反应详情

The present invention solves a number of the abovementioned problems and consists firstly of the lyophilisation of purified phages in the presence of a stabilising factor such as trehalose, most preferentially a modified probiotic extract. Lyophilisation is a popular storage method, i.e. of microbiota, which facilitates long-term viability [11]. The effectiveness of the lyophilisation of microorganisms is influenced by a series of factors such as the strain of microorganism, cell shape, the stage and rate of proliferation, medium composition, cellular composition, water and lipid content, cooling and heating rates and the duration of storage [12]. The composition of the medium in which the microorganisms are suspended prior to freezing is extremely important, as the addition of appropriate substances ensures viability following lyophilisation. Initially, glycerol and dimethylsulphoxide were used to protect microbial cells against destruction caused by freezing [13,14]. Protective substances are known, both of low and high molecular weight [15], which rapidly penetrate the cell (within 30 minutes of introduction), including methanol, ethanol, ethylene glycol, propylene glycol, dimethylformamide, methylacetamide and dimethylsulphoxide; as well as slowly penetrating ones like glycol and non penetrating such as glucose, trehalose, dextran, hydroxyethyl amylose, mannitol, sorbitol, albumin, gelatin, other proteins, polyethylene glycol, or polyvinyl alcohol. As a stabilizer and protectant, sorbitol is used in microbiology at concentrations of 1-36% (usu. 9%). Depending on the microorganisms, one observes variable effectiveness of sorbitol treatment. The use of 5% mannitol on organisms yields better effects than sorbitol treatment. Glucose is used at concentrations of 1-18% (usu. 4%). Increased bacterial viability had been noted previously with the use of glucose during storage at −20° C. This has been shown to be effective for T4 bacteriophages (along with saccharose), Enterobacter aerogenes, yeasts and Puccinia spores. The drawbacks of most of the above stabilisers are that these are either liquids, or toxic (i.e. methanol), or immunogenic (i.e. albumin, gelatin), or synthetic substances usually expensive to produce, or very reactive (i.e. glukoza). Trehalose, however, is a natural protectant, occurs in plants and baker's yeast cells (Saccharomyces cerevisiae) and is the only disaccharide to contain two water molecules in its crystal, and is chemically inert. It is used at concentrations of 5-19% (avg. 10%) as a protectant for various viruses, Lactobacillus bulgaricus and other microbiota [12]. Phage preparations can be maintained in a lyophilised form, but due to the loss of activity during lyophilisation, it is recommended to use stabilizers. The present invention relates to a method of stabilising lyophilised phages with a modified probiotic extract. First, purified phage preparations are made using known methods, such as the use of a detergent and ultrafiltration membranes [10] or on an immobilised polylysine column [16], if the detergent deactivates bacteriophages. During research on the conditions for stabilising phages during lyophilisation, it was shown that among the stabilizers used, including trehalose, glucose, sorbitol or probiotic extract, the best results were obtained for trehalose and the probiotic extract. A method according to the present invention lacks the heretofore known drawbacks of lysed phage storage. Storage of bacteriophages T4, Im11 or K1 for two months in lyophilised form with trehalose or probiotic extract at various concentrations guarantees the maintenance of activity levels. After six months of storage, the activity of lyophilised phages only decreased from 8.0×107 pfu/ml do 4.0×106 pfu/ml. This activity level did not decrease and stayed unchanged, when the lyophilisate was maintained in vials under a vacuum. Phage activity was highest at a 2-4% trehalose or extract content, and decreased at higher concentrations of and trehalose, extract or other stabilizers, which means that 2% stabiliser content is an optimum. Lower titres were obtained in 1% glucose. The lowest titres were obtained using sorbitol. Though all four reagents stabilised the phages while maintaining their activity, the best result was produced by the use of 2-4% trehalose or probiotic extract. The novelty of the present invention consists of the lyophilisation and storage of phages under a vacuum in the presence of a probiotic extract as a stabilizer, which is a marked improvement over previously patented methods of storing phage preparations. The method of producing pharmacological forms is simple and requires one step, namely a solution of purified phage molecules is supplemented with a probiotic extract, usu. of Bifidobacterium, to a cencentration of 2%, frozen and lyophilised and apportioned into vials under a vacuum. The present invention relates to a novel pharmacological form of phages, different to any patented thus far.

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2. additionto freezing is extremely important, as the addition of appropriate substances
3. customagainst destruction
4. customthe cell (within 30 minutes of introduction)