Patent GB-1101440-A (GB)
Title: Method for cultivating micro-organisms having exoenzymeproducing ability
IPC: C12N9/00
Filed: 1965-04-06
Abstract: 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 least 0.5% by weight in the case of cultivation in a solid medium.  Many species of micro-organisms are particularly mentioned, including bacteria, actinomycetes, yeasts and fungi.  Proteolytic enzyme-producing: Bacillus, Streptomyces and Aspergillus.  Lipolytic enzyme-producing: Candida, Mucor, Rhizopus and Aspergillus.  Amylolytic enzyme-producing: Rhizopus, Aspergillus and Bacillus.  Species of the following are also mentioned: Pseudomonas, Serratia, Clostridinium, Leuconostoc, Lactobacillus, Saccharomyces, Torula, Penicillium, 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 proteolytic, 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.ALSO: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 least 0.5% by weight in the case of cultivation in a solid medium. Many species of micro-organisms are particularly mentioned, including bacteria, actinomycetes, yeasts and fungi.  Proteolytic enzyme-producing: Bacillus, Streptomyces and Aspergillus.  Lipolytic enzyme-producing: Candida, Mucor, Rhizopus and Aspergillus. Amylolytic enzyme-producing: Rhizopus, Aspergillus and Bacillus. Species of the following are also mentioned: Pseudomonas, Serratia, Clostridium, Leuconostoc, Lactobacillus, Saccharomyces, Torula, Penicillium, Absidia, Fusarium and Sclerotinia.  It is stated that phosphatidyl choline, phosphatidyl ethanolamine and inositol have no appreciable ability in enhancing the production of enzymes.  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. 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. Examples are directed to comparisons of the proteolytic, 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.  Proteolytic enzyme activity is measured by mixing 1 ml. of the enzyme solution with 1 ml. of 2% milk casein solution containing 0.2 mol buffer solution of pH 7.0, incubating the mixture for 60 minutes at 35 DEG 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 Folin'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 5 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-Schoorl's 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.ALSO: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.