Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres

Definitions

• This invention relates to a process for the preparation of compositions comprising dried microbial cells in a stasis state, to such compositions and to living cultures prepared therefrom.
• US 3,897,307 discloses d) the use of a combination of an ascorbate compound and a glutamate or aspartate as stabiliser for lactic acid producing bacterial cells and (n) the use of certain sugars, particularly mositol at a concentration of 25mg/ml sample solution, as a cryoprotectant where such bacterial cells are freeze-dried.
• Mugnier et al, Applied and Environmental Microbiology, 1985, pp 108-114 discloses the use of polysaccharide gels in combination with certain nutritives, eg C and C compounds, as the matrix for
• a stabilised dried composition comprising microbial cells in a stasis state suspended in a collapsed matrix
• stasis state we mean that the cells are not metabolising, dividing or growing (but are recoverable f subjected to a suitable treatment)
• recovery we mean cells which on exposure to suitable conditions (xe rehydration and source of nutrient) are capable of growth and division.
• viable cells we mean cells which on exposure to suitable conditions de rehydration and source of nutrient) are capable of growth and division.
• the matrix has shrunk and become less porous allowing little penetration of low MW diffusive species into the matrix, eg it absorbs little water vapour on exposure to humid air;
• the matrix has experienced a temperature above its glass transition temperature (Tg) such that viscous flow thereof has occurred leading to a substantial reduction in surface area/volume ratio and encapsulating the cells m a low porosity protective coating.
• Tg glass transition temperature
• a process for the preparation of a stabilised dried composition comprising microbial cells in a stasis state suspended in a matrix which process comprises the steps of:
• A mixing the microbial cells w th an aqueous composition comprising the material from which the matrix will be derived;
• composition prepared n Step B is stored at a temperature below the Tg of the matrix, le the composition has a Tg above its anticipated storage temperature.
• the composition prepared in Step B is preferably dried further, so-called “secondary drying", to increase the Tg of the matrix such that the composition is stabilised to a broader range of storage conditions, le it can be stored at a higher temperature.
• the microbial cells of which the stabilised dried composition according to the present invention is comprised are preferably bacterial cells. However, we do not exclude the possibility that alternative microbial cells may be used, eg fungi, yeast, etc. Where the microbial cells are bacterial cells they are preferably Gram-negative bacterial cells although we do not exclude the possibility that they may be cells of a Gram-positive bacteria As examples of such Gram-negative cells may be mentioned inter alia Pseudomonas fluorescens, Escherich a coll and rhizosphere-assoc ated bacteria.
• Step A of the process according to the present invention is between 10 /ml and 10 /ml and preferably is between 10 /ml and 10 /ml.
• the material which is mixed with the microbial cells m Step A of the process according to the present invention is a polyhydroxy compound, eg a polyol such as mannitol, mositol, sorbitol, galactitol, or preferably a carbohydrate, more preferably a sacchande.
• a polyhydroxy compound eg a polyol such as mannitol, mositol, sorbitol, galactitol, or preferably a carbohydrate, more preferably a sacchande.
• the material is a sacchande it may be a di-sacchande, a tri-saccharide, an oligo-saccharide, or preferably a monosaccharide.
• mono-saccharides may be mentioned into al a hexoses, eg rhamnose, xylose, fructose, glucose, mannose and galactose.
• disaccharides may be mentioned inter alia maltose, lactose, trehalose and sucrose.
• a t ⁇ saccharide may be mentioned raff ose.
• oligosaccharides may be mentioned maltodextrms .
• the concentration of the polyhydroxy compound used in the mixture in Step A of the process according to the present invention is between lOmg/10 and lOOOmg/10 cells and preferably between 200mg/10 cells and 400mg/10 cells.
• concentrations from which a collapsed matrix can be prepared for a particular polyhydroxy compound For example, we have found that mositol has an optimum concentration at about 45 mg/ml, it causes massive cell damage above 60 mg/ml and does not collapse at below about 25 mg/ml.
• polyhydroxy compounds exhibit protective properties over a wide range of concentrations, whereas certain others above a critical concentration, which appears to be related to the solubility of the polyhydroxy compound m the aqueous medium, exhibit a detrimental effect.
• Figure 1 illustrates in the form of a graph the variation of viability of Pseudomonas fluorescens with mositol (additive) concentration when freeze-dried from water or 0.04M MgSO The vertical axis represents
• Figures 2 to 7 illustrate in the form of graphs the variation of viability of freeze-dried Pseudomonas fluorescens w th monosaccharide concentration for a range of monosaccha ⁇ des.
• Figure 8 illustrates m the form of a graph the variation of cell death rate (k ) with Tg of the matrix and the variation of Tg with relative humidity
• the left-hand vertical axis represents k
• the right-hand vertical axis represents Tg (°C)
• the horizontal axis represents relative humidity (%) .
• the black squares (•) on the graph connected by a solid line plot the cell death rate constant (k ) and the lozenges connected by a broken line plot the glass transition temperature (Tg) .
• the concentration at which the material is effective, ie it collapses without unduly damaging the cells, is dependent on a variety of factors, including inter alia: the volume fraction of the cells in the suspension in Step A; the inherent glass transition temperature of the polyhydroxy compound; the variation in glass transition temperature of the matrix as a function of water concentration therein; and the temperature to which the matrix is exposed during and after freeze-drying.
• the polyhydroxy compound may act as (i) a cryo-protectant at low temperature, particularly against damage by ice-particles during freeze-drying; and /or (ii) a lyo-protectant protecting against damage due to loss of water during drying and/or storing; and /or (iii) a nutrient source during recovery of the cell.
• the microbial cells for use in the process of the present invention may be grown in conventional growth media, eg nutrient broth or tryptone soya broth. They may be harvested at any convenient phase of growth, preferably at early stationary phase.
• a culture is grown in or on a suitable medium, eg liquid or solid plates, to give a desired cell concentration.
• the cells are isolated, typically by centrifugation. They are resuspended in an aqueous composition comprising the material which will form the matrix and optionally certain other additives as mentioned hereinafter.
• the microbial cells used in the process of the present invention are isolated from the growth medium, resuspended in a solution comprising polyhydroxy compound, suitable additives, etc and dried.
• a suitable aqueous medium eg aqueous MgSO solution, or preferably water, containing the polyhydroxy compound.
• the drying in Step B of the process according to the present invention may be carried out by, for example, evaporation, vacuum-drying, spray-drying, air-drying or preferably freeze-drying.
• Step B it is essential to achieve viscous flow during at least the drying step, Step B, or any subsequent step.
• the water content of the dried composition prepared in Step B is less than 15% w/w.
• the composition typically contains one or more suitable additives.
• suitable additives may be mentioned inter alia cryo-protectants, for example sugars or polymeric species, eg polyvinylalcohol, polyvinylpyrrolidone; lyo-protectants, for example sugars or polymeric species, eg polyvinyl alcohol, polyethylene glycol; or preferably anti-oxidants or so-called potentiators, eg ascorbate or glutamate.
• additives for example, so-called bulking agents, for example crystallising sugars, eg mannitol, and osmo-regulants, eg betaine, urea/trimethylamine-N-oxide, proline, sarcosine.
• bulking agents for example crystallising sugars, eg mannitol, and osmo-regulants, eg betaine, urea/trimethylamine-N-oxide, proline, sarcosine.
• compositions according to the present invention wherein the matrix comprises rhamnose.
• Pseudomonas fluorescens was cultured in standard media (double strength nutrient broth) and harvested in early stationary phase by centrifugation. The cell concentrate was resuspended in sterile water and a sufficient volume of an autoclave-sterilised, concentrated rhamnose solution was added to give approximately 200 aliquots of a final concentration of 200 mg of sugar to 2x10 cells in a total volume of 4ml water in 5ml capacity freeze-drying vials.
• the vials were loaded onto the temperature-controlled shelves of a freeze-drying apparatus and the shelf-temperature was driven to -30°C, freezing the contents of the vials and lowering their temperatures to -28°C to -30°C, over a two hour period. Vacuum was applied and primary drying was carried out over a period of 48 hours. The shelf temperature was raised to 0°C and secondary drying was allowed to occur for 24 hours. The vials were brought to room temperature and sealed under vacuum before removal from the freeze-drier.
• the vials were stored at 4°C in vacuo (Example 1) or in humidity controlled air at 21°C (Examples 2-6) .
• the samples were rehydrated in sterile water and viable bacterial cell numbers were determined by serial dilution in water followed by plating onto King's B growth medium. The number of colony-forming units (cfu) on the highest dilution plates was used to calculate the number of bacterial cells per unit volume which survived the freeze-drying and storage conditions .
• CT1 is a Comparative Test with no rhamnose present.
• CT2 is a Comparative Example with no rhamnose present.
• compositions according to the present invention wherein the matrix comprises rhamnose and magnesium sulphate.
• Examples 1-6 The procedure of Examples 1-6 was repeated except that the cell concentrate was resuspended in 0.04M magnesium sulphate instead of sterile water and rhamnose solution was then added.
• the viability of the cells immediately after freeze-drying was 5x10 .
• the freeze-dried cells were stored at the temperatures and for the periods of time shown in Table 3.
• compositions according to the present invention wherein sodium ascorbate and sodium glutamate are present in the matrix.
• Examples 1-6 The procedure of Examples 1-6 was repeated except that concentrated aqueous solutions of sodium ascorbate and sodium glutamate were added to the resuspended cells in water and rhamnose.
• the samples were stored in humid air at 21°C.
• Glucidex IT19 a commercial grade of maltodextrin
• sodium ascorbate sodium ascorbate
• Drying was terminated after approximately 18 hours, at which time the vacuum was of the order of 1 mbar.
• Materials prepared by this method appear as collapsed amorphous matrices with glass transitions exceeding 20°C (when samples were exposed to standard laboratory relative humidities) .

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• 1993
  • 1993-04-28 GB GB939308734A patent/GB9308734D0/en active Pending
• 1994
  • 1994-03-31 GB GB9406552A patent/GB9406552D0/en active Pending
  • 1994-04-18 PL PL94311297A patent/PL311297A1/xx unknown
  • 1994-04-18 CZ CZ952805A patent/CZ280595A3/cs unknown
  • 1994-04-18 CN CN94191905A patent/CN1121731A/zh active Pending
  • 1994-04-18 JP JP6523994A patent/JPH08509374A/ja active Pending
  • 1994-04-18 KR KR1019950704675A patent/KR960701986A/ko not_active IP Right Cessation
  • 1994-04-18 BR BR9406488A patent/BR9406488A/pt not_active Application Discontinuation
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• 1995
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