Title of Invention

"MODIFIED CULTURE MEDIUM AND PROCESS FOR THE PREPARATION THEREOF"

Abstract

The present invention relates to an unproved culture medium for the growth of human pathogenic yeast. The invention also relates to a process for the preparation of a culture medium for the growth of human pathogenic yeast. More particularly, the present invention also relates to a process for the culturing of microorganisms such as pathogenic yeast using the unproved culture medium of die invention.

Full Text

Field of the invention The present invention relates to a modified culture medium for the growth of human pathogenic yeast. The invention also relates to a process for the preparation of a culture medium for the growth of human pathogenic yeast. More particularly, the present invention also relates to a process for the culturing of microorganisms such as pathogenic yeast using the improved culture medium of the invention. Background of the invention In medicine, it is an important part of human health care systems to determine the amount and the species of a microorganism which has caused infections or contamination such that the correct method of treatment can be formulated. Similarly, in the food industry it is important to determine which microorganisms can contaminate or actually contaminate food products such that methods for achieving purity can be obtained. Various culture media are used in the field of the microbiology, particularly in the diagnosis of diseases to determine the nature of the microorganism which infects the patient and the susceptibility thereof to different anti-microbial drugs. In the prior art, the following culture media are known: a. Czapek-Doxbroth medium b. Dixon's Agar medium c. Panja's Medium d. Martin-Scott medium e. Sabouraud's glucose medium The constituents of the above culture media comprise: a. Czapek's medium = Sucrose (30gm), Sodium nitrate (3gm), Dipotassium phosphate (1gm), Magnesium sulphate (O.8gm), Ferrous sulphate (O.Olgmm), Potassium chloride (O.5gm), Yeast extract (2.5gm), Chloramphenicol (50gm), Cycloheximide (500gm) Tween20 (75ml) and Distilled Water (to 1 litre). b. Dixon's medium = Malt extract (3.6%), Peptone (0.6%), Oxoid (2%), Tween40 (1%), Glycerol (0.2%), Oleic Acid (0.2%), Agar (1.2%). c. Panja's medium= Minced Beeg (l00gm), Glycerine (15ml), Water (85ml), Fresh eggs (8-12), Gentian Violet (0.05ml). d. Martin-Scott's medium = Tauroglycocholate (10%),Oxopeptone(5%),Agar (3- 4%). e. Sabouraud's glucose medium = Glucose (40gm), peptone (10gm), Agar (18gm), and Distilled Water (to 1 liter). The Czapek-Doxbroth medium developed in 1964 is not advantageous since the cost of some of the components thereof namely Tween and cycloheximide is prohibitively high. The Dixon's Agar medium also developed in 1964 also contains Tween and is therefore very expensive. Another disadvantage of the Dixon's medium is that it contains oxoid which not only is available in limited quantities but also has a bad odor. Additionally, the use of oxoid in the Dixon's medium poses a problem in countries such as India where a majority of the population does not touch cattle or cattle based meat products due to religious reasons. The Martin-Scott medium utilizes oxopeptone and tauroglycolate which are not readily available thereby increasing the cost of the medium. Panja's medium contains egg and minced beef which again for religious or moral reasons is not acceptable to most users. Another disadvantage of the Panja's medium is that the since egg is used, the preparation of the medium takes at least three to lour days thereby limiting its use to situations where ready use mediums are not required. Another disadvantage of the medium is that the minced beef gets contaminated unless manufactured or stored under conditions of extreme hygiene which in turn increases the cost of the preparation of the medium. The medium also does not have a good odor which increases its lack of acceptability among users. Saburaud's Glucose Medium while being comparatively inexpensive to manufacture is unsuitable for the culturing of lipophilic yeast. U.S. Pat. No. 5,296,370 to Martin et al describes a very effective liquid culture medium for membrane repair of microbial cells, the disclosure of which is incorporated by reference. In this invention, the culture medium contains a non-selective complete medium to supply nutrients required by food pathogens, a yeast derivative, one or more antioxidants, an oxygen tension reducing agent and one or more fatty acids for membrane repair. The fatty acids are 10 to 25 carbon atoms in length and can have up to 4 double bonds and are used in amounts between 0.025 and 1.0 weight percent. The complete medium can include tryptic soy broth, nutrient broth, Eagles medium or yeast mold broth in an amount between about 2.5 and 20 weight percent. The antioxidants can include pyruvate, succinate, glutathione, catalase, selenium, albumin, glucose, BHA, BHT, ascorbic acid, lactic acid, uric acid, superoxide dismutase, glutathione peroxidase acid and n-acetyl cysteine, vitamin E and beta-carotine in amounts of about 0.10 to 1.0 percent by weight. The yeast derivative can be yeast extract, live yeast cell derivative, autolysed yeast, enzymatically treated yeast and mixtures thereof in amounts between about 0.1 and 1.0 weight percent. Various protein supplements such as casamino acids, brain-heart infusion proteins, proteases, peptones and mixtures thereof can be used in about between about 0.1 and 1.0 weight percent. Various preferred liquid media are described. GB1101440 discloses a method for cultivating microorganisms having exoenzyme producing ability. Exoenzyine-producing microorganisms are cultivated in a culture medium in the presence of phosphatidyl inositol, preferably in proportions of at least 0.05% by weight (e.g. between 0.1-1%) in a liquid culture medium and at least 0.5% by weight in the case of cultivation in a solid medium. Many species of microorganisms are particularly mentioned, including bacteria, actinomycetes, yeasts and fungi. Proteolytic enzyme-producing: Bacillus, Streptomyces and Aspergilhis. JLipolytic enzyme-producing: Candida, Mucor, Rhizopus and Aspergillus. Amylolytic enzyme-producing: Rhizopus, Aspergillus and Bacillus. Species of the following are also mentioned: Pseudomonas, Serratia, Cfostridinitun, Leuconostoc, LactobaciUus, Saccharomyces, Torula, Penicillhan, Absidia, Fusarium and Sclerotinia. The phosphatidyl inositol may be obtained from yeast, hexane-extracted crude soybean oil, or soya bean material such as defatted soybean, soybean flour and soybean meal, by procedures described in the specification. Examples are directed to comparisons of the proteotytic, lipolytic and amylolytic enzyme activities (as the case may be) produced by the cultivation of specified micro-organisms in specified culture media containing phosphatidyl inositol, with the corresponding enzyme activities obtained when employing culture media free from phosphatidyl inositol. Exoenzyme - producing micro-organisms are cultivated in a culture medium in the presence of phosphatidyl inositol, preferably in proportions of at least 0.05% by weight (e.g. between 0.1 - 1%) in a liquid culture medium and at feast 0.5% by weight in the case of cultivation in a solid medium. It is stated that phosphatidyl choline, phosphatidyl ethanokunine and inositol have no appreciable ability in enhancing the production of enzymes. Phosphatidyl inositol is extracted from soybean flakes with 92 vol. % ethanol at a high temperature (near the boiling point) or under elevated pressure, the extraction liquid is cooled to room temperature and the precipitated solid phase is separated by centrifuging; residual ethanol and any water are removed from the precipitate by treatment with anhydrous ethanol or acetone. The precipitate is purified by extraction with a mixed solvent composed of 2 parts chloroform and 1 part ethanol, followed by dialysis of the extraction liquid, pouring the product into 10 times its volume of a methyl acetat solution under violent stirring, and collecting and drying the resultant precipitate. The extraction of phosphatidyl inositol from yeast and from hexane-extracted crude soybean oil also is disclosed. Proteorytic enzyme activity is measured by mixing 1 ml. of the enzyme solution with I ml. of 2% milk casein solution containing 0.2 mol buffer solution of pH 7.0, incubating the mixture for 60 minutes at 35 DEC C., adding 4 ml. of 5% trichloroacetic acid to remove protein not yet decomposed, adding to 1 ml. of the filtrate 5 ml. of 0.4 mol sodium carbonate and 1 ml. of Folio's reagent, and computing the proteolytic enzyme activity as the optical density (660mm ) of the resulting liquid minus that of a control liquid. Lipolytic enzyme activity is measured by adding to 1 ml. of a non-diluted enzyme solution, 5 ml. of a homogenized mixture of 3 parts of 2% polyvinyl alcohol with 1 part olive oil, and 4 ml. of a 0.2 mol buffer solution of pH 5.0. The resultant mixture is incubated for 60 minutes at 37 DEG C., the reaction being stopped by the addition of 20 ml. of 1.1 alcohol-acetone mixture. The reaction mixture is titrated with N/20 sodium hydroxide solution and the lipolytic enzyme activity is taken as the titration value obtained as diminished by that for a control liquid. Amylolytic enzyme activity is deduced as follows. To 10 ml of enzyme solution and S ml. of 0.2 mol buffer solution of pH 4.8 there is added 20 ml. of 2% soluble starch solution and the mixture is incubated for 30 minutes at 40 DEG C. The reducing power of the reaction mixture is determined by Fehling-Lehmann-SchoorTs method (saccharifying power). In the course of the enzyme reaction 0.5 ml. of the reaction mixture is mixed with 5 ml. of iodine and potassium iodide solution and the incubation time required for this mixture to show the same degree of colour to standard colour of cobalt chloride solution is measured. Phosphatidyl inositol is extracted from soybean flakes with 92 vol. per cent ethanol at a high temperature (near the boiling point) or under elevated pressure, the extraction liquid is cooled to room temperature and the precipitated solid phase is separated by centrifuging; residual ethanol and any water are removed from the precipitate by treatment with anhydrous ethanol or acetone. The precipitate is purified by extraction with a mixed solvent composed of 2 parts chloroform and 1 part ethanol, followed by dialysis of the extraction liquid, pouring the product into 10 times its volume of a methyl acetate solution under violent stirring, and collecting and drying the resultant precipitate. The extraction of phosphatidyl inositol from yeast and from hexane-extracted crude soybean oil also is disclosed. We claim: 1. A modified culture medium comprising from 30 to 33 g of a sugar selected from sucrose, galactose and maltose, yeast extract from 6 to 8 g, peptone from 2 to 4 g, oleic acid from 2 to 5 ml and glycerol from 3 to 5 ml and the balance, if any, of conventional additives selected from antibiotics, nutrients and oils. 2. A culture medium as claimed in claim 1 wherein the culture medium is a broth. 3. A culture medium as claimed in any preceding claim wherein the conventional additive is growth medium such as agar-agar from 10 to 16 g. 4. A culture medium as claimed in any preceding claim wherein the conventional additive is vegetable oil selected from the group consisting of mustard oil, sunflower oil, sesame oil, linseed oil, coconut oil and olive oil from 15 to 25 ml. 5. A culture medium as claimed in any preceding claim wherein the conventional additive is one or more antibiotic from 5 to 10 µg. 6. A culture medium as claimed in claim 5 wherein the antibiotic is selected from streptomycin and penicillin, in an amount of 5 µg per antibiotic. 7. A process for the preparation of a modified culture medium as claimed in claim 1 and comprising from 30 to 33 g of a sugar selected from sucrose, galactose and maltose, yeast extract from 6 to 8 g, peptone from 2 to 4 g, oleic acid from 2 to 5 ml and glycerol from 3 to 5 ml and the balance, if any, of conventional additives selected from antibiotics, nutrients and oils, said process comprising intimately mixing said sugar, peptone, oleic acid, glycerol and yeast extract and autoclaving the mixture to obtain the modified culture medium the balance, if any, of conventional additive selected from antibiotics, nutrients and oils are added either prior to or subsequent to the autoclaving. 8. A process as claimed in claim 7 wherein the culture medium is prepared in the form of a broth. 9. A process as claimed in any preceding claim wherein the conventional additive is growth medium such as agar-agar from 10 to 16 g and is added to the mixture prior to autoclaving. 10. A process as claimed in any preceding claim wherein the conventional additive is growth medium such as agar-agar from 10 to 16 g and is added to the mixture subsequent to the autoclaving. 11. A process as claimed in any preceding claim wherein the conventional additive is vegetable oil selected from the group consisting of mustard oil, sunflower oil, sesame oil, linseed oil, coconut oil and olive oil from 15 to 25 ml. 12. A process as claimed in any preceding claim wherein the conventional additive is one or more antibiotic from 5 to 10 µg and is added to the mixture prior to the autoclaving. 13. A process as claimed in claim 12 wherein the antibiotic is selected from streptomycin and penicillin, in an amount of 5 µg per antibiotic. 14. A modified culture medium substantially as described hereinbefore and with reference to the foregoing examples. 15. A process for the preparation of a modified culture medium substantially as described hereinbefore and with reference to the foregoing examples.

Documents:

735-del-2002-abstract.pdf

735-del-2002-claims.pdf

735-del-2002-correspondence-others.pdf

735-del-2002-correspondence-po.pdf

735-del-2002-description (complete).pdf

735-del-2002-form-1.pdf

735-del-2002-form-19.pdf

735-del-2002-form-2.pdf

735-del-2002-form-3.pdf

735-del-2002-form-4.pdf

735-del-2002-gpa.pdf