutrient media a) to h). a)

BMH ad. 10, OmI-T ¹ BMH: biomass hydrolyzate glucose 2, Og · T ¹ biotin 20.0mg I " ¹¹ Add from sterile Vitamin B ₁ ioo, OMg · i ~ ¹ ' filtered stock solutions Agar Agar 20.0 g · T ¹¹ Aqua Dest. 1.01 BMH 10.0ml: T ¹ sucrose 4.0g · T ¹ BMH 20, OmI-T ¹ Succinic acid 2.5 g · T ¹ biotin 20.0Mg Γ ¹¹ Addition of sterile fil NAD 10.0Mg · i ~ ¹¹ trated stock solutions folic acid 10.0Mg-T ¹¹ Agar Agar 20, Og · T ¹ Distilled water 1.01 BMH 25, OmI-T ¹ citric acid 30, Og · T ¹ Vitamin B ₁ ad. 30.0Mg · I " ¹ ' Addition of sterile fil Vitamin B ₂ 10.0Mg · i ~ ¹⁾ trated stock solutions folic acid 10.0Mg · I " ¹ ' pantothenic acid 10.0Mg I " ¹¹ Agar Agar 15, Og-T ¹ Distilled water ad. 1.01 BMH 20.0ml · T ¹ corn starch 5, Og · T ¹ glucose 2, Og · T ¹ Agar Agar 15, Og-T ¹ Distilled water 1.01 BMH 15,0ml · T ¹ Ethanol (96%) ad. 2.0ml · T ¹¹ Addition of sterile fil biotin 20.0Mg · I " ¹¹ trated stock solutions Vitamin B ₁ 50.0Mg · I " ¹¹ Vitamin B ₆ 10.0Mg · Γ ¹¹ Agar Agar 20, Og · T ¹ Aqua Dest. 1.01 BMH ad. 20, OmI-T ¹ glycerin 10, OmI-T ¹ folic acid 10.0Mg · I " ¹¹ Addition of sterile fil Vitamin Bi ₂ 50.0Mg-I " ¹¹ trated stock solutions Agar Agar 20, Og · T ¹ Aqua of. 1.01 BMH 15, OmI-T ¹ NH ₄ -acetate 3, Og · T ¹ Agar Agar 20, Og · T ¹ Aqua Dest 1.01

Table 3

Growth of microorganisms on nutrient media a) to h)

Microorganism nutrient medium

a) b) c) d) e) f) g) h) _

E.coliB

B. Amylofaciens P. chrysogenum B. subtilis S. Hydroscopicus

Escherichia B)

Bacillus amylofaciens) incubation at 34 ° C, evaluation of growth after 24 hours

Bacillus subtilis)

Penicillium chrysogenum) _{incubation at 29} ° _c , _{evaluation after 3} days

Streptomyces hygroscopicus a. a a

+: weak growth + +: moderate growth + + +: good growth + + + +: very good growth

Claims (5)

1. A process for the preparation of a microbial nutrient medium, characterized by the use of sulfuric acid hydrolysis of biomehl or its from the slurry fermentation as underflow removed and concentrated by separation precursor biosludge concentrate and subsequent neutralization with ammonia produced hydrolyzates after clarification or filtration as organic complex nitrogen source. 2. The method according to claim 1, characterized by the sole use of biomehl hydrolyzate or hydrolyzate of biosludge concentrate as a complex organic nitrogen source. 3. The method according to claim 1, characterized by the use of biomehl hydrolyzate or hydrolyzate of biosludge concentrate in combination with carbon and energy sources without further addition of mineral salts and trace elements. 4. The method according to claim 1 to 3, characterized by the use of biomehl hydrolyzate or hydrolyzate of biosludge concentrate in an amount corresponding to an a-NH ₂ -N content of 1 (g · ml " ¹ to 1 000 ug · ml " ¹ , but preferably from 50 .mu.g.ml" corresponds to ¹ to 500 .mu.g ml ^-1 . 5. The method according to claim 3, characterized by the use of monohydric and polyhydric alcohols, monocarboxylic acids, dicarboxylic acids, oils, hydroxy-di and tricarboxylic acids, ketodicarboxylic acids, mono-, oligo- and polysaccharides, phenols, aromatic alcohols, aldehydes, ketones and Carboxylic acids and heterocyclic compounds as carbon and energy sources for combination with biomehl hydrolyzate or hydrolyzate of biosludge concentrate.