AU684072B2 - Viable bacteria - Google Patents

Description

OPI DATE 21/11/94 AOJP DATE 05/01/95 APPLN. ID 65104/94 111111111llllll PCT NUMBER PCT/GB94/0081 111111111111 IIllIIIII I 111 AU9465104 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5: (11) International Publication Number: WO 94/25564 C12N 1/04, 11/04 Al S1 (43) International Publication Date: 10 November 1994 (10.11.94) (21) International Application Number: (22) International Filing Date: Priority Data: 9308734.4 28 April 19 PCT/GB94/0081 18 April 1994 (18.04.94) 93 (28.04.93) (71) Applicant (for all designated States except US): ZENECA LIMITED [GB/GB]; 15 Stanhope Gate, London W1Y 6LN

(GB).

(72) Inventors; and Inventors/Applicants (for US only): RODHAM, David, Kirk [GB/GB]; 19 Moorland Drive, Abbots Lodge, Runcom, Cheshire WA7 6HL CANTWELL, John, Bumett [GB/GB]; 35 Sunnymede Drive, Lydiate, Merseyside L31 2NR (GB).

(74) Agents: HOUGHTON, Malcoln, John et al.; ICI Group Patents Services Dept., Shire Park, P.O. Box 6, Bessemer Road, Welwyn Garden City, Hertfordshire AL7 1HD (GB).

(54) Title: VIABLE BACTERIA (81) Designated States: AU, BB, BG, BR, BY, CA, CN, CZ, FI, GB, HU, JP, KP, KR, KZ, LK, LV, MG, MN, MW, NO, NZ, PL, RO, RU, SD, SI, SK, TT, UA, US, UZ, VN, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published With international search report.

68 40 72 S(57) Abstract Microbial cells are stabilised for storage in the form of a dried composition in a stasis state suspended in a collapsed matrix. The composition is prepared by mixing the microbial cells, for example Gram-negative bacterial cells such as Pseudomonas fluorescens, with an aqueous composition comprising a polyhydroxy compound, preferably a saccharide, from which the matrix will be derived, and drying the mixture under conditions such that viscous flow of the polyhydroxy compound occurs and the matrix collapses but does not unduly damage the cells. The glass transition temperature of the matrix is ideally above the temperature at which the composition is stored.

~ss br I~L~SPIC~ llgl WO 94/25564 PCT/GB94/00811 1 VIABLE BACTERIA 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.

Storage of viable cultures is a recognised problem in the art. For example, US 3,897,307 discloses the use of a combination of an ascorbate compound and a glutamate or aspartate as stabiliser for lactic acid producing bacterial cells and (ii) the use of certain sugars, particularly inositol 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 3 and C12 compounds, as the matrix for 1-3 6-12 freeze-dried bacterial cells. We have found that gel-forming polysaccharides do not collapse on freeze-drying.

Redway et al, Cryobiology, 1974, Vol 11, 73-79 examined certain monosaccharides (concentrations up to 150mg/2 ml sample) and related compounds as media for long-term survival of freeze-dried bacteria.

We have now found that where microbial cells are suspended in a certain matrix as hereinafter defined and dried under certain conditions the short-term viability thereof is improved and that where such dried systems are stored and rehydrated under certain conditions as hereinafter defined the long term viability of the microbial cells is improved.

We have further found surprisingly that collapse of the matrix in which the microbial cells are suspended does not lead to poor short-term viability.

provided a stabilised dried composition comprising microbia ells in a stasis state suspended in a collapsed matrix.

By "stabilised" we mean that the degr ion of the microbial cells is reduced (which degradation would 1 to a loss of recoverable viable cells) By "stasis stf we mean that the cells are not metabolising, dividing o rowing (but are recoverable if subjected to a suitable 4 .ment) According to the first aspect of the present invention there is provided a stabilised dried composition comprising microbial cells in a stasis state suspended in a collapsed matrix, in which the microbial cells are Gram-negative bacterial cells.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

By "stabilised" we mean that the degradation of the microbial cells is reduced (which degradation would lead to a loss of recoverable viable cells).

By "stasis state" we mean that the cells are not metabolising, dividing or growing (but are recoverable if subjected to a suitable treatment).

o t o* *o .o 4U So 0 0o By "recoverable" we mean cells which on exposure to suitable conditions (ie rehydration and source of nutrient) are capable of growth and division.

By "viable cells" we mean cells which on exposure to suitable conditions (ie rehydration and source of nutrient) are capable of growth and division.

By "collapsed" we mean i) that 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; and/or ii) 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 in a low porosity protective coating.

According to the second aspect of the present invention there is provided a process for the preparation of a stabilised dried composition comprising microbial cells, in which the microbial cells are Gram-negative bacterial cells, in a stasis state suspended in a matrix which process comprises the steps of: A: mixing the microbial cells with an aqueous composition comprising the material from which the matrix will be derived; B: drying the mixture under conditions such that viscous flow of the material occurs and 20 the matrix collapses but does not unduly damage the cells.

i Preferably the composition prepared in Step B is stored at a temperature below the Tg of the matrix, ie the composition has a Tg above its anticipated storage temperature.

Accordingly, the composition prepared in Step B is preferably dried further, so- 25 called "secondary drying", to increase the Tg of the matrix such that the composition is S*0 stabilised to a broader range of storage conditions, ie 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 Gram-negative bacterial cells.

WN CA.%WINWVORDfliWENDYArYPINGIOr54TDOC WO 94/25564 PCT/GB94/00811 3 Gram-positive bacteria. As examples of such Gram-negative cells may be mentioned inter alia Pseudomonas fluorescens, Escherichia coli and rhizosphere-associated bacteria.

The concentration of the microbial cells in the mixture prepared in Step A of the process according to the present invention is between 10 /ml and 1013/ml and preferably is between 1010/ml and 1011/ml.

The material which is mixed with the microbial cells in Step A of the process according to the present invention is a polyhydroxy compound, eg a polyol such as mannitol, inositol, sorbitol, galactitol, or preferably a carbohydrate, more preferably a saccharide.

Where the material is a saccharide it may be a di-saccharide, a tri-saccharide, an oligo-saccharide, or preferably a monosaccharide. As examples of mono-saccharides may be mentioned into alia hexoses, eg rhamnose, xylose, fructose, glucose, mannose and galactose. As examples of disaccharides may be mentioned inter alia maltose, lactose, trehalose and sucrose. As an example of a trisaccharide may be mentioned raffinose. As examples of oligosaccharides may be mentioned maltodextrins.

The concentration of the polyhydroxy compound used in the mixture in Step A of the process according to the present invention is between 10mg/10 and 1000mg/1010 cells and preferably between 200mg/1010 cell. and 400mg/10 0 cells. The skilled person will be able to find by simple experiment the concentrations from which a collapsed matrix can be prepared for a particular polyhydroxy compound. For example, we have found that inositol 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.

Certain of the 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 in the aqueous medium, exhibit a detrimental effect.

The present invention is further illustrated by reference to the accompanying drawings which illustrate, by way of example only, compositions according to the present invention.

In the drawings: Figure 1 illustrates in the form of a graph the variation of viability of Pseudomonas fluorescens with inositol (additive) concentration when WO 94/25564 PCT/GB94/00811 4 freeze-dried from water or 0.04M MgSO The vertical axis represents viability in parts per billion (ie 1E+09=10 ppb, 1E+08=10 ppb, etc) and the horizontal axis represents the concentration of the additive in milligrams per sample. The black squares on the graph plot inositol in magnesium sulphate and the circles plot inositol in water.

Figures 2 to 7 illustrate in the form of graphs the variation of viability of freeze-dried Pseudomonas fluorescens with monosaccharide concentration for a range of monosaccharides. The vertical axis represents viability in parts per billion, in numbers of billions (ie 1E+0=1 billion, 5E-1=0.5 billion, 2E-1=0.2 billion, etc) and the horizontal axis represents the sugar concentration in milligrams per sample. The monosaccharides illustrated are: Figure 2 galactose Figure 5 xylose Figure 3 fructose Figure 6 rhamnose Figure 4 glucose Figure 7 mannose Figure 8 illustrates in 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 1 the right-hand vertical axis represents Tg (OC) and the horizontal axis represents relative humidity The black squares on the graph connected by a solid line plot the cell death rate constant (kl) and the lozenges connected by a broken line plot the glass transition temperature (Tg).

In the drawings, viability is expressed as the number of viable bacteria per 109 viable bacteria in the original suspension, ie it represents the number of bacteria which survive from each one billion bacteria which were viable initially. It is represented as the parts per billion viability (ppb), ie 10 ppb is equivalent to 100% survival and is equivalent to 10% survival, etc.

From Figure 1 it can be seen that at low inositol concentrations the viability of the cells is maintained whereas at higher concentrations, eg greater than 100mg/sample (equivalent to 50mg/ml), cell viability rapidly decreases.

WO 94/25564 PCT/GB94/00811 5 From Figures 2 to 7 it can be seen that certain monosaccha: ides protect the cells from damage at low concentrations, eg less than mg/sample (equivalent to 5mg/ml) and that protection is substantially maintained at high concentrations, eg about 400 mg/sample (equivalent to 130mg/ml).

From Figure 8 it can be seen that where the Tg drops below the storage temperature (represented by the top horizontal line at 21-22 0 the rate of cell death (k increases significantly, illustrating the importance of maintaining a glassy state during storage.

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.

It will be appreciated that the polyhydroxy compound may act as 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 growr 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.

For example, 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.

Preferably, 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.

However, we do not exclude the possibility that the polyhydroxy compound and suitabl' ives, etc are added to the cells in the growth medium and the resulting mixture dried.

WO 94/25564 PCT/GB94/00811 6 Where the microbial cells are resuspended, they are resuspended in a suitable aqueous medium, eg aqueous MgSO 4 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 pref-ably freeze-drying.

As hereinbefore defined it is essential to achieve viscous flow during at least the drying step, Step B, or any subsequent step.

Typically the water content of the dried composition prepared in Step B is less than 15% w/w.

Where the drying in Step B comprises freeze-drying the composition typically contains one or more suitable additives. As examples of 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. We do not exclude the lossibility that other additives may be present, for example, so-called bulking agents, for example crystallising sugars, eg mannitol, and osmo-regulants, eg betaine, urea/trimethylamine-N-oxide, proline, sarcosine.

The present invention is further illustrated by reference to the following Examples.

EXAMPLES 1-6 These Examples illustrate 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 2x1010 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 0

C,

freezing the contents of the vials and lowering their temperatures to -28 0

C

to -30°C, over a two hour period. Vacuum was applied and primary drying WO 94/25564 PCT/GB94/00811 7 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 0 C in vacuo (Example 1) or in humidity controlled air at 21 0 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.

Immediately after freeze-drying the viability of the cells was 3x10 ppb.

The results from bacteria stored at 4 0 C in vacuo are shown in Table 1.

TABLE 1 Example Example Viability (ppb) No.

after storage for (weeks) 3 8 1 ND 5x10 CT1 0 ND: not determined CT1 is a Comparative Test with no rhamnose present.

From Table 1 it can be seen that the presence of a rhamnose matrix improves the viability of the bacteria substantially.

The results from bacteria stored at 21 0 C in controlled humidity chambers are shown in Table 2.

WO 94/25564 PCT/GB94/00811 -8 TABLE 2 Example Rel Viability (ppb) No. humidity after storage for (days) 13 27 34 2 1 8x107 3x10 4x107 3 4 3x10 7x10 2x10 4 9 9x107 5x10 4x10 7 7 6 23 4x10 4x107 8x10 6 44 3x10 1x10 3x10 CT2 1 103 CT2 is a Comparative Example with no rhamnose present.

From Table 2 it can be seen that the presence of a sugar substantially increases the viability even at low RH, ie Ex 2 compared with CT2.

EXAMPLES 7-10 These Examples illustrate compositions according to the present invention wherein the matrix comprises rhamnose and magnesium sulphate.

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.

8 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.

TABLE 3 Example Storage Viability (ppb) after days No. temp 50 100 175 365 7 -20 5x10 5x10 5x10 5x10 8 8 8 8 4 2x108 2x10 1x10 9 15 x10 8 5x10 7 7 20 2x10 ~i WO 94/25564 PCT/GB94/00811 9 It can be seen from Table 3 that the formulation provides substantial protection over a range of temperatures.

EXAMPLES 11-15 These Examples illustrate compositions according to t present invention wherein sodium ascorbate and sodium glutamate are present in the matrix.

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.

8 The viability of the cells immediately after freeze-drying was 2x10 ppb.

The samples were stored in humid air at 21 0

C.

TABLE 4 Example Relative Viability (ppb) Tg No. humidity after storage for (days) °C 34 128 8 8 11 0 1 10 1x10 8 12 14 24 7 13 30 4x10 14 7 6 14 40 9X10 5x10 12 7 3 53 3x10 3x10 8 From Examples 11 and 12 in Table 4 it can be seen that storing the dried cells at temperatures below the Tg of the matrix stabilises the cells for long periods.

The results indicate that the combination of collapsed matrix in a glassy state and the presence of an anti-oxidant provides a matrix which can stabilise the viable cells for long periods of time under relatively harsh environments.

EXAMPLE 16 A 4g pellet of Pseudomonas fluorescens cells, separated from culture 12 media by centrifugation and containing 5.10 viable cells, was mixed with 14g of a commercial grade of maltodextrin (Glucidex IT19) and 1.6g of sodium ascorbate and the material, initially at room temperature, dried WO 94/25564 PCT/GB94/00811 10 under reduced pressure, in which the evaporated water condensed onto an ice trap maintained at -50 0

C.

Drying was terminated after approximately 18 hours, at which time the vacuum was of the order of 1 mbar.

Samples rehydrated into pure water and plated onto Nutrient agar typically showed 50-90% recovery of viable cells.

Materials prepared by this method appear as collapsed amorphous matrices with glass transitions exceeding 20 0 C (when samples were exposed to standard laboratory relative humidities).

L

Claims (13)

1. A stabilised dried composition comprising Gram negative bacterial cells in a stasis state suspended in a collapsed matrix.

2. A composition according to claim 1 in which the Gram-negative cells are Pseudomonas fluorescens, Escherichia coli or rhizosphere-associated bacteria.

3. A process for the preparation of a stabilised dried composition comprising Gram-negative bacterial cells in a stasis state suspended in a matrix which process comprises the steps of: A: mixing the Gram-negative bacterial cells with an aqueous composition comprising the material from which the matrix will be derived; 15 B: drying the mixture under conditions such that viscous flow of the material occurs and the matrix collapses but does not unduly damage the cells.

4. A process according to claim 3 in which the composition prepared in step 20 B is dried further to increase the glass transition temperature of the matrix such that the composition is stabilised to a broader range of storage conditions.

5. A process according to claim 3 or claim 4 in which the concentration of the 25 Gram negative bacterial cells in the mixture prepared in Step A is between 4 /ml and 10 13 /ml.

6. A process according to claim 5 in which the concentration of the cells is between 1010/ml and 10 1 1/ml. II C!WINWORDIJAM~h NODELLTLWI((N9YJ3DO I- 12

7. A process according to any one of claims 3 to 6 in which the material which is mixed with the Gram-negative bacterial cells in Step A of the process is a polyhydroxy compound.

8. A process according to claim 7 in which the polyhydroxy compound is a monosacchariae.

9. A process according to claim 7 or claim 8 in which the concentration of the polyhydroxy compound used in the mixture in Step A is between 10mg/1010 and 1000mg/1010 cells.

A process according to claim 9 in which the concentration of the polyhydroxy compound is between 200mg/1010 cells. 15

11. A composition prepared according to the process of claim 3 which has a glass transition temperature above its anticipated storage temperature.

12. A composition according to claim 1 substantially as hereinbefore described with reference to any one of the examples.

13. A process according to claim 13 substantially as hereinbefore described with reference to any one of the examples. 0 DATED 3 September, 1997 PHILLIPS ORMONDE FITZPATRICK Attorneys for: ZENECA LIMITED 11 CAWINWORDJAC'KILEODELETa511MN94.DOC