GB2099454A - Nutrient media containing protein hydrolysate - Google Patents

Abstract

The present invention provides a nutrient medium for bacteriological purposes, consisting of or containing a protein hydrolysate obtained by the sulphuric acid hydrolysis of casein or of other natural proteinaceous products.

Description

SPECIFICATION Bacteriological nutrient media The present invention is concerned with bacteriological nutrient media which are useful for culturing bacteria, for general and special bacteriological diagnosis, for biochemical tasks, such as the isolation of metabolic products, and for the investigation into bacteria genetics.

The nutrient media previously used for culturing bacteria, for bacteriological diagnosis and for isolating bacterial toxins and antigens essentially contain high molecular weight proteins, for example enzymatically split proteins (peptones), extracts obtained by the autolysis of yeast or meat extracts or meat broth as main nutrient materials. The proportion of low molecular weight nutrient materials, for example amino acids, in such products is relatively low so that for a good and rapid bacterial growth, a correspondingly large amount of total nutrient is necessary. It is known that high molecular weight proteins and ballast materials, such as are present in peptones, can have a toxic action on bacteria and that too high a proportion of ballast material has an inhibiting action on bacterial growth.The isolation of bacterial toxins and antigens is impaired, for example, by a high proportion of ballast material and by the amount of high molecular weight proteins present. Bacteriological nutrient media for the above mentioned fields of use are also prepared by the addition of individual amino acids but, in this form, they are too expensive for most fields of use.

The protein hydrolysates obtained by hydrochloric acid hydrolysis hitherto used for similar purposes contain, due to their method of production, comparatively large amounts of sodium chloride, which influences the ionic strength of the final nutrient media, as well as a higher residual peptide content than protein hydrolysates produced by sulphuric acid hydrolysis (pressure hydrolysis).

It is an object of the present invention to use protein hydrolysates obtained by the sulphuric acid hydrolysis of casein or other natural protein products, either alone or as an additive to peptone, yeast extract or meat extract nutrient media or to appropriate combinations of peptone-yeast extract or similar nutrient media or in combination with definite sources of energy, such as glucose. The use of these products containing high molecular weight ballast materials can thereby be markedly reduced and the quality of the nutrient media for the above-mentioned fields of use is improved.

Thus, according to the present invention, there is provided a nutrient medium for bacteriological purposes, consisting of or containing a protein hydrolysate obtained by the sulphuric acid hydrolysis of casein or of other natural proteinaceous products Although the nutrient media according to the present invention have a markedly reduced proportion of nutrient material, they display the same or better growth properties for bacteria than the previously used nutrient media. Due to the reduction of the content of ballast materials in these nutrient media, metabolic performances are easier to control and bacterial metabolic products can be isolated more simply.

Such nutrient media can be obtained by combining sulphuric acid protein hydrolysates with glucose or similarly utilisable carbohydrates, vitamins and trace elements, with peptones, yeast extracts, meat extracts, meat broth and the like.

The following Examples 1 to 3 describe some of the new base nutrient media and the following Table gives the growth factors for a few species of bacteria on these new base nutrient media in comparison with previously used peptone nutrient media. On the basis of these new base nutrient media, there can be prepared most of the nutrient media required for bacteriological diagnosis (e.g.

selective nutrient media, nutrient media for the "variegated series" and nutrient media for lysotyping), for biochemical investigations and for bacterial genetics. The following Example 4 describes the composition of a selective nutrient medium which, because of its weak selective properties and its very wide diagnostic possibilities, is very well suited for the preliminary diagnosis of pathogens in urine, -foodstuffs and other materials to be investigated. The following Example 5 describes the composition of a nutrient medium for lysotyping Staphylococcus aureus. On this nutrient medium, the phagelysis can be demonstrated with great constancy and thus the lysotype can be demonstrated.The following Example 6 describes a nutrient medium for detecting and obtaining E. Coli haemolysin which, in contradistinction to the previously employed meat broth nutrient media, can be standardised. The following Example 7 describes a nutrient medium which can be used for the production of lysostaphin from the Staphylococcus aureus strain RNC.

The Examples here set out can be appropriately broadened in scope. However, the base nutrient media described in Examples 1 to 3, also in combination with glucose, vitamins and trace elements, can be incorporated as in an assembly system as nutrients in nutrient media for the most varied fields of use, for example for the production of inoculation materials and for antibiotic production.

EXAMPLE 1 Base nutrient medium peptone 3.0 g./litre sulphuric acid protein hydrolysate 4.0 litre yeast extract 2) 0.5-1.0 litre sodium chloride 5.0 g./litre distilled water 1000 ml.

) pancreatic casein peptone; mixed peptone (meat, fish, gelatine peptone); blood peptone and the like.

2) "Bramsch" yeast extract (DDR), yeast extract from feed yeast (Russian preparation), yeast autolysate from VEB Impfstoffwerk Dessau' DDR.

EXAMPLE 2 Protein hydrolysate-yeast extract base nutrient medium sulphuric acid protein hydrolysate 4.0 litre yeast extract "Bramsch" 1.5 litre sodium chloride 5.0 litre distilled water 1000 ml.

EXAMPLE 3 Protein hydrolysate-yeast extract-meat extract base nutrient medium sulphuric acid protein hydrolysate 4.0 g./litre yeast extract "Bramsch" 1.0 g./litre meat extract 3) "Chemex" (Munchen, Fed. Rep. of Germany) 1.0 g,/litre sodium chloride 5.0 litre distilled water 1000 ml.

3) use can also be made of Liebig's meat extract or meat broth (cattle, horse)Snstead of distilled water for the dissolving.

Example 4 Bile-chrysoidine-glycerol medium (BCG) BCG base: nutient agar II (SIFIN) (containing 10 g.

pancreatic peptone, 10 g. agar for culture media and 5 g. sodium chloride) 25 g.

yeast extract (Bramsch) 5 g.

sulphuric acid protein hydrolysate (VEB Berlin Chemie) 2 g.

ferric ammonium citrate 2 g.

sodium thiosulphate pentahydrate, analytically pure 3 g.

chrysoidine 40 mg.

bovine bile (dried) 8 g.

distilled water 1 litre Dissolve and sterilise for 2 hours in a steam vessel (pH 7.3-7.5) and add 29 ml. BCG solution.

BCG solution glycerol (2nd DDR Pharmacopoeia) 1 litre ethanol 0.4 litre bromothymbol blue 11.0 g.

urea, analytically pure 50.0 g.

EXAMPLE 5 Nutrient medium for lysotyping Staphylococci peptone from casein, pancreatic 4.0 g./litre sulphuric acid protein hydrolysate 6.0 litre yeast extract "Bramsch" 2.0 g./litre sodium chloride 5.0 g./litre agar-agar 15.0 g./litre sodium carbonate, analytically pure 0.32 g./litre L-tryptophane 0.064 g./litre distilled water 1000 ml.

EXAMPLE 6 Nutrient medium for detecting and obtaining E. coli haemolysine sulphuric acid protein hydrolysate 20.0 g./litre sodium chloride 5.0 g./litre glucose (2nd DDR Pharmacopoeia) 2.0 g./litre meat extract "Chemex" 3J 20.0 litre agar-agar 15.0 g./litre magnesium sulphate heptahydrate, analytically pure 0.29 g./litre manganese sulphate tetrahydrate, analytically pure 0.014 litre ferric ammonium citrate 0.004 g./litre vitamin B1 0.002 g./litre vitamin B6 0.002 litre EXAMPLE 6 - Continued nicotinamide 0.002 litre distilled water 1000 ml.

3) see legend to Example 3.

EXAMPLE 7 Nutrient medium for the production oflysostaphine from Staph. aureus RNC nutrient broth I (SEVAC, Czechoslovakia) with 5.0 g. peptone and 3.0 g. meat extract/litre 8.0 g./litre sulphuric acid protein hydrolysate 2.0 g./litre sodium hydrogen phosphate dihydrate, analytically pure 5.0 g./litre sodium chloride 1.0 g./litre distilled water 1000 ml.

TABLE Growth factors 1) of selected bacterial strains grown in nutrient media according to Examples 1, 2 and 3, in comparison with growth in a nutrient medium containing 20 g. pacreatic peptone/litre, after 4 and 8 hours

Example 1 Example 2 Example 3 bacterial strain 4h. 8h. 4h. 8h. 4h. 8h.

E.coli 1.05 1.14 1.15 1.11 1.24 1.16 54127 E. coli 1.16 1.14 1.20 1.21 1.12 1.09 35033 Pseudomonas 1.17 1.08 1.21 1.01 1.27 1.07 Elsworth Staph v aureus 1.16 1.22 1.50 1.13 1.58 1.08 78/71 Staph.

aureus 1.20 - 1.26 1.20 1.13 1.33 1.27 1413 growth in test nutrient medium 1) growth factor= growth in 20 g. pancreatic peptone medium The extinction values of shake cultures at370C. are measured.

Claims (6)

1. A nutrient medium for bacteriological purposes, consisting of or containing a protein hydrolysate obtained by the sulphuric acid hydrolysis of casein or of other natural proteinaceous products.

2. A nutrient medium according to claim 1, comprising the sulphuric acid protein hydrolysate and a nutrient medium produced from high molecular weight nutrient materials.

3. A nutrient medium according to claim 1 or 2, which additionally comprises at least one member selected from peptones, yeast extracts, meat extracts and glucose.

4. A nutrient medium according to any of the preceding claims, which additionally comprises at least one member selected from haemin, vitamins and lecithin.

5. A nutrient medium according to any of the preceding claims, whenever adapted for use in bacteriological diagnosis, biochemical investigations, bacterial genetics, the product of inoculation materials and the production of antibiotics.

6. A nutrient medium according to claim 1, substantially as hereinbefore described and exemplified.